Human Vascular Smooth Cells Research Articles

LncRNA ANRIL regulates cell proliferation and migration via sponging miR-339-5p and regulating FRS2 expression in atherosclerosis.

Atherosclerosis (AS), with high risk of stroke or cerebrovascular disease, is one of the most common causes of death worldwide. Increasing evidence shows that long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is related to atherothrombotic stroke susceptibility and contributes to AS progression. However, the underlying mechanism was not explained yet. Human aorta vascular smooth muscle cells (HA-VSMCs) and human umbilical vein endothelial cells (HUVECs) were treated with oxidized Low Density Lipoprotein (ox-LDL) and considered as AS cell models. Quantitative reverse transcriptase PCR (qRT-PCR) and Western blotting were employed to investigate the mRNA and protein expression level, respectively. Microscopic examination through fluorescent in situ hybridization (FISH) was used to determine the location of ANRIL. Cell proliferation and migration assays were demonstrated to evaluate the functional role of ANRIL in AS. Potential target of ANRIL was determined using Luciferase reporter assay and RNA immunoprecipitation (RIP). ANRIL was upregulated and miR-399-5p was down-regulated in both human atherosclerotic plaques and ox-LDL-induced cells. ANRIL was located in cytoplasm and promoted cell proliferation and migration by sponging miR-399-5p. Further analysis identified fibroblast growth factor receptor substrate 2 (FRS2) as a direct target of miR-399-5p. Finally, RAS/RAF/ERK signal pathway was proved to be involved in the regulation of ANRIL on the progression of AS. These results revealed the underlying mechanism that ANRIL promoted AS progression by sponging miR-399-5p and regulating RAS/RAF/ERK signal pathway, suggesting that ANRIL might be a potential target for the therapeutic strategy of AS.

Inflammation and Vascular Calcification Causing Effects of Oxidized HDL are Attenuated by Adiponectin in Human Vascular Smooth Muscle Cells.

The role of oxidized high- density lipoprotein (oxHDL) and the protective effects of adiponectin in terms of vascular calcification is not well-established. This study was conducted to investigate the effects of oxHDL with regard to inflammation and vascular calcification and to determine the protective role of adiponectin in attenuating the detrimental effects of oxHDL. Cell viability, mineralization, and calcification assays were conducted to optimize the concentration of oxHDL. Then, human vascular smooth muscle cells (HAoVSMCs) were incubated with β-glycerophosphate, HDL, oxHDL, adiponectin, or the combination of oxHDL with adiponectin for 24 h. Protein expression of IL-6, TNF-α, osterix, RUNX2, ALP, type 1 collagen, osteopontin, osteocalcin, WNT-5a, NF-ĸβ(p65), cAMP and STAT-3 were measured by ELISA kits. OxHDL induced vascular calcification by promoting the formation of mineralization nodules and calcium deposits in HAoVSMCs. This was accompanied by an increased secretion of IL-6, osterix, WNT-5a and NF-ĸβ (p65). Interestingly, these detrimental effects of oxHDL were suppressed by adiponectin. Besides, incubation of adiponectin alone on HAoVSMCs showed a reduction of inflammatory cytokines, osteoblastic markers (RUNX2, osterix and osteopontin), WNT-5a and NF-ĸβ (p65). This study exhibits the ability of oxHDL in inducing inflammation and vascular calcification and these detrimental effects of oxHDL can be attenuated by adiponectin.

Cyclooxygenase-2 expression in atherosclerosis tissues and the effect of celecoxib on proliferation and apoptosis of human vascular smooth muscle cells

Objective To investigate the expression of cyclooxygenase-2 (COX-2) in atherosclerosis tissues and the effect of selective COX-2 inhibitor celeboxib on proliferation and apoptosis of human vascular smooth muscle cells (VSMCs).Methods Immunohistochemistry for COX-2 was performed on 22 cases of formalin-fixed,paraffin-embedded human atherosclerosis tissues.Cell growth rate was assessed by methyl thiazol tetrazolium (MTT) assay,and apoptosis was examined by flow cytometry.Results COX-2 protein was detected in 21 of 22 (95％) human atherosclerosis tissues.COX-2 was decreased in VSMCs when treated with celecoxib in vitro.After cells were treated with increasing concentrations of celecoxib (0,10,20,40 and 80 μmol/L) for 24 h,the cell growth rate was 100.00％,90.06％,81.38％,75.41％ and 68.90％ respectively,and the early apoptosis ratio was 0.02％,0.62％,0.92％,1.17％ and 1.63％ respectively.Conclusion COX-2 is highly expressed in human atherosclerosis tissues,and the celecoxib inhibited proliferation and induced apoptosis of VSMCs in a dose-dependent manner. Key words: Cyclooxygenase-2 ; Celecoxib ; Atherosclerosis; Vascular smooth muscle cells; Proliferation ; Apoptosis

The role of Toll like receptor 4 pathway in palmitate-induced interleukin-6 expression in human aortic vascular smooth muscle cells

The recombinant adenovirus Toll like receptor 4 (TLR4) shRNA vector (pGSadeno-TLR4) was constructed and transfected into human aortic vascular smooth muscle cells (HA-VSMC).After HA-VSMC were treated with palmitate or different signaling pathway inhibitors,the mRNA and protein levels of interleukin-6 (IL-6)and NF-κB activity were tested with real-time PCR and ELISA,respectively.The results showed that palmitate increased mRNA and protein levels of IL-6 in HA-VSMC in a dose-dependent manner.The expression of IL-6 mRNA reached peak after treatment with 400 μmol/L of palmitate for 6 h,being 10.43 fold of control (P＜0.01).Treatment with 400 pmol/L of palmitate for 24 h maximally upregulated the protein level of IL-6,which was 2.18 fold of control (P＜0.01).NF-κB inhibitor parthenolide markedly inhibited palmitate-stimulated increased in IL-6 mRNA level by 65％ and protein level by 59％ (both P＜0.01).Protein kinase C (PKC) inhibitor chlerythrine suppressed palmitateinduced IL-6 mRNA expression by 24％ and IL-6 protein level by 28％.By contrast,extracellular signal-regulated protein kinase inhibitor PD98059 and phosphatidylinositol 3-kinase inhibitor wortmannin had no effect on the induction of IL-6 by palmitate.Blockade of TLR4 with pGSadeno-TLR4 significantly suppressed palmitate-induced IL-6 mRNA expression by 72％ and IL-6 protein expression by 75％ (both P＜0.01),along with decrease of NF-κB p65 activity decreased by 62％.These results suggest that TLR4/NF-κB and PKC pathways mediate palmitate-induced IL-6 expression in HA-VSMC. Key words: Human aortic vascular smooth muscle cells; Palmitate; Toll like receptor 4 ; Intedeukin-6

Modified explant technique for the culture of human vascular smooth muscle cells

Objective To establish an effective method of human vascular smooth muscle cells in vitro culture.Methods Medial layer of human aorta was separated under sterile conditions,then cut into small pieces (about 1 mm3).After pretreatment with 0.1％ type Ⅰ collagenase,the tissues were transferred into flask and cultured in DMEM containing 20％ fetal bovine serum,penicillin (100 U/ml) and streptomycin (100 g/L).The cell morphology was observed under the inverted microscopy.Smooth muscle cell specific protein (α-SMA) was identified by using immumofluorescence methods.Results Some smooth muscle cells started growing from explants in 4 to 5 days.One to 2 weeks later,the cells grew to confluence by migrated cells.Immumofluorescence staining showed positive expression of α-SMA (fluorescence intensity (++)-(+++)).Conclusion Modified explant technique by pretreating tissues with collagenase was an effective method to get vascular smooth muscle cells in vitro. Key words: Aorta; Vascular smooth muscle cells; Cell culture

Bupivacaine blocks BK Ca channels by a fast open mechanism in human vascular smooth muscle cells

Bupivacaine blocks BK Ca channels by a fast open mechanism in human vascular smooth muscle cells

Gene transfer of human endothelial nitric oxide synthase inhibits proliferation of human vascular smooth muscle cells and the mechanism

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis. Key words: Endothelial nitric oxide synthase; Cell cycle; p27

Construction of the recombinant human endothelial nitric oxide synthase gene adenovirus and its expression in human vascular smooth muscle cells

Objective To construct recombinant adenovirus vector carrying the human endothelial nitric oxide synthase gene (heNOS) and detect its expression in human vascular smooth muscle cells in vitro. Methods heNOS was cloned by polymerase chain reaction (PCR) using plasmid PMSCV-heNOS,heNOS cDNA was inserted into adenovirus shuttle plasmid-PSUCMV to generate a recombinant plasmid PSUCMV-heNOS, transfected into E. coli DH5α, and positive clones were correctly constructed and confirmed by DNA sequencing analysis and restriction endonucleas analysis. The plasmid PSUCMV-heNOS was co-transfected with pBHGE3 in 293 cells to generate replication-defective recombinant adenovirus vector carrying heNOS gene. Human vascular smooth muscle cells were transfected by Ad-heNOS. The effect on the proliferation of human vascular smooth muscle cells was investigated by MTT assay. The expression of heNOS was detected by immunohistochemistry staining and Western blotting. Results ( 1 ) PCR, restrictive digestion, and sequencing revealed the successful construction of the recombinant adenovirus vector carrying heNOS gene and its titer was 3.5 × 1010 PFU/ml; (2) It was confirmed that the human vascular smooth muscle cells cultivated in vitro were characterized by morphology and immunohistochemistry strain of α-actin; (3) At 120 h, the A570 values in multiplicity of infection (MOI) 50, 150,250,300,450 were respectively 1. 410 ±0. 081, 1. 357 ±0. 150, 1.303 ±0. 311,0. 995 ±0. 248 and 0. 731 ±0. 101. The proliferation of human vascular smooth muscle cells was significantly and stably inhibited with MOI 300 of AdCMVheNOS ( P ＜ 0. 01 ). Immunostaining and Western blotting with heNOS in engineered cells were positive.Conclusion The successful construction of AdCMV-heNOS and expression may provide an opportunity for the gene therapy of vascular intimal hyperplasia. Key words: Nitric oxide synthase; Vascular smooth muscle cells; Gene transfection

Isolation and culture of human umbilical artery smooth muscle cells and gene transfection of TFPI gene

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor （TFPI） gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid （1,2,and 3 μg/ml,respectively）and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection. Key words: Human smooth muscle cell; Tissue factor pathway inhibitor; Restenosis

Adipophilin increases lipid accumulation in human vascular smooth muscle cells

Objective To observe the effect of acetylated low-density lipoprotein (AcLDL) on the expression of adipophilin and the effect of adipophilin on AcLDL uptake and lipid accumulation in human vascular smooth muscle cells (HVSMCs)in order to approach the role played by adipophilin in genesis of macroangiopathy. Methodse HVSMCs were treated with various amount of AcLDL. Adipophilin expression levels were detected by Northern blot and Western blot. The effects of adipophilin on AcLDL uptake and lipid accumulation in HVSMCs were observed by the methods of siRNA, flow cytometry, enzymatic method and oil red stain. Results AcLDL dose-dependently increased adipophilin expression in HVSMCs. Silence adipophilin by siRNA decreased AcLDL uptake (decreasing by 38.7%, P<0. 05) and lipids accumulation (tfiglyceride and total cholesterol decreasing by 30.6% and 29.8% respectively, both P<0. 01) in HVSMCs, Conclusion Adipophilin is able to increase AcLDL uptake and lipid accumulation in HVSMCs, suggesting that it might play a role in enhancing atherosclerosis. Key words: Adipophilin; Human vascular smooth muscle ceils (HVSMCs) ; Acetylated low-density lipoprotein(AcLDL) ; Lipid accumulation

Post-transcriptional regulation of CRLR expression during hypoxia

Adrenomedullin and CGRP are two potent vasodilator peptides, and their receptors are formed by heterodimerization of the CRLR and a RAMP molecule. Hypoxia is associated with many diseases of the cardiovascular system. It was recently shown that the human CRLR gene promoter contains an HIF-1α regulatory element, and that CRLR mRNA was increased by hypoxia in human endothelial cells. In the present work, we have assessed the effect of hypoxia on CRLR expression both in vivo and in vitro using two different experimental models. We have also investigated the effect of hypoxia on RAMP expression. (1) We analyzed the effects of a chronic hypobaric hypoxia on rat ventricle expression of RAMPs and CRLR. (2) Acute hypoxia was studied in human vascular smooth cells from coronary artery (CASMC) exposed for 6 h to 2% O 2. RT-PCR was used to analyze the mRNA expression, and protein levels were determined by Western blotting. A sharp increase in HIF-1α protein levels was induced by hypoxia in CASMC, and 3.5-fold rise of the CRLR protein occurred after 1 h of hypoxia in face of unchanged mRNA levels. The CRLR mRNA levels were only elevated later. A clear decrease of the CRLR protein level occurred after 3 and 6 h of hypoxia. Thus, acute hypoxia in CASMC induced a rapid change of the CRLR protein amount independently of changes in the CRLR mRNA. This finding suggested a major post-transcriptional effect of hypoxia on CRLR expression in CASMC. RAMP2 and adrenomedullin mRNAs were increased after 4 h, but no change was observed for RAMP1. Chronic hypoxia in rats enhanced both mRNA and protein levels of the three RAMPs and CRLR in right and left ventricles. Together, our in vivo and in vitro data suggested that hypoxia up-regulates both adrenomedullin and its receptor (CRLR/RAMP2) to enhance the signaling at the target cell.

Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa.

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.

Agonist-induced polyphosphoinositide breakdown in cultured human endothelial and vascular smooth muscle cells.

Human aortic endothelial cells and smooth cells (SMC) from human aorta and coronary arteries were grown in culture. Subcultured vascular SMC retained several important features of human vascular SMC in situ, for example, vimentin-type intermediate filaments, smooth muscle myosin, a well-developed microfilament system, and expression of caldesmon protein involved in the regulation of contraction in smooth muscle. Aortic endothelial cells were shown to possess functional receptors to histamine, thrombin, serotonin, acetylcholine, bradykinin, platelet activating factor (PAF), angiotensin II, vasopressin, prostaglandin E2 (PGE2), and U46619, a stable analog of thromboxane A2. All these substances stimulated polyphosphoinositide (PPI) breakdown in endothelium. Thrombin, histamine, and PAF were the most potent activators. The response of aortic SMC to the same panel of agonists were different. Serotonin, histamine, and angiotensin II produced higher levels of inositol phosphates (IP, IP2, IP3) in SMC than in endothelium. Responses to acetylcholine, bradykinin, and PGE2 were weak and inferior to those of endothelial cells. Other agents evoked approximately equivalent responses in both cell types. Coronary artery SMC resembled aortic SMC in the high extent of PPI hydrolysis after stimulation with serotonin and histamine. The complete inability of angiotensin II and vasopressin to cause accumulation of inositol phosphates in coronary SMC contrasted with the presence of functional receptors to these hormones on aortic SMC. We conclude that the effect of vasoactive agents on human vascular cells may be realized via activation of PPI hydrolysis. Agonists with reported strong vasoconstrictor action seem to stimulate preferential PPI hydrolysis in SMC, whereas endothelium-dependent relaxers cause more pronounced PPI breakdown in endothelial cells. Peculiarities of angiotensin II and vasopressin receptor expression and/or coupling in human aorta and coronary artery SMC may be relevant for understanding the selective action of agonists on human vessels.