Effect of calcitonin gene-related peptide on proliferation of vascular smooth muscle cells

AIM: To elucidate the effect of calcitonin gene-related peptide (CCRP) in the therapy of atherosclerosis.METHODS:Effect of CGRP on cell cycle kinetics of cultured vascular smooth muscle cells(HA-VSMC) was investigated by flow cytometry. The expression of cyclins D₁ and E required for initiation of S phase were also studied by immunochemistry method. RESULT: CGRP was shown to arrest VSMC in the G₀/G₁ phase of cell cycle and reduced expression of cyclins D₁ and E. CONCLUSION:CGRP inhibits proliferation of HA-VSMC by arresting cells in G₁ phase via limiting accumulation of cyclin D₁ and E. It might play a role in the therapy of atherosclerosis.     

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

AIM：To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.     

Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.     

Effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells

AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E₂) than that in VSMCs without 17β-E₂ pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.     

Changes of plasma and myocardial calcitonin gene-related peptide in myocardial stunning rats

AIM: To investigate changes of calcitonin gene-related peptide(CGRP) in myocardial stunning rats. METHODS: Rat in vivo myocardial stunning model was used. CGRP content in plasma and myocardium were determined by radioimmunoassay. RESULTS: Plasma level of CGRP increased significantly (P＜0.01), but in left ventricular myocardium CGRP decreased obviously (P＜0.05) in myocardial stunning group compared with the control group. CONCLUSION: CGRP content in the left ventricular myocardium was negatively correlated with plasma CGRP.     

Lentivirus-mediated calcitonin gene-related peptide transfection enhances endothelial differentiation of rat bone marrow mesenchymal stem cells

AIM：To study the effect of calcitonin gene-related peptide (CGRP) gene transfection mediated by lentivirus on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to endothelial cells. METHODS：Rat bone marrow MSCs were isolated by density gradient centrifugation combined with adherence method. Recombinant lentivirus vector carrying CGRP gene (Lenti-CGRP) was transfected into the MSCs. The secretion of CGRP in culture supernatants of the transfected MSCs was detected using ELISA method. The cells at passage 3 were divided into three groups: CGRP group (MSCs transfected with Lenti-CGRP), CGRP+CGRP8-37 (an antagonist of CGRP receptor) group and control group (MSCs transfected with PBS). The differentiation of the MSCs was detected by immunocytochemical staining for CD31 and factor Ⅷ-related antigen. The proliferation of the cells was measured by cell counting, and the angiogenic ability of the cells was analyzed using Matrigel assay. RESULTS：The proportion of CD31-and factor Ⅷ-related antigen-positive cells in CGRP and CGRP+CGRP8-37 groups was larger than that in control group (P<0.05). The numbers of the cells in CGRP and CGRP+CGRP8-37 groups were significantly increased compared with control group (P<0.05). Lumen-like structures were observed in CGRP and CGRP+CGRP8-37 groups. The above indexes in CGRP+CGRP8-37 group were reduced compared with CGRP group. CONCLUSION: Transfection with CGRP gene induces rat bone marrow MSCs to differentiate into endothelial cells and enhances their proliferation, suggesting that CGRP may play a role in the regulation of angiogenesis.     

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability,migration and phenotypic changes

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs). METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs. Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion. The cell viability was measured by MTT assay. The migration ability was detected by scratch assay. The mRNA and protein expression of SM22α was determined by RT-PCR, immunocytochemical staining and Western blot. RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05), the migration ability was increased (P<0.05) and the expression of SM22α at mRNA and protein level was decreased (P<0.05). Compared with negative control lentiviral group, the cell viability in 27nt-miRNA over-expression group was decreased (P<0.05), the migration ability was decreased (P<0.05), and the mRNA and protein expression of SM22α was increased (P<0.05). While in anti-27nt-miRNA group, the cell viability was increased(P<0.05), the migration ability was increased (P<0.05), and the mRNA and protein expression of SM22α was decreased (P<0.05). CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

Effect of fibrinogen,fibrin and fibrin degradation products on the proliferation and migration of human vascular smooth muscle cells

AIM: To investigate the effect of fibrinogen (Fg), fibrin (Fb) and fibrin degradation products (FDPs) on the proliferation and migration of human vascular smooth muscle cells (VSMC).METHODS: The effects of Fg, Fb and FDPs on the proliferation of VSMC were observed by means of cell counting and MTT test, migration assays were performed using the wounding model and the transwell cell culture apparatus.RESULTS: Fg itself did not stimulate the proliferation of VSMC, but stimulated VSMC migration. Fb and FDPs both stimulated the proliferation and migration of VSMC, meanwhile the effect of Fb was in a dose-dependent manner.CONCLUSION: Fb, in particular FDPs, may play an important role by stimulating the proliferation and migration of VSMC in restenosis and atherogenesis.     

Expression of recombinant myocardin and its effect on human bone marrow mesenchymal stem cell reprogramming into vascular smooth muscle cells

AIM: To optimize the expression and purification of recombinant myocardin, and to explore the effect of recombinant myocardin on the transdifferentiation of human bone marrow mesenchymal stem cells (hBMSCs) into vascular smooth muscle cells (vSMCs). METHODS: The prokaryotic expression vector for myocardin was constructed. The best ways to induce the expression of myocardin recombinant protein and to optimize the isolation and purification methods were explored. Nano-transducers were applied to encapsulate myocardin to form nano-protein complexes. Nano-myocardin was transducted into hBMSCs and the protein transduction efficiency was measured. After transduction of the protein was successful, smooth muscle myosin heavy chain, a specific marker of vSMCs, was detected, and the transformation of hBMSCs into vSMCs was also observed. RESULTS: Compared with the traditional methods, the expression of myocardin recombinant protein was higher by high-concentration induction method, and the new elution method was able to obtain more myocardin recombinant protein. The nano-myocardin complexes were successfully transduced into the hBMSCs and induced the transformation of hBMSCs into vSMCs. CONCLUSION: The recombinant myocardin was efficiently transduced into the cells and reprogrammed hBMSCs into vSMCs.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

Effects of Chlamydia pneumoniae infection on adhesion and migration of vascular smooth muscle cells

AIM: To investigate the effects of Chlamydia pneumoniae (C.pn) infection on the adhesion and migration of vascular smooth muscle cells (VSMCs). METHODS: Primary VSMCs, isolated from the aorta of SD rats, was infected with C.pn after the culture and propagation of C.pn in the HEp-2 cells. The morphological characteristics of C.pn inclusions in VSMCs were examined under the fluorescence microscope with acridine orange (AO) staining. The specific DNA fragment of C.pn was identified by polymerase chain reaction (PCR). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of VSMCs to collagen I. Wound-healing assay and Transwell assay were performed to detect the effects of C.pn infection on VSMCs migration. RESULTS: The C.pn inclusions and infection spots were observed in VSMCs under fluorescence microscope with AO staining. The inclusions were larger than infection spots in volume, but were much lower in count. A 437 bp-specific fragment of C.pn DNA was detected in C.pn-infected VSMCs by PCR. In the cell adhesion assay, the absorbance values in C.pn infection group were far higher than those in control group 2 h after infection (P<0.01). The cell adhesion ratio in C.pn infection group was 134.38%. The migration distance of VSMCs infected with C.pn was significantly longer than that of the control cells 24 h after infection by a wound-healing assay (P<0.05). More migratory VSMCs infected with C.pn for 24 h were found in a Transwell assay than those in control group (P<0.01). CONCLUSION: C.pn infection significantly promotes the adhesion and migration of VSMCs.     

miR-17 regulates senescence of rat vascular smooth muscle cells

AIM: To investigate the effect of microRNA-17 (miR-17) on the senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanism.METHODS: The medial layer of the thoracic aorta was collected from the SD rats and isolated for primary culture. VSMCs were identified by immunofluorescence staining. The VSMCs were collected at the 4th~6th generations, and then the miR-17 mimics and miR-17 inhibitor were transfected into the VSMCs by liposome method. After 24 h, the cell senescence was induced by D-galactose. The VSMCs were divided into the following 6 groups:aging induction+miR-17 mimics (A-miR-17) group, aging induction+miR-17 inhibitor (A-anti-miR-17) group, A-control group, normal (N)+miR-17-mimics (N-miR-17) group, N-anti-miR-17 group, and N-control group. On day 3 after the addition of D-galactose, the senescence of VSMCs was observed with β-galactosidase staining. The expression of miR-17, p16 and p21 was detected by RT-qPCR and immunohistochemistry. RESULTS: miR-17 expression in the VSMCs was significantly lower in A-control group than that in N-control group (P<0.01). Compared with A-control group, the expression of miR-17 in the VSMCs was significantly increased in A-miR-17 group (P<0.01), while that was significantly decreased in A-anti-miR-17 group (P<0.01). The number of β-galactosidase positive staining cells in A-anti-miR-17 group was significantly higher than that in A-miR-17 group (P<0.01). The expression of p21 at mRNA and protein levels in the VSMCs was significantly lower in A-miR-17 group than that in A-control group (P<0.01), and the expressions of p21 at mRNA and protein levels was significantly higher in A-anti-miR-17 group than that in A-miR-17 group (P<0.01). CONCLUSION: miR-17 inhibits rat VSMCs senescence induced by D-galactose, the underlying mechanism is associated with the inhibition of p21 expression.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effect of nicotine on systolic and diastolic function of rat aortic smooth muscle cells

AIM:To observe the effects of nicotine on systolic and diastolic function of rat aortic vascular smooth muscle cells (VSMCs). METHODS:The primary rat aortic VSMCs were cultured in vitro. After exposed to nicotine at different concentrations for 24 h, the cytoskeleton of the VSMCs was stained with rhodamine-phalloidin,the photographs of the VSMCs in different experimental groups were taken and the surface area was measured to reflect the cell contractility. Collagen contraction method was also used to determine the effect of nicotine on the contractility of rat aortic VSMCs. RESULTS:The primary rat aortic VSMCs were successfully cultured. After the VSMCs were treated with nicotine (0.1 μmol/L, 1 μmol/L, 10 μmol/L and 100 μmol/L) for 24 h, the skeleton showed a significant contraction, and the cell plate shape was obviously enhanced in a concentration-dependent manner. The results showed that 10 μmol/L was the optimal concentration of nicotine for VSMCs (P<0.01). The collagen contraction method also showed that 10 μmol/L nicotine contracted the rat aortic VSMCs. With the increase in the nicotine action time, the maximum contraction effect was observed at 60 min (P<0.01). CONCLUSION:Nicotine has a strong contractile effect on VSMCs of rat aorta, and its contractile effect is dependent on concentration and time.