Background
The excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which result in pulmonary vascular remodeling, are crucial patholo... More
Background
The excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which result in pulmonary vascular remodeling, are crucial pathological features of pulmonary arterial hypertension (PAH). Protein S-nitrosylation (SNO), which is a modification by which a nitric oxide (NO) group is added to a cysteine residue, has been shown to play a critical role in PASMC proliferation and PAH, but the underlying mechanism remains largely unknown.
Methods
Using a combination of membrane and nuclear localization analyses, iodoTMT switch assays, molecular biology techniques, and in vitro and in vivo approaches, we investigated the effects of ANXA2 SNO at cysteine 133 (Cys133) on PASMC proliferation and migration in PAH.
Results
ANXA2 protein expression was increased in PASMCs from rats with PAH. ANXA2 knockdown significantly inhibited excessive PASMC proliferation and migration. Additionally, inhibiting ANXA2 or conditionally knocking down ANXA2 in PASMCs ameliorated pulmonary vascular remodeling in experimental models of PAH. Moreover, we found that the NO donor S-nitrosoglutathione (GSNO) mediated the SNO of ANXA2 at Cys133 under hypoxic conditions. S-nitrosylated Cys133 (SNO-Cys133) of ANXA2 inhibited hypoxia-mediated PASMC proliferation and migration by regulating the subcellular localization of ANXA2 in cells. In addition, SNO-Cys133 of ANXA2 regulated the WNT pathway by inhibiting ANXA2 phosphorylation at Tyr24. Finally, SNO-Cys133 of ANXA2 ameliorated pulmonary vascular remodeling and improved RV function in vivo.
Conclusions
SNO-Cys133 of ANXA2 suppressed ANXA2 relocation and the WNT pathway to ameliorate pulmonary vascular remodeling in PAH. 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
Marfan syndrome (MFS), caused by mutations in the FBN1 gene, predisposes individuals to thoracic aortic aneurysm (TAA), a life-threatening complication. Recent studies ha... More
Marfan syndrome (MFS), caused by mutations in the FBN1 gene, predisposes individuals to thoracic aortic aneurysm (TAA), a life-threatening complication. Recent studies have suggested that dysregulated mechanosignaling in aortic smooth muscle cells (SMCs) plays a pivotal role in TAA pathogenesis in MFS. However, the key molecular drivers remain largely undefined. Here we identify fibroblast growth factor 12 (FGF12) as a novel mediator of aberrant mechanosignaling in aortic SMCs during TAA formation in MFS. FGF12 is markedly upregulated in aortic SMCs of thoracic aneurysmal aortas from Fbn1C1039G/+ MFS mice and from patients with MFS. Mechanistically, FGF12 expression is induced by transforming growth factor-β/SMAD signaling and by cyclic mechanical stretch in aortic SMCs. FGF12 upregulates the expression of angiotensin II (AngII) and AngII type 1 receptor (AT1R), thereby activating the AngII/AT1R signaling pathway. FGF12-induced AT1R activation promotes aberrant mechanosignaling, as indicated by increased RhoA-GTP levels, stress fiber formation, focal adhesion assembly and focal adhesion kinase phosphorylation, ultimately leading to increased aortic SMC stiffness. In vivo studies using Fgf12 heterozygous (Fgf12+/−) mice reveal that Fgf12 haploinsufficiency significantly ameliorates AngII/β-aminopropionitrile-induced TAA formation, accompanied by reduced AT1R signaling and attenuation of aberrant mechanosignaling in the thoracic aortas. Furthermore, in Fbn1C1039G/+ MFS mice, Fgf12 haploinsufficiency (Fgf12+/−Fbn1C1039G/+) substantially mitigates TAA progression and arterial stiffening, while alleviating dysregulated mechanosignaling in thoracic aortic SMCs. Collectively, these findings identify FGF12 as a critical regulator of aberrant mechanosignaling in aortic SMCs and a key contributor to TAA formation in MFS. Less
CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration... More
CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques. Less
Beta-3 adrenergic receptor (β3AR) agonists have been shown to produce vasodilation and prevention of ventricular remodeling in different conditions. Given that these bio... More
Beta-3 adrenergic receptor (β3AR) agonists have been shown to produce vasodilation and prevention of ventricular remodeling in different conditions. Given that these biological functions are critical in pulmonary hypertension (PH), we aimed to demonstrate a beneficial effect of β3AR agonists in PH. An experimental study in pigs (n = 34) with chronic PH created by pulmonary vein banding was designed to evaluate the acute hemodynamic effect and the long-term effect of β3AR agonists on hemodynamics, vascular remodeling and RV performance in chronic PH. Ex vivo human experiments were performed to explore the expression of β3AR mRNA and the vasodilator response of β3AR agonists in pulmonary arteries. Single intravenous administration of the β3AR agonist BRL37344 produced a significant acute reduction in PVR, and two-weeks treatment with two different β3AR selective agonists, intravenous BRL37344 or oral mirabegron, resulted in a significant reduction in PVR (median of −2.0 Wood units/m2 for BRL37344 vs. +1.5 for vehicle, p = 0.04; and −1.8 Wood units/m2 for mirabegron vs. +1.6 for vehicle, p = 0.002) associated with a significant improvement in magnetic resonance-measured RV performance. Histological markers of pulmonary vascular proliferation (p27 and Ki67) were significantly attenuated in β3AR agonists-treated pigs. β3AR was expressed in human pulmonary arteries and β3AR agonists produced vasodilatation. β3AR agonists produced a significant reduction in PVR and improved RV performance in experimental PH, emerging as a potential novel approach for treating patients with chronic PH. Less
Urotensin II (UII) is a potent vasoactive peptide and mitogenic agent to induce proliferation of various cells including vascular smooth muscle cells (VSMCs). In this stu... More
Urotensin II (UII) is a potent vasoactive peptide and mitogenic agent to induce proliferation of various cells including vascular smooth muscle cells (VSMCs). In this study, we examined the effects of a novel UII receptor (UT) antagonist, KR-36676, on vasoconstriction of aorta and proliferation of aortic SMCs. In rat aorta, UII-induced vasoconstriction was significantly inhibited by KR-36676 in a concentration-dependent manner. In primary human aortic SMCs (hAoSMCs), UII-induced cell proliferation was significantly inhibited by KR-36676 in a concentration-dependent manner. In addition, KR-36676 decreased UII-induced phosphorylation of ERK, and UII-induced cell proliferation was also significantly inhibited by a known ERK inhibitor U0126. In mouse carotid ligation model, intimal thickening of carotid artery was dramatically suppressed by oral treatment with KR-36676 (30 mg/ kg/day) for 4 weeks compared to vehicle-treated group. From these results, it is indicated that KR-36676 suppress UII-induced proliferation of VSMCs at least partially through inhibition of ERK activation, and that it also attenuates UII-induced vasoconstriction and vascular neointima formation. Our study suggest that KR-36676 may be an attractive candidate for the pharmacological management of vascular dysfunction.
Keywords: ERK; KR-36676; Proliferation; Smooth muscle; Urotensin II; Urotensin II receptor antagonist. Less
Angelica sinensis has been used to attenuate cold-induced cutaneous vasospasm syndrome, such as Raynaud’s disease and frostbite, in China for many years. Ferulic acid (... More
Angelica sinensis has been used to attenuate cold-induced cutaneous vasospasm syndrome, such as Raynaud’s disease and frostbite, in China for many years. Ferulic acid (PubChem CID: 445858) and Z-ligustilide (PubChem CID: 529865), two major components extracted from Angelica sinensis, had been reported to inhibit vasoconstriction induced by vasoconstrictors. In this study, the pharmacological interaction in regulating cold-induced vascular smooth muscle cell contraction via cold-sensing protein TRPM8 and TRPA1 was analyzed between ferulic acid and Z-ligustilide. Pharmacological interaction on inhibiting [Ca2+]i influx evoked by TRPM8 agonist WS-12 or TRPA1 agonist ASP 7663 as well as cold-induced upregulation of TRPM8 was determined using isobolographic analysis. The isobolograms demonstrated that the combinations investigated in this study produced a synergistic interaction. Combination effect of two components in inhibiting RhoA activation and phosphorylation of MLC20 induced by WS-12 or ASP 7663 was also being quantified. These findings suggest that the therapeutic effect of Angelica sinensis on cold-induced vasospasm may be partially attributed to combinational effect, via TRPM8 and TPRA1 way, between ferulic acid and Z-ligustilide. Less
The construction of a smooth muscle layer for blood vessel through electrospinning method plays a key role in vascular tissue engineering. However, smooth muscle cells (S... More
The construction of a smooth muscle layer for blood vessel through electrospinning method plays a key role in vascular tissue engineering. However, smooth muscle cells (SMCs) penetration into the electrospun graft to form a smooth muscle layer is limited due to the dense packing of fibers and lack of inducing factors. In this paper, silk fibroin/poly (L-lactide-ε-caplacton) (SF/PLLA-CL) vascular graft loaded with platelet-rich growth factor (PRGF) was fabricated by electrospinning. The in vitro results showed that SMCs cultured in the graft grew fast, and the incorporation of PRGF could induce deeper SMCs infiltrating compared to the SF/PLLA-CL graft alone. Mechanical properties measurement showed that PRGF-incorporated graft had proper tensile stress, suture retention strength, burst pressure and compliance which could match the demand of native blood vessel. The success in the fabrication of PRGF-incorporated SF/PLLA-CL graft to induce fast SMCs growth and their strong penetration into graft has important application for tissue-engineered blood vessels. Less
The present study aimed to determine the expression of microRNA-146a (miR-146a) in the plasma of children with asthma, and to investigate the effect of miR-146a on the pr... More
The present study aimed to determine the expression of microRNA-146a (miR-146a) in the plasma of children with asthma, and to investigate the effect of miR-146a on the proliferation and apoptosis of bronchial smooth muscle cells (BSMCs). Reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of miR-146a mimics and its inhibitor. A Cell Counting kit-8 assay was performed to examine the proliferation of BSMCs. Caspase-3/7 activity was determined using a Caspase-Glo 3/7 kit. To measure the expression levels of proteins associated with apoptosis, western blotting was performed. The target gene of miR-146a was identified using a dual-luciferase reporter assay. The plasma levels of miR-146a in children with asthma were significantly higher compared with those in healthy children. Enhanced miR-146a expression inhibited the proliferation of BSMCs. BSMC apoptosis was promoted by miR-146a. The mechanism underlying the miR-146a-induced promotion of BSMC apoptosis may be its direct targeting of epidermal growth factor receptor (EGFR), which affects downstream signaling pathways. In conclusion, miR-146a expression in asthma inhibits the proliferation and promotes the apoptosis of BSMCs by direct targeting of EGFR. Keywords: microRNA-146a, bronchial smooth muscle cell, proliferation, apoptosis Less
Hypoxic pulmonary hypertension (PH) is a common disease characterized by a disturbance to the balance of apoptosis and cell proliferation in pulmonary artery smooth muscl... More
Hypoxic pulmonary hypertension (PH) is a common disease characterized by a disturbance to the balance of apoptosis and cell proliferation in pulmonary artery smooth muscle cells (PASMCs). The anti-apoptotic protein, survivin, has been observed to be upregulated in pulmonary arteries (PAs) of chronic hypoxia-induced PH rats. The present study aimed to investigate the therapeutic potential of sepantronium bromide (YM155), a selective survivin inhibitor, on hypoxic human PASMCs and examine the potential underlying mechanisms. Cultured human PASMCs (HPASMCs) were randomly divided into the following groups: i) Normoxia (N); ii) normoxia + 100 nmol/l YM155 (NY100); iii) hypoxia (H); iv) hypoxia + 1 nmol/l YM155 (HY1); v) hypoxia + 10 nmol/l YM155 (HY10); and hypoxia + 100 nmol/l YM155 (HY100) groups. The cells were exposed to the different conditions for 24 h, according to the group. Cell viability was then determined using a Cell Counting Kit‑8 assay, and apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. The expression levels of survivin were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry and Western blot analyses. The expression levels of the voltage-dependent K+ (Kv) channels, Kv1.5 and Kv2.1, were measured using RT-qPCR and Western blotting. Cell proliferation in the hypoxic PASMCs was significantly increased by hypoxia, however, apoptosis of the HPASMCs was suppressed, the expression of survivin were upregulated and the expression levels of Kv1.5 and Kv2.1 were downregulated. YM155 treatment ameliorated the hypoxia‑induced increase in cell proliferation and expression of survivin in a concentration‑dependent manner, increased apoptosis, and increased the expression levels of Kv1.5 and Kv2.1 (P<0.05). By contrast, YM155 treatment in normoxic HPASMCs had no significant effects on proliferation, apoptosis, or the expression levels of survivin and Kv channels in the PASMCs. The present study is the first, to the best of our knowledge, to demonstrate that YM155, a selective survivin inhibitor, has a beneficial therapeutic effect on hypoxic HPASMCs, and that YM155 induces a pro‑apoptotic effect by downregulating the apoptosis inhibitor, survivin, possibly through a Kv channel-mediated mechanism. Less
Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved ... More
Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol. Less
Background Illicium verum Hook. fil. Illiciaceae (Illicium v.) has been traditionally used in herbal medicine for treating many inflammatory diseases, including skin infl... More
Background Illicium verum Hook. fil. Illiciaceae (Illicium v.) has been traditionally used in herbal medicine for treating many inflammatory diseases, including skin inflammation and rheumatism. We investigated its use as a preventive agent against inflammatory and vascular diseases in a murine model of atherosclerosis using apolipoprotein E-knockout (ApoE−/−) mice fed on a high-fat diet (HFD). Methods We investigated the effect of Illicium v. on cytotoxicity, NF-κB activity, and adhesion molecule expression in TNF-α – stimulated HASMCs (Human Aortic smooth muscle cells). ApoE−/−mice, fed a HFD and treated daily for 12 weeks by oral administration of either Illicium v. (100 or 200 mg/kg) or atorvastatin (10 mg/kg), were evaluated for atherosclerotic lesions and inflammatory responses by performing Oil red O and iNOS staining, respectively. Expression of inflammatory cytokines (i.e., NF-κB, TNF-α, IL-1β, COX, IκB-α, Iκκ-α/β) and adhesion molecules in the aorta were measured by western blot analysis. Results In TNF-α-stimulated HASMCs, Illicium v. treatment decreased NF-κB transcriptional activity, and NF-κB protein levels were reduced in a dose-dependent manner over a range of 10–100 μg/mL Illicium v. Also, Illicium v. attenuated the expression of adhesion molecules that are responsible for inflammation in these cells. In animal experiments, treatment with Illicium v. or atorvastatin counteracted the characteristic changes in body weight, blood pressure, and lipid levels seen in HFD-fed ApoE−/− mice. In addition, Illicium v. treatment reduced aortic atherosclerotic plaque lesions and the immunoreactivity of iNOS activation. The aortic expression of inflammatory adhesion molecules and cytokines (TNF-α, IL-1β, NF-κB, COX, IκB-α, Iκκ-α/β), which is characteristic of HFD-fed ApoE−/− mice, was attenuated by 12-week treatment with daily oral administration of Illicium v. or atorvastatin, and the most potent effect was seen with the herbal tincture. Conclusions The beneficial effects of Illicium v. are consistent with a significant decrease in the iNOS-mediated inflammatory response, resulting in reduction of inflammation-associated gene expression. Treatment with Illicium v. may be the basis of a novel therapeutic strategy for hyperlipidemia-atherosclerosis. Less
Background Gastroschisis (GS) is a congenital abdominal wall defect that results in the development of GS-related intestinal dysfunction (GRID). Transforming growth facto... More
Background Gastroschisis (GS) is a congenital abdominal wall defect that results in the development of GS-related intestinal dysfunction (GRID). Transforming growth factor-β, a pro-inflammatory cytokine, has been shown to cause organ dysfunction through alterations in vascular and airway smooth muscle. The purpose of this study was to evaluate the effects of TGF-β3 on intestinal smooth muscle function and contractile gene expression. Methods Archived human intestinal tissue was analyzed using immunohistochemistry and RT-PCR for TGF-β isoforms and markers of smooth muscle gene and micro-RNA contractile phenotype. Intestinal motility was measured in neonatal rats ± TGF-β3 (0.2 and 1 mg/kg). Human intestinal smooth muscle cells (hiSMCs) were incubated with fetal bovine serum ±100 ng/ml of TGF-β 3 isoforms for 6, 24 and 72 h. The effects of TGF-β3 on motility, hiSMC contractility and hiSMC contractile phenotype gene and micro-RNA expression were measured using transit, collagen gel contraction assay and RT-PCR analysis. Data are expressed as mean ± SEM, ANOVA (n = 6–7/group). Results GS infants had increased immunostaining of TGF-β3 and elevated levels of micro-RNA 143 & 145 in the intestinal smooth muscle. Rats had significantly decreased intestinal transit when exposed to TGF-β3 in a dose-dependent manner compared with Sham animals. TGF-β3 significantly increased hiSMC gel contraction and contractile protein gene and micro-RNA expression. Conclusion TGF-β3 contributed to intestinal dysfunction at the organ level, increased contraction at the cellular level and elevated contractile gene expression at the molecular level. A hyper-contractile response may play a role in the persistent intestinal dysfunction seen in GRID. Keywords: Gastroschisis, Intestinal dysfunction, Smooth muscle, Contraction Less
Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. ... More
Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)‑1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti‑inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL‑1β‑induced inflammation‑activated endothelial cell (EC)‑smooth muscle cell (SMC)‑mononuclear cell (MC) co‑culture model was established, based on the changes in a series of atherosclerosis‑associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low‑density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti‑inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed. 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
The root of Cynanchum wilfordii (C. wilfordii) contains several biologically active compounds which have been used as traditional medicines in Asia. In the present study,... More
The root of Cynanchum wilfordii (C. wilfordii) contains several biologically active compounds which have been used as traditional medicines in Asia. In the present study, we evaluated the anti-inflammatory effects of an ethanol root extract of C. wilfordii (CWE) on tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs). The inhibitory effects of CWE on vascular cell adhesion molecule (VCAM)-1 expression under an optimum extraction condition were examined. CWE suppressed the expression of VCAM-1 and ICAM-1 and the adhesion of THP-1 monocytes to the TNF-α-stimulated HASMCs. Consistent with the in vitro observations, CWE inhibited the aortic expression of ICAM-1 and VCAM-1 in atherogenic diet-fed mice. CWE also downregulated the expression of nuclear factor-κB (NF-κB p65) and its uclear translocation in the stimulated HASMCs. In order to identify the active components in CWE, we re-extracted CWE using several solvents, and found that the ethyl acetate fraction was the most effective in suppressing the expression of VCAM-1 and ICAM-1. Four major acetophenones were purified from the ethyl acetate fraction, and two components, p-hydroxyacetophenone and cynandione A, potently inhibited the expression of ICAM-1 and VCAM-1 in the stimulated HASMCs. We assessed and determined the amounts of these two active components from CWE, and our results suggested that the root of C. wilfordii and its two bioactive acetophenones may be used for the prevention and treatment of atherosclerosis and vascular inflammatory diseases. Less
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we exami... More
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)-mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP-9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP-9, MMP-1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP-9 activity and mRNA transcripts for MMP-9, MMP-1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP-9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF-treated VSMCs of both groups. Additionally, the magnitude in decreased MMP-9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP-9 gene with siRNA in EGF-treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP-9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis. 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
Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascula... More
Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD). To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease. Less
