Abstract:Aim To investigate the effect of miR-92a-3p_R +1 and miR-92a-1-5p on phenotypic transformation and proliferation of rat vascular smooth muscle cells (VSMC) induced by oxidized low-density lipoprotein (ox-LDL) in the ERK 1/2 signaling pathway. Methods VSMC isolated from rats, and ox-LDL (50 mg/L) was used to induce VSMC.ERK1/2 inhibitors U0126(10 μmol/L)was used for intervention. The miRNA microarray profiling was performed using small RNA sequencing analysis. CCK-8 method and Brdu flow cytometry were used to detect VSMC proliferation. Immunofluorescence assay was performed to detect the expression of SM22α protein in VSMC. Western blot was used to detect the expression changes of ERK1/2 pathway signal molecules (ERK, p-ERK), phenotype marker protein SM22α and proliferation associated proteins such as PCNA, cyclin D1, p21 and p27. Results Under ox-LDL induction, the expressions of miR-92a-3p_R +1 and miR-92a-1-5p in VSMC were significantly up-regulated. After the intervention of ERK1/2 inhibitors U0126, the phosphorylation level of ERK1/2 was inhibited, the corresponding VSMC miR-92a-3p_R +1 and miR-92a-1-5p expression significantly lowered (P<0.05). Therefore, the study speculated that ERK1/2 signaling pathway may affect the phenotypic transformation and proliferation of VSMC by regulating the expression of miR-92a. After inhibiting the ERK1/2 signaling pathway, the proliferation of VSMC was significantly reduced, and the expression of SM22α was up-regulated (P<0.05). The expression of PCNA and cyclin D1 was down-regulated and the expression of p21 and p27 proteins were up-regulated (P<0.05). This indicated that the phenotypic transformation and proliferation were significantly inhibited. Conclusion Mir-92a-3p_R +1 and miR-92a-1-5p play important roles in the ERK1/ 2 signaling pathway that ox-LDL induces phenotypic transformation and proliferation of VSMC.

Abstract:Aim To investigate the effect of alisol A 24-acetate on the phenotypic modulation of vascular smooth muscle cells（VSMC）as well as the expression of matrix metalloproteinase-9（MMP-9） in ox-LDL-induced rats vascular smooth muscle cells （VSMC）, and their correlation with ERK1/2 pathway. Methods VSMC isolated from the thoracic aorta of rats were induced by ox-LDL（50 mg/L） and intervened by alisol A 24-acetate （10 mg/L）. Immunocytochemistry was performed to detect the expression of SM22α protein.RT-PCR was performed to detect MMP-9 mRNA expression and Western blot was applied to detect the expressions of MMP-9, p-ERK and t-ERK. Results The contractive phenotypic specificity protein SM22α in the VSMC induced by ox-LDL was inhibited, VSMCs changed the phenotype from constriction to synthesis, the expression of phosphorylated ERK （p-ERK） and MMP-9 in ox-LDL group were elevated compared with the control group（P<0.05）. Alisol A 24-acetate.partially inversed the effects of ox-LDL on VSMC （P<0.05）. Conclusion Alisol A 24-acetate inhibited the phenotype transformation of VSMC induced by ox-LDL，and the mechanism maybe has correlation with ERK1/2 pathway.

Abstract:Aim To investigate the phenotypic modulation and proliferation changes of vascular smooth muscle cell(VSMC)and vascular adventitia fibroblasts(VAF)in the vein graft restenosis model.To evaluate the effects of the phenotypic modulation and proliferation changes of VSMC and VAF in vascular remodeling.Methods To create the pig autogenous vein grafts restenosis model,specimens were harvested at three time points that was 7th,14th and 30th day.Histomorphometrical and immunohistochemical approach were used to detect the changes of vascular remodeling and the expression of proliferation cell nuclear(PCNA),smooth muscle α-actin(SM-α-actin)and osteopontin(OPN).Results ①At 7th day after operation,the neointima formed and continuously thickened,whose thickness reached maximum at 30th day postoperation.Internal elastic lamina(IELA)gradually decreased after operation,and at 30th day group luminal area reduced minimally(P<0.05).Remodeling index and external elastic lamina(EELA)slightly increased at 7th day group,but reduced gradually after that,which at 14th day and 30th day group decreased distinctly(P<0.05).②According to PCNA stain,the proliferation index of VAF and VSMC increased significantly at 7th day after operation,which reached peak at 14th day,whereas the expression of PCNA reverted toward the baseline at 30th day after operation.③According to SM-α-actin stain,at 7th day after operation,the positive cell area decreased in the medium,but adventitia and neointimal were positive;At 14th day,the positive cell area decreased significantly in the medium,however,the positive area of adventitia and neointimal increased significantly;At 30th day positive expression of the medium recovered gradually,and the positive area of adventitia was less than that at 14th day,but the positive expression in the neointimal has no significant changes compared with 14th day.④OPN stain showed that the positive expression was significant in the medium at 7th day after operation.At 14th day,the positive expression of VSMC was less than that at 7th day in the medium,but that of neointimal increased significantly,which reached peak.At 30th day,there was a less positive part of VSMC in the medium,and the positive expression of VSMC in the neointimal decreased significantly compared with 14th day.OPN was negative in adventitia at different time after operation.Conclusion The phenotypic modulation and proliferation changes of VSMC and VAF are the important factors on vascular remodeling,which takes part in and accelerates the course of vein restenosis.

Abstract:Aim To discuss the regulation of expression of GATA-6 by nitric oxide/cGMP-dependent protein kinase I in vascular smooth muscle cells(VSMC) phenotypic modulation. Methods Cultured wistar rat aortic VSMC were used as an experimental model.Cell growth was determined by MTT assay.The mRNA and protein expression of GATA-6 and smooth muscle myosin heavy chain(sm-MHC) were assayed by RTPCR and Western blot analysis. Results The results showed that phenylephrineinduced proliferation of VSMC was inhibited by S-nitroso-N-Acetylpenicillamine (SNAP) and Sp-8-pCPT-cGMPs,but promoted by Rp-8-pCPT-cGMPs.The expression of GATA-6 and sm-MHC mRNA and protein were increased by SNAP and Sp-8-pCPT-cGMPs,and decreased by Rp-8-pCPT-cGMPs. Conclusions NO/PKG I can up-regulate the expression of GATA-6 and sm-MHC at the transcriptional and translational level.These findings demonstrate that NO/PKG I pathway is involved in the maintenance of the differentiated phenotype in VSMC.

Abstract:Aim To explore phenotypic modulation of vascular smooth muscle cells(VSMC) and change of p38,mitogen-activated protein kinase phosphatase-1(MKP-1) expression after intimal injury. Methods The model of vascular restenosis established by intimal injury of rabbit carotid arteries was used.Immunohistochemistry,Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR) were used to detect the changes of proliferation cell nuclear antigen(PCNA),smooth muscle α-actin(SM α-actin),p38 protein,and MKP-1 protein as well as its mRNA of sham-injured arteries and injured arteries at different time points. Results PCNA was negative in the medium and endothelium in shaminjured arteries.Positive cell rate of PCNA was gradually increased at 1～14 days in the medium and at 5～14 days in the neointima after injury,but it declined gradually after 28 days.Positive cell rate of PCNA in the neointima was slightly more than that in the medium at different time points.SM α-actin was positive in the medium,negative in the endothelium in sham-injured arteries.SM α-actin initially decreased in the medium at 1 day,it was minimal at 3 days after injury,but it increased gradually after 5 days.Positive expression of SM α-actin in the neointima was slightly lower than that in the medium.p38 was negative or feeble positive in the medium in sham-injured arteries.p38 was continuously increased at 1～35 days after injury.Positive expression of p38 in the neointima was higher than that in the medium.There was positive relationship between change of p38 and that of PCNA in the vascular wall at different time points after injury.MKP-1 was feeble positive or positive in sham-injured arteries.MKP-1 initially decreased at 1 day and increased graduallv from 14～28th day,but it was still lower than that in the sham-injured arteries on 35th day after injury.There was negative relationship between change of MKP-1 and that of PCNA in the vascular wall at different time points after injury. Conclusion There was close relationship between phenotypic modulation and proliferation ability of VSMC.p38 and MKP-1 participated in phenotypic modulation of VSMC and its regulation after intimal injury.

Abstract:Aim To investigate the relationship between tetrandrine(Tet) on prevention and treatment of restenosis and phenotypic modulation of vascular smooth muscle cells(VSMC) as well as its signal transduction pathway after vascular intimal injury. Methods HE staining was used to analyse vascular morphology change at sham-injured,injured and Tet-treated group 28 days.Immunohistochemistry and Western blot were respectively used to detect the expression change of smooth muscle α-actin(SM α-actin) and proliferation cell nuclear antigen(PCNA),p38 in injured group and Tet group at 7,14 and 28 days after balloon injury. Results The every layer structure in vascular wall of sham-injured artery was intact.Neointimal area was obviously increased and lumen area was notably decreased in injured group.In the Tet group the intimal proliferation was showed but notably weaker than that of the injured group and lumen area notably increased.Compared with the injured group,the expression of SM α-actin,PCNA and p38 in vascular wall of the Tet group was not significantly different and neointimal proliferation degree was also basicly the same at 7 days after injury.The expression of PCNA and p38 in Tet group was all significantly lower than that in injured group in vascular wall at 14 and 28 days after injury.However,expression of SM α-actin in Tet group was slightly higher than that in injured group at 14 days, and not significantly different between the two groups at 28 days. Conclusion Tet could reduce neointimal hyperplasia by inhibiting VSMC phenotypic modulation and its signal transduction.

Abstract:Aim To explore the effects of Astragalus membranaceus and Angelica sinensis on vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation. Methods Basic fibroblast growth factor (bFGF) was used as a stimulus and the effects of Astragalus membranaceus and Angelica sinensis on VSMC differentiation, DNA synthesis and proto-oncogene expression in VSMC induced by bFGF were observed. Results bFGF could downregulate the expression of smooth muscle (SM) α-actin, induce the expression of smooth muscle embryonic myosin heavy chain and osteopontin, stimulate the expression of c-jun gene and promote the DNA synthesis. Both Astragalus membranaceus and Angelica sinensis could inhibit VSMC phenotypic modulation and DNA synthesis induced by bFGF, and result in the effective inhibition of VSMC proliferation. The mechanisms were related with the inhibition of c-jun gene expression stimulated by bFGF. Compared with Astragalus membranaceus, Angelica sinensis was more effective in inhibiting the VSMC phenotypic conversion and proliferation. Conclusions All results above reflected multi-site effects of Astragalus membranaceus and Angelica sinensis on VSMC phenotypic conversion and proliferation, and revealed their new pharmacological mechanisms.