Abstract:Aim To observe whether the proliferation and apoptosis of mouse vascular smooth muscle cells(VSMC) were induced by stretch stress (SS) via PKCα phosphorylation and to investigate the role of berberine (BBR) and related mechanism on the process. Methods The experiment was divided into six groups:normal control(NC) group, BBR group, PKCα inhibitor (Go6976) group, SS group, SS+BBR group and SS+Go6976 group. VSMC were pretreated with BBR or Go6976 for 1 h, and stimulated for 15 min with SS at 10% amplitude. PKCα and MAPK (ERK, JNK, p38) phosphorylation were measured by Western blot. After pretreated by BBR or Go6976, and SS stimulation for 1 h, the proliferation (Ki67) and apoptosis (TUNEL) of VSMC were measured by immunofluorescence. Results Compared with the NC group, SS could increase the phosphorylation levels of PKCα and MAPK(ERK, JNK, p38) (P＜0.05), and increase the levels of VSMC proliferation and apoptosis (P＜0.05). BBR could inhibit the phosphorylation levels of PKCα and MAPK (P＜0.05), while inhibiting VSMC proliferation and apoptosis (P＜0.05); Go6976 could inhibit PKCα, ERK and JNK phosphorylation (P＜0.05), but had no effect on the phosphorylation increase of p38, while inhibiting VSMC proliferation and apoptosis (P＜0.05). Conclusion BBR inhibits the phosphorylation of PKCα and MAPK (ERK, JNK, p38) induced by SS, and further inhibits the proliferation and apoptosis of VSMC.

Abstract:Aim To investigate the effect of jaranol on platelet derived growth factor-BB (PDGF-BB)-induced proliferation and migration of vascular smooth muscle cell(VSMC) as well as its possible mechanism. Methods Human aortic vascular smooth muscle cells (HA-VSMC)were treated with 25 μg/L PDGF-BB to induce proliferation and migration. The cells were divided into normal control group (NC group), PDGF-BB group, PDGF-BB+jaranol (0,0 and 40 μmol/L) group, DMSO group. Cell counting Kit-8 (CCK-8) assay was used to assess proliferation ability of HA-VSMC, and Transwell assay was used to examin the migration ability of HA-VSMC. Western blot was used to detect the expression of autophagy related proteins including p62, LC3, mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR). Results After treatment with PDGF-BB, the proliferation and migration ability of HA-VSMC was notably upregulated (P＜0.01), while jaranol showed a significant and concentration-dependent inhibition effect on PDGF-BB-induced proliferation and migration of HA-VSMC (P＜0.05). Western blot results showed that compared with the PDGF-BB group, the protein expression levels of LC3Ⅰ and mTOR were significantly decreased in PDGF-BB+jaranol (40 μmol/L) group (P＜0.05); The expression levels of LC3Ⅱ/LC3Ⅰ, p62 and p-mTOR proteins were significantly increased (P＜0.05), and the effect of jaranol(40 μmol/L) and compound C on AMPK/mTOR signaling pathway of HA-VSMC induced by PDGF-BB was consistent. Conclusion Jaranol can inhibit the abnormal proliferation and migration of HA-VSMC invoked by PDGF-BB, its mechanism may be related to its inhibition effect of PDGF-BB-induced autophagy of HA-VSMC by regulating AMPK/mTOR signaling pathway.

Abstract:Aim To observe whether mechanical stretch stress (SS) induces the proliferation and migration of mouse vascular smooth muscle cells (VSMC) through PKCδ activation, and to further explore the effect and mechanism of berberine (BBR) on the proliferation and migration of VSMC. Methods Mouse VSMC in vitro were divided into six groups:negative control group (NC), berberine group (BBR), PKCδ inhibitor Sotrastaurin group (Sotras), SS group, SS+BBR group and SS+Sotras group. The cultured VSMC in each group,which were pretreated with BBR or Sotrastaurin or ddH2O for 1h, followed by SS (10% tensile strength) for different time or no treatment for control, were collected for the detection of PKCδ phosphorylation, proliferation and migration by Western blot, immunofluorescence and scratch assay respectively. Results Immunofluorescence and cell scratch test results showed that compared with NC group, SS stimulation significantly increased Ki67 positive level in VSMC by 469%(P＜0.05, n＝3), and reduced scratch width by 54.9%(P＜0.05, n＝3),while BBR and Sotrastaurin could significantly inhibit the changes caused by SS, Ki67 positive level decreased by 66.9% and 80.2%(P＜0.05, n＝3), and scratch width increased by 79.4% and 120.1%(P＜0.05, n＝3)compared with SS group, respectively. Meanwhile, Western blot results showed that SS stimulation induced a time-dependent increase in PKCδ phosphorylation compared with NC group, with the most significant increase of 97.5%(P＜0.05,n＝3) at 30 min, which also inhibited by berberine in a concentration-dependent manner, and the effect was the most significant at 200 μmol/L, with a decrease of about 37.6% (P＜0.05, n＝3). Conclusion Berberine inhibits SS-induced vascular smooth muscle cell proliferation and migration by inhibiting PKCδ phosphorylation.

Abstract:Aim To explore a novel experimental method for detecting cardiomyocyte proliferation at the live cell level, and to exclude false positive cells caused by multinucleate cardiomyocytes, so as to provide experimental reference for accurate detection of cardiomyocytes proliferation. Methods Use the cell division cyclin 20 (CDC20) and spastic paraplegia 20 (SPG20) as the molecular beacons targets. These molecular beacons allow for CDC20 and SPG20 detection in live cells, through Lipo-2000 transfection. The double positive cells are the proliferating ones that undergo cytokinesis. Results When the cells did not proliferate, the probes could not detect the target sequences and the cells cannot be detected by the fluorescence. When the nuclei replicated and divided without cytokinesis, the sequences could not be detected by both two probes at the same time, and the cells also could not be detected. When cells underwent mitosis with complete cytokinesis proliferation, both two fluorescence probes could be detected simultaneously. Conclusion The molecular beacons-based approach targeting CDC20 and SPG20 identified a proliferative subset of cardiomyocytes, which could exclude false positive problems of cell proliferation caused by nuclear proliferation and improve the accuracy of cell proliferation detection.

Abstract:Aim To investigate the effect of glycogen synthase kinase 3β(GSK3β)inhibition on the proliferation, migration and adhesion of endothelial progenitor cells(EPC)in atherosclerotic mice. Methods Atherosclerotic mice model were established. Mononuclear cells were isolated and cultured from bone marrow in atherosclerotic mice (atherosclerotic group) and wild type mice (normal control group). EPC in logarithmic phase were transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β(GKS3β-KM)(gene transfer group) in atherosclerotic mice. The number and biological functions of EPC, such as proliferation, migration and adhesion were assessed by cells count, MTT assay, modified Boydens chamber migrationtrial and adhesion test. Results Compared with normal control group, the number of EPC was observably reduced and proliferation, migration and adhesion capacities of EPC were markedly impaired in atherosclerotic mice(P＜0.01,n＝5). Compared with atherosclerotic group, the number (P＜0.01,n＝5)and biological functions of EPC including proliferation (P＜0.01,n＝5), migration (P＜0.01,n＝5) and adhesion were significantly enhanced in gene transfer group (P＜0.01,n＝5). Conclusion GSK3β inhibition could improve impaired proliferation, migration and adhesion capabilities of EPC in atherosclerotic mice.

Abstract:Aim To observe the effect of miR-206 on proliferation of vascular endothelial cell and its mechanism, and provide a new therapeutic target for clinical arteriosclerosis. Methods The arterial endothelial tissue on lesion site and normal endothelial tissue were collected from the patients who were diagnosed as diabetic lower limb atherosclerosis by first time in clinical pathology. The expression of miR-206 was detected by qPCR. Normal HUVEC cell lines, HUVEC transfected with miR-206-mimics and HUVEC transfected with miR-206-inhibitor were studied through fundamental experiment, and the transfection efficiency were observed. In the three groups, CCK8 was used to detect the proliferation status, transwell was used to evaluate the migration ability, flow cytometry was used to detect cell cycle and Western blot was used to observe expression of Cx43. Results The mRNA expression of miR-206 on lesion site of lower limb artery (shin peroneal artery) in the patients with diabetic arteriosclerosis was significantly increased compared with normal endothelial tissue(P<0.05); The expression of miR-206 significantly increased (P<0.05) after transfected with miR-206-mimics, while it was significantly reduced (P<0.05) after transfected with miR-206-inhibitor. Compared with normal HUVEC and HUVEC transfected with miR-206-inhibitor, the cell proliferation rate was significantly lower in HUVEC transfected with miR-206-mimics for 24 h (P<0.05). After HUVEC was transfected with miR-206-mimics, the cell migration capacity was significantly lower than normal HUVEC group (P<0.05), and the cell cycle was blocked during G2 phase; while the cell migration capacity was higher in HUVEC transfected with miR-206-inhibitor group than that of normal HUVEC group (P<0.05). Cx43 expression was significantly reduced in miR-206-mimics group but significantly increased in miR-206-inhibitor group. Conclusion miR-206 can inhibit the proliferation of vascular endothelial cells, block endothelial cells in G2 phase and suppress their migration, and it may affect proliferation of endothelial cells by regulating Cx43 expression.

Abstract:Aim To study the role and mechanism of dehydroepiandrosterone (DHEA) in the proliferation of vascular smooth muscle cells (VSMCs) induced by angiotensin Ⅱ (AngⅡ). Methods VSMCs were cultured with AngⅡ in vitro, then treated with DHEA. The effect of DHEA on the proliferation of VSMCs induced by AngⅡ was detected by MTS and cell counting. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effects of DHEA on the expressions of proliferating cell nuclear antigen (PCNA), Krüppel-like factor 5 (KLF5) and inducible nitric oxide synthase (iNOS) in AngⅡ-induced VSMCs. The effect of DHEA on the concentration of peroxynitrite anion (ONOO－) in the medium of AngⅡ-induced VSMCs was examined by enzyme-linked immunosorbent assay (ELISA). Endogenous iNOS was knocked down by transfecting VSMCs with iNOS-specific siRNA (si-iNOS) or nonspecific siRNA (si-Ctrl), and then treated the cells with or without DHEA; Western blot was performed to examine the expression of PCNA. Results The results of MTS and cell counting showed that DHEA could significantly inhibit the proliferation of VSMCs induced by AngⅡ (P＜0.05). The results of qRT-PCR and Western blot showed that DHEA significantly inhibited the expressions of PCNA, KLF5 and iNOS in VSMCs induced by AngⅡ at the level of protein and mRNA (P＜0.05). ELISA results showed that DHEA could reduce the concentration of ONOO－ in culture medium of VSMCs treated with AngⅡ (P＜0.05). Western blot results suggested that DHEA had no effect on AngⅡ-induced PCNA protein expression after knocked down endogenous iNOS (P＞0.05). Conclusion DHEA may inhibit the proliferation of VSMCs induced by AngⅡ probably through down-regulation of KLF5 expression by inhibiting ONOO－ produced by iNOS.

Abstract:Background and Aim Vascular smooth muscle cell (VSMC) apoptosis plays a critical role in the pathogenesis of many angiocalcified diseases. Recent studies have shown that lysine methyltransferase SET8 was involved in regulating cell proliferation and apoptosis. The purpose of this study was to explore whether SET8 can influence calcification by regulating proliferation and apoptosis of VSMC. Methods VSMC were obtained from rat thoracic aorta, and then randomly divided into control group, the empty plasmid group and SET8-shRNA group. Transfection was performed with cationic lipid vectors (LipofectamineTM2000) on VSMC. Calcium deposition was measured by alizarin red staining and calcium content measurement; The proliferation of VSMC in vitro was measured by MTT assay. The expressions of proliferation-related genes survivin and apoptosis-related genes caspase-3 were determined by real-time PCR and Western blot assay. Results The expression of SET8 protein in VSMC was effectively inhibited by SET8-shRNA. The calcium content was decreased in cells after SET8-shRNA transfection. Proliferation of VSMC was inhibited after transfected with SET8-shRNA 12 h, 24 h, 36 h and 48 h (P<0.05). The expressions of survivin was decreased in cells, but expressions of caspase-3 was increased after SET8-shRNA transfection (P<0.05 for all). Conclusion Interfering with SET8 can increase the expression of apoptosis gene and inhibit the expression of proliferative genes, and SET8 may be involved in regulating the calcification of rat blood vessels.

Abstract:In the process of atherosclerosis, media vascular smooth muscle cell phenotype transform, migrate and proliferate, enter the vascular intima, and participate in the formation of fibrous caps and neovascularization of atherosclerotic plaques. In this review, the current research progress on the role of vascular smooth muscle cell phenotype transformation to atherosclerosis is reviewed.

Abstract:Aim To investigate the inhibitory effect of berberine on the proliferation of human umbilical vein endothelial cell (HUVEC) induced by oxidized low density lipoprotein (ox-LDL) and the changes of related signaling pathways. Methods HUVEC was cultured in vitro, and the proliferation of HUVEC was stimulated by ox-LDL and intervened with berberine at different concentrations. The inhibitory effect of berberine on proliferation of HUVEC was detected by cell counting kit 8 and EdU incorporation experiment. The expressions of related genes and proteins and the changes of signaling pathway were analyzed by real-time quantitative PCR and Western blot. Results In a concentration dependent manner, berberine (1~50 mg/L) significantly inhibited the HUVEC proliferation induced by ox-LDL, and reduced the expressions of proliferating cell nuclear antigen (PCNA), nuclear factor κB (NF-κB) and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). These effects were associated with decreased phosphorylation levels of PI3K/Akt, ERK1/2 and p38MAPK signaling pathways. Conclusion Berberine inhibits the proliferation of HUVEC induced by ox-LDL through the down-regulation of PCNA, NF-κB and LOX-1 expressions. PI3K/Akt, ERK1/2 and p38MAPK signaling pathways are involved in this process.