Endothelial Cell Growth Supplement

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

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

Tumor angiogenesis plays very important roles for tumorigenesis, tumor development, metastasis, and prognosis. Targeting T1/T2 dual modality magnetic resonance (MR) imagi... More

Tumor angiogenesis plays very important roles for tumorigenesis, tumor development, metastasis, and prognosis. Targeting T1/T2 dual modality magnetic resonance (MR) imaging of the tumor vascular endothelial cells (TVECs) with MR molecular probes can greatly improve diagnostic sensitivity and specificity, as well as helping to make an early diagnosis of tumor at the preclinical stage. In this study, a new T1 and T2 dual modality nanoprobe was successfully fabricated. The prepared nanoprobe comprise peptides CL 1555, poly(ε-caprolactone)-blockpoly(ethylene glycol) amphiphilic copolymer shell, and dozens of manganese ferrite (MnFe2 O4 ) nanoparticle core. The results showed that the hydrophobic MnFe2 O4 nanoparticles were of uniform spheroidal appearance and narrow size distribution. Due to the self-assembled nanomicelles structure, the prepared probes were of high relaxivity of 281.7 mM−1 s−1 , which was much higher than that of MnFe2 O4 nanoparticles (67.5 mM−1 s−1 ). After being grafted with the targeted CD105 peptide CL 1555, the nanomicelles can combine TVECs specifically and make the labeled TVECs dark in T2 -weighted MR imaging. With the passage on, the Mn2+ ions were released from MnFe2 O4 and the size decreased gradually, making the signal intensity of the second and third passage of labeled TVECs increased in T1 -weighted MR imaging. Our results demonstrate that CL-poly(ethylene glycol)-MnFe2 O4 can conjugate TVECs and induce dark and bright contrast in MR imaging, and act as a novel molecular probe for T1 - and T2 -enhanced MR imaging of tumor angiogenesis. Keywords: CL 1555, CL-PEG-MnFe2 O4 , TVECs, CD105, tumor angiogenesis Less

Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles i... More

Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles in liver injury and repair, but their role in the underlying pathogenetic mechanisms of non-alcoholic fatty liver disease remains undefined. Here, we evaluated the effects of steatosis on LSEC gene expression in a murine model of non-alcoholic fatty liver disease and an immortalized LSEC line. Using microarray we identified distinct gene expression profiles following exposure to free fatty acids. Gene pathway analysis showed a number of differentially expressed genes including those involved in lipid metabolism and signaling and inflammation. Interestingly, in contrast to hepatocytes, fatty acids led to decreased expression of pro-inflammatory chemokines including CCL2 (MCP-1), CXCL10 and CXCL16 in both primary and LSEC cell lines. Chemokine downregulation translated into a significant inhibition of monocyte migration and LSECs isolated from steatotic livers demonstrated a similar shift towards an anti-inflammatory phenotype. Overall, these pathways may represent a compensatory mechanism to reverse the liver damage associated with non-alcoholic fatty liver disease. 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

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(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

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

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

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

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

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of ... More

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of this study was to investigate whether UII affects the development of atherosclerosis in cholesterol-fed rabbits. UII was infused for 16 weeks through an osmotic mini-pump into male Japanese White rabbits fed on a high-cholesterol diet. Plasma lipids and body weight were measured every 4 weeks. Aortic atherosclerotic lesions along with cellular components, collagen fibers, matrix metalloproteinase-1 and -9 were examined. Moreover, vulnerability index of atherosclerotic plaques was evaluated. UII infusion significantly increased atherosclerotic lesions within the entire aorta by 21% over the control (P = 0.013). Atherosclerotic lesions were increased by 24% in the aortic arch (P = 0.005), 11% in the thoracic aorta (P = 0.054) and 18% in the abdominal aorta (P = 0.035). These increases occurred without changes in plasma levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or body weight. Immunohistochemical staining revealed that macrophages and matrix metalloproteinase-9 were significantly enhanced by 2.2-fold and 1.6-fold in UII group. In vitro studies demonstrated that UII up-regulated the expression of vascular cell adhesion protein-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells, which was inhibited by the UII receptor antagonist urantide. In conclusion, our results showed that UII promotes the development of atherosclerotic lesions and destabilizes atherosclerotic plaques in cholesterol-fed rabbits. Less

Background: Autophagy is important for cells to degrade protein aggregates and organelles. Our preliminary study suggests that ischemia/reperfusion in rabbit hearts promo... More

Background: Autophagy is important for cells to degrade protein aggregates and organelles. Our preliminary study suggests that ischemia/reperfusion in rabbit hearts promoted autophagic myocardial injury, resulting in no-reflow phenomenon. In this study, we sought to further understand the mechanism and outcome of the upregulation of autophagy in ischemia/reperfusion. 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: 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

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

Aims: Conventional revascularization strategies or drug therapies for ischemic heart disease (IHD) are designed for reperfusion of coronary arteries to salvage cardiomyoc... More

Aims: Conventional revascularization strategies or drug therapies for ischemic heart disease (IHD) are designed for reperfusion of coronary arteries to salvage cardiomyocytes, but occasionally, myocardial reperfusion injury can occur because of microcirculatory dysfunction. Therefore, a more microcirculation-friendly strategy should be explored to overcome and compensate for the shortcomings of conventional strategies. In this work, we investigated the proangiogenic effect of S-Propargyl-Cysteine (SPRC), a novel water-soluble modulator of endogenous hydrogen sulfide, and elucidated the possible mechanisms involved to provide an experimental basis for angiogenesis-mediated drug therapy for IHD. Results: SPRC promoted cell proliferation, adhesion, migration, and tube formation of primary human umbilical vein endothelial cells (HUVEC) and increased angiogenesis in the rat aortic ring and Matrigel plug models. In a mouse model of hindlimb ischemia and a rat model of myocardial ischemia, SPRC also promoted angiogenesis after ligation of the left femoral artery or coronary artery to ameliorate ischemic conditions. In primary HUVEC, STAT3 phosphorylation was significantly induced after SPRC treatment. The critical roles of STAT3 in mediating the proangiogenic effect of SPRC were confirmed by RNA interference. Co-crystallization excluded the possible direct interaction between SPRC and STAT3, whereas co-immunoprecipitation revealed an enhanced interaction between VEGFR2 and STAT3 after SPRC treatment. Meanwhile, immunofluorescence and chromatin immunoprecipitation showed that SPRC induced the nuclear translocation of STAT3, followed by transcriptional activation of downstream promoters, particularly the Vegf promoter. Innovation and conclusion: We present a novel STAT3-mediated mechanism in SPRC-induced angiogenesis and demonstrate the therapeutic potential of SPRC in ischemic disease through angiogenesis promotion. 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

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: Autophagy is important for cells to degrade protein aggregates and organelles. Our preliminary study suggests that ischemia/reperfusion in rabbit hearts promo... More

Background: Autophagy is important for cells to degrade protein aggregates and organelles. Our preliminary study suggests that ischemia/reperfusion in rabbit hearts promoted autophagic myocardial injury, resulting in no-reflow phenomenon. In this study, we sought to further understand the mechanism and outcome of the upregulation of autophagy in ischemia/reperfusion. 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

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

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of ... More

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of this study was to investigate whether UII affects the development of atherosclerosis in cholesterol-fed rabbits. UII was infused for 16 weeks through an osmotic mini-pump into male Japanese White rabbits fed on a high-cholesterol diet. Plasma lipids and body weight were measured every 4 weeks. Aortic atherosclerotic lesions along with cellular components, collagen fibers, matrix metalloproteinase-1 and -9 were examined. Moreover, vulnerability index of atherosclerotic plaques was evaluated. UII infusion significantly increased atherosclerotic lesions within the entire aorta by 21% over the control (P = 0.013). Atherosclerotic lesions were increased by 24% in the aortic arch (P = 0.005), 11% in the thoracic aorta (P = 0.054) and 18% in the abdominal aorta (P = 0.035). These increases occurred without changes in plasma levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or body weight. Immunohistochemical staining revealed that macrophages and matrix metalloproteinase-9 were significantly enhanced by 2.2-fold and 1.6-fold in UII group. In vitro studies demonstrated that UII up-regulated the expression of vascular cell adhesion protein-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells, which was inhibited by the UII receptor antagonist urantide. In conclusion, our results showed that UII promotes the development of atherosclerotic lesions and destabilizes atherosclerotic plaques in cholesterol-fed rabbits. Less

Aims: Conventional revascularization strategies or drug therapies for ischemic heart disease (IHD) are designed for reperfusion of coronary arteries to salvage cardiomyoc... More

Aims: Conventional revascularization strategies or drug therapies for ischemic heart disease (IHD) are designed for reperfusion of coronary arteries to salvage cardiomyocytes, but occasionally, myocardial reperfusion injury can occur because of microcirculatory dysfunction. Therefore, a more microcirculation-friendly strategy should be explored to overcome and compensate for the shortcomings of conventional strategies. In this work, we investigated the proangiogenic effect of S-Propargyl-Cysteine (SPRC), a novel water-soluble modulator of endogenous hydrogen sulfide, and elucidated the possible mechanisms involved to provide an experimental basis for angiogenesis-mediated drug therapy for IHD. Results: SPRC promoted cell proliferation, adhesion, migration, and tube formation of primary human umbilical vein endothelial cells (HUVEC) and increased angiogenesis in the rat aortic ring and Matrigel plug models. In a mouse model of hindlimb ischemia and a rat model of myocardial ischemia, SPRC also promoted angiogenesis after ligation of the left femoral artery or coronary artery to ameliorate ischemic conditions. In primary HUVEC, STAT3 phosphorylation was significantly induced after SPRC treatment. The critical roles of STAT3 in mediating the proangiogenic effect of SPRC were confirmed by RNA interference. Co-crystallization excluded the possible direct interaction between SPRC and STAT3, whereas co-immunoprecipitation revealed an enhanced interaction between VEGFR2 and STAT3 after SPRC treatment. Meanwhile, immunofluorescence and chromatin immunoprecipitation showed that SPRC induced the nuclear translocation of STAT3, followed by transcriptional activation of downstream promoters, particularly the Vegf promoter. Innovation and conclusion: We present a novel STAT3-mediated mechanism in SPRC-induced angiogenesis and demonstrate the therapeutic potential of SPRC in ischemic disease through angiogenesis promotion. 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

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

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

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

Restenosis after intraluminal or open vascular reconstruction remains an important clinical problem. Vascular endothelial cell (EC) injury induced by oxidative stress pla... More

Restenosis after intraluminal or open vascular reconstruction remains an important clinical problem. Vascular endothelial cell (EC) injury induced by oxidative stress plays an important role in the development of intimal hyperplasia. In this study, we sought to evaluate the protective effects of Bcl-xl overexpression in vitro on oxidative stress-induced EC injury and the role of the Akt/endothelial nitric oxide synthase (eNOS) pathway. Human umbilical vein endothelial cells (HUVECs) exposed to hydrogen peroxide (H2O2, 0.5 mM) were used as the experimental oxidative stress model. The Bcl-xl gene was transferred into HUVECs through recombinant adenovirus vector pAdxsi-GFP-Bcl-xl before oxidative treatment. Cell apoptosis was evaluated by Annexin V/propidium iodide and Hoechst staining, caspase-7 and PARP cleavage. Cell viability was assessed using the cell counting kit-8 assay, proliferating cell nuclear antigen (PCNA) immunocytochemical detection and the scratching assay. Expressions of Akt, phospho-Akt and eNOS were detected by Western blotting. Our results showed that H2O2 induced apoptosis and decreased the cell viability of HUVECs. Bcl-xl overexpression significantly protected cells from H2O2-induced cell damage and apoptosis and maintained the cell function. Furthermore, the level of phospho-Akt and eNOS protein expression was significantly elevated when pretreated with Bcl-xl gene transferring. These findings suggest that Bcl-xl overexpression exerts an anti-apoptotic and protective effect on EC function. The Akt/eNOS signaling pathway is probably involved in these processes. Keywords: vascular endothelial cells, oxidative stress, intimal hyperplasia, gene therapy, apoptosis Less

Restenosis after intraluminal or open vascular reconstruction remains an important clinical problem. Vascular endothelial cell (EC) injury induced by oxidative stress pla... More

Restenosis after intraluminal or open vascular reconstruction remains an important clinical problem. Vascular endothelial cell (EC) injury induced by oxidative stress plays an important role in the development of intimal hyperplasia. In this study, we sought to evaluate the protective effects of Bcl-xl overexpression in vitro on oxidative stress-induced EC injury and the role of the Akt/endothelial nitric oxide synthase (eNOS) pathway. Human umbilical vein endothelial cells (HUVECs) exposed to hydrogen peroxide (H2O2, 0.5 mM) were used as the experimental oxidative stress model. The Bcl-xl gene was transferred into HUVECs through recombinant adenovirus vector pAdxsi-GFP-Bcl-xl before oxidative treatment. Cell apoptosis was evaluated by Annexin V/propidium iodide and Hoechst staining, caspase-7 and PARP cleavage. Cell viability was assessed using the cell counting kit-8 assay, proliferating cell nuclear antigen (PCNA) immunocytochemical detection and the scratching assay. Expressions of Akt, phospho-Akt and eNOS were detected by Western blotting. Our results showed that H2O2 induced apoptosis and decreased the cell viability of HUVECs. Bcl-xl overexpression significantly protected cells from H2O2-induced cell damage and apoptosis and maintained the cell function. Furthermore, the level of phospho-Akt and eNOS protein expression was significantly elevated when pretreated with Bcl-xl gene transferring. These findings suggest that Bcl-xl overexpression exerts an anti-apoptotic and protective effect on EC function. The Akt/eNOS signaling pathway is probably involved in these processes. Keywords: vascular endothelial cells, oxidative stress, intimal hyperplasia, gene therapy, apoptosis 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

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

The NAD+-dependent deacetylases Sirt1 and Sirt2 mediate cellular stress responses and are highly expressed in vascular endothelial cells. In contrast to the well-document... More

The NAD+-dependent deacetylases Sirt1 and Sirt2 mediate cellular stress responses and are highly expressed in vascular endothelial cells. In contrast to the well-documented protective actions of Sirt1, the role of endothelial Sirt2 remains unknown. Using cDNA microarray and PCR validation, we examined global gene expression changes in response to Sirt2 knock down in primary human umbilical vein endothelial cells under oxidative stress. We found that Sirt2 knock down changed expression of 340 genes, which are mainly involved in cellular processes including actin binding, cellular amino acid metabolic process, transmembrane receptor protein serine/threonine kinase signaling, ferrous iron transport, protein transport and localization, cell morphogenesis, and functions associated with endosome membrane and the trans-Golgi network. These genes and associated functions were largely non-overlapping with those altered by Sirt1 knock down. Moreover, we showed that pharmacological inhibition of Sirt2 attenuated oxidant-induced cell toxicity in endothelial cells. These suggest that Sirt2 is functionally important in endothelial cells under oxidative stress, and may have a primarily distinct role as compared to Sirt1. Our results may provide a basis for future studies aiming to dissect the specific signaling pathway(s) that mediates specific Sirt2 functions in endothelial cells. Keywords: Sirt1, Sirt2, endothelial cell, oxidative stress, functional genomics, microarray 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

Objectives: Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone reg... More

Objectives: Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. For these reasons, the aim of this work was to develop a co-culture system to study in detail any time-dependent interactions between human mesenchymal stem cells (HMSC) and human dermal microvascular endothelial cells (HDMEC), co-cultured in a 2D system, for 35 days. Materials and methods: HMSC and HDMEC were co-cultured at a ratio of 1:4, respectively. Single-cell cultures were used as controls. Cell viability/proliferation was assessed using MTT, DNA quantification and calcein-AM assays. Cell morphology was monitored using confocal microscopy, and real time PCR was performed. Alkaline phosphatase activity and histochemical staining were evaluated. Matrix mineralization assays were also performed. Results: Cells were able to grow in characteristic patterns maintaining their viability and phenotype expression throughout culture time, compared to HMSC and HDMEC monocultures. HMSC differentiation seemed to be enhanced in the co-culture conditions, since it was observed an over expression of osteogenesis-related genes, and of ALP activity. Furthermore, presence of calcium phosphate deposits was also confirmed. Conclusions: This work reports in detail the interactions between HMSC and HDMEC in a long-term co-culture 2D system. Endothelial and mesenchymal stem cells cultured in the present co-culture conditions ensured proliferation and phenotype differentiation of cell types, osteogenesis stimulation and over-expression of angiogenesis-related genes, in the same culture system. It is believed that the present work can lead to significant developments for bone tissue regeneration and cell biology studies. © 2012 Blackwell Publishing Ltd. Less

Background: One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain... More

Background: One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions. Results: Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS. Conclusions: Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance. Less

In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro... More

In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood-Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope=0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1beta) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion. Less

Purpose: A biotechnologic breakthrough for the study of drug permeability across the blood-brain barrier (BBB) would be the use of a reproducible in vitro model that reca... More

Purpose: A biotechnologic breakthrough for the study of drug permeability across the blood-brain barrier (BBB) would be the use of a reproducible in vitro model that recapitulates the functional, structural, and pathologic properties of the BBB in situ. We developed a humanized dynamic in vitro BBB model (DIV-BBB) based on cocultures of human microvascular endothelial cells (HBMECs) from "normal" and drug-resistant epileptic brain tissue with human brain astrocytes (HAs) from epilepsy patients or controls. Methods: HBMECs and HAs were cocultured for 28 days in polypropylene capillaries. HBMECs were exposed to physiologic levels of shear stress generated by intraluminal flow. Permeability to [3H]sucrose, [14C]phenytoin, and [14C]diazepam was measured in control and drug-resistant DIV-BBB with and without pretreatment with the MDR1 inhibitor XR9576. BBB integrity was monitored by transendothelial electrical resistance measurements (TEERs). Cell growth and viability were assessed by measurement of glucose consumption and lactate production. Results: PSucrose and TEER values did not depend on the origin of the endothelium used (epileptic or normal). PPhenytoin was 10-fold less (1.54 x 10(-6) cm/s) in drug-resistant BBB models than in controls (1.74 x 10(-5) cm/s). MDR1 blockade with XR9576 was effective (3.5-fold increase) only in drug-resistant cultures. PDiazepam in control and drug-resistant DIV-BBB was not affected by XR9576 and did not depend on the epileptic or control origin of endothelia. The overall contribution of epileptic glia to pharmacoresistance was negligible. Conclusions: These results show that, for the substances used, the humanized DIV-BBB recapitulates the physiologic permeability properties of the BBB in vivo and is also capable of mimicking a drug-resistant BBB phenotype. 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