Endothelial Cell Medium

The recombinant vesicular stomatitis virus–vectored Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine, while effective and well-tolerated, exhibits notable reac... More

The recombinant vesicular stomatitis virus–vectored Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine, while effective and well-tolerated, exhibits notable reactogenicity, manifesting in expected adverse events (AEs), such as fever, headache, and pain, along with rare, unexpected AEs, including skin lesions, cutaneous vasculitis, and transient arthritis. The presence or absence of AEs following rVSVΔG-ZEBOV-GP vaccination is associated with a specific innate plasma signature. This study aims to elucidate in vitro the tropism of the vaccine for different cell types derived from tissues previously reported to be involved in the unexpected AEs. Upon in vitro infection with rVSVΔG-ZEBOV-GP, various cell types, such as synoviocytes, fibroblasts, keratinocytes, and endothelial cells (except chondrocytes), demonstrate productive infection, which in dermal fibroblasts triggered the release of many innate plasma signature markers, including keratinocytes’ pro-inflammatory and proapoptotic cytokines such as OSM and TRAIL. Infected monocytes from buffy coats, activated by infection, produce most innate plasma signature markers. In co-culture, rVSVΔG-ZEBOV-GP-infected monocytes serve as a source to synoviocyte infection, resulting in distinct kinetics modulation in innate biomarkers (transcription and secretion) and upregulation of specific genes, such as NEDD8 and SIGLEC-1, which have been associated with inflammatory arthritis in animal models. Altogether, our work, based on in vitro studies, provides insights into the possible mechanisms of rVSVΔG-ZEBOV-GP underlying the observed reactogenicity by showing tropism of the vaccine for off-target cells derived from AE-affected compartments (skin, joints, vessels). Furthermore, in vitro interaction with infected monocytes modulates the innate response of synoviocytes. Less

Introduction: Exposure to indoor aerosols that include fine particulate matter 2.5 (PM2.5) and microorganisms has been implicated in various health issues, including neur... More

Introduction: Exposure to indoor aerosols that include fine particulate matter 2.5 (PM2.5) and microorganisms has been implicated in various health issues, including neurodegenerative diseases. Objectives: The major components of indoor aerosols could induce the blood–brain barrier (BBB) leakiness and β amyloid (Aβ) aggregation, potentially exacerbating Alzheimer’s disease (AD) pathology. Methods: The main components of aerosols were collected by an intelligent sampler and an airborne microorganism sampler, respectively. The PM2.5 was characterized with SEM and UV–vis spectrophotometer. The microorganisms were identified by 16S rRNA gene sequencing. The Aβ aggregation was studied by thioflavin T kinetic assay and circular dichroism spectroscopy. The BBB models were constructed by seeding astrocytes and human brain microvascular endothelial cells on the membrane of transwell inserts. Moreover, the BBB leakiness induced by PM2.5, Staphylococcus aureus (S. aureus), and the Aβ aggregates was evaluated by immunofluorescence imaging and transwell assay both in vitro and in vivo. Results: The PM2.5 owns the size of 112 ± 35.41 nm and the surface charge of −0.125 mV. PM2.5 and S. aureus can independently disrupt the BBB integrity both in vitro and in vivo by down-regulating adherens and tight junction proteins including zonula occludens-1, VE-cadherin, occludin, and claudin-5. Furthermore, PM2.5 and S. aureus accelerated Aβ aggregation into neurotoxic oligomers and fibrils. In combined exposures, PM2.5 + Aβ or S. aureus + Aβ act synergistically to exacerbate BBB permeability and cytotoxicity of endothelial cells, astrocytes, and neuron cells, creating a vicious cycle of the BBB dysfunction and neurodegeneration. Conclusions: These findings establish PM2.5 and S. aureus as dual environmental drivers of BBB compromise and Aβ pathology, offering novel mechanistic insights and emphasizing the urgent need for strategies to mitigate indoor aerosol-related health risks for AD patients. Less

Porous metallic platforms are promising as polymer-free drug-eluting stent carriers, but the intrinsic bioactivity of the carrier material itself is often overlooked. Thi... More

Porous metallic platforms are promising as polymer-free drug-eluting stent carriers, but the intrinsic bioactivity of the carrier material itself is often overlooked. This study systematically investigated the microstructure-dependent biological effects of porous nickel-titanium (NiTi) alloys fabricated by metal injection molding with a powder space-holder. We created NiTi samples with controlled porosities of 0% (P0), 20.8% (P20), and 34.9% (P40). Microstructural characterization revealed that the high-porosity alloys possessed a highly interconnected pore network, which significantly amplified the effective surface area of the as-sintered pore walls containing Ni-rich precipitates. This architectural feature led to a direct correlation between porosity and nickel (Ni²⁺) ion release, while titanium release remained negligible. In vitro, the released Ni²⁺ ions induced a dose-dependent inhibition of proliferation and migration and promoted apoptosis in both human endothelial cells and smooth muscle cells (SMCs). This intrinsic "cytotoxicity" translated into a significant therapeutic effect in a rat aortic implantation model, where increased porosity led to a marked, dose-dependent reduction in neointimal formation. The most porous P40 group exhibited the strongest anti-restenotic effect, driven by a non-selective inhibition of both SMC accumulation and re-endothelialization. Despite this potent local bioactivity, all materials demonstrated excellent hemocompatibility and systemic safety. These findings reveal that the inherent properties of porous NiTi can be harnessed as a "drug-free" therapeutic modality, opening a new paradigm for designing functional vascular implants where bioactivity is controlled through material architecture. Less

Chronic kidney disease (CKD) is characterized not only by progressive fibrosis but also by systemic endothelial dysfunction and inflammation. Platelets, traditionally rec... More

Chronic kidney disease (CKD) is characterized not only by progressive fibrosis but also by systemic endothelial dysfunction and inflammation. Platelets, traditionally recognized for their role in hemostasis, also serve as key modulators of endothelial activation and immune cell recruitment. Platelet activation is commonly observed in patients with CKD and contributes to the proinflammatory environment. Although platelet-endothelial interactions are well-characterized in cardiovascular disease, their role in renal endothelial dysfunction and inflammation remains poorly understood. To investigate this, we used the unilateral ureteral obstruction (UUO) model in mice, to examine how platelet activation influences endothelial responses and monocyte/macrophage recruitment in the early phase of renal fibrosis development. Platelet depletion reduced the number of infiltrating macrophages in kidney tissue, decreased expression of endothelial activation and inflammation markers, and preserved the peritubular capillary (PTC) integrity. Further in vitro studies using human umbilical vein endothelial cells (HUVECs) showed that activated platelets induced endothelial dysfunction and inflammation, in line with the in vivo findings. To recapitulate the vascular microenvironment, we performed a shear flow-based transmigration assay. Monocyte adhesion and transendothelial migration significantly increased when endothelial cells were pretreated with activated platelets compared to unstimulated controls. Moreover, the presence of platelets on the inflamed endothelium further enhanced monocyte migration, suggesting a synergistic effect in promoting immune cell recruitment. Collectively, our findings highlight that activated platelets contribute to endothelial dysfunction, inflammation, and monocyte infiltration in early kidney injury, suggesting their potential as a therapeutic target to mitigate microvascular injury and preserve renal vascular integrity in kidney disease. Less

Background: Glucocorticoids (GCs) are a key pathogenic factor in steroid-induced avascular necrosis of the femoral head (SANFH). GCs can directly damage bone microvascula... More

Background: Glucocorticoids (GCs) are a key pathogenic factor in steroid-induced avascular necrosis of the femoral head (SANFH). GCs can directly damage bone microvascular endothelial cells (BMECs), leading to impaired intraosseous blood supply. Recent studies suggest the Hippo signaling pathway may be involved in the pathogenesis of SANFH; however, its role in vascular endothelial repair and angiogenesis remains unclear. This study aims to investigate the therapeutic effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on SANFH, with a particular focus on their protective or reparative mechanisms on BMECs. Methods: In vivo, a SANFH mouse model is established and divided into NC, MPS, and hUC-MSCs groups, followed by Micro-CT imagin, hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) (n = 8 per group). In vitro, BMECs are divided into NC, dexamethasone (Dex), hUC-MSCs, and Fer-1 groups to analyze cellular biological behaviors. Target protein expression is assessed using Western blotting and immunofluorescence microscopy. Ferroptosis-related markers are detected via biochemical assays. Mitochondrial ultrastructural changes are observed using transmission electron microscopy. Results: In vivo, the MPS group exhibited significant bone cavitation, sparse trabeculae, and disrupted trabecular architecture in the femoral head. The hUC-MSCs group showed marked improvement in bone microstructure, HE staining showed a significant decrease in the empty lacunae rate in the femoral head, and IHC results revealed markedly increased expression of cluster of differentiation 31 (CD31) and vascular endothelial growth factor (VEGF). In vitro, Dex stimulation suppressed BMECs proliferation. In Dex-treated cells, levels of intracellular reactive oxygen species (ROS), lipid peroxides, ferrous ion (Fe2+), malondialdehyde (MDA), acyl-CoA synthetase long chain family member 4 (ACSL4) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) were all increased, while expression of glutathione (GSH) and glutathione Peroxidase 4 (GPX4) was reduced. Transmission electron microscopy revealed plasma membrane rupture and reduction or loss of mitochondrial cristae. Furthermore, Dex promoted Hippo-mediated phosphorylation of Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ), upregulated NOX4 expression, and suppressed CD31 and VEGF expression. Following hUC-MSCs treatment, BMECs demonstrated enhanced proliferation, migration, and tube-forming capacity. Cellular GSH and GPX4 levels increased, antioxidant capacity was restored, peroxide accumulation decreased, and cells were protected from ferroptosis-effects comparable to those in the Fer-1 group. Additionally, hUC-MSCs inhibited YAP/TAZ phosphorylation and promoted elevated expression of CD31 and VEGF. Conclusions: These findings suggest that hUC-MSCs may attenuate Dex-induced ferroptosis in BMECs, enhance BMEC migration and angiogenesis, and improve femoral head microstructure in SANFH through modulation of the Hippo-YAP/TAZ signaling pathway. This study provides novel insights into the therapeutic potential of hUC-MSCs for SANFH. Keywords: human umbilical cord mesenchymal stem cells; steroid-induced osteonecrosis of the femoral head; microvascular endothelial cells; ferroptosis; Hippo pathway Less

Ménière’s disease (MD) is thought to involve dysfunction of the blood–labyrinth barrier, but circulating mechanisms of endothelial injury remain poorly understood. ... More

Ménière’s disease (MD) is thought to involve dysfunction of the blood–labyrinth barrier, but circulating mechanisms of endothelial injury remain poorly understood. The present study investigated whether cell-free DNA (cfDNA) and inflammatory mediators in plasma contribute to vascular stress and barrier disruption in MD. cfDNA levels were significantly elevated in plasma from patients compared with plasma from healthy controls. Exposure of primary human stria vascularis endothelial cell monolayers to plasma from MD patients led to decreased transepithelial electrical resistance and a significant increase in FITC-dextran permeability, indicating impaired barrier function. MD plasma also induced higher lactate dehydrogenase release and pronounced F-actin disorganization with reduced syndecan-1 expression, consistent with endothelial cytotoxicity and glycocalyx degradation. DNase I partially reversed these effects, implicating extracellular DNA as a key driver. Furthermore, IL-1β, CCL3 (MIP-1α), and CCL27 were elevated in MD plasma. Collectively, our data support a model in which cfDNA and inflammatory mediators cooperatively induce endothelial injury, cytoskeletal remodeling, and glycocalyx shedding, leading to blood–labyrinth barrier weakening. Targeting extracellular DNA or glycocalyx preservation may represent a novel strategy to protect inner ear vascular integrity and modify disease progression in MD, and cfDNA-related readouts may be promising biomarkers of endothelial damage. Keywords: Ménière’s disease; cell-free DNA; blood–labyrinth barrier; cytokine; endothelial glycocalyx Less

Humans are increasingly exposed to "eco-friendly" biodegradable microplastic pollution, whose usage in packaging and medical applications is growing exponentially. The bi... More

Humans are increasingly exposed to "eco-friendly" biodegradable microplastic pollution, whose usage in packaging and medical applications is growing exponentially. The bioplastic polylactic acid (PLA) has recently been demonstrated to release large quantities of oligomeric lactic acid (OLA) nanoplastics causing adverse health effects. No research has reported on intrauterine biodistribution of OLA, and how gestational exposure may impact on early development of the fetus. Here, we reveal that OLA plastics can readily breach the placental barrier and accumulate in various fetal organs in a mouse model. Gestational exposure to environmentally relevant dose of OLA impairs vasculature development, causing intrauterine growth restriction in the pups. Mechanistically, OLA causes blockage of the vascular endothelial growth factor pathway and abnormal physiological development of placenta, which is mediated by the obstruction of transcription factor GATA2 translocation into the nucleus. This study highlights the potential developmental health effect of oligomer nanoparticles released from biodegradable PLA plastic. Less

Background Endothelial cells play a central role in the pathogenesis of sepsis. Currently, effective therapeutic options for sepsis remain limited. Baicalein (BAI) is a ... More

Background Endothelial cells play a central role in the pathogenesis of sepsis. Currently, effective therapeutic options for sepsis remain limited. Baicalein (BAI) is a compound with multiple bioactivities. This study aims to investigate the protective effects of BAI against lipopolysaccharide (LPS)-induced endothelial cell injury and to explore the underlying molecular mechanisms. Methods Bioinformatics tools, including RNA sequencing (RNA-seq), immune cell infiltration analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, were utilized to explore the molecular mechanisms of BAI in human umbilical vein endothelial cells (HUVECs) induced by LPS. The LPS-induced HUVECs model was used to assess the effects of BAI through CCK8 assays, cell permeability assays, and RT-qPCR. Results RNA-seq analysis revealed a set of differentially expressed genes (DEGs) shared between the Control group vs LPS group and LPS vs Baicalein group, including vascular cell adhesion molecule 1 (VCAM1), phosphoinositide-specific phospholipase C X-domain containing 1 (PLCXD1), and MIR3142 host gene (MIR3142HG), a long non-coding RNA. GO, KEGG, and Reactome enrichment analyses indicated that the DEGs were primarily enriched in TNF signaling, NF-κB signaling, and immune regulation pathways. Molecular docking and molecular dynamics simulation analyses revealed that BAI exhibits a strong binding affinity for key targets VCAM1 and PLCXD1. ROC analysis revealed that these core genes, VCAM1 and PLCXD1, exhibited significant diagnostic potential for sepsis. The cell experimental results demonstrated that BAI significantly alleviated the expression levels of inflammatory markers (such as IL-6 and IL-1β) and reduced endothelial cell permeability induced by LPS in HUVECs. Conclusion BAI may alleviate LPS-induced endothelial cell injury by modulating the inflammatory response and immune microenvironment through the regulation of MIR3142HG, VCAM1, and PLCXD1 targets. This study provides new molecular targets and theoretical insights for sepsis therapy. Less

Forkhead box P1 (Foxp1) plays a key role in endothelial preservation and the suppression of inflammatory responses. However, its exact function in valvular calcification ... More

Forkhead box P1 (Foxp1) plays a key role in endothelial preservation and the suppression of inflammatory responses. However, its exact function in valvular calcification (VC) of chronic kidney disease (CKD) remains unclear. Our previous work showed that parathyroid hormone (PTH) contributes to CKD-related VC by driving valvular endothelial-to-mesenchymal transition (EndMT). Whether Foxp1 inhibits PTH-induced EndMT remains determined. This study investigates whether Foxp1 reduces VC by restraining EndMT and delineates the underlying mechanisms. The Foxp1-KI mice were crossed with the Cdh5-Cre (ERT2) mice to generate endothelial-specific Foxp1 knock-in mice (named Foxp1EC-OE). Here, it was found that Foxp1EC-OE mice exhibited reduced valvular calcification compared with CKD mice. Foxp1 overexpression suppressed valvular EndMT and inhibited Notch signaling by binding the Jagged-1 promoter and repressing its transcription. This led to decreased TGF-β1 secretion, thereby attenuating osteogenic transition of valvular interstitial cells. Additionally, Foxp1 restored endothelial integrity and reduced high mobility group box 1 protein (HMGB1)-driven macrophage infiltration. These findings identify Foxp1 as a key regulator of VC via Notch-mediated EndMT inhibition, revealing regulatory nodes in the Jagged-1/Notch pathway that may be amenable to future pharmacological targeting for CKD-related VC. Less

This study investigated whether quercetin protects endothelial cells from Oxidized Low-Density Lipoprotein (Ox-LDL)-induced injury by inhibiting ferroptosis via the sirtu... More

This study investigated whether quercetin protects endothelial cells from Oxidized Low-Density Lipoprotein (Ox-LDL)-induced injury by inhibiting ferroptosis via the sirtuin 3 (SIRT3)/AMP-activated protein kinase (AMPK) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with Ox-LDL either in the presence or absence of quercetin, and transfected with SIRT3 siRNA. Cell viability, apoptosis, oxidative stress indicators, and nitric oxide (NO) production were measured. Ferroptosis-related proteins and signaling pathway proteins were analyzed by Western blot. Quercetin improved cell viability, reduced apoptosis, and attenuated oxidative injury as well as ferroptosis among Ox-LDL-stimulated HUVECs. Mechanistically, quercetin upregulated the ferroptosis-protective factors glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) while downregulating the pro-ferroptotic enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4), thereby restoring antioxidant capacity and resistance to ferroptosis. In addition, quercetin markedly increased SIRT3 expression and AMPK phosphorylation, whereas SIRT3 silencing significantly attenuated both the molecular alterations and the associated functional improvements. Quercetin protected endothelial cells against Ox-LDL-provoked injury via suppressing ferroptosis through SIRT3/AMPK pathway activation. These findings identify a previously unrecognized SIRT3/AMPK-dependent mechanism underlying quercetin-mediated endothelial protection and provide mechanistic insight relevant to atherosclerosis. Less

Chronic kidney disease (CKD) is associated with inflammation and cardiovascular complications and is partly exacerbated by the uremic toxin indoxyl sulfate (IS). IS is kn... More

Chronic kidney disease (CKD) is associated with inflammation and cardiovascular complications and is partly exacerbated by the uremic toxin indoxyl sulfate (IS). IS is known to activate the aryl hydrocarbon receptor (AhR) to promote vascular inflammation. On the other hand, avenanthramide C (Ave), an oat-derived polyphenol, has antioxidative and anti-inflammatory properties. Therefore, we investigated whether Ave can suppress IS-induced inflammatory responses. Less

Background Aging is characterized by progressive immunosenescence and inflammaging, in which impaired gut barrier and dysregulated mucosal immunity exacerbate systemic s... More

Background Aging is characterized by progressive immunosenescence and inflammaging, in which impaired gut barrier and dysregulated mucosal immunity exacerbate systemic senescence. While probiotics modulate gut health, their role in mitigating age-related immune dysfunction via specific microbial metabolites remains unclear. This study aims to investigate the effects of Bacillus velezensis DS2, a novel probiotic, in alleviating inflammaging, with a focus on tryptophan-metabolic signaling and immune regulation. Results In senescent endothelial cells, DS2-sourced indole-3-lactic acid (ILA) activated aryl hydrocarbon receptor (AhR) signaling. This activation reduced the expression of senescence markers (p16, γ-H2A.X) and decreased the levels of pro-inflammatory molecules (ICAM-1, VCAM-1). In aged mice, DS2 supplementation increased the abundance of beneficial bacteria, including Lactobacillus and Ligilactobacillus. DS2 administration also elevated plasma ILA and IL-22 levels, and reduced intestinal permeability. This was evidenced by the expansion of IL-22-producing type 3 innate lymphoid cells (ILC3s) and activation of the AhR-IL-22 axis. Consequently, DS2 enhanced intestinal barrier integrity and mitigated systemic inflammation (TNF-α, IL-6). Exogenous ILA was sufficient to recapitulate these benefits by potentiating gut mucosal immunity and attenuating inflammaging, as all these effects were abolished by the AhR antagonist CH223191. Conclusions We demonstrate that DS2 mitigates inflammaging by producing ILA, which acts as a key metabolite to activate the AhR-IL-22-ILC3 axis. Our findings highlight the potential of targeting the gut-immune axis with specific probiotics as a novel strategy against age-related immune decline. Less

Pulmonary hypertension of the newborn (PHN) is a common pulmonary vascular disease in newborns, affecting the prognosis of affected infants and even leading to death. Cur... More

Pulmonary hypertension of the newborn (PHN) is a common pulmonary vascular disease in newborns, affecting the prognosis of affected infants and even leading to death. Currently, there is still no specific targeted prevention and treatment program available. In recent years, research on the regulatory mechanism of endothelial-to-mesenchymal transition (EndMT) has become a hot topic, with the interplay between miRNAs and the Wnt signaling pathway playing an important role. MiR-29 is highly expressed in distal pulmonary arteriolar smooth muscle cells and endothelial cells. In miR-29 gene knockout mice, pathological changes in lung tissue can be observed, indicating an obstruction in the maturation of smooth muscle cells in the distal pulmonary arterioles. LRP6 is an important Wnt co-receptor in the Wnt signaling pathway, and the Targetscan website predicts a binding site between miR-29 and LRP6. Therefore, our studies establish a role for decreased miR29a in the endothelial mesenchymal transition through activation of the LRP6-Wnt signaling pathway in neonatal hypoxia-mediated PAH. By generating hypoxic newborn rat models of pulmonary artery hypertension and hypoxic injury models of pulmonary vascular endothelial cells, we observed the presence of EndMT in the model group, as well as the activation of the LRP6 and Wnt/β-catenin signaling pathways and the differential expression of the miR-29 family, particularly miR-29a. Subsequently, We used cell transfection technology to overexpress/knock down miR-29a and LRP6, respectively, to observe changes in EndMT-related indicators and successfully found that miR-29a and LRP6 can negatively and positively regulate the occurrence of EndMT, respectively. Finally, the dual-luciferase reporter gene assay confirmed that LRP6 can directly bind to miR-29a-3p. These findings provide experimental evidence of the potential of miR-29 as a novel target for the prevention and treatment of PHN. Less

Objective Lupus nephritis (LN) is characterised by renal endothelial dysfunction, which contributes to progressive kidney injury. Endothelial nitric oxide synthase (eNOS)... More

Objective Lupus nephritis (LN) is characterised by renal endothelial dysfunction, which contributes to progressive kidney injury. Endothelial nitric oxide synthase (eNOS) plays a modulating role in LN, as genetic ablation of the eNOS enzyme worsens disease. Serum from patients with active LN induces uncoupling of eNOS homodimers, leading to superoxide (SO) rather than nitric oxide (NO) production by eNOS. This uncoupling is reversed with L-sepiapterin (L-Sep). This study was designed to further examine changes in gene expression in glomerular endothelial cells induced by LN serum and whether treatment with L-Sep can ameliorate these changes. Less

Background Achieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize ne... More

Background Achieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize new vessels. This study investigates the in vivo effects of Sema4D-Plexin-B1 signaling on stem cells from human exfoliated deciduous teeth (SHED)-supported angiogenesis, focusing on its mechanism in PDGF-BB secretion. We also explore macrophages as an endogenous source of Sema4D for vascular stabilization. Methods The in vivo Matrigel plug angiogenesis assay was conducted to examine the impact of Sema4D on vessel formation and stabilization supported by SHED. Knockdown of Plexin-B1 in human umbilical vein endothelial cells (HUVECs) and the application of PDGFR-β inhibitors were utilized to explore the fundamental regulatory mechanisms. Furthermore, the m6A methylation levels of total RNA and the expression of Methyltransferase-like 3 (METTL3) were assessed under conditions of Sema4D treatment in vitro. An ELISA was employed to measure the levels of Sema4D in the supernatants derived from THP-1 cell-mediated macrophages. Additionally, a three-dimensional vasculature-on-a-chip microfluidic device was employed to investigate the role of M2c macrophage-derived Sema4D in the stabilization of vascular structures. Results Sema4D induced the formation of a greater number of perfused vessels by HUVECs and enhanced the coverage of these vessels by SM22α-positive SHED (SM22α+SHED). Conversely, the knockdown of the Plexin-B1 receptor in HUVECs or inhibition of PDGFR-β reversed the Sema4D-induced vascular stabilization, thereby confirming the regulatory role of the Plexin-B1/PDGF-BB axis in the recruitment of mural cells mediated by Sema4D. Mechanistically, Sema4D was found to upregulate the expression of methyltransferases, specifically METTL3, and to elevate the level of m6A modification in HUVECs. This modification was determined to be critical for enhancing PDGF-BB secretion, suggesting that Sema4D activates an epigenetic regulatory mechanism. Additionally, we investigated the secretion of Sema4D by various macrophage phenotypes, identifying that M2c macrophages secrete significant levels of Sema4D. This secretion recruits SM22α+SHED as mural cells by inducing endothelial PDGF production on a vasculature-on-a-chip platform, indicating a potential role for macrophages in facilitating vascular stabilization. Conclusions Sema4D acts on Plexin-B1, inducing METTL3-mediated PDGF-BB secretion to recruit SHED to stabilize vessels. Macrophages could be a key source of Sema4D for vascular stabilization. Less

In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored ho... More

In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored how T lymphocytes promote breast cancer cell penetration through the blood brain barrier (BBB). An in vitro BBB model was employed to study the effects of T lymphocytes on BBB trespassing capacity of three different breast carcinoma cell lines. Differential protein expression was explored by comparing the proteomes of the breast cancer cells before and after co-culture with activated T lymphocytes using liquid chromatography-mass spectrometry (LC-MS). siRNA was used to silence protein expression in the breast cancer cells to study contribution to in vitro BBB passage. Furthermore, protein expression in primary breast cancer tissues was explored and related to brain-metastatic potential. Co-culturing with activated T lymphocytes or their conditioned medium (CM) resulted in increased passage through the in vitro BBB. The effects were less for cell line MDA-MB-231-B2M2 (brain affinity) as compared to MDA-MB-231 and SK-BR-7. Mass spectrometry-based proteomics revealed significant alterations in the expression of 35 proteins by the breast cancer cell lines upon T cell contact. Among the proteins is coronin-1 A, a protein related to cell motility. Knockdown of CORO1A in the breast cancer cells reduced their ability to cross the artificial BBB to 60%. The effects were significantly less for the cell line derived from breast cancer with affinity for brain. The expression of coronin-1A was confirmed by immunohistochemistry and RT-PCR of 52 breast cancer samples of patients with metastasized breast cancers, with and without brain locations. Lastly, CORO1A upregulation was validated in a publicly available mRNA expression database from 204 primary breast cancers with known metastatic sites. We conclude that T lymphocytes trigger cancer cells to express proteins including coronin-1A that enable the cancer cells to cross an in vitro BBB. In addition, a prominent role of coronin-1A in the formation of cerebral metastases in breast cancer patients is strongly suggestive by its upregulation in tissue samples of breast cancer patients with brain metastases. Less

Introduction: Diabetic nephropathy (DN) is a long-term loss of renal function occurring in the diabetic patients, leading to 5 million deaths in 2015, and this number is ... More

Introduction: Diabetic nephropathy (DN) is a long-term loss of renal function occurring in the diabetic patients, leading to 5 million deaths in 2015, and this number is dramatically growing annually. Due to unsatisfied outcome of current treatment, there is urgent need to develop more effective therapeutic drugs for DN. Less

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular event... More

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular events are poorly understood. Herein, we show that bortezomib exposure leads to microglia activation and cognitive impairment, this occurs along with decreased nuclear translocation of TFEB (transcription factor EB), which is linked to macroautophagy/autophagy disorder, STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL23A (interleukin 23 subunit alpha) expression. Pharmacological enhancement of TFEB nuclear translocation by digoxin restores lysosomal function and reduces STAT3-dependent endothelial IL23A secretion. As a consequence, we found that brain endothelial-specific ablation of Il23a ameliorated both microglia activation and cognitive dysfunction. Thus, the endothelial TFEB-STAT3-IL23A axis in the brain represents a critical cellular event for initiating bortezomib-mediated aberrant microglial activation and synapse engulfment. Our results suggest the reversal of TFEB nuclear translocation may provide a novel therapeutic approach to prevent symptoms of cognitive dysfunction during clinical use of bortezomib. Less

Biodegradable shape memory polymers (SMP) with suitable transition temperatures (Tr) and mechanical properties are highly demanded in biomedical field as deployable medic... More

Biodegradable shape memory polymers (SMP) with suitable transition temperatures (Tr) and mechanical properties are highly demanded in biomedical field as deployable medical devices. Herein, we report a 4D printing shape memory Inferior Vena Cava Filters (IVCFs), an implantation device, which could prevent the fatal pulmonary embolism, to exemplify the applicability of the biodegradable shape memory polymer in biomedical device field. The IVCF composed of poly(glycerol sebacate) acrylate-co-hydroxyethyl methacrylate (PGSA-co-HEMA) was digital light processing (DLP) 3D printed. The appropriate mechanical property and Tr = 37.8 °C, which is close to human body temperature, was tailored by tuning the ratio of the raw material. PGSA-PHEMA presents an excellent cytocompatibility, hemocompatibility and histocompatibility as implants. Besides, in vitro degradation results indicate the biodegradability but withhold the mechanical properties within the service time. Furthermore, the simulated filter deploying and fully emboli interception verifies the successful realization of the concept of rapid, minimally invasive and controllable implantation of the 4D printing of IVCFs through the SMP transformation process, and the feasibility of the filter as well. Therefore, this work provides a new biocompatible SMP and offers a new strategy for developing deployable medical devices. Less

RNA-binding proteins (RBPs) are involved in the regulation of RNA splicing, stability, and localization. How RBPs control the development of atherosclerosis, is not fully... More

RNA-binding proteins (RBPs) are involved in the regulation of RNA splicing, stability, and localization. How RBPs control the development of atherosclerosis, is not fully understood. To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in atherosclerosis. We made a comprehensive work to integrate analyses of differentially expressed genes, including differential RBPs, and variable splicing characteristics related to different stages of atherosclerosis in dataset GSE104140. A total of 3712 differentially expressed genes (DEGs) were identified, including 2921 upregulated genes and 791 downregulated genes. Further analysis screened out 54 RBP genes, and 434 AS genes overlapped DEGs. We selected high expression ten RBP genes (SAMHD1, DDX60 L, TLR7, RBM47, MYEF2, RNASE6, PARP12, APOBEC3G, SMAD9, and RNASE1) for co-expression analysis. Meanwhile, we found seven regulated alternative splicing genes (RASGs) (ABI1, FXR1, CHID1, PLEC, PRKACB, BNIP2, PPP3CB) that could be regulated by RBPs. The co-expression network was used to further elucidate the regulatory and interaction relationship between RBPs and AS genes. Apoptotic process and innate immune response, revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to atherosclerosis. In addition, 26 of the 344 alternative splicing genes regulated by the above 10 RBPs were transcription factors (TFs), We selected high expression nine TFs (TFDP1, RBBP7, STAT2, CREB5, ERG, ELF1, HMGN3, BCLAF1, and ZEB2) for co-expression analysis. The target genes of these TFs were mainly enriched in inflammatory and immune response pathways that were associated with atherosclerosis. indicating that AS abnormalities of these TFs may have a function in atherosclerosis. Furthermore, the expression of differentially expressed RBPs and the alternative splicing events of AS genes was validated by qRT-PCR in umbilical vein endothelial cells (HUVEC). The results showed that RBM47 were remarkedly difference in HUVEC treated with ox-LDL and the splicing ratio of AS in BCLAF1which is regulated by RBM47 significantly changed. In conclusion, the differentially expressed RBPs identified in our analysis may play important roles in the development of atherosclerosis by regulating the AS of these TF genes. Less

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence ... More

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV. Less

Trans, trans-2,4-decadienal (tt-DDE) is a highly reactive unsaturated aldehyde that widely present in foods. This study aimed to investigate the toxic effects of tt-DDE e... More

Trans, trans-2,4-decadienal (tt-DDE) is a highly reactive unsaturated aldehyde that widely present in foods. This study aimed to investigate the toxic effects of tt-DDE exposure on human umbilical vein endothelial cells (HUVECs) and the possible protective effects of green tea catechins. tt-DDE exposure induced pyroptosis and NLRP3 inflammasome activation in endothelial cells, as evidenced by increased LDH release and PI-positive cells, and elevated protein expressions of NLRP3, cleaved caspase-1, and GSDMD-N. NLRP3 inhibitor (MCC950) efficiently suppressed the tt-DDE-induced NLRP3 inflammasome activation and pyroptosis. Additionally, epigallocatechin-3-gallate (EGCG) showed the strongest activity among the four green tea catechins, which significantly alleviated tt-DDE-induced cytotoxicity. Moreover, EGCG effectively attenuated tt-DDE-induced endothelial cell pyroptosis and dysfunction by inhibiting NLRP3 inflammasome. Results of cultured mesenteric arteries further confirmed that EGCG prevented tt-DDE-induced endothelial dysfunction and pyroptosis. These results provide novel insights into tt-DDE-induced endothelial injury, and demonstrate the protective role of EGCG against tt-DDE-associated endothelial toxicity. Less

Background Neonatal hypoxic ischemic encephalopathy (HIE) is a main factor of neonatal death and permanent neurologic deficit. This study sought to investigate the functi... More

Background Neonatal hypoxic ischemic encephalopathy (HIE) is a main factor of neonatal death and permanent neurologic deficit. This study sought to investigate the functional role of hsa_circ_0007706 (circ_0007706) in modulating neonatal HIE. Methods In vitro HIE cell model was established in hBMVECs under the condition of oxygen‑glucose deprivation/reperfusion (OGD/R) treatment. qRT-PCR analysis was utilized for detecting the level of circ_0007706, microRNA-579–3p (miR-579–3p) and TNF receptor-associated factor 6 (TRAF6). RNase R treatment and Oligo (dT) 18 primers were employed to verify the features of circ_0007706, and nucleocytoplasmic separation was conducted for determining the location of circ_0007706. CCK-8 assay, EdU assay, and flow cytometry were carried out to measure cell proliferation and apoptosis, respectively. The protein expression of Bax, Bcl-2 and TRAF6 was detected using western blot. Meanwhile, the levels of the pro-inflammatory factors were determined via ELISA. SOD activity and MDA level were assessed via the respective kits. Besides, dual-luciferase reporter assay and RNA pull-down were used to identify the association between miR-579–3p and circ_0007706 or TRAF6. Results Circ_0007706 was elevated in HIE newborns and OGD/R cell model. Knockdown of circ_0007706 greatly alleviated OGD/R-induced injury, inflammatory response and oxidative stress. We found that miR-579–3p was a direct target of circ_0007706, and miR-579–3p inhibitor could reverse the impact of circ_0007706 knockdown on OGD/R-caused cell damage in hBMVECs. In addition, miR-579–3p directly interacted with TRAF6, and the protective effects of miR-579–3p on OGD/R-induced injury in hBMVECs were harbored by TRAF6 overexpression. Our data indicated that circ_0007706 knockdown could downregulate the expression of TRAF6 by sponging miR-579–3p in OGD/R-treated hBMVECs. Conclusion This study demonstrated that circ_0007706 knockdown assuaged HIE-induced injury by decreasing TRAF6 expression via targeting miR-579–3p. Less

Angiogenesis is a significant pathogenic characteristic of diabetic microangiopathy. Advanced glycation end products (AGEs) are considerably elevated in diabetic tissues ... More

Angiogenesis is a significant pathogenic characteristic of diabetic microangiopathy. Advanced glycation end products (AGEs) are considerably elevated in diabetic tissues and can affect vascular endothelial cell shape and function. Regulation of the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway is a critical mechanism in the regulation of angiogenesis, and VEGFR2 activity can be modified by post-translational changes. However, little research has been conducted on the control of small ubiquitin-related modifier (SUMO)-mediated VEGFR2 alterations. The current study investigated this using human umbilical vein endothelial cells (HUVECs) in conjunction with immunoblotting and immunofluorescence. AGEs increased Nrf2 translocation to the nucleus and promoted VEGFR2 expression. They also increased the expression of sentrin/SUMO-specific protease 6 (SENP6), which de-SUMOylated VEGFR2, and immunofluorescence indicated a reduction in VEGFR2 accumulation in the Golgi and increased VEGFR2 transport from the Golgi to the cell membrane surface via the coatomer protein complex subunit beta 2. VEGFR2 on the cell membrane was linked to VEGF generated by pericytes, triggering the VEGF signaling cascade. In conclusion, this study demonstrates that SENP6 regulates VEGFR2 trafficking from the Golgi to the endothelial cell surface. The SENP6-VEGFR2 pathway plays a critical role in pathological angiogenesis. Less

Autophagy is closely associated with atherosclerosis and other cardiovascular diseases (CVD). Compound Danshen prescription is widely used as a clinical antiatherosclerot... More

Autophagy is closely associated with atherosclerosis and other cardiovascular diseases (CVD). Compound Danshen prescription is widely used as a clinical antiatherosclerotic drug. In our previous studies, we have shown that the combined active component, ginsenoside Rg1-notoginsenoside R1-protocatechualdehyde (RRP), can effectively alleviate endothelial dysfunction and reduce atherosclerotic plaques. However, the association between cellular senescence, caused by reduced autophagy, and atherosclerosis remains unclear. In this study, we investigated whether RRP can enhance autophagy and alleviate cell senescence through the AMPK pathway. Our results showed that RRP reduced the secretion of inflammatory factors in the serum of atherosclerotic mice, enhanced autophagy, and alleviated aortic aging in mice, thus reducing atherosclerotic plaques. In human aortic endothelial cells (HAECs), RRP effectively enhanced autophagy and inhibited senescence by activating the AMPK pathway. When AMPKα was silenced, the effect of RRP was inhibited, thus reversing its antiaging effect. Overall, our results show that RRP regulates autophagy through the AMPK pathway, thereby inhibiting cell senescence and alleviating the progression of atherosclerosis, suggesting that RRP may be a potential candidate drug for the treatment of atherosclerosis. Less

Background The activation of HIF-1α/CXCR4 pathway in liver sinusoidal endothelial cells (LSECs) could downregulate CXCR7, leading to the capillarization of LSECs to prom... More

Background The activation of HIF-1α/CXCR4 pathway in liver sinusoidal endothelial cells (LSECs) could downregulate CXCR7, leading to the capillarization of LSECs to promote hepatic fibrosis. However, the mechanism between CXCR4 and CXCR7 is still undefined. The aim is to investigate the role of PDGF-BB in the dedifferentiation of LSECs and hepatic stellate cells (HSCs) activation. Methods The activation of HIF-1α/CXCR4 pathway in two kinds of liver fibrosis models were observed. The effects of HIF-1α, CXCR4, PDGF-BB on the dedifferentiation of LSECs were investigated by using the inhibitors of HIF-1α, CXCR4 or PDGFR-β separately or transfecting with a CXCR4 knockdown lentiviral vector. In addition, the relationship between LSECs and HSCs was demonstrated by co-culture of LSECs and HSCs using the transwell chamber. Results CXCR4 upregulation and CXCR7 downregulation were accompanied by LSECs capillarization and HSCs activation both in CCl4-induced and BDL-induced fibrotic liver. In vitro, downregulation of HIF-1α significantly descreased CXCR4 and CD31 expression, and enhanced the expressions of CXCR7, CD44 and LYVE1. Downregulation of CXCR4 in LSECs significantly downregulated PDGF-BB, PDGFR-β and CD31, and enhanced CXCR7, CD44 and LYVE1 expression, while the expression of HIF-1α did not change significantly. STI571, a PDGF receptor inhibitor, could significantly downregulate PDGFR-β and increase the expression of CXCR7 to inhibit the dedifferentiation of LSECs. In addition, alleviateion the dedifferentiation of LSECs could decrease the expression of PDGFR-β of HSCs, then inhibiting the activation of HSCs. Conclusions This study revealed that HIF-1α/CXCR4/PDGF-BB/CXCR7 axis promoted the dedifferentiation of LSECs, consequently triggering HSCs activation and liver fibrosis.Background The activation of HIF-1α/CXCR4 pathway in liver sinusoidal endothelial cells (LSECs) could downregulate CXCR7, leading to the capillarization of LSECs to promote hepatic fibrosis. However, the mechanism between CXCR4 and CXCR7 is still undefined. The aim is to investigate the role of PDGF-BB in the dedifferentiation of LSECs and hepatic stellate cells (HSCs) activation. Methods The activation of HIF-1α/CXCR4 pathway in two kinds of liver fibrosis models were observed. The effects of HIF-1α, CXCR4, PDGF-BB on the dedifferentiation of LSECs were investigated by using the inhibitors of HIF-1α, CXCR4 or PDGFR-β separately or transfecting with a CXCR4 knockdown lentiviral vector. In addition, the relationship between LSECs and HSCs was demonstrated by co-culture of LSECs and HSCs using the transwell chamber. Results CXCR4 upregulation and CXCR7 downregulation were accompanied by LSECs capillarization and HSCs activation both in CCl4-induced and BDL-induced fibrotic liver. In vitro, downregulation of HIF-1α significantly descreased CXCR4 and CD31 expression, and enhanced the expressions of CXCR7, CD44 and LYVE1. Downregulation of CXCR4 in LSECs significantly downregulated PDGF-BB, PDGFR-β and CD31, and enhanced CXCR7, CD44 and LYVE1 expression, while the expression of HIF-1α did not change significantly. STI571, a PDGF receptor inhibitor, could significantly downregulate PDGFR-β and increase the expression of CXCR7 to inhibit the dedifferentiation of LSECs. In addition, alleviateion the dedifferentiation of LSECs could decrease the expression of PDGFR-β of HSCs, then inhibiting the activation of HSCs. Conclusions This study revealed that HIF-1α/CXCR4/PDGF-BB/CXCR7 axis promoted the dedifferentiation of LSECs, consequently triggering HSCs activation and liver fibrosis. Less

Background Distraction osteogenesis (DO) is an efficacious but lengthy procedure to reconstruct segmental bone defects under the principle of tension-stress, during which... More

Background Distraction osteogenesis (DO) is an efficacious but lengthy procedure to reconstruct segmental bone defects under the principle of tension-stress, during which the periosteum-mediated mechanical stimulation plays a pivotal role. Inspired by the dynamic process of DO and the mechanical stimulation of periosteum, a new design of bionic periosteum was developed to simulate the mechanical transduction of natural periosteum for the application in DO procedure. Methods In this study, an injectable organic-inorganic hybrid hydrogel was developed based on a novel combination of the PEGylated poly (glycerol sebacate) (PEGS) polymer network and in situ formed CaP nanoparticles (ICPNs). Rat bone marrow mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cultured and tested in vitro to evaluate biocompatibility, cell adhesion, proliferation, and pro-osteogenic and pro-angiogenic activity. In vivo experiments were conducted in the rat tibial model of distraction osteogenesis. Results The developed nanocomposite hydrogels exhibited excellent injectability, robust bone adhesion, superior stretchability, and enhanced osteogenic activity. The results of in vitro and in vivo studies showed that PEGS/ICPN hydrogels could promote new bone formation and mineralization during the dynamic distraction process through the synergistic effects of angiogenesis and osteogenesis. Conclusions This periosteum-inspired nanocomposite hydrogel represents a mechanobiology approach for effectively restoring large bone defects through the dynamic DO process. Less

Air pollution induces neurodegeneration, including cognitive deficits, neuroinflammation, and disruption of the blood–brain barrier. The mechanisms underlying air pollu... More

Air pollution induces neurodegeneration, including cognitive deficits, neuroinflammation, and disruption of the blood–brain barrier. The mechanisms underlying air pollution-mediated neurodegeneration have not yet been fully elucidated given the limited knowledge on intercellular interactions. A brain-on-a-chip platform is presented comprising neurons, glia, and brain endothelial cells (bECs; neuro-glia-vascular, NGV) and diesel exhaust particle (DEP)-induced neurodegeneration is evaluated with a particular focus on the intercellular interactions. DEP exposure in the NGV model yields Alzheimer's disease-like signatures, including amyloid beta accumulation, tau phosphorylation, hydrogen peroxide (H2O2)/reactive oxygen species (ROS) production, and neuronal cell death. bEC-secreted granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates microglial activation and the overproduction of H2O2/ROS in microglia, suggesting that the bEC-microglia-neuron is a neurodegeneration cascade. Pharmacological inhibition at each step of the cascade, including GM-CSF neutralization, microglial activation suppression, and ROS scavenging, prohibits neurodegeneration in the NGV model. Therefore, intercellular interactions should be further studied of air pollution-induced neurodegeneration. Less

Purpose Angiogenesis is involved in many pathological and physiological processes and is mainly driven by hypoxia. Karyopherin subunit alpha 2 (KPNA2), a member of the nu... More

Purpose Angiogenesis is involved in many pathological and physiological processes and is mainly driven by hypoxia. Karyopherin subunit alpha 2 (KPNA2), a member of the nuclear transport protein family, was recently shown to be induced by hypoxia in various types of tumours, so we aimed to investigate the role and mechanism of KPNA2 in angiogenesis under hypoxia. Materials and methods After overexpression or knockdown of KPNA2 in human umbilical vein endothelial cells (HUVEC) by adenovirus vector infection, the tube formation, proliferation and migration of HUVEC under hypoxia were detected by tubule formation assay, 5-ethynyl-2′-deoxyuridine (EdU) staining and Transwell assay, respectively. After overexpression or knockdown of KPNA2 in a murine hindlimb ischemia model by local injection of purified adenovirus vector into the gastrocnemius muscle, blood flow changes were examined with a laser Doppler system. Changes in KPNA2-binding proteins under hypoxia were detected by immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP). The effect of KPNA2 on signal transducer and activator of transcription 3 (STAT3) was detected by Western blotting and quantitative RT‒PCR. Results KPNA2 was upregulated in the HUVEC hypoxia model and murine hindlimb ischemia model. Overexpression of KPNA2 increased the proliferation, migration and tube formation of HUVEC under hypoxia, while knockdown of KPNA2 reduced the proliferation, migration and tube formation of HUVEC. Overexpression of KPNA2 promoted the restoration of blood flow in the murine hindlimb ischemia model, while knockout of KPNA2 inhibited the restoration of blood flow in the murine hindlimb ischemia model. Mechanistically, hypoxia promoted the binding of STAT3 to KPNA2. Overexpression of KPNA2 promoted STAT3 phosphorylation and then upregulated vascular endothelial growth factor (VEGF) and angiopoietin 2(ANGPT2), whereas knockdown of KPNA2 inhibited STAT3 phosphorylation and then downregulated VEGF and ANGPT2. Conclusion Our study demonstrates that hypoxia promotes the binding of STAT3 to KPNA2 and KPNA2 promotes angiogenesis under hypoxia by promoting the binding of STAT3 and JAK1 and regulating STAT3 phosphorylation. Less

Testicular endothelial cells have been found to play an important role in spermatogenesis and fertility, but their mechanism is obscure. Exosomes released by various cell... More

Testicular endothelial cells have been found to play an important role in spermatogenesis and fertility, but their mechanism is obscure. Exosomes released by various cells are recognized as cell–cell communication mediators during the initiation and progression of many diseases. Therefore, the current study aimed to investigate the protein and miRNA components of human testicular endothelial cell-derived exosomes (HTEC-Exos) and to explore their potential effects on spermatogenesis. In this study, HTEC-Exos were first isolated by the ultracentrifugation method, and then identified by nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting. The characteristics of HTEC-Exos were examined by liquid chromatography–mass spectrometry and microRNA (miRNA) chip analysis. Bioinformatics analysis was performed to explore the potential role of the exosomal content on spermatogenesis. A total of 945 proteins were identified, 11 of which were closely related to spermatogenesis. A total of 2578 miRNAs were identified. Among them, 30 miRNAs demonstrated potential associations with male reproductive disorders, such as azoospermia, and spermatogenesis disorders. In particular, 11 out of these 30 miRNAs have been proven to be involved in spermatogenesis based on available evidence. This study provides a global view of the proteins and miRNAs from HTEC-Exos, suggesting that HTEC-Exos may function as potential effectors during the process of spermatogenesis. Less

Background: The kava-kava plant (Piper methysticum) is traditionally consumed by the pacific islanders and has been linked to be involved in several biological activities... More

Background: The kava-kava plant (Piper methysticum) is traditionally consumed by the pacific islanders and has been linked to be involved in several biological activities. Flavokawain B is a unique chalcone, which can be found in the roots of the kava-kava plant. In this study, the operational mechanism of the anti-cancer activity of a synthetic Flavokawain B (FKB) on two breast cancer cell lines, MCF-7 and MDA-MB231 was investigated. Method: Several in vitro assays were attempted such as MTT, flow cytometry of cell cycle analysis, annexin V analysis, and JC-1 analysis to detect apoptosis. Moreover, in vitro metastasis assays were also performed such as transwell migration assay, invasion assay, rat aorta ring and HUVEC tube formation. Molecular analysis of related genes and proteins were conducted using real-time PCR and proteome profiler analysis. Results: Based on our results, apoptosis was induced when both MCF-7 and MDA-MB231 were treated with FKB. A significant G2/M arrest was seen in MDA-MB231 cells. Additionally, FKB also inhibited the in vitro migration and invasion in MDA-MB231 cells in a dose dependent manner. Moreover, FKB can be a potential inhibitor in angiogenesis as it suppressed the formation of vessels in HUVEC cells as well as in the ex-vivo rat aortic ring assay. Conclusion: Our findings suggested that FKB also regulated several receptor tyrosine kinases. Overall, FKB is not only a potential candidate to be an anti-cancer agent, but as an anti-metastatic agent as well. Less

Carbon plasma nanocoatings with controlled fraction of sp3-C bonding were deposited on TiO2 nanorod arrays (TNAs) by DC magnetic-filtered cathodic vacuum arc deposition (... More

Carbon plasma nanocoatings with controlled fraction of sp3-C bonding were deposited on TiO2 nanorod arrays (TNAs) by DC magnetic-filtered cathodic vacuum arc deposition (FCVAD). The cytocompatibility of TNA/carbon nanocomposites was systematically investigated. Human umbilical vein endothelial cells (HUVECs) were cultured on the nanocomposites for 4, 24, and 72 h in vitro. It was found that plasma-treated TNAs exhibited excellent cell viability as compared to the untreated. Importantly, our results show that cellular responses positively correlate with the sp3-C content. The cells cultured on high sp3-C-contented substrates exhibit better attachment, shape configuration, and proliferation. These findings indicate that the nanocomposites with high sp3-C content possessed superior cytocompatibility. Notably, the nanocomposites drastically reduced platelet adhesion and activation in our previous studies. Taken together, these findings suggest the TNA/carbon scaffold may serve as a guide for the design of multi-functionality devices that promotes endothelialization and improves hemocompatibility. Less

Around 7-17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cro... More

Around 7-17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells from reaching the brain could provide a relevant and important strategy. For that purpose, an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells' adhesion capacities. Less

Diabetic retinopathy, a leading cause of visual loss and blindness, is characterized by microvascular dysfunction. Hyperglycemiais considered the major pathogenic factor ... More

Diabetic retinopathy, a leading cause of visual loss and blindness, is characterized by microvascular dysfunction. Hyperglycemiais considered the major pathogenic factor for diabetic retinopathy and is associated with increased oxidative stress in the retina.In this study, we investigated the potential protective effects of Panax notoginseng Saponins (PNS) in retinal capillary endothelialcells (RCECs) exposed to high glucose conditions. We found a pronounced increase in cell viability in rat RCECs incubated withboth PNS and high glucose (30 mM) for 48 h or 72 h. The increased viability was accompanied by reduced intracellular hydrogenperoxide (H2O2) and superoxide (O2−), decreased mitochondrial reactive oxygen species (ROS), and lowered malondialdehyde(MDA) levels. PNS also increased the activities of total superoxide dismutase (SOD), MnSOD, catalase (CAT), and glutathioneperoxidase (GSH-PX). The glutathione (GSH) content also increased after PNS treatment. Furthermore, PNS reduced NADPHoxidase 4 (Nox4) expression. These results indicate that PNS exerts a protective effect against high glucose-induced injury in RCECs,which may be partially attributed to its antioxidative function. Less

Ectopic apoptosis of vascular cells plays a critical role in the early stage development of diabetic retinopathy (DR). Uncoupling protein 2 (UCP2) is a mitochondrial modu... More

Ectopic apoptosis of vascular cells plays a critical role in the early stage development of diabetic retinopathy (DR). Uncoupling protein 2 (UCP2) is a mitochondrial modulator which protects against endothelial dysfunction. However, the role which UCP2 plays in endothelial apoptosis and its association with DR was unclear. In the present study, we investigated whether UCP2 functioned as an inhibitor of DR in endothelial cells. Firstly, we noted that in UCP2‑knockout mice retinal cell death and damage in vivo was similar to that of db/db diabetic mice. Additionally, UCP2 knockdown induced caspase-3 activation and exaggerated high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, adenovirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs and HG-induced caspase-3 activation. Furthermore, HG treatment resulted in the opening of the permeability transition pore (PTP) and liberation of cytochrome c from mitochondria to the cytosol in HUVECs. Notably, UCP2 overexpression inhibited these processes. Furthermore, adenovirus-mediated UCP2 overexpression led to a significant increase in intracellular nitric oxide (NO) levels and a decrease in reactive oxygen species (ROS) generation in HUVECs. Collectively, these data suggest that UCP2 plays an anti-apoptotic role in endothelial cells. Thus, we suggest that approaches which augment UCP2 expression in vascular endothelial cells aid in preventing the early stage development and progression of DR. Less

Background CC-chemokine receptor 7 (CCR7), which plays an important role in cell directional movement, is highly expressed in various cancers and positively related to ly... More

Background CC-chemokine receptor 7 (CCR7), which plays an important role in cell directional movement, is highly expressed in various cancers and positively related to lymph node metastasis. The inflammatory cytokine tumour necrosis factor (TNF)-α promotes tumour progression and lymph node metastasis in gallbladder cancer (GBC). However, the expression of CCR7 in GBC is unclear, and its role in the TNF-α-induced lymphatic metastasis of GBC requires further research. Methods The expression of CCR7 in clinical samples was detected by immunohistochemistry, and the relationship between CCR7 and clinicopathological factors or the TNF-α level of the bile was analyzed. After treatment with various concentrations of TNF-α, CCR7 expression in GBC cell lines was measured by Western blotting. The relative luciferase reporter assay, site-directed mutagenesis and chromatin immunoprecipitation were used to analyze the promoter activity and transcriptional regulation of CCR7. MAPKs inhibitors were used to explore the upstream signalling molecules of AP-1. We established a NOZ cell line stably expressing lentiviral CCR7 shRNA that effectively silenced the expression of CCR7, and to determine the role of TNF-α - CCR7 axis in the migration of GBC cells to the lymphatic system by transwell assays and animal experiments. Results CCR7 was highly expressed in GBC samples. Higher expression of CCR7 was associated with American Joint Committee on Cancer (AJCC) staging and lymph node metastasis. Moreover, we found that CCR7 expression in GBC tissue was positively correlated with the levels of TNF-α in the bile, and that TNF-α enhanced the promoter activity and protein expression of CCR7 through the “ERK1/2-AP-1” and “JNK-AP-1” pathways. Finally, we revealed that TNF-α could promote GBC cell migration to lymphatic endothelial cells or lymph nodes through upregulation of CCR7 in vitro and in vivo. Conclusions Our study suggests that CCR7 is highly expressed in GBC, and mediates the TNF-α-induced lymphatic metastasis of GBC through the “TNF-α - ERK1/2 - AP-1 - CCR7” and “TNF-α - JNK - AP-1 - CCR7” pathways. Less

Background Tumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gall... More

Background Tumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined. Methods The expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment. Results TNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo. Conclusion Taken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway. Keyword: Gallbladder cancer, TNF-α, VEGF-D, Lymphatic metastasis Less

Background: Claudin‑5, claudin‑9, and claudin‑11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeabilit... More

Background: Claudin‑5, claudin‑9, and claudin‑11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin‑5, claudin‑9, and claudin‑11 in hypoxia‑stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported. Methods: In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin‑5, claudin‑9, and claudin‑11 was confirmed. Then, the protein content and distribution of claudin‑9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin‑9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up‑regulates claudin‑9 in hypoxia‑stimulated HCMECs. Results: We found that hypoxia‑suppressed claudin‑9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia‑suppressed claudin‑9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high‑dose TXL promoted the cytomembrane localization of claudin‑9 in hypoxia‑stimulated HCMECs, with attenuation of cell injury. Furthermore, high‑dose TXL elevated the hypoxia‑inhibited H3K9ac in the claudin‑9 gene promoter (F = 37.766; P = 0.000), activating claudin‑9 transcription. Conclusions: The results manifested that TXL reversed the hypoxia‑suppressed claudin‑9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs Less

Objectives. Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented th... More

Objectives. Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented that endothelial cells and osteoblasts play critical roles in multiple phases of bone healing. However, the biological effects of G-CSF on these cells remain controversial. This study aimed to investigate the influence of G-CSF at various concentrations on endothelial cells and osteoblasts. Materials and Methods. Human umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) were treated with G-CSF at 1000, 100, 10, and 0 ng/mL, respectively. The capacity of cell proliferation, migration, and tube formation of HUVECs was evaluated at 72, 8, and 6 hours after treatment, respectively. The capacity of proliferation, differentiation, and mineralization of hOBs was evaluated at 24 hours, 72 hours, and 21 days after treatment, respectively. Results. HUVECs treated with 100 and 1000 ng/mL G-CSF showed a significantly higher value comparing with controls in migration assay (p < 0.001, p < 0.01, resp.); the group treated with 1000 ng/mL G-CSF showed a significantly lower value on tube formation. No significant difference was detected in groups of hOBs. Conclusions. G-CSF showed favorable effects only on the migration of HUVECs, and no direct influence was found on hOBs. Less

The protective effect of high‑density lipoprotein (HDL) on endothelial function is impaired in patients with type 2 diabetes mellitus (T2DM), which may result in athero... More

The protective effect of high‑density lipoprotein (HDL) on endothelial function is impaired in patients with type 2 diabetes mellitus (T2DM), which may result in atherosclerotic complications. Naoxintong (NXT) is a compound preparation that includes Radix Astragali, Angelicae sinensis, Radix Paeoniae Rubra and Ligusticum wallichii. It is widely administered in China to prevent atherosclerotic complications. In the present study, NXT was administered to 69 patients with T2DM. HDLs were isolated from patient blood samples prior to and following the intervention. In vitro endothelial functions of HDL, including proliferation, migration, angiogenesis, and anti‑apoptosis were investigated by bromodeoxyuridine, wound healing, Transwell and Matrigel tube formation assays on human umbilical vein endothelial cells (HUVECs). The results from the present study demonstrated that HUVECs treated with HDL isolated from diabetic patients following NXT therapy exhibited increased proliferative effects (10‑27%; P<0.05), and improved migration ability (15‑35%; P<0.05), anti‑apoptotic function (23‑34%; P<0.05) and angiogenesis (30‑54%; P<0.001). Furthermore, the phosphorylation levels of Akt (26‑36%; P<0.01) and extracellular signal‑regulated kinase (16‑80%; P<0.01) were increased following NXT therapy. The present in vitro study demonstrates that the protective effect of HDL on endothelial function is markedly impaired in diabetic patients who tend to develop atherosclerosis, and the impaired function may be partly abrogated by NXT. Less

Background Recent discoveries of the atherosclerosis-related miRNAs shed new light on the treatment of cardiovascular diseases. Of note, miR-106b ~ 25 cluster and miR-17 ... More

Background Recent discoveries of the atherosclerosis-related miRNAs shed new light on the treatment of cardiovascular diseases. Of note, miR-106b ~ 25 cluster and miR-17 ~ 92 cluster are paralogs. Up till now, plenty of researches have shown the role of miR-17 ~ 92 cluster in tumor and atherosclerosis, but miR-106b ~ 25 cluster has stayed mysterious in atherosclerosis field. This study was designed to investigate how miR-106b functions in the atherosclerosis-related angiogenesis and to explore the functioning processes of miR-106b, so as to seek out a new target for the treatment of atherosclerosis. Methods Up and down regulation of miR-106b expression was achieved through transfection in HUVECs so as to investigate the function of miR-106b. Next we predicted the target genes of miR-106b and detected them using qRT-PCR and Western blot technique. At last, luciferase assay was conducted to verify the direct target gene of miR-106b. Data are expressed as mean ± SEM. Two treatment groups were compared by Mann–Whitney U test or student’s t-test. Results were considered statistically significant when P < 0.05. Results The results showed miR-106b up-regulation groups formed less tubes than control groups while the down-regulation groups showed the opposite. Meanwhile, no obvious effect on apoptosis was observed in endothelial cells. Next we predicted the target genes of miR-106b and finally settled down to MAPK14 (Mitogen-Activated Protein Kinase), STAT3 (Signal Transducers and Activators of Transcription 3), JAK1(Janus Kinase 1) and VEGFA(Vascular Endothelial Growth Factor A) as candidate target genes. Our results revealed over-expressed miR-106b represses STAT3 expression, while miR-106b inhibition resulted in STAT3 up-regulation. Ultimately, luciferase assay confirmed STAT3 mRNA is the direct target of miR-106b. Conclusions Our research demonstrated that miR-106b modulate angiogenesis in endothelial cells through affecting expression of STAT3, which occurs by direct target action. Therefore, we affirmed that miR-106b exerts an anti-angiogenic effect in endothelial cells via STAT3-involved signaling pathway, via directly targeting STAT3. Less

Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and an... More

Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-α)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches including a cell viability assay, reverse transcription and quantitative polymerase chain reaction, western blot, and immunofluorescence staining. We showed that TNF-α induced CD40 expression and ROS production in cultured HUVECs, which were attenuated by resveratrol treatment. Also, resveratrol increased the expression of sirtuin 1 (SIRT1); and repression of SIRT1 by small-interfering RNA (siRNA) and the SIRT1 inhibitor Ex527 reduced the inhibitory effects of resveratrol on CD40 expression and ROS generation. In addition, resveratrol downregulated the levels of p65 and phospho-p38 MAPK, but this inhibitory effect was attenuated by the suppression of SIRT1 activity. Moreover, the p38 MAPK inhibitor SD203580 and the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC) achieved similar repressive effects as resveratrol on TNF-α-induced ROS generation and CD40 expression. Thus, our study provides a mechanistic link between resveratrol and the activation of SIRT1, the latter of which is involved in resveratrol-mediated repression of the p38 MAPK/NF-κB pathway and ROS production in TNF-α-treated HUVECs. Less

Angiogenesis is a critical process in the development of tumor malignancy and occurs at various stages of tumor progression. Interleukin-8 (IL-8) is a pro-angiogenic fact... More

Angiogenesis is a critical process in the development of tumor malignancy and occurs at various stages of tumor progression. Interleukin-8 (IL-8) is a pro-angiogenic factor produced by tumor-infiltrating macrophages that has been revealed to facilitate the development of angiogenesis in various cancers. However, whether IL-8 activates angiogenesis in gastric cancer remains unclear. The present study investigated the effect of IL-8 on the migration and canalization capacities of human umbilical vein endothelial cells (HUVECs). In addition, the protein and messenger RNA (mRNA) expression of selected angiogenesis markers, consisting of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1 and VEGFR-2, were assessed in the HUVECs. The HUVECs were co-cultured with human gastric cancer SGC7901 cells and exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1.0 ng/ml). The migration and canalization abilities of the cells were detected by Transwell chamber and tube formation assays. Protein expression was detected using immunofluorescence and western blot analysis, and mRNA levels were assessed using reverse transcription quantitative polymerase chain reaction. The protein and mRNA levels of VEGF-A, VEGFR-1 and VEGFR-2 were measured in HUVECs cultured for 24 h. IL-8 at concentrations of 0.5, 0.8 and 1.0 ng/ml significantly promoted HUVEC cell migration (P=0.005, P=0.001 and P<0.001, respectively) and tube formation (P=0.039, P=0.003 and P<0.001, respectively). IL-8 at concentrations of 0.2, 0.5, 0.8 and 1.0 ng/ml significantly elevated the protein levels of VEGF-A (P<0.001) and VEGFR-2 (P=0.034, P<0.001, P<0.001 and P<0.001, respectively). IL-8 at concentrations of 0.8 and 1.0 ng/ml significantly elevated the protein levels of VEGF-1 (P=0.037 and P=0.002, respectively). Similarly, IL-8 at concentrations of 0.5, 0.8 and 1.0 ng/ml significantly upregulated the mRNA levels of VEGF-A (P=0.046, P=0.001 and P<0.001, respectively) and VEGFR-1 (P=0.042, P<0.001 and P<0.001, respectively). IL-8 at concentrations of 0.2, 0.5, 0.8 and 1.0 ng/ml significantly upregulated the mRNA levels of VEGFR-2 (P=0.003, P=0.005, P<0.001 and P<0.001, respectively). In conclusion, IL-8 may be a potent promoter of angiogenesis in gastric cancer. Keywords: angiogenesis; gastric cancer; interleukin-8; vascular endothelial growth factor receptor-1; vascular endothelial growth factor receptor-2; vascular endothelial growth factor-A. Less

Objective To assess the feasibility of ultrasound molecular imaging in the early diagnosis of liver ischemia-reperfusion injury (IRI) using a nanoscale contrast agent tar... More

Objective To assess the feasibility of ultrasound molecular imaging in the early diagnosis of liver ischemia-reperfusion injury (IRI) using a nanoscale contrast agent targeting anti-intracellular adhesion molecule-1 (anti-ICAM-1). Methods The targeted nanobubbles containing anti-ICAM-1 antibody were prepared using the avidin-biotin binding method. Human hepatic sinusoidal endothelial cells (HHSECs) were cultured at the circumstances of hypoxia/reoxygenation (H/R) and low temperature. The rabbit liver IRI model (I/R group) was established using the Pringle’s maneuver. The time-intensity curve of the liver contrast ultrasonographic images was plotted and the peak intensity, time to peak, and time of duration were calculated. Results The size of the targeted nanobubbles were 148.15 ± 39.75 nm and the concentration was 3.6–7.4 × 109/ml, and bound well with the H/R HHSECs. Animal contrast enhanced ultrasound images showed that the peak intensity and time of duration of the targeted nanobubbles were significantly higher than that of common nanobubbles in the I/R group, and the peak intensity and time of duration of the targeted nanobubbles in the I/R group were also significantly higher than that in the SO group. Conclusion The targeted nanobubbles have small particle size, stable characteristic, and good targeting ability, which can assess hepatic ischemia-reperfusion injury specifically, noninvasively, and quantitatively at the molecular level. Less

The microenvironment of a tumour is an important factor in ovarian cancer metastasis. The present study aimed to simulate the in vivo microenvironment of an ovarian carci... More

The microenvironment of a tumour is an important factor in ovarian cancer metastasis. The present study aimed to simulate the in vivo microenvironment of an ovarian carcinoma using a co‑culture system consisting of human lymphatic endothelial cells (HLECs) and human ovarian carcinoma cells with directional high lymphatic metastasis (SKOV3-PM4s) in order to investigate the role of both cell types in ovarian carcinoma metastasis. The SKOV3-PM4s cultured in the HLEC-conditioned medium exhibited increased numbers of pseudopodia and mitotic figures, proliferated at a faster rate and exhibited enhanced invasion and migratory abilities. Furthermore, the HLECs cultured in SKOV3‑PM4‑conditioned medium exhibited significant morphological alterations and vacuolisation of the cytoplasm, as well as increased invasion, migratory and tube forming abilities. In addition, spontaneous fusion of the SKOV3-PM4s and HLECs was observed in the co‑culture system using laser confocal microscopy. The gelatin zymography assay demonstrated that matrix metalloproteinase-2, which was downregulated in the SKOV3-PM4s, was upregulated in the co‑culture system. The results of the present study suggested that the invasion ability of the SKOV3‑PM4s was increased in the in vitro co‑culture system of SKOV3-PM4 and HLECs. Therefore, alterations in the cell microenvironment may represent a novel strategy for ovarian cancer therapy. Less

Background Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transfor... More

Background Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor β, and other signals, but the detailed molecular mechanisms remain unclear. Methods and Results Small RNA sequencing initially identified miR‐342‐5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR‐342‐5p targeted endoglin and modulated transforming growth factor β signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR‐342‐5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads–based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR‐342‐5p agomir in P3 pups. Moreover, miR‐342‐5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial–mesenchymal transition. Transfection of endoglin at least partially reversed endothelial–mesenchymal transition induced by miR‐342‐5p. The expression of miR‐342‐5p was upregulated by transforming growth factor β, and inhibition of miR‐342‐5p attenuated the inhibitory effects of transforming growth factor β on lumen formation and sprouting by ECs. In addition, VEGF repressed miR‐342‐5p expression, and transfection of miR‐342‐5p repressed VEGFR2 and VEGFR3 expression and VEGF‐triggered Akt phosphorylation in ECs. miR‐342‐5p repressed angiogenesis in a laser‐induced choroidal neovascularization model in mice, highlighting its clinical potential. Conclusions miR‐342‐5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways. Less

Melittin, a significant constituent of Apis mellifera (honeybee) venom, is a water-soluble toxic peptide that has traditionally been used as an antitumor agent. However, ... More

Melittin, a significant constituent of Apis mellifera (honeybee) venom, is a water-soluble toxic peptide that has traditionally been used as an antitumor agent. However, the underlying mechanisms by which it inhibits tumor cell growth and angiogenesis remain to be elucidated. In the present study, screening for increased cathepsin S (Cat S) expression levels was performed in MHCC97-H cells and various other hepatocellular carcinoma cell lines by reverse transcription-polymerase chain reaction and western blot analysis. A pcDNA3.1-small hairpin RNA (shRNA)-Cat S vector was stably transfected into MHCC97-H cells (shRNA/MHCC97-H) in order to knockdown the expression of Cat S. The effects resulting from the inhibition of Cat S-induced proliferation, invasion and angiogenesis by melittin were examined using cell proliferation, cell viability, flat plate colony formation, migration, wound healing, Transwell migration and ELISA assays. In order to substantiate the evidence for melittin-mediated inhibition of Cat S-induced angiogenesis, Cat S RNA was transfected into primary human umbilical vein endothelial cells (Cat S-HUVECs) to induce overexpression of the Cat S gene. The effects of melittin on HUVECs were examined using Transwell migration and tube formation assays. The findings demonstrated that melittin was able to significantly suppress MHCC97-H cell (Mock/MHCC97-H) proliferation, invasion and angiogenesis, as well as capillary tube formation of Cat S-HUVECs, in a dose-dependent manner. However, proliferation, invasion and angiogenesis in shRNA/MHCC97-H and in native HUVECs (Mock-HUVECs) were unaffected. In addition, melittin specifically decreased the expression of phosphorylated (activated) Cat S, and components of the vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2)/mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK)1/2 signaling pathway in Mock/MHCC97-H cells. In conclusion, the inhibition of tumor cell growth and anti-angiogenic activity exerted by melittin may be associated with anti-Cat S actions, via the inhibition of VEGF-A/VEGFR-2/MEK1/ERK1/2 signaling. Keywords: melittin, cathepsin S, human hepatocellular carcinoma, invasion, angiogenesis Less

Background Endothelial-mesenchymal transition (EndoMT) is a major source of myofibroblast formation in kidney fibrosis. Our previous study showed a profibrotic role for m... More

Background Endothelial-mesenchymal transition (EndoMT) is a major source of myofibroblast formation in kidney fibrosis. Our previous study showed a profibrotic role for matrix metalloproteinase 9 (MMP-9) in kidney fibrosis via induction of epithelial-mesenchymal transition (EMT). Inhibition of MMP-9 activity reduced kidney fibrosis in murine unilateral ureteral obstruction. This study investigated whether MMP-9 also plays a role in EndoMT in human glomerular endothelial cells. Results TGF-β1 (10 or 20 ng/ml) induced EndoMT in HKGECs as shown by morphological changes. In addition, VE-cadherin and CD31 were significantly downregulated, whereas α-SMA, vimentin, and N-cadherin were upregulated. RT-PCR revealed that Snail, a known inducer of EMT, was upregulated. The MMP inhibitor GM6001 abrogated TGF-β1-induced EndoMT. Zymography indicated that MMP-9 was also upregulated in TGF-β1-treated HKGECs. Recombinant MMP-9 (2 μg/ml) induced EndoMT in HKGECs via Notch signaling, as evidenced by increased formation of the Notch intracellular domain (NICD) and decreased Notch 1. Inhibition of MMP-9 activity by its inhibitor showed a dose-dependent response in preventing TGF-β1-induced α-SMA and NICD in HKGECs, whereas inhibition of Notch signaling by γ-secretase inhibitor (GSI) blocked rMMP-9-induced EndoMT. Conclusions Taken together, our results demonstrate that MMP-9 plays an important role in TGF-β1-induced EndoMT via upregulation of Notch signaling in HKGECs. Less

Pygo2 has been discovered as an important Wnt signaling component contributing to the activation of Wnt-target gene transcription. In the present study, we discovered tha... More

Pygo2 has been discovered as an important Wnt signaling component contributing to the activation of Wnt-target gene transcription. In the present study, we discovered that Pygo2 mRNA and protein levels were up-regulated in the majority of (152/209) human brain glioma tissues and five glioma cell lines, and significantly correlated with the age, the WHO tumor classification and poor patient survival. The histone methyltransferase complex components (WDR5, Ash2, and menin, but not CXCC1 or NCOA6) were down-regulated at the promoter loci of Wnt target genes after Pygo2 knockdown, and this was accompanied by the down-regulation of Wnt/β-catenin pathway activity. Further, we demonstrated that the involvement of Pygo2 in the activation of the Wnt pathway in human glioma progression is through up-regulation of the H3K4me3 (but not H3K4me2) by promoting the recruitment of the histone methyltransferase MLL1/MLL2 complex to Wnt target gene promoters. Thus, our study provided evidence that Pygo2 functions as a novel prognostic marker and represents a potential therapeutic target. Less

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal... More

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption. Less

Background Previous studies have shown that high glucose (HG) induced endothelial cell (EC) damage via a phenotypic transition of EC. There is increasing evidence suggest... More

Background Previous studies have shown that high glucose (HG) induced endothelial cell (EC) damage via a phenotypic transition of EC. There is increasing evidence suggesting the role of inflammatory cytokines in mediated HG-induced EC damage. However, little is known about the potential role of interleukin-1β (IL-1β) in the process. The aim of present study was to investigate whether IL-1β mediated HG–induced phenotypic transition in human aortic endothelial cells (HAECs) and to determine the possible underlying mechanism. Methods Primary HAECs were exposed to normal glucose (NG, 5.5 nM), high glucose (HG,30 nM), IL-1β (10 ng/ml), HG + IL-1β (10 ng/ml) and HG + anti-IL-1β antibodies (1000 ng/ml) or HG + IL-1β small interfering RNA (siRNA). Pathological changes were investigated using confocal microscopy and electron microscopy. Confocal microscopy was performed to detect the co-expression of CD31 and fibroblast specific protein 1 (FSP1). To study the effect of protein kinase C-β (PKCβ) activation on IL-1β in HAECs, HAECs were stimulated with 30 nM PMA (PKCβ activator) and 0.3 μM PKCβ inhibition (LY317615) for 48 h in the NG or HG group. The expressions of PKCβ and IL-1β were detected by RT-PCR and Western blot. And the concentration of IL-1β in the supernatant of HAECs was measured by ELISA. The expressions of FSP1, a-SMA and CD31 were detected by Western blot. Results It was shown that the HG resulted in significant increase in the expressions of PKCβ and IL-1β in dose-and time-dependent manners. The HG or exogenous IL-1β alone inhibited the expression of CD31 and markly increased the expressions of FSP1 and α-SMA. Furthermore, we observed that the HG and IL-1β synergistically increased FSP1 and a-SMA expressions compared with the HG or IL-1β alone group (P < 0.05). Confocal microscopy revealed a colocalization of CD31 and FSP1 and that some cells acquired spindle-shaped morphologies and a loss of CD31 staining. Electron microscopy showed that the HG resulted in the increased microfilamentation and a roughened endoplasmic reticulum structure in the cytoplasm. However, the changes above were attenuated by the intervention of anti-IL-1β antibodies or IL-1β siRNA (P < 0.05). In addition, the PMA induced the expressions of PKCβ and IL-1β in HAECs. The PKCβ activation may mediate the effect of the HG on IL-1β production, which could be attenuated by the PKCβ selective inhibitor (LY317615) (P < 0.05). Conclusions Our findings suggested that HG-induced phenotypic transition of HAECs might require IL-β activation via the PKCβ pathway. Less

Inhibition of angiogenesis is considered as one of the desirable pathways for the treatment of tumor growth and metastasis. Herein we demonstrated that a series of pyridi... More

Inhibition of angiogenesis is considered as one of the desirable pathways for the treatment of tumor growth and metastasis. Herein we demonstrated that a series of pyridinyl-thiazolyl carboxamide derivatives were designed, synthesized and examined against angiogenesis through a colony formation and migration assays of human umbilical vein endothelial cells (HUVECs) in vitro. A structure-activity relationship (SAR) study was carried out and optimization toward this series of compounds resulted in the discovery of N-(3-methoxyphenyl)-4-methyl-2-(2-propyl-4-pyridinyl)thiazole-5-carboxamide (3k). The results indicated that compound 3k showed similar or better effects compared to Vandetanib in suppressing HUVECs colony formation and migration as well as VEGF-induced angiogenesis in the aortic ring spreading model and chick embryo chorioallantoic membrane (CAM) model. More importantly, compound 3k also strongly blocked tumor growth with the dosage of 30 mg/kg/day, and subsequent mechanism exploration suggested that this series of compounds took effect mainly through angiogenesis signaling pathways. Together, these results suggested compound 3k may serve as a lead for a novel class of angiogenesis inhibitors for cancer treatments. Less

Emerging evidence has suggested the critical role of endothelial to mesenchymal transition (EndMT) in fibrotic diseases. The present study was designed to examine whether... More

Emerging evidence has suggested the critical role of endothelial to mesenchymal transition (EndMT) in fibrotic diseases. The present study was designed to examine whether EndMT is involved in arsenic trioxide (As2O3)-induced cardiac fibrosis and to explore the underlying mechanisms. Cardiac dysfunction was observed in rats after exposure to As2O3 for 15 days using echocardiography, and the deposition of collagen was detected by Masson’s trichrome staining and electron microscope. EndMT was indicated by the loss of endothelial cell markers (VE-cadherin and CD31) and the acquisition of mesenchymal cell markers (α-SMA and FSP1) determined by RT-PCR at the mRNA level and Western blot and immunofluorescence analysis at the protein level. In the in-vitro experiments, endothelial cells acquired a spindle-shaped morphology accompanying downregulation of the endothelial cell markers and upregulation of the mesenchymal cell markers when exposed to As2O3. As2O3 activated the AKT/GSK-3β/Snail signaling pathway, and blocking this pathway with PI3K inhibitor (LY294002) abolished EndMT in As2O3-treated endothelial cells. Our results highlight that As2O3 is an EndMT-promoting factor during cardiac fibrosis, suggesting that targeting EndMT is beneficial for preventing As2O3-induced cardiac toxicity. Less

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects o... More

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). In vivo, edema was more serious in FES mice compared with the control group. The expression of AQP1 and the wet-to-dry lung weight ratio (W/D) in the FES group were significantly increased compared with the control group. At the same time, inhibition of AQP1 decreased the pathological damage resulting from pulmonary edema. Then we performed a study in vitro to investigate whether AQP1 was induced by FFA release in FES. The mRNA and protein level of AQP1 were increased by FFAs in a dose- and time-dependent manner in PMVECs. In addition, the up-regulation of AQP1 was blocked by the inhibitor of p38 kinase, implicating the p38 MAPK pathway as involved in the FFA-induced AQP1 up-regulation in PMVECs. Our results demonstrate that AQP1 may play important roles in pulmonary edema induced by FES and can be regarded as a new therapy target for treatment of pulmonary edema induced by FES. Less

Background: Apoptosis of endothelial cells (ECs) plays a key role in the development of atherosclerosis and there are also evidence indicated that phosphatase and tensin ... More

Background: Apoptosis of endothelial cells (ECs) plays a key role in the development of atherosclerosis and there are also evidence indicated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a viable target in therapeutic approaches to prevent vascular ECs apoptosis. Aberrant miR-106b-5p expression has been reported in the plasma of patients with unstable atherosclerotic plaques. However, the role and underlying mechanism of miR-106-5p in the genesis of atherosclerosis have not been addressed. In this study, we explored the anti-apoptotic role of miR-106-5p by regulating PTEN expression in vascular ECs. Methods: Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the expression levels of miR-106b-5p in human atherosclerotic plaques and normal vascular tissues. Human umbilical vein endothelial cells (HUVEC) were transfected with miR-106b-5p mimic or negative control mimic, and apoptosis was induced by serum starvation and tumor necrosis factor-α (TNF-α) treat. Western blotting and real-time RT-PCR experiments were used to detect PTEN expression levels and TNF-α-induced apoptosis was evaluated by the activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Results: The expression of miR-106b-5p was significantly downregulated in plaques than in normal vascular tissues. TNF-α significantly downregulated miR-106b-5p expression levels and upregulated activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Overexpression of miR-106b-5p with miR-106b-5p mimic inhibited PTEN expression and TNF-α-induced apoptosis in HUVEC. Luciferase reporter assays confirmed that miR-106b-5p binds to PTEN mRNA 3’ untranslated region site. Conclusion: MiR-106b-5p could inhibit the expression of PTEN in vascular ECs, which could block TNF-α-induced activation of caspase-3, thus prevent ECs apoptosis in atherosclerosis diseases. Less

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver functi... More

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver function. Here we show a functional conservation of miR-122 in the TGFβ pathway: miR-122 target site is present in the mouse but not human TGFβR1, whereas a noncanonical target site is present in the TGFβ1 5′UTR in humans and other primates. Experimental switch of the miR-122 target between the receptor TGFβR1 and the ligand TGFβ1 changes the metastatic properties of mouse and human liver cancer cells. High expression of TGFβ1 in human primary liver tumours is associated with poor survival. We identify over 50 other miRNAs orthogonally targeting ligand/receptor pairs in humans and mice, suggesting that these are evolutionarily common events. These results reveal an evolutionary mechanism for miRNA-mediated gene regulation underlying species-specific physiological or pathological phenotype and provide a potentially valuable strategy for treating liver-associated diseases. Less

Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effec... More

Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In the present study, SCH rat models were established in thyroidectomized Wistar rats that were given l-T4 daily. The results showed that in vivo, the expression of osteopontin (OPN) vascular cell adhesion molecule (VCAM-1), and levels of integrin αvβ3 in the aortic tissue in SCH and Hypothyroidism (CH) groups was higher than in the control group. However, the effect in the SCH group was higher than in the CH group. In vitro, results showed that different concentration and time gradients of TSH stimulation could increase the expression of OPN, VCAM-1, and integrin αvβ3, and this was accompanied by extracellular signal regulated kinase 1/2 (Erk1/2) and Akt activation in human umbilical vein endothelial cells (HUVECs). TSH induced elevation of these proatherosclerotic factors was partially suppressed by a specific Akt inhibitor but not by a specific Erk inhibitor. Findings suggested that the endothelial dysfunction caused by SCH was related to increased proatherosclerotic factors induced by TSH via Akt activation. Less

Background Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1)... More

Background Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) play an important role in the development of these diseases. Mitogen-activated protein kinase (MAPK) p38/activated protein kinase 2 (MK2) regulates the expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells (HPMECs) stimulated by tumor necrosis factor-α (TNF-α); however, the underlying molecular mechanism remains unclear. Here, we show that human antigen R (HuR), an RNA binding protein which binds preferentially to AU-rich elements (AREs) and stabilizes mRNAs, regulates TNF-α-induced ICAM-1 expression in the MK2/HuR signaling pathway. Method MK2 and HuR were silenced respectively in HPMECs and then HPMECs were stimulatied with TNF-α. Nucleo-cytoplasmic shuttling of HuR was detected by subcellular fractionation and confocal microscopy in MK2 knockdown HPMECs. In HuR silencing cells, protein and mRNA levels of ICAM-1 and IL-8 were measured by western blot analysis, ELISA and real-time PCR; mRNA stabilization were measured by real-time PCR after actinomycin D (ActD) blocking transcription. Furthermore, we performed neutrophil adhesion assay to assess the adhering capacity after HuR silencing. Results MK2 were subjected to a knockdown by interfering RNA, the mRNA and protein levels of HuR in human pulmonary microvascular endothelial cells (HPMECs) were not affected. However, after the stimulation of TNF-α, silencing MK2 inhibited HuR accumulation to cytoplasm from nucleus in HPMECs. Consequently, knockdown of HuR by RNA interference in HPMECs, there was reduction in the stability of ICAM-1 mRNA and ICAM-1 protein level. This event was accompanied by a decrease in the adhesion of neutrophils towards HPMECs. Nevertheless, HuR silencing had no effect on the mRNA and protein levels of IL-8. Conclusion These results indicate that MK2 post-transcriptionally regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs. Less

TGF-β-induced endothelial-to-mesenchymal transition (EndoMT) is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role i... More

TGF-β-induced endothelial-to-mesenchymal transition (EndoMT) is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role in the pathogenesis of various fibrotic processes. Endothelin-1 (ET-1) has been implicated in the development of tissue fibrosis but its participation in TGF-β-induced EndoMT has not been studied. Here we evaluated the role of ET-1 on TGF-β1-induced EndoMT in immunopurified CD31+/CD102+ murine lung microvascular endothelial cells. The expression levels of α-smooth muscle actin (α-SMA), of relevant profibrotic genes, and of various transcription factors involved in the EndoMT process were assessed employing quantitative RT-PCR, immunofluorescence histology and Western blot analysis. TGF-β1 caused potent induction of EndoMT whereas ET-1 alone had a minimal effect. However, ET-1 potentiated TGF-β1-induced EndoMT and TGF-β1-stimulated expression of mesenchymal cell specific and profibrotic genes and proteins. ET-1 also induced expression of the TGF-β receptor 1 and 2 genes, suggesting a plausible autocrine mechanism to potentiate TGF-β-mediated EndoMT and fibrosis. Stimulation of TGF-β1-induced skin and lung fibrosis by ET-1 was confirmed in vivo in an animal model of TGF-β1-induced tissue fibrosis. These results suggest a novel role for ET-1 in the establishment and progression of tissue fibrosis. Less

Abstract: Hydrogen sulfide (H2S) functions as a protective gas transmitter in various physiological and pathological processes, but the lack of ideal donors severely hamp... More

Abstract: Hydrogen sulfide (H2S) functions as a protective gas transmitter in various physiological and pathological processes, but the lack of ideal donors severely hampers the clinical application of H2S. This study aims to construct a controlled release H2S donor and evaluate its protective effect on graft endothelium. Mesoporous silica nanoparticles (MSNs) were synthesized using the sol–gel method and loaded with diallyl trisulfide (DATS), an H2S-releasing agent named DATS-MSN. In vitro experiments showed that DATS-MSN could alleviate endothelial cell inflammation and enhance endothelial cell proliferation and migration. In vivo experiments demonstrated that the apoptosis of graft endothelium was mitigated in the presence of DATS-MSN. Our results indicated that DATS-MSN, releasing H2S in a controlled release fashion, could serve as an ideal H2S donor. Keywords: inflammatory response, rejection, cellular uptake, proliferation, cardiac allograft vasculopathy Less

The present study investigated whether atorvastatin antagonizes the visfatin-induced expression of inflammatory mediators in human coronary artery endothelial cells (HCAE... More

The present study investigated whether atorvastatin antagonizes the visfatin-induced expression of inflammatory mediators in human coronary artery endothelial cells (HCAECs). Several analysis methods, such as reverse transcription-quantitative polymerase chain reaction, western blot analysis and H2DCFDA incubation, were used in the present study. The data showed that atorvastatin decreased the visfatin-induced expression of interleukin (IL)-6 and IL-8 in HCAECs. In addition, atorvastatin inhibited the visfatin-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in HCAECs. In addition, the present study found that atorvastatin inhibited the visfatin-activated nuclear factor-κB (NF-κB) signal pathway by preventing extracellular signal-regulated kinase phosphorylation in HCAECs. Atorvastatin significantly inhibited visfatin-induced NF-κB activity via the upregulation of reactive oxygen species production. Atorvastatin, a visfatin antagonist (FK866) and an NF-κB inhibitor (BAY11-7082) decreased the visfatin-induced expression of inflammatory mediators via the upregulation of NF-κB activation in HCAECs. These results suggest that atorvastatin may inhibit the visfatin‑induced upregulation of inflammatory mediators through blocking the NF-κB signal pathway. The findings of the present study provide a potential use for atorvastatin and visfatin in the pathogenesis of HCAEC dysfunction. This knowledge may contribute to the development of novel therapies for atherosclerosis. Less

It has been shown that forced expression of four mouse stem cell factors (OCT4, Sox2, Klf4, and c-Myc) changed the phenotype of rat endothelial cells to vascular progenit... More

It has been shown that forced expression of four mouse stem cell factors (OCT4, Sox2, Klf4, and c-Myc) changed the phenotype of rat endothelial cells to vascular progenitor cells. The present study aimed to explore whether the expression of OCT4 alone might change the phenotype of human umbilical vein endothelial cells (HUVECs) to endothelial progenitor cells and, if so, to examine the possible mechanism involved. A Matrigel-based in vitro angiogenesis assay was used to evaluate the angiogenesis of the cells; the gene expression profile was analyzed by an oligonucleotide probe-based gene array chip and validated by RT-QPCR. The cellular functions of the mRNAs altered by OCT4 were analyzed with Gene Ontology. We found that induced ectopic expression of mouse OCT4 in HUVECs significantly enhanced angiogenesis of the cells, broadly changed the gene expression profile and particularly increased the expression of CD133, CD34, and VEGFR2 (KDR) which are characteristic marker molecules for endothelial progenitor cells (EPCs). Furthermore by analyzing the cellular functions that were targeted by the mRNAs altered by OCT4 we found that stem cell maintenance and cell differentiation were among the top functional response targeted by up-regulated and down-regulated mRNAs upon forced expression of OCT4. These results support the argument that OCT4 remodels the phenotype of HUVECs from endothelial cells to EPCs by up-regulating the genes responsible for stem cell maintenance and down-regulating the genes for cell differentiation. Key words: Endothelial Progenitor Cells; Human Umbilical Vein Endothelial Cells; Angiogenesis; Gene Expression; Octamer-binding transcription factor 4. Less

Background Three-dimensional (3-D) cultures of cancer cells can potentially bridge the gap between 2-D drug screening and in vivo xenografts. The objective of this study ... More

Background Three-dimensional (3-D) cultures of cancer cells can potentially bridge the gap between 2-D drug screening and in vivo xenografts. The objective of this study was to characterize the cellular and extracellular matrix characteristics of spheroids composed of human lung epithelial cells (epi), pulmonary vascular endothelial (endo) cells, and human marrow-derived mesenchymal stems cells (MSCs). Methods Spheroids composed of epi/endo/MSCs, termed herein as synthetic tumor microenvironment mimics (STEMs), were prepared by the hanging drop method. Cellular composition and distribution in the STEMs was characterized using fluorescence microscopy. Induction of reactive oxygen species and upregulation of efflux transporters was quantified using fluorometry and PCR, respectively, and phenotypic markers were qualitatively assessed using immunohistochemistry. Results STEMs exhibited three unique characteristics not captured in other spheroid cultures namely, the presence of a spheroid core devoid of epithelial cells and primarily composed of MSCs, a small viable population of endothelial cells hypothesized to be closely associated with MSCs within the hypoxic core, and discrete regions with high expression for vimentin and cytokeratin-18, whose co-expression is co-related with enhanced metastasis. Although cells within STEMs show elevated levels of reactive oxygen species and mRNA for ABC-B1, an efflux transporter associated with drug resistance, they exhibited only modest resistance to paclitaxel and gemcitabine in comparison to 2-D tri-cultures. Conclusions The epi/endo/MSC spheroid model described herein offers a promising platform for understanding tumor biology and drug testing in vitro. Less

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endo... More

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs. Less

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progres... More

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease. Less

In contrast to blood capillaries, lymphatic capillaries in peripheral tissues are composed of a single-cell layer of lymphatic endothelial cells (LECs) without a covering... More

In contrast to blood capillaries, lymphatic capillaries in peripheral tissues are composed of a single-cell layer of lymphatic endothelial cells (LECs) without a covering of mural cells. However, in lymphatic malformations, the enlarged lymphatic vessels were covered with mural cells. This study aimed to understand the molecular mechanism of differences between human dermal lymphatic endothelial cells (HDLECs) and human umbilical vein endothelial cells (HUVECs) and to determine the changes of LECs in the pathological condition of lymphatic malformation. Results showed that HDLECs exhibited lower expression of endothelial proteins, including VE-cadherin and CD31, than HUVECs; HDLECs also showed higher expression of mesenchymal proteins, including α-SMA, SM22α, calponin, and epithelial mesenchymal transition-related transcription factor Slug, than HUVECs. Likewise, HDLECs displayed higher permeability and weaker recruitment of SMCs than HUVECs; HDLECs also exhibited low PDGF-BB expression. TGF-β2 treatment and FGF2 depletion enhanced mesenchymal marker expression with increased permeability and reduced SMC recruitment. By contrast, Slug depletion in HDLECs enhanced VE-cadherin expression, inhibited α-SMA expression, decreased permeability, and enhanced PDGF-BB expression. These results suggested that HDLECs were in a mesenchymal status, which contributed to their functions and might determine their identities. Our data also revealed that miR143/145 was implicated in the mesenchymal status of HDLECs. In lymphatic malformations (LMs) treated with OK-432 sclerotherapy, immunohistochemistry results showed that Prox1 expression was reduced and mural cell investment was increased; these results indicated that LECs lost their mesenchymal status after OK-432 treatment was administered. The decreased mesenchymal status of LECs in LMs may induce dilated vessel constriction, which could be the mechanism of OK-432 sclerotherapy Less

Securing an adequate blood supply for the survival of cell transplants is critical for a successful outcome in tissue

Curcumin, a natural polyphenol compound from the perennial herb Curcuma longa, has been proved to be beneficial for tumor-bearing animals through inhibiting tumor neovasc... More

Curcumin, a natural polyphenol compound from the perennial herb Curcuma longa, has been proved to be beneficial for tumor-bearing animals through inhibiting tumor neovasculature formation, but the underlying mechanisms are unclear. Here, we aim to test whether curcumin affects VEGF-VEGFR2 signaling pathway and attenuates defective hematopoiesis induced by VEGF in tumor model. We demonstrated that curcumin inhibited proliferation, migration of HUVEC under VEGF stimulation and caused HUVEC apoptosis, and blocked VEGFR2 activation and its downstream signaling pathways in vitro. Furthermore, in VEGF over-expressing tumor model, curcumin significantly inhibited the tumor growth accelerated by VEGF in a dose-dependent manner and improved anemia and extramedullary hematopoiesis in livers and spleens of tumor-bearing mice induced by tumor-derived VEGF. Immunohistochemical analysis showed that curcumin normalized vasculature structures of livers and reduced tumor microvessel density. ELISA revealed that curcumin suppressed VEGF secretion from tumor cells both in vitro and in vivo. Survival analysis showed that curcumin significantly improved survival ability of VEGF tumor-bearing mice. Taken together, these findings establish curcumin as a modulator of VEGF and VEGF-VEGFR2 signaling pathway, with potential implication for improving the quality of life of cancer patients. Less

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immu... More

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. METHODS: Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including fulllength transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. RESULTS: HLSECs internalized HCV, independent of cell–cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid–inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogenassociated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN l3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8þ T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. CONCLUSIONS: Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity. Less

Background Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathoge... More

Background Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. Methods Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots. Results The manual and automated quantification showed strong, positive correlation (r2 = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells. Conclusions The assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells. Keywords: Plasmodium falciparum, Cytoadhesion, Chinese hamster ovary cells, Endothelial cells, Field isolates Less

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tu... More

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma. Less

Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (... More

Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases. Keywords: chemotaxis; colorectal cancer; hepatic sinusoidal endothelial cell; macrophage migration inhibitory factor; metastasis. Less

The aim of this study was to evaluate the angiogenic capacity and proteolytic mechanism of coculture using human amniotic mesenchymal stem cells (hAMSCs) with human umbil... More

The aim of this study was to evaluate the angiogenic capacity and proteolytic mechanism of coculture using human amniotic mesenchymal stem cells (hAMSCs) with human umbilical vein endothelial cells (HUVECs) in vivo and in vitro by comparing to those of coculture using bone marrow mesenchymal stem cells with HUVEC. For the in vivo experiment, cells (HUVEC-monoculture, HUVEC-hAMSC coculture, and HUVEC-BMMSC coculture) were seeded in fibrin gels and injected subcutaneously in nude mice. The samples were collected on days 7 and 14 and histologically analyzed by H&E and CD31 staining. CD31-positive staining percentage and vessel-like structure (VLS) density were evaluated as quantitative parameters for angiogenesis. The increases of CD31-positive staining area and VLS density in both HUVEC-hAMSC group and HUVEC-BMMSC group were found between two time points, while obvious decline of those was observed in HUVEC-only group. For the in vitro experiment, we utilized the same 3D culture model to investigate the proteolytic mechanism related to capillary formation. Intensive vascular networks formed by HUVECs were associated with hAMSCs or BMMSCs and related to MMP2 and MMP9. In conclusion, hAMSCs shared similar capacity and proteolytic mechanism with BMMSCs on neovascularization. Less

Background: Dysfunction of vascular endothelium is implicated in many pathological situations. Cytoskeleton plays an importance role in vascular endothelial permeability ... More

Background: Dysfunction of vascular endothelium is implicated in many pathological situations. Cytoskeleton plays an importance role in vascular endothelial permeability barrier and inflammatory response. Many Chinese herbs have the endothelial protective effect, of which, "Astragalus membranaceus" is a highly valued herb for treatment of cardiovascular and renal diseases in traditional Chinese medicine, In this study, we tested whether calycosin-7-O-β-D-glucoside (Calycosin), a main effective monomer component of "Astragalus membranaceus", could protect endothelial cells from bacterial endotoxin (LPS)-induced cell injury. Methods: Endothelial cell injury was induced by exposing human umbilical vein endothelial cells (HUVECs) to LPS. The effects of calycosin on LPS-induced changes in cell viability, apoptosis rate, cell migration, nitric oxide synthase (NOS), generationof intracellular reactive oxygen species (ROS) and cytoskeleton organization were determined. Microarray assay was employed to screen the possible gene expression change. Based on the results of microarray assay, the expression profile of genes involved in Rho/ROCK pathway and AKT pathway were further evaluated with quantitative real-time RT-PCR or western blot methods. Results: Calycosin improved cell viability, suppressed apoptosis and protected the cells from LPS-induced reduction in cell migration and generation of ROS, protein level of NOS at a comparable magnitude to that of Y27632 and valsartan. Similar to Y27632 and valsartan, Calycosin, also neutralized LPS-induced actomyosin contraction and vinculin protein aggregation. Microarray assay, real-time PCR and western blot results revealed that LPS induced expression of FN, ITG A5, RhoA, PI3K (or PIP2 in western blotting), FAK, VEGF and VEGF R2, and inhibited expression of MLCP. We believed multiple pathways involved in the regulation of calycosin on HUVECs. Calycosin are considered to be able to activate MLCP through promoting the generation of NO, decreasing PMLC, suppressing the cytoskeleton remodeling caused by activation of Rho/ROCK pathway and inhibiting AKT pathway by decreasing VEGF, VEGF R2 and PI3K level. Conclusion: Calycosin protected HUVEC from LPS-induced endothelial injury, possibly through suppression of Rho/ROCK pathway and regulation of AKT pathway. Less

Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestr... More

Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology. Less

Abstract: The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the... More

Abstract: The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 µg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%–60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy. Keywords: tumor microenvironment, nanocarriers, proteoglycans, polysaccharides, passive targeting Less

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum vene... More

Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), NG-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity. Keywords: Apocynum venetum, nitric oxide, endothelium, vasorelaxation, antihypertensive medicinal herb Less

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less

Background Tumor lymphangiogenesis plays an important role in promoting growth and metastasis of tumors, but no antilymphangiogenic agent is used clinically. Based on the... More

Background Tumor lymphangiogenesis plays an important role in promoting growth and metastasis of tumors, but no antilymphangiogenic agent is used clinically. Based on the effect of norcantharidin (NCTD) on lymphangiogenesis of human lymphatic endothelial cells (LECs), we firstly investigated the antilymphangiogenic activity of NCTD as a tumor lymphangiogenic inhibitor for human colonic adenocarcinomas (HCACs). Methods In vivo and in vitro experiments to determine the effects of NCTD on tumor growth and lymphangiogenesis of the in-situ colonic xenografts in nude mice, and lymphatic tube formation of the three-dimensional (3-D) of the co-culture system of HCAC HT-29 cells and LECs were done. Proliferation, apoptosis, migration, invasion, Ki-67, Bcl-2 and cell cycle of LECs and the co-culture system in vitro were respectively determined. Streparidin-peroxidase staining, SABC, western blotting and RT-PCR were respectively used to examine the expression of LYVE-1, D2-40, CK20 (including their LMVD), and VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in vitro and in vivo. Results NCTD inhibited tumor growth and lymphangiogenesis of the in-situ colonic xenografts in vivo, and these observations were confirmed by facts that lymphatic tube formation, proliferation, apoptosis, migration, invasion, S-phase cell cycle, and Ki-67 and Bcl-2 expression in vitro, and LYVE-1, D2-40, CK20 expression and their LMVD in vitro and in vivo were inhibited and affected. Furthermore, the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 at protein/mRNA levels in the process of lymphatic tube formation in vitro and tumor lymphangiogenesis in vivo was downregulated; NCTD in combination with mF4-31C1 or Sorafenib enhanced these effects. Conclusions NCTD inhibits tumor growth and lymphangiogenesis of HCACs through “multi-points priming” mechanisms i.e. affecting related malignant phenotypes, inhibiting Ki-67 and Bcl-2 expression, inducing S-phase cell cycle arrest, and directly or indirectly downregulating VEGF-A,-C,-D/VEGFR-2,-3 signaling pathways. The present finding strongly suggests that NCTD could serve as a potential antilymphangiogenic agent for tumor lymphangiogenesis and is of importance to explore NCTD is used for antitumor metastatic comprehensive therapy for HCACs. Keywords: Colonic neoplasm, Norcantharidin, Tumor growth, Lymphangiogenesis, Antilymphangiogenic therapy Less

Amyloid beta-peptides (Aβ) are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature... More

Amyloid beta-peptides (Aβ) are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer׳s disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ1–40 (fAβ1–40) were observed. The results show that fAβ1–40-induced cytotoxicity in human brain microvascular endothelial cells (hBMECs) can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation. Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to fAβ1–40. In conclusion, quercetin protects hBMECs from fAβ1–40-induced toxicity. Less

Background Human immunodeficiency virus (HIV) infection leads to decreased reverse cholesterol transport (RCT) in macrophages, and Nef mediated down-regulation and redist... More

Background Human immunodeficiency virus (HIV) infection leads to decreased reverse cholesterol transport (RCT) in macrophages, and Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA1) are identified as key factors for this effect. This may partially explain the increased risk of atherosclerosis in HIV infected individuals. Since endothelial dysfunction is key in the initial stages of atherosclerosis, we sought to determine whether RCT was affected in human aortic endothelial cells (HAECs). Results We found that apoA-I does not significantly stimulate cholesterol efflux in HAECs while cholesterol efflux to high-density lipoprotein (HDL) was dramatically reduced in HAECs co-cultured with HIV infected cells. Studies with wild type and Nef defective HIV revealed no significant differences suggesting that multiple factors are working perhaps in concert with Nef to affect cholesterol efflux to HDL from HAECs. Interestingly, treating HAECs with recombinant Nef showed similar effect in HDL mediated cholesterol efflux as observed in HAECs co-cultured with HIV infected cells. Using a detergent-free based subcellular fractionation approach, we demonstrated that exposure of HAECs to HIV infected cells or Nef alone disrupts caveolin 1 (Cav-1) subcellular trafficking upon HDL stimulation. Moreover, Nef significantly enhanced tyrosine 14 phosphorylation of Cav-1 which may have an impact on recycling of Cav-1 and caveolae. Conclusion These results suggest that HIV interferes with cholesterol efflux by HDL in HAECs through the disruption of Cav-1s’ cellular distribution and that multiple factors are involved, possibly including Nef, for the inhibition of HDL mediated cholesterol efflux and alteration of cellular distribution of Cav-1. Less

Background: The mechanisms by which bacterial ligands alter angiogenesis remain unknown. Results: Lipopolysaccharide-mediated Angiopoietin-2-dependent autocrine angiogene... More

Background: The mechanisms by which bacterial ligands alter angiogenesis remain unknown. Results: Lipopolysaccharide-mediated Angiopoietin-2-dependent autocrine angiogenesis in lung endothelial cells is regulated by NADPH oxidase 2. Conclusion: Endothelial Nox2 regulates Angiopoietin-2-dependent angiogenesis. Significance: This study presents new data regarding the regulation of proinflammatory angiogenesis. Keywords: Angiogenesis, Endothelial Cell, LPS, Lung Injury, NADPH Oxidase, Angiopoeitin-2 Less

Lymphatic metastasis is a major progression route of gastric cancer. Interleukin-8 (IL-8), as an inflammatory cytokine, is induced by Helicobacter pylori infection and is... More

Lymphatic metastasis is a major progression route of gastric cancer. Interleukin-8 (IL-8), as an inflammatory cytokine, is induced by Helicobacter pylori infection and is strongly associated with gastric cancer development and metastasis. The blood and lymphatic systems are similar in their function and gene expression profiles. It has been proposed that IL-8 activates angiogenesis. However, the direct role of IL-8 in lymphangiogenesis in gastric cancer remains unclear. We investigated the effect of IL-8 on the growth of human lymphatic endothelial cells (LECs). In addition, protein and mRNA expression of selected lymphangiogenesis markers was assessed in these cells. LECs were co-cultured with gastric cancer SGC7901 cells and exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1.0 ng/ml). The Cell Counting Kit-8 was used to evaluate LEC proliferation (cultured for 1-6 days). Then, protein (immunofluorescence and western blotting) and mRNA [quantitative transcription-polymerase chain reaction (qPCR)] levels were measured in samples obtained from the 24-h cultured cells, for lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), vascular endothelial growth factor (VEGF)-C, VEGF-D and vascular endothelial growth factor receptor-3 (VEGFR-3). The data presented herein demonstrated that IL-8 promotes the proliferation of LECs and enhances the protein and mRNA expression of LYVE-1. Notably, IL-8 inhibited VEGF-C, VEGF-D and VEGFR-3 protein expression as well as VEGF-D and VEGFR-3 mRNA expression. These findings suggest that IL-8 may be a potent inducer of LECs, although this effect does not appear to involve the VEGF-C/VEGF-D and VEGFR-3 signaling pathway. Less

Creating a long-lasting and functional vasculature represents one of the most fundamental challenges in tissue engineering. VEGF has been widely accepted as a potent angi... More

Creating a long-lasting and functional vasculature represents one of the most fundamental challenges in tissue engineering. VEGF has been widely accepted as a potent angiogenic factor involved in the early stages of blood vessel formation. In this study, fibrous scaffolds that consist of PCL and gelatin fibers were fabricated. The gelatin fibers were further functionalized by heparin immobilization, which provides binding sites for VEGF and thus enables the sustained release of VEGF. In vitro release test confirms the sustained releasing profile of VEGF, and stable release was observed over a time period of 25 days. In vitro cell assay indicates that VEGF release significantly promoted the proliferation of endothelial cells. More importantly, in vivo subcutaneous implantation reflects that vascularization has been effectively enhanced in the PCL/gelatin scaffolds compared with the PCL counterpart due to the sustained release of VEGF. Therefore, the heparinized PCL/gelatin scaffolds developed in this study may be a promising candidate for regeneration of complex tissues with sufficient vascularization. Less

Mitochondrial injury and dysfunction, a significant feature in metabolic syndrome, triggers endothelial cell dysfunction and cell death. Increasing evidence suggests that... More

Mitochondrial injury and dysfunction, a significant feature in metabolic syndrome, triggers endothelial cell dysfunction and cell death. Increasing evidence suggests that mitophagy, a process of autophagic turnover of damaged mitochondria, maintains mitochondrial integrity. PINK1 (phosphatase and tensin homolog (PTEN)-induced putative kinase 1) and Parkin signaling is a key pathway in mitophagy control. In this study, we examined whether this pathway could protect mitochondria under metabolic stress. We found that palmitic acid (PA) induced significant mitophagy and activated PINK1 and Parkin in endothelial cells. Knocking down PINK1 or Parkin reduced mitophagy, leading to impaired clearance of damaged mitochondria and intracellular accumulation of mitochondrial fragments. Furthermore, PINK1 and Parkin prevented PA-induced mitochondrial dysfunction, ROS production and apoptosis. Finally, we show that PINK1 and Parkin were up-regulated in vascular wall of obese mice and diabetic mice. Our study demonstrates that PINK1-Parkin pathway is activated in response to metabolic stress. Through induction of mitophagy, this pathway protects mitochondrial integrity and prevents metabolic stress-induced endothelial injury. Less

Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and... More

Inflammatory breast cancer (IBC) is an aggressive and invasive tumor, accounting for 2.5% of all breast cancer cases, and characterized by rapid progression, regional and distant metastases, younger age of onset, and lower overall survival. Presently, there are no effective therapies against IBC and a paucity of model systems. Our aim was to develop a clinically relevant IBC model that would allow investigations on the role of chemokine receptors in IBC metastasis. Primary cultures of tumor cells were isolated from pleural exudates of an IBC patient and grown as spheres or monolayers. We developed a human xenograft model where patient-derived IBC cells, stably transduced with lentiviral vectors expressing fluorescent and bioluminescent markers, were inoculated directly into the left ventricle of mice. Our in vivo data show that these IBC cells (FC-IBC02A) are able to seed and proliferate into various organs, including brain, lungs, lymph nodes, and bone, closely replicating the metastatic spread observed in IBC patients. Moreover, cells were able to generate tumors when grafted in the mammary fat pad of mice. RT-PCR and microscopy studies revealed expression of both CXCR4 and ACKR3 receptors in FC-IBC02A cells. Furthermore, CXCL12 (the endogenous chemokine ligand of these receptors) induced transendothelial migration of these cells and stimulated signaling pathways involved in cell survival and migration - an effect reduced by CXCR4 or ACKR3 antagonists. This new model can be used to develop chemokine-based pharmacological approaches against the IBC metastatic process. This work also provides the first evidence of ACKR3 expression in IBC cells. Less

Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis ... More

Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism responsible, at least in part, for macrophage invasion during arteriogenesis. Less

Cigarette smoking, a major independent risk factor of atherosclerosis, can cause oxidative and inflammatory damage of vascular tissue. Heme oxygenase-1 (HO-1) is an endog... More

Cigarette smoking, a major independent risk factor of atherosclerosis, can cause oxidative and inflammatory damage of vascular tissue. Heme oxygenase-1 (HO-1) is an endogenous cytoprotective enzyme with an anti-oxidant role in cells. The aim of the present study was to investigate whether HO-1 was able to protect vascular and endothelial cells from the oxidative damage induced by cigarette smoking. It was observed that cigarette smoking was able to induce the generation of the reactive oxygen species (ROS) in carotid arteries of rats. Hemin, a widely used HO-1 inducer, was able to reduce the generation of ROS. In addition, when human umbilical vein endothelial cells (HUVECs) were cultured in the serum of smoking rats, this was able to increase ROS, and the protective effect of hemin was also observed in this system. In conclusion, the present study demonstrated that cigarette smoking causes oxidative damage of vascular cells and HUVECs by inducing the generation of ROS, while HO-1 has an anti-oxidant effect in this course. This also implied that hemin, an inducer of HO-1, may have potential therapeutic applicability in the prevention of vascular diseases caused by cigarette smoking. Less

Background: Mesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial g... More

Background: Mesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood. Methods: We introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence. Results: MSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF antibody or with both HGF and VEGF antibodies. Additionally, the effects of MSC-CM and VEGF/HGF on reducing endothelial paracellular and transcellular permeability were weakened when HPMECs were pretreated with the Rac1 inhibitor NSC23766. Conclusion: HGF secreted by MSCs protects the endothelial barrier function; however, VEGF secreted by MSCs may synergize with HGF to stabilise endothelial cell barrier function. Rac1 is the pathway by which MSC-secreted VEGF and HGF regulate endothelial permeability. Less

To investigate the effect of endostar on specific angiogenesis induced by human hepatocellular carcinoma, this research systematically elucidated the inhibitory effect on... More

To investigate the effect of endostar on specific angiogenesis induced by human hepatocellular carcinoma, this research systematically elucidated the inhibitory effect on HepG2-induced angiogenesis by endostar from 50 ng/mL to 50000 ng/mL. We employed fluorescence quantitative Boyden chamber analysis, wound-healing assay, flow cytometry examination using a coculture system, quantitative analysis of tube formation, and in vivo Matrigel plug assay induced by HCC conditioned media (HCM) and HepG2 compared with normal hepatocyte conditioned media (NCM) and L02. Then, we found that endostar as a tumor angiogenesis inhibitor could potently inhibit human umbilical vein endothelial cell (HUVEC) migration in response to HCM after four- to six-hour action, inhibit HCM-induced HUVEC migration to the lesion part in a dose-dependent manner between 50 ng/mL and 5000 ng/mL at 24 hours, and reduce HUVEC proliferation in a dose-dependent fashion. Endostar inhibited HepG2-induced tube formation of HUVECs which peaked at 50 ng/mL. In vivo Matrigel plug formation was also significantly reduced by endostar in HepG2 inducing system rather than in L02 inducing system. It could be concluded that, at cell level, endostar inhibited the angiogenesis-related biological behaviors of HUVEC in response to HCC, including migration, adhesion proliferation, and tube formation. At animal level, endostar inhibited the angiogenesis in response to HCC in Matrigel matrix. Less

The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) wh... More

The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity before implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVECs) under hypoxia promotes the formation of endothelial tubules and a blood vessel network. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, whereas SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α and VEGF, and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture Petri dishes were detected, suggesting that VEGF secreted by SCAP might be used by HUVEC to accelerate the formation of vessel-like structures. Taken together, we revealed that artificial hypoxia stimulates angiogenic responses in SCAP for possible use in engineering dental pulp replacements. Our results may help to delineate the optimal therapeutic target to promote angiogenesis so that future bioengineered pulp replacements integrate faster and permanently within the host. Less

Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic... More

Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as “metabolic memory.” Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how “metabolic memory” would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of “metabolic memory” of cellular senescence (senescent “memory”). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent “memory.” In contrast, senescent “memory” was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of “metabolic memory.” Furthermore, we found that RSV or MET treatment prevented senescent “memory” by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent “memory.” In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET treatment could enhance SIRT1-mediated signaling and thus protect against senescent “memory” independent of their glucose lowering mechanisms. Therefore, they may serve as promising therapeutic drugs against the development of “metabolic memory.” Less

Objective(s): This study was designed to investigate the effect of receptor for advanced glycation end products (RAGE), S100A12 and C-reactive protein (CRP) on the releas... More

Objective(s): This study was designed to investigate the effect of receptor for advanced glycation end products (RAGE), S100A12 and C-reactive protein (CRP) on the release of circulating endothelial cells (CECs) from human coronary artery endothelial cells (HCAECs). Materials and Methods: HCAECs were cultured in increasing concentration of CRP (0, 12.5, 25, 50μg/ml) or S100A12 protein (0, 4, 10, 25μg/ml) for 24 hr. CECs were measured by flow cytometry. Small interfering RNA (siRNA) was designed to decrease RAGE level. Fluorescence microscopy and real-time quantitative polymerase chain reaction were used to assess the efficiency of siRNA silencing RAGE. The release of CECs from HCAECs was further evaluated by flow cytometry. Results: CRP caused a significant increase in the release of CECs from HCAECs. The number of CECs increased by about 2-fold in 25 μg/ml CRP-treated group compared to the control group (12.22% compared to 6.82%, P=0.032). But S100A12 failed to increase the release of CECs from HCAECs. Blockade of RAGE by siRNA significantly decreased the release of CECs induced by CRP (13.22% of CRP group compared to 8.77% of CRP+siRNA group, P=0.017). Conclusion: RAGE is involved in the release of CECs induced by CRP, and the effect can be attenuated by silencing RAGE. RAGE may play an important role in endothelial dysfunction in cardiovascular disease. Inhibition of RAGE may be a therapeutic target for coronary artery lesions in Kawasaki disease. Keywords: CECs, CRP, HCAECs, RAGE, S100A12 Less

Background: High mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. ... More

Background: High mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. In recent years, HMGB1 has been identified as a critical “late” pro-inflammatory mediator due to its unique secretion pattern and lethal effects in sepsis. Therefore, preventing the active release and inhibiting the pro-inflammatory activity of HMGB1 become promising strategies for the treatment of sepsis. Here, we reported the therapeutic effects of Gu-4, a lactosyl derivative, on sepsis and the underlying molecular mechanisms. Methodology/Principal Findings: In an experimental rat model of sepsis caused by cecal ligation and puncture (CLP), Gu-4 administration prominently attenuated lung injury and improved the survival of the septic animals, which was positively correlated with the decrease of the serum HMGB1 level. Using RAW264.7 macrophage cell line, we further showed that Gu-4 significantly suppressed the lipopolysaccharide (LPS)-induced release and cytoplasmic translocation of HMGB1. Moreover, Gu-4 not only dose-dependently attenuated recombinant human (rhHMGB1)-induced production of TNF-α, IL-6, and IL-1β in THP-1 cells, but also greatly inhibited the adhesion of rhHMGB1-challenged THP-1 cells to HUVECs. Analyses of flow cytometry demonstrated that Gu-4 could effectively reduce the activation of CD11b elicited by rhHMGB1. Western blot analyses revealed that Gu-4 treatment could partially block the rhHMGB1-induced activation of ERK and NF-κB signalings. Meanwhile, CD11b knockdown also obviously attenuated the rhHMGB1-induced phosphorylations of ERK and IKKα/β. Conclusions/Significance: Taken together, our results suggest that Gu-4 possesses a therapeutic potential in the treatment of sepsis probably via inhibiting the LPS-induced release of HMGB1 from macrophages and via suppressing the pro-inflammatory activity of HMGB1. Less

Magnesium (Mg) based alloys are the most advanced cardiovascular stent materials. This new generation of stent scaffold is currently under clinical evaluation with encour... More

Magnesium (Mg) based alloys are the most advanced cardiovascular stent materials. This new generation of stent scaffold is currently under clinical evaluation with encouraging outcomes. All these Mg alloys contain a certain amount of rare earth (RE) elements though the exact composition is not yet disclosed. RE alloying can usually enhance the mechanical strength of different metal alloys but their toxicity might be an issue for medical applications. It is still unclear how RE elements will affect the magnesium (Mg) alloys intended for stent materials as a whole. In this study, we evaluated MgZnCaY-1RE, MgZnCaY-2RE, MgYZr-1RE, and MgZnYZr-1RE alloys for cardiovascular stents applications regarding their mechanical strength, corrosion resistance, hemolysis, platelet adhesion/activation, and endothelial biocompatibility. The mechanical properties of all alloys were significantly improved. Potentiodynamic polarization showed that the corrosion resistance of four alloys was at least 3–10 times higher than that of pure Mg control. Hemolysis test revealed that all the materials were non-hemolytic while little to moderate platelet adhesion was found on all materials surface. No significant cytotoxicity was observed in human aorta endothelial cells cultured with magnesium alloy extract solution for up to seven days. Direct endothelialization test showed that all the alloys possess significantly better capability to sustain endothelial cell attachment and growth. The results demonstrated the promising potential of these alloys for stent material applications in the future. Less

Angiogenesis is crucial for tumor development, growth and metastasis. Vascular endothelial growth factor (VEGF) has been implicated in promoting solid tumor growth and me... More

Angiogenesis is crucial for tumor development, growth and metastasis. Vascular endothelial growth factor (VEGF) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis, and blocking the activity of VEGF can starve tumors. Avastin, which is a humanized anti-VEGF antibody, has been successfully applied in clinics since 2004. However, the price of Avastin is extremely high for Chinese people. Here, we report a novel human anti-VEGF neutralizing antibody, MIL60, which shows an affinity comparable to that of Avastin (the KD value of MIL60 was 44.5 pM, while that of Avastin was 42.7 pM). MIL60 displays favorable actions in inhibiting VEGF-triggered endothelial cell proliferation (the IC50 value of MIL60 was 31±6.4 ng/ml and that of Avastin was 47±8.1 ng/ml), migration (8 µg/ml or 0.8 µg/ml MIL60 versus the control: P<0.05) and tube formation (2 µg/ml or 0.2 µg/ml MIL60 versus the control: P<0.05) via the VEGFR2 signaling pathway. Moreover, MIL60 was shown to inhibit tumor growth and angiogenesis in vivo in xenograft models of human colon carcinoma and ovarian cancer using immunotherapy and immunohistochemistry analysis (MIL60 versus N.S.: P=0.0007; Avastin versus N.S.: P=0.00046). These data suggest that MIL60 is a potential therapeutic, anti-angiogenic agent. Our work provides a novel anti-VEGF antibody, which can be considered an anti-tumor antibody candidate and a new option for patients with various cancers. Less

Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of infla... More

Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of inflammatory and cardiovascular diseases. We have previously reported that TMP acts as a potent antioxidant protecting endothelial cells against high glucose-induced damages. However, the molecular mechanism responsible for the antioxidant effect of TMP remains to be elucidated. In this study, we show that TMP increases nitric oxide production in endothelial cells and promotes endothelium-dependent relaxation in rate aortic rings. The antioxidant effect of TMP appears attributable to its ability to activate the mitochondrial biogenesis, as reflected in an up-regulation of complex III and amelioration of mitochondrial membrane potential. Furthermore, TMP is able to reverse high glucose-induced suppression of SIRT1 and the biogenesis-related factors, including PGC-1α, NRF1 and TFAM, suggesting a new molecular mechanism underlying the protective effect of TMP on the endothelium. Less

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protecti... More

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙−) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways. Less

Nanoparticles (NPs) constitute an important medium for the targeted delivery of cancer therapeutics. Targeting of NPs to a specific cell type is traditionally achieved th... More

Nanoparticles (NPs) constitute an important medium for the targeted delivery of cancer therapeutics. Targeting of NPs to a specific cell type is traditionally achieved through the modification of the NP surface with peptides, aptamers, or other motifs that specifically recognize a cell-surface receptor, leading to internalization of NPs via clathrin and caveolae-mediated endocytosis. We have discovered that modifying the NP surface with anionic polyelectrolytes of varying lipophilicity can regulate the uptake of lipid NPs by endothelial and epithelial cells. Furthermore, we report the finding that synthetic polyelectrolytes composed of an aromatic sulfonic acid backbone exhibit specific affinity for caveolae of endothelial cells. By exploiting the higher expression of caveolae in endothelial cells in comparison with epithelial cells, a purely physiochemical approach to the targeted uptake of lipid NPs to endothelial cells is demonstrated. The ability to confer preferential affinity for NPs to cell surface domains by varying the charge and lipophilic characteristics of an NP surface offers a general means of achieving targeted delivery without the need for receptor-ligand-type targeting strategies. Keywords: aromatic polysulfonates; polyanion; polystyrene sulfonate; vasculature. Less

Background: Vascular endothelial growth factor (VEGF) is a key angiogenic factors. It plays an important role in both physiologic and pathologic angiogenesis and increase... More

Background: Vascular endothelial growth factor (VEGF) is a key angiogenic factors. It plays an important role in both physiologic and pathologic angiogenesis and increases permeability across the vessels. Using antibody phage display technology, we obtained a novel anti-VEGFA IgG, named as FD006. In this study, the pharmacological characteristics and efficacy of FD006 in corneal neovascularization (CoNV) were evaluated. Results: FD006 was predicted to have similar binding mode to bevacizumab. Experimental analysis showed that the binding ability of FD006 seemed a little stronger than bevacizumab, for the EC50 of FD006 to bind VEGF analyzed by ELISA was about 0.037 μg/mL while that of bevacizumab was 0.18 μg/mL. Binding kinetics assays showed similar results that FD006 possessed 2-fold higher affinity to bind VEGF than bevacizumab due to slower dissociation rate of FD006; meanwhile, FD006 inhibited the VEGF-induced proliferation of HUVEC with an IC50 value of 0.031 ± 0.0064 μg/ml, which seemed similar or a litter better than bevacizumab (0.047 ± 0.0081 μg/ml). The subconjunctival administration of FD006, bevacizumab or dexamethasone could significantly inhibit the growth of CoNV contrasting to N.S (p Less

OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Ka... More

OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and angiogenesis-related diseases. Keywords: Rat Aortic Ring Assay, Cotton Pellet Granuloma, Tail Flick Assay, Tube Formation Assay, Cell Migration Assay, Cytokine Inhibition Assay Less

Background: Glycation of high-density lipoprotein (HDL) decreases its ability to induce cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in endoth... More

Background: Glycation of high-density lipoprotein (HDL) decreases its ability to induce cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in endothelial cells. Whether lipid content of HDL, especially sphingosine-1-phosphate (S1P), plays any specific role in restoring the protective function of HDL in type 2 diabetes mellitus (T2DM) is still unknown. Methods and results: Immunochemical techniques demonstrated that glycated HDL loses its protective function of regulating COX-2 expression compared with diabetic HDL. We proved that the lipid content, especially phospholipid content differed between diabetic HDL and glycated HDL. Levels of HDL-c-bound S1P were increased in T2DM compared with control subjects as detected by UPLC-MS/MS (HDL-c-bound S1P in control subjects vs. T2DM: 309.1 ± 13.71 pmol/mg vs. 382.1 ± 24.45 pmol/mg, P < 0.05). Additionally, mRNA levels of S1P lyase enzymes and S1P phosphatase 1/2 were decreased in peripheral blood by real-time PCR. Antagonist of S1P receptor 1 and 3 (S1PR1/3) diminished the functional difference between apoHDL&PL (HDL containing the protein components and phospholipids) and diabetic apoHDL&PL (diabetic HDL containing the protein components and phospholipids). With different doses of S1P reconstituted on glycated HDL, its function in inducing the COX-2 expression was restored to the same level as diabetic HDL. The mechanism of S1P reconstituted HDL (rHDL) in the process of regulating COX-2 expression involved the phosphorylation of ERK/MAPK-CREB signal pathway. Conclusion/significance: S1P harbored on HDL is the main factor which restores its protective function in endothelial cells in T2DM. S1P and its receptors are potential therapeutic targets in ameliorating the vascular dysfunction in T2DM. Less

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glyc... More

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport. Less

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, ... More

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals. Less

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the ... More

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the development of renal fibrosis. Quercetin (QC), a natural component of foods, protects against renal injury. Here, we explored the possible mechanisms that are responsible for ADMA-induced renal fibrosis and the protective effect of QC. We found that ADMA treatment activated the endoplasmic reticulum (ER) stress sensor proteins phosphorylated protein kinase RNA-activated-like ER kinase (PERK) and inositol requiring-1α (IRE1), which correspondingly induced C/EBP homologous protein (CHOP) expression and phosphorylated c-Jun N-terminal kinase (JNK) phosphorylation in glomerular endothelial cells (GEnCs). Following this, ADMA promoted ER stress-induced apoptosis and resulted in transforming growth factor β (TGF-β) expression in GEnCs. SP600125, an inhibitor of JNK, and CHOP siRNA protected against ADMA-induced cell apoptosis and TGF-β expression. QC prevented ADMA-induced PERK and IRE1 apoptotic ER stress pathway activation. Also, ADMA-induced GEnCs apoptosis and TGF-β expression was reduced by QC. Overexpression of CHOP blocked QC-mediated protection from apoptosis in ER stressed cells. Overall, these observations indicate that ADMA may induce GEnCs apoptosis and TGF-β expression by targeting the PERK-CHOP and IRE1-JNK pathway. In addition, drugs such as QC targeting ER stress may hold great promise for the development of novel therapies against ADMA-induced renal fibrosis. Less

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (D... More

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells. Less

Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular en... More

Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular endothelium or form rosettes with uninfected erythrocytes to survive in vivo by sequestering IE in the microvasculature and avoiding splenic clearance mechanisms. Both rosetting and cytoadherence are mediated by the parasite-derived IE surface protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting and cytoadherence have been widely studied as separate entities; however, the ability of rosetting P. falciparum strains to cytoadhere has received little attention. Here, we show that IE of the IT/R29 strain expressing a rosette-mediating PfEMP1 variant (IT4var09) cytoadhere in vitro to a human brain microvascular endothelial cell line (HBEC-5i). Cytoadherence was inhibited by heparin and by treatment of HBEC-5i with heparinase III, suggesting that the endothelial receptors for IE binding are heparan sulfate proteoglycans. Antibodies to the N-terminal regions of the IT4var09 PfEMP1 variant (NTS-DBL1α and DBL2γ domains) specifically inhibited and reversed cytoadherence down to low concentrations (<10 μg/ml of total IgG). Surface plasmon resonance experiments showed that the NTS-DBLα and DBL2γ domains bind strongly to heparin, with half-maximal binding at a concentration of ∼0.5 μM in both cases. Therefore, cytoadherence of IT/R29 IE is distinct from rosetting, which is primarily mediated by NTS-DBL1α interactions with complement receptor 1. These data show that IT4var09-expressing parasites are capable of dual interactions with both endothelial cells and uninfected erythrocytes via distinct receptor-ligand interactions. Less

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (D... More

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells. Less

Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular en... More

Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular endothelium or form rosettes with uninfected erythrocytes to survive in vivo by sequestering IE in the microvasculature and avoiding splenic clearance mechanisms. Both rosetting and cytoadherence are mediated by the parasite-derived IE surface protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting and cytoadherence have been widely studied as separate entities; however, the ability of rosetting P. falciparum strains to cytoadhere has received little attention. Here, we show that IE of the IT/R29 strain expressing a rosette-mediating PfEMP1 variant (IT4var09) cytoadhere in vitro to a human brain microvascular endothelial cell line (HBEC-5i). Cytoadherence was inhibited by heparin and by treatment of HBEC-5i with heparinase III, suggesting that the endothelial receptors for IE binding are heparan sulfate proteoglycans. Antibodies to the N-terminal regions of the IT4var09 PfEMP1 variant (NTS-DBL1α and DBL2γ domains) specifically inhibited and reversed cytoadherence down to low concentrations (<10 μg/ml of total IgG). Surface plasmon resonance experiments showed that the NTS-DBLα and DBL2γ domains bind strongly to heparin, with half-maximal binding at a concentration of ∼0.5 μM in both cases. Therefore, cytoadherence of IT/R29 IE is distinct from rosetting, which is primarily mediated by NTS-DBL1α interactions with complement receptor 1. These data show that IT4var09-expressing parasites are capable of dual interactions with both endothelial cells and uninfected erythrocytes via distinct receptor-ligand interactions. Less

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the ... More

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the development of renal fibrosis. Quercetin (QC), a natural component of foods, protects against renal injury. Here, we explored the possible mechanisms that are responsible for ADMA-induced renal fibrosis and the protective effect of QC. We found that ADMA treatment activated the endoplasmic reticulum (ER) stress sensor proteins phosphorylated protein kinase RNA-activated-like ER kinase (PERK) and inositol requiring-1α (IRE1), which correspondingly induced C/EBP homologous protein (CHOP) expression and phosphorylated c-Jun N-terminal kinase (JNK) phosphorylation in glomerular endothelial cells (GEnCs). Following this, ADMA promoted ER stress-induced apoptosis and resulted in transforming growth factor β (TGF-β) expression in GEnCs. SP600125, an inhibitor of JNK, and CHOP siRNA protected against ADMA-induced cell apoptosis and TGF-β expression. QC prevented ADMA-induced PERK and IRE1 apoptotic ER stress pathway activation. Also, ADMA-induced GEnCs apoptosis and TGF-β expression was reduced by QC. Overexpression of CHOP blocked QC-mediated protection from apoptosis in ER stressed cells. Overall, these observations indicate that ADMA may induce GEnCs apoptosis and TGF-β expression by targeting the PERK-CHOP and IRE1-JNK pathway. In addition, drugs such as QC targeting ER stress may hold great promise for the development of novel therapies against ADMA-induced renal fibrosis. Less

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, ... More

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals. Less

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glyc... More

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport. Less

Background: Glycation of high-density lipoprotein (HDL) decreases its ability to induce cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in endoth... More

Background: Glycation of high-density lipoprotein (HDL) decreases its ability to induce cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in endothelial cells. Whether lipid content of HDL, especially sphingosine-1-phosphate (S1P), plays any specific role in restoring the protective function of HDL in type 2 diabetes mellitus (T2DM) is still unknown. Methods and results: Immunochemical techniques demonstrated that glycated HDL loses its protective function of regulating COX-2 expression compared with diabetic HDL. We proved that the lipid content, especially phospholipid content differed between diabetic HDL and glycated HDL. Levels of HDL-c-bound S1P were increased in T2DM compared with control subjects as detected by UPLC-MS/MS (HDL-c-bound S1P in control subjects vs. T2DM: 309.1 ± 13.71 pmol/mg vs. 382.1 ± 24.45 pmol/mg, P < 0.05). Additionally, mRNA levels of S1P lyase enzymes and S1P phosphatase 1/2 were decreased in peripheral blood by real-time PCR. Antagonist of S1P receptor 1 and 3 (S1PR1/3) diminished the functional difference between apoHDL&PL (HDL containing the protein components and phospholipids) and diabetic apoHDL&PL (diabetic HDL containing the protein components and phospholipids). With different doses of S1P reconstituted on glycated HDL, its function in inducing the COX-2 expression was restored to the same level as diabetic HDL. The mechanism of S1P reconstituted HDL (rHDL) in the process of regulating COX-2 expression involved the phosphorylation of ERK/MAPK-CREB signal pathway. Conclusion/significance: S1P harbored on HDL is the main factor which restores its protective function in endothelial cells in T2DM. S1P and its receptors are potential therapeutic targets in ameliorating the vascular dysfunction in T2DM. Less

Nanoparticles (NPs) constitute an important medium for the targeted delivery of cancer therapeutics. Targeting of NPs to a specific cell type is traditionally achieved th... More

Nanoparticles (NPs) constitute an important medium for the targeted delivery of cancer therapeutics. Targeting of NPs to a specific cell type is traditionally achieved through the modification of the NP surface with peptides, aptamers, or other motifs that specifically recognize a cell-surface receptor, leading to internalization of NPs via clathrin and caveolae-mediated endocytosis. We have discovered that modifying the NP surface with anionic polyelectrolytes of varying lipophilicity can regulate the uptake of lipid NPs by endothelial and epithelial cells. Furthermore, we report the finding that synthetic polyelectrolytes composed of an aromatic sulfonic acid backbone exhibit specific affinity for caveolae of endothelial cells. By exploiting the higher expression of caveolae in endothelial cells in comparison with epithelial cells, a purely physiochemical approach to the targeted uptake of lipid NPs to endothelial cells is demonstrated. The ability to confer preferential affinity for NPs to cell surface domains by varying the charge and lipophilic characteristics of an NP surface offers a general means of achieving targeted delivery without the need for receptor-ligand-type targeting strategies. Keywords: aromatic polysulfonates; polyanion; polystyrene sulfonate; vasculature. Less

Background: Vascular endothelial growth factor (VEGF) is a key angiogenic factors. It plays an important role in both physiologic and pathologic angiogenesis and increase... More

Background: Vascular endothelial growth factor (VEGF) is a key angiogenic factors. It plays an important role in both physiologic and pathologic angiogenesis and increases permeability across the vessels. Using antibody phage display technology, we obtained a novel anti-VEGFA IgG, named as FD006. In this study, the pharmacological characteristics and efficacy of FD006 in corneal neovascularization (CoNV) were evaluated. Results: FD006 was predicted to have similar binding mode to bevacizumab. Experimental analysis showed that the binding ability of FD006 seemed a little stronger than bevacizumab, for the EC50 of FD006 to bind VEGF analyzed by ELISA was about 0.037 μg/mL while that of bevacizumab was 0.18 μg/mL. Binding kinetics assays showed similar results that FD006 possessed 2-fold higher affinity to bind VEGF than bevacizumab due to slower dissociation rate of FD006; meanwhile, FD006 inhibited the VEGF-induced proliferation of HUVEC with an IC50 value of 0.031 ± 0.0064 μg/ml, which seemed similar or a litter better than bevacizumab (0.047 ± 0.0081 μg/ml). The subconjunctival administration of FD006, bevacizumab or dexamethasone could significantly inhibit the growth of CoNV contrasting to N.S (p Less

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protecti... More

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙−) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways. Less

Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of infla... More

Tetramethylpyrazine (TMP) is an active compound isolated from a Chinese herbal prescription that is widely used in traditional Chinese medicine for the treatment of inflammatory and cardiovascular diseases. We have previously reported that TMP acts as a potent antioxidant protecting endothelial cells against high glucose-induced damages. However, the molecular mechanism responsible for the antioxidant effect of TMP remains to be elucidated. In this study, we show that TMP increases nitric oxide production in endothelial cells and promotes endothelium-dependent relaxation in rate aortic rings. The antioxidant effect of TMP appears attributable to its ability to activate the mitochondrial biogenesis, as reflected in an up-regulation of complex III and amelioration of mitochondrial membrane potential. Furthermore, TMP is able to reverse high glucose-induced suppression of SIRT1 and the biogenesis-related factors, including PGC-1α, NRF1 and TFAM, suggesting a new molecular mechanism underlying the protective effect of TMP on the endothelium. Less

Angiogenesis is crucial for tumor development, growth and metastasis. Vascular endothelial growth factor (VEGF) has been implicated in promoting solid tumor growth and me... More

Angiogenesis is crucial for tumor development, growth and metastasis. Vascular endothelial growth factor (VEGF) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis, and blocking the activity of VEGF can starve tumors. Avastin, which is a humanized anti-VEGF antibody, has been successfully applied in clinics since 2004. However, the price of Avastin is extremely high for Chinese people. Here, we report a novel human anti-VEGF neutralizing antibody, MIL60, which shows an affinity comparable to that of Avastin (the KD value of MIL60 was 44.5 pM, while that of Avastin was 42.7 pM). MIL60 displays favorable actions in inhibiting VEGF-triggered endothelial cell proliferation (the IC50 value of MIL60 was 31±6.4 ng/ml and that of Avastin was 47±8.1 ng/ml), migration (8 µg/ml or 0.8 µg/ml MIL60 versus the control: P<0.05) and tube formation (2 µg/ml or 0.2 µg/ml MIL60 versus the control: P<0.05) via the VEGFR2 signaling pathway. Moreover, MIL60 was shown to inhibit tumor growth and angiogenesis in vivo in xenograft models of human colon carcinoma and ovarian cancer using immunotherapy and immunohistochemistry analysis (MIL60 versus N.S.: P=0.0007; Avastin versus N.S.: P=0.00046). These data suggest that MIL60 is a potential therapeutic, anti-angiogenic agent. Our work provides a novel anti-VEGF antibody, which can be considered an anti-tumor antibody candidate and a new option for patients with various cancers. Less

Magnesium (Mg) based alloys are the most advanced cardiovascular stent materials. This new generation of stent scaffold is currently under clinical evaluation with encour... More

Magnesium (Mg) based alloys are the most advanced cardiovascular stent materials. This new generation of stent scaffold is currently under clinical evaluation with encouraging outcomes. All these Mg alloys contain a certain amount of rare earth (RE) elements though the exact composition is not yet disclosed. RE alloying can usually enhance the mechanical strength of different metal alloys but their toxicity might be an issue for medical applications. It is still unclear how RE elements will affect the magnesium (Mg) alloys intended for stent materials as a whole. In this study, we evaluated MgZnCaY-1RE, MgZnCaY-2RE, MgYZr-1RE, and MgZnYZr-1RE alloys for cardiovascular stents applications regarding their mechanical strength, corrosion resistance, hemolysis, platelet adhesion/activation, and endothelial biocompatibility. The mechanical properties of all alloys were significantly improved. Potentiodynamic polarization showed that the corrosion resistance of four alloys was at least 3–10 times higher than that of pure Mg control. Hemolysis test revealed that all the materials were non-hemolytic while little to moderate platelet adhesion was found on all materials surface. No significant cytotoxicity was observed in human aorta endothelial cells cultured with magnesium alloy extract solution for up to seven days. Direct endothelialization test showed that all the alloys possess significantly better capability to sustain endothelial cell attachment and growth. The results demonstrated the promising potential of these alloys for stent material applications in the future. Less

Background: High mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. ... More

Background: High mobility group box 1(HMGB1) was first recognized as a nuclear protein that increased the chromatin remodeling and regulates transcription of many genes. In recent years, HMGB1 has been identified as a critical “late” pro-inflammatory mediator due to its unique secretion pattern and lethal effects in sepsis. Therefore, preventing the active release and inhibiting the pro-inflammatory activity of HMGB1 become promising strategies for the treatment of sepsis. Here, we reported the therapeutic effects of Gu-4, a lactosyl derivative, on sepsis and the underlying molecular mechanisms. Methodology/Principal Findings: In an experimental rat model of sepsis caused by cecal ligation and puncture (CLP), Gu-4 administration prominently attenuated lung injury and improved the survival of the septic animals, which was positively correlated with the decrease of the serum HMGB1 level. Using RAW264.7 macrophage cell line, we further showed that Gu-4 significantly suppressed the lipopolysaccharide (LPS)-induced release and cytoplasmic translocation of HMGB1. Moreover, Gu-4 not only dose-dependently attenuated recombinant human (rhHMGB1)-induced production of TNF-α, IL-6, and IL-1β in THP-1 cells, but also greatly inhibited the adhesion of rhHMGB1-challenged THP-1 cells to HUVECs. Analyses of flow cytometry demonstrated that Gu-4 could effectively reduce the activation of CD11b elicited by rhHMGB1. Western blot analyses revealed that Gu-4 treatment could partially block the rhHMGB1-induced activation of ERK and NF-κB signalings. Meanwhile, CD11b knockdown also obviously attenuated the rhHMGB1-induced phosphorylations of ERK and IKKα/β. Conclusions/Significance: Taken together, our results suggest that Gu-4 possesses a therapeutic potential in the treatment of sepsis probably via inhibiting the LPS-induced release of HMGB1 from macrophages and via suppressing the pro-inflammatory activity of HMGB1. Less

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intr... More

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro L-arginine methyl ester (L-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by L-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta. Less

Background: The study of the cerebrovascular physiology is crucial to understand the pathogenesis of neurological disease and the pharmacokinetic of drugs. Appropriate mo... More

Background: The study of the cerebrovascular physiology is crucial to understand the pathogenesis of neurological disease and the pharmacokinetic of drugs. Appropriate models in vitro often fail to represent in vivo physiology. To address these issues we propose the use of a novel artificial vascular system that closely mimics capillary and venous segments of human cerebrovasculature while also allowing for an extensive control of the experimental variables and their manipulation. Results: Using hollow fiber technology, we modified an existing dynamic artificial model of the blood-brain barrier (BBB) (DIV-capillary) to encompass the distal post-capillary (DIV-venules) segments of the brain circulatory system. This artificial brain vascular system is comprised of a BBB module serially connected to a venule segment. A pump generates a pulsatile flow with arterial pressure feeding the system. The perfusate of the capillary module achieves levels of shear stress, pressure, and flow rate comparable to what observed in situ. Endothelial cell exposure to flow and abluminal astrocytic stimuli allowed for the formation of a highly selective capillary BBB with a trans-endothelial electrical resistance (TEER; >700 ohm cm2) and sucrose permeability (< 1X10-u cm/sec) comparable to in vivo. The venule module, which attempted to reproduce features of the hemodynamic microenvironment of venules, was perfused by media resulting in shear stress and intraluminal pressure levels lower than those found in capillaries. Because of altered cellular and hemodynamic factors, venule segments present a less stringent vascular bed (TEER <250 Ohm cm2; Psucrose > 1X10-4 cm/sec) than that of the BBB. Abluminal human brain vascular smooth muscle cells were used to reproduce the venular abluminal cell composition. Conclusion: The unique characteristics afforded by the DIV-BBB in combination with a venule segment will realistically expand our ability to dissect and study the physiological and functional behavior of distinct segments of the human cerebrovascular network. Less

Background: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, increases the activity of NF-κB (NF-κB) and then induces the expres... More

Background: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, increases the activity of NF-κB (NF-κB) and then induces the expression of intercellular adhesion molecule-1 (ICAM-1). However, the mechanisms regulating ADMA-induced NF-κB activation are unknown. This study investigated the function of actin cytoskeleton for ADMA-induced NF-κB activation and ICAM-1 expression in endothelial cells. Less

Background: Dysfunctional high-density lipoprotein (HDL) may have pro-inflammatory effects on the endothelial cells,which causes atherosclerosis in type 2 diabetes mellit... More

Background: Dysfunctional high-density lipoprotein (HDL) may have pro-inflammatory effects on the endothelial cells,which causes atherosclerosis in type 2 diabetes mellitus (T2DM). HDL is a major carrier of sphingosine-1-phosphate (S1P) in plasma while S1P exhibits multiple biological activities. However, potential role of HDL and S1P in T2DM remains unexplored. We hypothesized that diabetic HDL with higher contents of S1P exerts beneficial effects on the vascular system. Methods: Subjects with T2DM with or without proved large arteries atherosclerosis and normal controls (n=15 for each group) were recruited in the present study. HDL was isolated from the subjects by ultracentrifugation. The levels of HDL-associated S1P were determined by UPLC-MS/MS. The protective function of diabetic HDL and S1P was evaluated by measuring cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release by human umbilical vein endothelial cells (HUVECs) using western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Results: The S1P levels in isolated HDL were significantly increased in T2DM subjects compared with controls (235.6 ± 13.4 vs 195.0 ± 6.4 ng/mg, P< 0.05). The diabetic HDL exerted greater protective effects on inducing COX-2 expression and PGI-2 release by HUVECs than those of control HDL (p < 0.05, p < 0.01, respectively). Pertussis toxin, a common inhibitor of G-couple protein receptors, and VPC 23019, an antagonist of S1P receptor 1 and 3 significantly attenuated HDL-induced COX-2 expression and PGI-2 release. Conclusions: Diabetic HDL carries higher level of S1P compared with normal HDL, which has the potential to contribute to protective effects on endothelial cells by inducing COX-2 expression and PGI-2 release. These findings provide a new insight of S1P function in T2DM patients, possibly leading to a new therapeutic target. Less

Aims: It has been shown that nerve growth factor-β (NGF-β) promoted the initiation and progression of many tumors, and we have previously demonstrated that the expressi... More

Aims: It has been shown that nerve growth factor-β (NGF-β) promoted the initiation and progression of many tumors, and we have previously demonstrated that the expression of NGF-β was associated with tumor stage, nerve infiltration and lymph node metastasis in human hilar cholangiocarcinoma. However, whether NGF-β promotes tumor progression in human cholangiocarcinoma requires further investigation. Therefore, we aimed to determine the effects of NGF-β on the progression of human cholangiocarcinoma. Methods: Human cholangiocarcinoma QBC939 stable cell lines with over-expressed or silenced NGF-β genes were generated with pEGFP-N1-NGF-β and pGPU6/GFP/Neo-NGF-β-shRNA recombinant plasmids. Cell proliferation assay, colony formation assay, cell cycle analysis, apoptosis assay and tumorigenicity assay were performed to evaluate the role of NGF-β in the progression of human cholangiocarcinoma. In addition, human lymphatic endothelial cells were co-cultured with QBC939 culture supernatants, and the cell proliferation and migration abilities of the lymphatic endothelial cells were evaluated. Results: Forced expression of NGF-β in QBC939 cell lines promoted proliferation, colony formation and tumorigenicity in these cells and inhibited the apoptosis. However, down-regulation of NGF-β inhibited proliferation, colony formation and tumorigenicity, and increased the apoptotic rate of QBC939 cells. In addition, the NGF-β gain-of-function induced a high expression of vascular endothelial growth factor C and enhanced the proliferation and migration of lymphatic endothelial cells, while NGF-β loss-of-function showed opposite effects. Conclusions: We concluded that NGF-β promoted tumor progression in human cholangiocarcinoma QBC939 cells. Our results provided a new concept to understand the role of NGF-β in cholangiocarcinoma progression, and might provide important information for the development of new targeted therapies in human cholangiocarcinoma. Less

Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). D... More

Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). Decreasing bioavailable copper has been used as an anti-angiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression) and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4+ T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes. Less

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and em... More

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy. Less

During acute inflammation, functioning lymphatics are believed to reduce edema and to provide a transiting route for immune cells, but the extent at which the dermal lymp... More

During acute inflammation, functioning lymphatics are believed to reduce edema and to provide a transiting route for immune cells, but the extent at which the dermal lymphatic remodeling impacts lymphatic transport or the factors regulating these changes remains unclear. Herein we quantify the increase in lymphatic endothelial cells (LECs) and examine the expression of pro-angiogenenic and lymphangiogenic factors during acute cutaneous hypersensitivity (CHS). We found that LECs actively proliferate during CHS but that this proliferation does not affect the lymphatic vessel density. Instead, lymphatic remodeling is accompanied by lymphatic vessel leakiness and lower ejection of lymph fluid, which is observed only in the proximal lymphatic vessel draining the inflamed area. LECs and the immune cells release growth factors and cytokines during inflammation, which impact the lymphatic microenvironment and function. We identified that FGF-2, PLGF-2, HGF, EGF, and KC/CXCL17 are differentially expressed within tissues during acute CHS, but both VEGF-C and VEGF-D levels do not significantly change. Our results indicate that VEGF-C and VEGF-D are not the only players and other factors may be responsible for the LECs proliferation and altered lymphatic function in acute CHS. Less

Background: Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate ... More

Background: Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract. Less

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR-146a and miR-146b a... More

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR-146a and miR-146b are induced in endothelial cells upon exposure to pro-inflammatory cytokines. Despite the rapid transcriptional induction of the miR-146a/b loci, which is in part mediated by EGR-3, miR-146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down-regulation of inflammatory gene expression. We demonstrate that miR-146 negatively regulates inflammation. Over-expression of miR-146a blunts endothelial activation, while knock-down of miR-146a/b in vitro or deletion of miR-146a in mice has the opposite effect. MiR-146 represses the pro-inflammatory NF-κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR-146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro-inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases. Less

Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth... More

Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth factor-responsive gene, and its protein product, VGF, is characterized by the presence of partially cleaved products. One of the VGF-derived peptides is TLQP-21, which is composed of 21 amino acids (residues 556-576). Past studies have reported that TLQP-21 could stimulate insulin secretion in pancreatic cells and protect these cells from apoptosis, which suggests that TLQP-21 has a potential function in diabetes therapy. Here, we explore the protective role of TLQP-21 against the high glucose-mediated injury of vascular endothelial cells. Using human umbilical vascular endothelial cells (HUVECs), we demonstrated that TLQP-21 (10 or 50 nM) dose-dependently prevented apoptosis under high-glucose (30 mmol/L) conditions (the normal glucose concentration is 5.6 mmol/L). TLQP-21 enhanced the expression of NAPDH, resulting in upregulation of glutathione (GSH) and a reduction in the levels of reactive oxygen species (ROS). TLQP-21 also upregulated the expression of glucose-6-phosphate dehydrogenase (G6PD), which is known as the main source of NADPH. Knockdown of G6PD almost completely blocked the increase of NADPH induced by TLQP-21, indicating that TLQP-21 functions mainly through G6PD to promote NADPH generation. In conclusion, TLQP-21 could increase G6PD expression, which in turn may increase the synthesis of NADPH and GSH, thereby partially restoring the redox status of vascular endothelial cells under high glucose injury. We propose that TLQP-21 is a promising drug for diabetes therapy. Less

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesi... More

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis. Less

We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric st... More

We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric stimulation. The HDEN device, with which drastic pH change during the electroporation was avoided,was demonstrated to be highly effective for transfection of not only DNA plasmids and small interfering RNAs (siRNA), but also a small molecular anti-cancer drug, into cells in adjustable volumes of cell suspension. The method constitutes a very flexible electroporation approach in a wide range of in vitro or ex vivo scenarios in various tubes, standard multi-well plates as well as flow chambers. Less

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angioge... More

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT), is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research. Less

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus... More

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3(+) structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival. Less

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angioge... More

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angiogenesis has been considered as an essential event in perpetuating inflammatory and immune responses, as well as supporting pannus growth and development of RA, inhibition of angiogenesis has been proposed as a novel therapeutic strategy for RA. Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, has been extensively used in treatment of RA patients. It also acts as a small molecule inhibitor of tumor angiogenesis in several cancer types. However, it is unclear whether triptolide possesses an anti-angiogenic effect in RA. To address this problem, we constructed collagen-induced arthritis (CIA) model using DA rats by the injection of bovine type II collagen. Then, CIA rats were treated with triptolide (11–45 µg/kg/day) starting on the day 1 after first immunization. The arthritis scores (P<0.05) and the arthritis incidence (P<0.05) of inflamed joints were both significantly decreased in triptolide-treated CIA rats compared to vehicle CIA rats. More interestingly, doses of 11∼45 µg/kg triptolide could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints (all P<0.05). Moreover, triptolide inhibited matrigel-induced cell adhesion of HFLS–RA and HUVEC. It also disrupted tube formation of HUVEC on matrigel and suppressed the VEGF-induced chemotactic migration of HFLS–RA and HUVEC, respectively. Furthermore, triptolide significantly reduced the expression of angiogenic activators including TNF-α, IL-17, VEGF, VEGFR, Ang-1, Ang-2 and Tie2, as well as suppressed the IL1-β-induced phosphorylated of ERK, p38 and JNK at protein levels. In conclusion, our data suggest for the first time that triptolide may possess anti-angiogenic effect in RA both in vivo and in vitro assay systems by downregulating the angiogenic activators and inhibiting the activation of mitogen-activated protein kinase downstream signal pathway. Less

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angioge... More

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angiogenesis has been considered as an essential event in perpetuating inflammatory and immune responses, as well as supporting pannus growth and development of RA, inhibition of angiogenesis has been proposed as a novel therapeutic strategy for RA. Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, has been extensively used in treatment of RA patients. It also acts as a small molecule inhibitor of tumor angiogenesis in several cancer types. However, it is unclear whether triptolide possesses an anti-angiogenic effect in RA. To address this problem, we constructed collagen-induced arthritis (CIA) model using DA rats by the injection of bovine type II collagen. Then, CIA rats were treated with triptolide (11–45 µg/kg/day) starting on the day 1 after first immunization. The arthritis scores (P<0.05) and the arthritis incidence (P<0.05) of inflamed joints were both significantly decreased in triptolide-treated CIA rats compared to vehicle CIA rats. More interestingly, doses of 11∼45 µg/kg triptolide could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints (all P<0.05). Moreover, triptolide inhibited matrigel-induced cell adhesion of HFLS–RA and HUVEC. It also disrupted tube formation of HUVEC on matrigel and suppressed the VEGF-induced chemotactic migration of HFLS–RA and HUVEC, respectively. Furthermore, triptolide significantly reduced the expression of angiogenic activators including TNF-α, IL-17, VEGF, VEGFR, Ang-1, Ang-2 and Tie2, as well as suppressed the IL1-β-induced phosphorylated of ERK, p38 and JNK at protein levels. In conclusion, our data suggest for the first time that triptolide may possess anti-angiogenic effect in RA both in vivo and in vitro assay systems by downregulating the angiogenic activators and inhibiting the activation of mitogen-activated protein kinase downstream signal pathway. Less

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus... More

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3(+) structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival. Less

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angioge... More

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT), is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research. Less

We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric st... More

We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric stimulation. The HDEN device, with which drastic pH change during the electroporation was avoided,was demonstrated to be highly effective for transfection of not only DNA plasmids and small interfering RNAs (siRNA), but also a small molecular anti-cancer drug, into cells in adjustable volumes of cell suspension. The method constitutes a very flexible electroporation approach in a wide range of in vitro or ex vivo scenarios in various tubes, standard multi-well plates as well as flow chambers. Less

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesi... More

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis. Less

Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth... More

Hyperglycemia causes oxidative stress that could damage vascular endothelial cells, leading to cardiovascular complications. The Vgf gene was identified as a nerve growth factor-responsive gene, and its protein product, VGF, is characterized by the presence of partially cleaved products. One of the VGF-derived peptides is TLQP-21, which is composed of 21 amino acids (residues 556-576). Past studies have reported that TLQP-21 could stimulate insulin secretion in pancreatic cells and protect these cells from apoptosis, which suggests that TLQP-21 has a potential function in diabetes therapy. Here, we explore the protective role of TLQP-21 against the high glucose-mediated injury of vascular endothelial cells. Using human umbilical vascular endothelial cells (HUVECs), we demonstrated that TLQP-21 (10 or 50 nM) dose-dependently prevented apoptosis under high-glucose (30 mmol/L) conditions (the normal glucose concentration is 5.6 mmol/L). TLQP-21 enhanced the expression of NAPDH, resulting in upregulation of glutathione (GSH) and a reduction in the levels of reactive oxygen species (ROS). TLQP-21 also upregulated the expression of glucose-6-phosphate dehydrogenase (G6PD), which is known as the main source of NADPH. Knockdown of G6PD almost completely blocked the increase of NADPH induced by TLQP-21, indicating that TLQP-21 functions mainly through G6PD to promote NADPH generation. In conclusion, TLQP-21 could increase G6PD expression, which in turn may increase the synthesis of NADPH and GSH, thereby partially restoring the redox status of vascular endothelial cells under high glucose injury. We propose that TLQP-21 is a promising drug for diabetes therapy. Less

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infec... More

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7, and ITvar19) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria. Less

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CN... More

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia. Less

Wide applications and extreme potential of metal oxide nanoparticles (NPs) increase occupational and public exposure and may yield extraordinary hazards for human health.... More

Wide applications and extreme potential of metal oxide nanoparticles (NPs) increase occupational and public exposure and may yield extraordinary hazards for human health. Exposure to NPs has a risk for dysfunction of the vascular endothelial cells. The objective of this study was to assess the cytotoxicity of six metal oxide NPs to human cardiac microvascular endothelial cells (HCMECs) in vitro. Metal oxide NPs used in this study included zinc oxide (ZnO), iron(III) oxide (Fe(2)O(3)), iron(II,III) oxide (Fe(3)O(4)), magnesium oxide (MgO), aluminum oxide (Al(2)O(3)), and copper(II) oxide (CuO). The cell viability, membrane leakage of lactate dehydrogenase, intracellular reactive oxygen species, permeability of plasma membrane, and expression of inflammatory markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, macrophage cationic peptide-1, and interleukin-8 in HCMECs were assessed under controlled and exposed conditions (12-24 h and 0.001-100 μg/ml of exposure). The results indicated that Fe(2)O(3), Fe(3)O(4), and Al(2)O(3) NPs did not have significant effects on cytotoxicity, permeability, and inflammation response in HCMECs at any of the concentrations tested. ZnO, CuO, and MgO NPs produced the cytotoxicity at the concentration-dependent and time-dependent manner, and elicited the permeability and inflammation response in HCMECs. These results demonstrated that cytotoxicity, permeability, and inflammation in vascular endothelial cells following exposure to metal oxide nanoparticles depended on particle composition, concentration, and exposure time. Less

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synt... More

Arginase constrains endothelial nitric oxide synthase activity by competing for the common substrate, L -Arginine. We have recently shown that inducible nitric oxide synthase (NOS2) S-nitrosates and activates arginase 1 (Arg1) leading to age-associated vascular dysfunction. Here, we demonstrate that a direct interaction of Arg1 with NOS2 is necessary for its S-nitrosation. The specific domain of NOS2 that mediates this interaction is identified. Disruption of this interaction in human aortic endothelial cells prevents Arg1 S-nitrosation and activation. Thus, disruption of NOS2-Arg1 interaction may represent a therapeutic strategy to attenuate age related vascular endothelial dysfunction. Less

CD200 is a highly glycosylated cell surface protein containing two immunoglobulin superfamily domains in the extracellular region and performs immunosuppressive activitie... More

CD200 is a highly glycosylated cell surface protein containing two immunoglobulin superfamily domains in the extracellular region and performs immunosuppressive activities. It is widely distributed in various tissues including the vascular endothelium. We report here the distribution of CD200 in rat endothelia from different vascular beds. Endothelial CD200 immunoreactivity was weakly expressed in most arteries but was intensely expressed in the arterioles, most veins and venules, as well as continuous and fenestrated capillaries. The distribution of CD200 in the sinusoidal and lymphatic endothelia was variable. Immunoelectron microscopic studies revealed that endothelial CD200 varied considerably not only in different microvasculatures but also in the membrane domains at the subcellular level. Endothelial CD200 expression was differentially regulated by lipopolysaccharide in cell types both in vivo and in vitro. Functional assessments of endothelial CD200 suggested that the physical binding between CD200 and CD200 receptor (CD200R) was involved in T-cell adhesion to the endothelium but not in macrophage-endothelium interaction. In the latter, however, CD200 agonist, a synthetic peptide from complementarity-determining region 3 of mouse CD200, may trigger CD200R signaling in macrophages to suppress their adhesion to the endothelium. Our findings demonstrate that the distribution, subcellular localization, and lipopolysaccharide-regulation of endothelial CD200 are heterogeneous, and provide evidence elucidating the functional roles of endothelial CD200 during tissue inflammation. Less

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with dis... More

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Abeta on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Abeta structure, suggesting that the toxic effect of the Abeta assemblies goes beyond mere multimerization. Less

Damage to endothelial cells is a key event in the pathogenesis of atherosclerosis and vascular disease. This study aimed to determine whether free fatty acids (FFAs) indu... More

Damage to endothelial cells is a key event in the pathogenesis of atherosclerosis and vascular disease. This study aimed to determine whether free fatty acids (FFAs) induced oxidative stress and apoptosis in human brain microvascular endothelial cells (HBMVECs) in vitro and, if so, which signalling pathway mediated these effects. After culture in different concentrations of FFAs for 24 - 72 h, cell viability/proliferation was determined using a cell counting kit, apoptosis was detected by measuring caspase-3 activity and by using annexin V-conjugated fluorescein isothiocyanate/propidium iodide staining, and oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). The HBMVECs exposed to FFAs showed significantly decreased cell proliferation, increased apoptosis and ROS levels, and decreased MMP. In conclusion, the results showed that high levels of FFAs induced oxidative stress, which damaged HBMVECs and resulted in apoptosis. Less

Endothelial cells regulate vascular integrity and express complement binding proteins including gC1qR/p33 (gC1qR), which recognize C1q, a subunit of the first component o... More

Endothelial cells regulate vascular integrity and express complement binding proteins including gC1qR/p33 (gC1qR), which recognize C1q, a subunit of the first component of the classical complement pathway. Experiments were performed to investigate classical complement pathway activation on resting endothelial cells and endothelial cells exposed to shear stress. C1q deposition and C4 activation (C4d) were demonstrated by solid phase ELISA and flow cytometry on human microvascular and umbilical vein endothelial cells after exposure to serum or plasma. C4d deposition was accompanied by downstream complement activation including C3b and C5b-9 deposition. C4 activation failed to occur in C1q depleted serum, but was not affected by Factor B depleted serum, confirming classical complement pathway activation. Moreover, C4 activation occurred following exposure of endothelial cells to purified C1 and C4, in the absence of other plasma proteins, and in the absence of detectable cell surface IgG and IgM. Shear stress (18 dynes/cm2) increased C1q (n=9, p<0.05) and C4d (n=9, p<0.05) deposition approximately two-fold, and enhanced endothelial cell gC1qR expression (n=7, p<0.05). Treatment of endothelial cells with anti gC1qR monoclonal antibody F(ab')2 fragments reduced C4d deposition by approximately 20% (n=5, p<0.05). These data demonstrate direct classical complement pathway activation on endothelial cells. gC1qR appears to play a minor but definable role, whereas cell surface IgG or IgM are not required. Less

Cerebral amyloid angiopathy is a common pathological feature of patients with Alzheimer's disease (AD) and it is also the hallmark of individuals with a rare autosomal do... More

Cerebral amyloid angiopathy is a common pathological feature of patients with Alzheimer's disease (AD) and it is also the hallmark of individuals with a rare autosomal dominant disorder known as hereditary cerebral hemorrhage with amyloidosis-Dutch type. We have shown previously that wild type A(beta) peptides are anti-angiogenic both in vitro and in vivo and could contribute to the compromised cerebrovascular architecture observed in AD. In the present study, we investigated the potential anti-angiogenic activity of the Dutch A(beta)(1-40) (E22Q) peptide. We show that compared to wild type A(beta), freshly solubilized Dutch A(beta) peptide more potently inhibits the formation of capillary structures induced by plating human brain microvascular endothelial cells onto a reconstituted basement membrane. Aggregated/fibrillar preparations of wild type A(beta) and Dutch A(beta) do not appear to be anti-angiogenic in this assay. The stronger anti-angiogenic activity of the Dutch A(beta) compared to wild type A(beta) appears to be related to the increased formation of low molecular weight A(beta) oligomers in the culture medium surrounding human brain microvascular endothelial cells. Using oligonucleotide microarray analysis of human brain microvascular endothelial cells, followed by a genome-scale computational analysis with the Ingenuity Pathways Knowledge Base, networks of genes affected by an anti-angiogenic dose of Dutch A(beta) were identified. This analysis highlights that several biological networks involved in angiogenesis, tumorigenesis, atherosclerosis, cellular migration and proliferation are disrupted in human brain microvascular endothelial cells exposed to Dutch A(beta). Altogether, these data provide new molecular clues regarding the pathological activity of Dutch A(beta) peptide in the cerebrovasculature. Less

l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been show... More

l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors. Less