Vascular Muscle Cell Proliferation Research Articles

Alpha-2-macroglobulin is involved in the occurrence of early-onset pre-eclampsia via its negative impact on uterine spiral artery remodeling and placental angiogenesis

BackgroundPre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood.MethodsHuman placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague–Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors.ResultsIn this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFβ1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats.ConclusionsOur data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

Resistin-like molecule beta augments phenotypic modulation of human aortic smooth muscle cell triggered by high glucose

Vascular muscle cells (VSMCs) participate in the pathophysiology of atherosclerosis. Resistin-like molecule beta (Relmβ) contributes to atherosclerosis development by activating macrophage. This study aims to investigate whether Relmβ regulates VSMC phenotypic modulation under high glucose environment. Human aortic vascular smooth muscle cells were cultured and treated with Relmβ in the presence or absence of high glucose. VSMC phenotypic modulation was assessed by expression of related markers. The migration of VSMCs was detected by wound healing assay and transwell assay. The proliferation of VSMCs was measured using CCK-8 assay. In this study, we observed that Relmβ modulated VSMC phenotypic modulation by down-regulating expression of smooth muscle α-actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), and calponin while up-regulating expression of osteopontin (OPN). Relmβ increased the expression of inflammatory genes in VSMCs. Relmβ also augmented VSMCs migration as well as proliferation. It is worth noting that all the effects of VSMCs were enhanced upon high glucose stimulation. The phosphorylation levels of p38MAPK and ERK1/2 were increased by co-treatment with Relmβ and high glucose. The p38 MAPK pathway inhibitor RWJ64809 and pERK1/2 inhibitor PD98059 significantly inhibited the proliferation of VSMCs induced by Relmβ and high glucose. Our results provide evidence that Relmβ augments phenotypic modulation and migration of human aortic smooth muscle cell induced by high glucose. Relmβ might be a potential target for treatment of atherosclerosis induced by hyperglycemia.

Bazedoxifene exhibits anti-inflammation and anti-atherosclerotic effects via inhibition of IL-6/IL-6R/STAT3 signaling

Atherosclerosis is regarded as chronic inflammatory disease. The IL-6/STAT3 pathway plays an important role in inflammation. We previously described a small-molecule compound, Bazedoxifene, which target IL-6/STAT3 pathway and has been approved for clinical use for osteoporosis in postmenopausal women. The aim of this study is to evaluate the effect of Bazedoxifene in the progression of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice.Five-week-old male ApoE−/− mice were fed with High-fat diet (HFD) containing 5 mg/kg Bazedoxifene or a matching control for 12 weeks. Oil red O (ORO) staining was used to detect plaque size; immunohistochemical staining was used to detect the presence of endothelial cells, vascular muscle cells and phosphorylated STAT3 (P-STAT3) in localized plaques. The potential underlying mechanisms in human umbilical vein endothelial cells (HUVECs) and vascular muscle cells (VSMCs) was detected by Western blot analysis, Wound healing assay and Elisa assay. In the ApoE−/− mice fed with HFD, daily Bazedoxifene administration effectively attenuated atherosclerotic plaque area (P < 0.01), down-regulated IL-6 levels (P < 0.01), decreased STAT3 phosphorylation, reduced VSMCs proliferation and increased endothelial coverage in aortic vessels. Interestingly, we found HUVECs lack of membrane IL-6 receptor (IL-6R) compared to VSMCs (P < 0.01). Furthermore, we found that the soluble IL-6 receptor (sIL6R) participates in the activation of STAT3 induced by IL-6 or TNF-α in HUVECs and primary HUVECs. Bazedoxifene did not inhibit the growth of HUVECs while suppressing the proliferation of VSMCs.Bazedoxifene is an attractive novel therapeutic reagent for atherosclerosis diseases. This mechanism may be partially attributed to regulating IL-6/IL-6R/STAT3 signaling pathway.

WD Repeat Domain 1 Deficiency Inhibits Neointima Formation in Mice Carotid Artery by Modulation of Smooth Muscle Cell Migration and Proliferation.

The migration, dedifferentiation, and proliferation of vascular smooth muscle cells (VSMCs) are responsible for intimal hyperplasia, but the mechanism of this process has not been elucidated. WD repeat domain 1 (WDR1) promotes actin-depolymerizing factor (ADF)/cofilin-mediated depolymerization of actin filaments (F-actin). The role of WDR1 in neointima formation and progression is still unknown. A model of intimal thickening was constructed by ligating the left common carotid artery in Wdr1 deletion mice, and H&E staining showed that Wdr1 deficiency significantly inhibits neointima formation. We also report that STAT3 promotes the proliferation and migration of VSMCs by directly promoting WDR1 transcription. Mechanistically, we clarified that WDR1 promotes the proliferation and migration of VSMCs and neointima formation is regulated by the activation of the JAK2/STAT3/WDR1 axis.

Inhibitory effects of herbal decoction Ojeoksan on proliferation and migration in vascular smooth muscle cells.

The proliferation of vascular smooth muscle cells plays a crucial role in pathogenesis of cardiovascular disease. The principal objective of this study was to determine the effects of Ojeoksan (OJS) on human aortic smooth muscle cell (HASMC) proliferation induced by tumor necrosis factor α (TNF-aα). Thymidine incorporation after TNF-α treatment was increased and this effect was inhibited significantly by OJS treatment. HASMC proliferation and migration by kinetic live cell imaging were also reduced by treatment with OJS. TNF-α induced the expression of cyclins/cyclin-dependent kinases (CDKs) and reduced the expression of p21waf1/cip1/p27kip1. However, OJS also attenuated the expression of TNF-α-induced cell-cycle regulatory proteins. The results of Western blot analysis demonstrated that the TNF-α treated HASMC secreted gelatinases, probably including MMP-2/-9, which may be involved in the invasion and migration of HASMC. Additionally, OJS suppressed the mRNA expression levels of matrix metalloproteinase-2/-9 (MMP-2/-9) in a dose-dependent manner. OJS inhibited the production of TNF-α-induced hydrogen peroxide (H2O2) and the formation of DCF-sensitive intracellular reactive oxygen species (ROS). Further, OJS suppressed the nuclear translocation and phosphorylation of inhibitor of kappa B-α (IκB-α) of nuclear factor κB (NF-κB) under TNF-α conditions. Our results demonstrate that OJS exerts inhibitory effects on TNF-α-induced HASMC proliferation and migration, suggesting the involvement of the inhibition of both MMP-2 and MMP-9 expressions, and the downregulation of ROS/NF-κB signaling. Thus, herbal decoction OJS may be a possible therapeutic approach to the inhibition of cardiovascular disease including atherosclerosis.

Inhibitory effect of irisin on vasular smooth muscle cell inflammation and proliferation by regulating autophagy through adenosine monophosphate-actived protein kinase/mammalian target of rapamycin pathway

Objective To explore the underlining mechanisms of irisin inhibiting inflammation and proliferation of vascular smooth muscle cells (VSMCs). Methods The cultured primary rat aortic VSMCs were pre-incubated with irisin and then treated with platelet derived growth factor-BB (PDGF-BB). Enzyme linked immunosorbent assay (ELISA) and methyl thiazol tetrazolium (MTT) assay were used to measure the VSMCs inflammation and proliferation, respectively. The protein expression and phosphorylation of adenosine monophosphate-actived protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway related molecule AMPK/p-AMPK (Thr172), acetyl-CoA carboxylase (ACC)/phosphorylated (p)-ACC (Thr308), mTOR/p-mTOR (Ser2448), ribosomal protein S6 kinase (p70SK6)/p-70SK6 (Thr389), and autophay related molecule microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3Ⅱ and nucleoporin p62 (P62) were detected by Western blotting. Results After treatment with PDGF-BB, VSMCs proliferation dramatically increased, and the inflammatory cytokines IL-1β (37.9±8.5 vs. 10.8±2.9, t=18.620, P<0.01), IL-6 (57.4±14.1 vs. 32.4±8.7, t=6.250, P<0.01), MCP-1 (158.3±39.1 vs. 57.2±12.5, t=14.280, P<0.01), ICAM-1 (371.8±77.3 vs. 160.4±39.8, t=10.850, P<0.01), tumor necrosis factor-α (TNF-α) (16.3±3.8 vs. 7.8±2.4, t=7.390, P<0.01) in the cell supernatant were significantly up-regulated. The protein expression levels of AMPK (0.39±0.11 vs. 0.83±0.18, t=14.250, P<0.01), p-AMPK (0.25±0.03 vs. 0.49±0.12, t=12.370, P<0.01), ACC (0.54±0.16 vs. 1.07±0.24, t=10.670, P<0.01), p-ACC (0.38±0.07 vs. 0.97±0.30, t=18.130, P<0.01), LC3Ⅰ (0.67±0.21 vs. 0.67±0.21, t=5.780, P<0.01) and LC3Ⅱ (0.42±0.13 vs. 0.93±0.38, t=6.820, P<0.01) were down-regulated, mTOR (2.57±0.53 vs. 1.23±0.25, t=21.350, P<0.01), and p-mTOR (1.35±0.0.37 vs. 0.57±0.12, t=8.870, P<0.01), p70SK6 (1.85±0.39 vs. 0.81±0.19, t=7.170, P<0.01), p-70SK6 (0.69±0.22 vs. 0.38±0.11, t=9.260, P<0.01) and P62 (1.24±0.38 vs. 0.58±0.14, t=5.270, P<0.01) were up-regulated. However, pre-incubation with irisin and then stimulation with PDGF-BB could significantly decrease VSMCs proliferation [(122.14±9.42)%, t=4.970, P<0.01], and significantly down-regulate the inflammatory cytokines IL-1β (17.38±4.10, t=6.740, P<0.01), IL-6 (34.9±8.9, t=2.760, P<0.05), MCP-1 (47.28±9.20, t=16.270, P<0.01), ICAM-1 (169.2±55.8, t=7.780, P<0.01) and TNF-α (6.9±2.0, t=8.180, P<0.01) in the cell supernatant. The protein expression levels of AMPK (0.72±0.26, t=2.670, P<0.05), p-AMPK (0.46±0.13, t=3.280, P<0.05), ACC (0.95±0.28, t=5.870, P<0.01), p-ACC (0.78±0.25, t=3.140, P<0.01), LC3Ⅰ (1.34±0.35, t=1.960, P<0.05) and LC3Ⅱ (1.03±0.19, t=7.260, P<0.01) were up-regulated, and mTOR (1.16±0.33, t=6.910, P<0.01), p-mTOR (0.66±0.18, t=6.830, P<0.01), p70SK6 (1.12±0.34, t=2.360, P<0.05), p-70SK6 (0.37±0.12, t=3.120, P<0.01) and P62 (0.68±0.18, t=3.360, P<0.05) were down-regulated. Conclusion Irisin could inhibit the inflammation and proliferation of VSMCs possibly by up-regulating autophagy through activation of the AMPK/mTOR signaling pathway. Key words: Irisin; Adenosine monophosphate-actived protein kinase/mammalian target of rapamycin signaling pathway; Autophagy; Vascualar smooth muscle cell proliferation; Vein graft stenosis

GLP-1 attenuates Ang II-induced proliferation and migration in rat aorta smooth muscle cells via inhibition of the RhoA/ROCK2 signaling pathway.

Glucagon-like peptide 1 (GLP-1), a neuroendocrine hormone produced by the gastrointestinal tract, plays a significant role in blood glucose regulation; drugs derived from GLP-1 are currently used for the treatment of type 2 diabetes. In addition to regulating glucose homeostasis, the protective effects of GLP-1 on the cardiovascular system are also of interest. However, the vascular protective mechanisms of GLP-1 remain unclear. The present study was designed to evaluate the role of GLP-1 in the proliferation and migration of vascular smooth muscle cells, and the underlying mechanisms. In this study, proliferation, migration, cyclin D1 expression, and phosphorylation of MLC, as well as RhoA and Rho-associated coiled-coil forming protein kinase 2 (ROCK2) expression, were increased in rat aorta smooth muscle cells (RASMCs) following incubation with angiotensin II (Ang II). These effects were significantly attenuated by GLP-1, forskolin (a cAMP activator) and Y-27632 (a ROCK2 inhibitor). However, H89 (a PKA inhibitor) inhibited the action of GLP-1, both in terms of inhibition of RASMC proliferation and migration, and RHOA/ROCK2 expression. These results indicate that GLP-1 inhibits Ang II-induced RASMC proliferation and migration via the cAMP/PKA/RhoA/ROCK2 signaling pathway. Our data suggest that GLP-1 should be considered for use in the clinical treatment of cardiovascular diseases, in addition to its current use in the treatment of diabetes mellitus.

Effect of angiotensin1-7 on intimal hyperplasia in autogenous vein graft in rats

Objective Angiotensin1-7 (Ang1-7) and ITS inhibitor A779 were used to study the effect of Ang1-7 on intimal hyperplasia in autogenous vein graft. Methods The transplantation models of the external jugular vein to the abdominal aorta were established in 40 Specific Pathogen Free SD male rats. All models were randomly divided into control group, gel group, Ang1-7 group and A779 group (10 rats each). The adventitia of the transplanted veins was evenly sprayed with normal saline, Poloxamer 407, Poloxamer 407 mixed with Ang 1-7, Poloxamer 407 mixed with A779 respectively in each group forementioned. 5 rats form each group were sacrificed at 14th and 28th day respectively. The transplanted vein was intercepted and then stained with hematoxylin and eosin (HE). The thickness of the inner membrane (I) and medium membrane (M) of the vein grafts were measured by computer, and I/M ratio was calculated. The expression of proliferating cell nuclear antigen (PCNA), extracellular regulated protein kinase-1 (ERK-1) and angiotensin Ⅱ receptor 1 (AT1R) was determined by immunohistochemistry. Results (1)14th and 28th Day, the intimal hyperplasia of vein graft in Ang1-7 group [(59.26±7.51), (62.50±5.91) μm] was lighter than that in the other groups(P=0.001, respectively). (2)14th Day, the I/M ratio of vein graft in Ang1-7 group (0.67±0.05) was lower compare to that in other groups (P=0.001, 0.006, 0.001). 28th Day, the I/M ratio of vein graft in Ang1-7 group (0.71±0.14) was lower than that in the other groups(P=0.001, respectively). (3)There was statistically differences in the intimal thickness of vein graft between A779 group and control group at 14th, 28th day(P=0.007, 0.001). (4)14th Day, the expression of PCNA in the Ang1-7 group (21.80±7.05) was lower than that in other groups (P=0.011, 0.016, 0.001). 28th Day, the PCNA expression in the Ang1-7 group (12.80±5.31) was lower than that in the other groups (P=0.001, 0.002, 0.001). (5)14th Day, the express of ERK1 in vein graft Ang1-7 group (57.00±5.79) was lower than that in other groups (P=0.002, 0.003, 0.001). 28th Day, the express of ERK1 in vein graft Ang1-7 group(50.20±8.79) was lower than that in other groups (P=0.024, 0.017, 0.001). (6)14th Day, the express of AT1R in vein graft Ang1-7 group (27.20±6.87) was lower than that in other groups (P=0.001, 0.003, 0.001). 28th Day, the express of PCNA in vein graft Ang1-7 group(20.60±4.83) was lower than that in other groups (P=0.001, respectively). Conclusion The main cells resulting in intimal hyperplasia may be derived from the proliferation and migration of vascular smooth muscle cells in the medium membrane. Ang1-7 can significantly inhibit the expression of AT1R in the bridging vein, reduce its role in promoting endometrial proliferation, thereby reduce the proliferation and migration of VSMCs. Ang1-7 can also inhibit the expression of ERK in the bridging vein after vein transplantation; RAS and MAPK signal systems are involved in the intimal hyperplasia of vein graft. Key words: Angiotensin1-7; Vein graft; Intimal hyperplasia; Renin angiotensin system

Influence of endothelin-1 on nitric oxide and hydrogen sulfide pathways in vascular smooth muscle cells in rats

Objective To explore the effect of different concentrations of endothelin-1 (ET-1) on the endogenous nitric oxide (NO) and hydrogen sulfide (H2S) pathways of vascular smooth muscle cells (A7r5 cell lines)in rats. Methods A7r5 cell lines were divided into the control group and the experimental group.ET-1 at a concentration of 10-8 -10-6 mol/L was added into the experimental group, and as for the control group, the same volume of sterile phosphate buffered saline (PBS) buffer solution was added.The content of NO and H2S in A7r5 cell lines was detected by fluorescent NO probe and H2S probe after ET-1 stimulation for 48 h, respectively.The content of NO in the supernatant was measured by NO assay kit at 48 h of the incubation.The content of H2S in the supernatant was measured by polarographic H2S sensor at 48 h of the incubation.The expressions of inducible nitric oxide synthase (NOS2), endothelial nitric oxide synthase (NOS3), cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) and proliferating cell nuclear antigen (PCNA) were detected by the Western blot method. Results The relative fluorescence intensity of the content of NO in the A7r5 cell lines of ET-1 10-8, 10-7 and 10-6 mol/L groups (0.078±0.080, 0.075±0.002, 0.056±0.009) was markedly lower than that in the control group(0.094±0.061), and the differences were statistically significant(F=15.248, P 0.05). The relative fluorescence intensity of the content of H2S in the A7r5 cell lines of ET-1 10-8, 10-7 and 10-6 mol/L groups (0.063±0.002, 0.056±0.008, 0.042±0.009) was markedly lower than that in the control group (0.082±0.006), and the differences were statistically significant(F=16.297, P 0.05). The data showed that PCNA protein expression in the 10-7 mol/L ET-1 group(0.686±0.180)significantly increased compared with that of the control group(0.437±0.191), and the difference was statistically significant (t=-2.840, P<0.01). Conclusion ET-1 stimulation can lead to the proliferation of vascular smooth muscle cells and down-regulate its endogenous NO and H2S pathways. Key words: Endothelin-1; Nitric oxide; Hydrogen sulfide; Vascular smooth muscle cells; Rat

Inhibitory effects of solid lipid nanoparticles of rhynchophylline on proliferation of rat vasculars mooth muscle cells induced by TGF-β1

Objective To observe the effect of Rhy-SLN on the proliferation of rat vascular smooth muscle cells (VSMC) induced by TGF-β1, and explore the mechanism. Methods The primary culture of rat thoracic aortic vascular smooth muscle cells was studied by tissue block culture method. The cells were divided into the control group, TGF-β1 group, TGF-β1+ the high, medium and low dosage groups of Rhy-SLN. In addition to the control group, the cells of the other groups were involved in the intervention of TGF-β1 of 20 g/L, and the high, medium and low dosage groups of Rhy-SLN cells were involved in the intervention of 25, 50, 100 mg/L of the hook teng solid lipid nanoparticles. After 24 hours of culture, MTT assay was used to detect cell proliferation inhibition rate in each group, and the cell cycle was detected by flow cytometry. The expression of c-myc and c-Fos protein in each group was detected by Western blot method. Results Compared with the TGF-β1 group, the absorbance value (0.457 ± 0.046 vs. 0.975 ± 0.049) of TGF-β1+ rhy-sln high dose group significantly decreased (P<0.01); the number of S phase cells (15.87% ± 2.47%, 15.23% ± 1.69%, 17.02% ± 2.87% vs. 38.58% ± 2.68%) of TGF-β1+ rhy-sln in each dose group significantly decreased (P<0.01); The c-myc (48.65 ± 3.65, 50.69 ± 4.16, 55.29 ± 3.67 vs. 68.21 ± 3.25) and c-Fos (38.78 ± 4.25, 43.56 ± 3.69, 46.58 ± 3.57 vs. 66.54 ± 4.09) of the TGF-β1+ rhy-sln each dose group significantly decreased (P<0.01). Conclusions The Rhy-SLN can inhibit the proliferation of VSMC in rats induced by TGF-β1.Its mechanism is related to the conversion of G0/G1 phase to the S phase and the expression of the reduction of c-myc and c-fos protein. Key words: Rhynchophylline; Solid lipid nanoparticles; Transforming growth factor beta; Proto-oncogene proteins c-fos; Proto-oncogene proteins c-myc; Muscle, smooth, vascular; Cell proliferation; Rat

Therapeutic effect of mesenchymal stem cell on pulmonary hypertension and its influence on tumor necrosis factor α/nuclear factor of activated T cells

Objective To illustrate that tumor necrosis factor-α(TNF-α)/ nuclear factor of activated T cells pathway is the main function way that mesenchymal stem cells(MSC) inhibit proliferation of pulmonary vascular smooth muscle cells during treating pulmonary hypertension(PH). Methods MSC from human umbilical cord was used to treat PH rat models induced by monocrotaline.Rats were divided into the control group, the PH model group and the MSC group.The general conditions of the rats were observed.Haemodynamics was detected.Pathological sections and immunohistochemistry method were used to detect the lung structure and tissue changes.Changing conditions of TNF-α/NFAT were detected. Results Compared with rats in the PH model group, the general conditions of the MSC group tended to be normal evidently: the right ventricular systolic pressure (RVSP) dropped [(30.37±3.13) mmHg vs.(47.90±3.45) mmHg, 1 mmHg=0.133 kPa], the aortic pressure (MAoP) increased [(115.03±16.01) mmHg vs.(92.78±16.28 mmHg)], the thickening condition of arterial intima-media was evidently relieved [(17.22±1.21)% vs.(31.68±2.26)%], the plasma TNF-α level decreased obviously [(842±76) ng/L vs.(245±24) ng/L], and the lung tissue TNF-α level decreased (0.172±0.024 vs.0.248±0.051), and all the differences were statistically significant (all P<0.05). The activation of pulmonary artery NFATc2 in the MSC treatment group was apparently inhibited. Conclusions MSC therapy may perform the treating effect in PH by inhibiting the over-proliferation of inflammation related pulmonary vascular smooth muscle cells via TNF-α/NFAT pathway. Key words: Mesenchymal stem cell; Pulmonary hypertension; Tumor necrosis factor; Nuclear factor of activated T cells

Effects of small interfering RNA silencing in epidermal growth factor receptor on proliferation and migration of vascular smooth muscle cells

Objective To investigate the effect of silencing epidermal growth factor receptor (EGFR) by small interfering RNA (siRNA) interference on the proliferation and migration of vascular smooth muscle cells (VSMCs), and the related mechanisms. Methods VSMCs of rats were isolated and cultured. VSMCs were infected with EGFR siRNA lentiviral vectors. The cells were divided into siRNA-EGFR group, siRNA-negative control group and blank control group. The cell proliferation abilities were detected by 5’-bromo-2’-deoxyuridine (5-BrdU) method. The cell migration abilities in different transfection groups were examined by Transwell chamber and scratch assays. The expression levels of EGFR, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 genes in different transfection groups were detected using real-time quantitative polymerase chain reaction (Real-time PCR) technology. The expression levels of EGFR, p-ERK, p-JNK and p-p38 proteins in different transfection groups were detected by Western blotting. Results The number of migration cells in the siRNA-EGFR group was 48.5±6.1, which were significantly lower than the siRNA-negative control group and blank control group, which were 86.2±7.2 and 88.3±7.6; the difference was statistically significant (F=37.261, P=0.000). Compared with 0 h, after 24 h, the migration rate of VSMC in the siRNA-EGFR group was (35.7±3.8)%, which was significantly lower than that of siRNA-EGFR group and blank group, which were (74.2±5.7)% and (75.5±6.1)%; the differences were statistically significant (F=40.315, P=0.000). The relative expression levels of ERK, JNK and p38 mRNA in the siRNA-EGFR group (0.53±0.09, 0.47±0.11, 0.41±0.07) were lower than the siRNA-negative control group (0.82±0.12, 0.64±0.10, 0.68±0.09) and blank control group (0.85±0.14, 0.66±0.13, 0.70±0.12), the differences were statistically significant (F=15.391, 12.817, 11.264, P=0.000, 0.000, 0.000). The relative expression levels of p-ERK, p-JNK and p-p38 proteins in the siRNA-EGFR group (0.46±0.09, 0.38±0.06, 0.33±0.06) were lower than the siRNA-negative control group (0.71±0.12, 0.57±0.08, 0.52±0.07) and blank control group (0.73±0.13, 0.56±0.07, 0.54±0.09), the differences were statistically significant (F=20.152, 14.375, 13.843, P=0.000, 0.000, 0.000). Conclusion Specific inhibition of EGFR could effectively reduce proliferation and migration capacities of VSMCs, which might be related to inhibition of mitogen-activated protein kinase signaling pathway. Key words: Vascular smooth muscle cells; Epidermal growth factor receptor; Small interference; Cell proliferation; Cell migration

Preliminary study of artificial synthetic E-selectin inhibits the proliferation mechanism of vascular smooth muscle cells in rats

Objective To preliminary study the mechanism of artificial synthetic E-selectin inhibiting the proliferation of vascular smooth muscle cells in rats. Methods Ninety adult male SD rats were randomly divided into 3 groups: a control group, an operation group, and a treatment group (n=30 in each group). The right common carotid artery was made a longitudinal incision and interrupted suture in the operation group and the treatment group, and the control group did not make vascular incision and suture. Artificial synthetic E-selectin and the same amount of saline were injected into the femoral veins of the rats respectively in the treatment group and the operation group after vascular suture. On day 1, 3, 7, and 14 after procedure, ELISA was used to detect the concentrations of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The vascular tissue at the suture was taken. Western blot and RT-PCR were used to detect the protein level of NF-κB p65 and mRNA expression. Electrophoretic mobility shift assay was used to detect the NF-κB transcriptional activity. Immunohistochemical staining was used to detect the positive cell rate of proliferating cell nuclear antigen (PCNA). Results The concentrations of serum TNF-α and IL-6 and the levels of NF-κB p65 protein and mRNA began to increase at day 1 after vascular suture in the operation group and the treatment group. They reached the peak at day 3, and they were still higher than those in the control group at day 7 (P 0.05). The NF-κB transcription activity in the operation group was higher than that in the treatment group at day 3 after suture (P<0.01). The PCNA positive cells began to express at day 3 after vascular suture in the operation group and the treatment group. The expression of PCNA positive cells in the control group was not obvious or no expression. There were significant differences among the 3 groups (P<0.05). They still expressed high at day 7 and 14 (P<0.01), while the expression of PCNA positive cells was lower than that in the treatment group over the same period (P<0.01). Conclusion Artificial synthetic E-selectin can inhibit the proliferation of vascular smooth muscle cells by inhibiting NF-κB activity. Key words: E-selectin; NF-kappa B; Myocytes, smooth muscle; Cell proliferation; Rats; Vessels

Testosterone suppresses rat vascular smooth muscle cell phenotypic transition and proliferation

Objective To investigate the inhibitory effect of testosterone on oxidized low-density lipoproteins(ox-LDL)-stimulated phenotypic transition and proliferation of vascular smooth muscle cells(VSMCs)in vitro, and to explore its possible mechanisms. Methods Rat VSMCs cultured in vitro were divided into control group, ox-LDL group(50 μg/ml ox-LDL), fetal bovine serum(FBS)group(10% FBS), and testosterone groups(5×10-8 or 5×10-7mol/L testosterone plus 50 μg/ml ox-LDL). The effect of testosterone on ox-LDL-induced proliferation of VSMCs was explored by WST-1 assay. The cell cycle distribution was determined using flow cytometry. Western blotting was used to detect the expressions of mitofusin2(Mfn2), phosphorylated extracellular signal-regulated kinases 1/2(p-ERK1/2), proliferating cell nuclear antigen(PCNA), α-smooth muscle actin(α-SMA), and osteopontin(OPN). Results Compared with control group, the proliferation of VSMCs was promoted by ox-LDL, the number of VSMCs decreased in G0/G1 phase and increased in S phase significantly, the expression levels of Mfn2 and α-SMA were significantly reduced, and the expression levels of p-ERK1/2, PCNA, and OPN were significantly raised in ox-LDL group. Compared with ox-LDL group, the proliferation of VSMCs was inhibited, the number of VSMCs increased in G0/G1 phase and decreased in S phase in two testosterone groups, along with the increased expressions of Mfn2 and α-SMA, and the descended expressions of p-ERK1/2, PCNA, and OPN. Conclusions Testosterone inhibits phenotypic transition and proliferation of VSMCs induced by ox-LDL in vitro, which may be related to the up-regulated expression of Mfn 2 and the suppression of ERK1/2 pathway.(Chin J Endocrinol Metab, 2015, 31: 806-809) Key words: Testosterone; Vascular smooth muscle cell; Mitofusin2; Phenotype; Proliferation

MicroRNA-10b Induces Vascular Muscle Cell Proliferation Through Akt Pathway by Targeting TIP30.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes significantly to the pathogenesis of atherosclerosis. MiR-10b has recently emerged as a critical mediator in regulating cell proliferation in many diseases. In our study, miR-10b expression was up-regulated in VSMCs isolated from atherosclerotic plaques, as well as in PDGFstimulated VSMCs.Overexpression of miR-10b promoted cell proliferation of VSMCs. Furthermore, we identified the Tat-interacting protein 30 (TIP30) as a direct target gene of miR-10b. TIP30 was down-regulated in VSMCs isolated from atherosclerosis plaques, as well as in proliferative VSMCs. Knockdown of TIP30 promoted VSMCs proliferation. In addition, miR-10b induced TIP30 down-regulation was accompanied by increased Akt phosphorylation. Akt was critical for miR-10b-mediated VSMCs proliferation. Our results demonstrated that miR-10b contributed to abnormal VSMCs proliferation through inhibiting the Akt pathway by targeting TIP30 in atherosclerosis. The modulation of miR-10b in VSMCs provides a potential target for the therapy of atherosclerosis.

Hydroxysafflor yellow A inhibits rat vascular smooth muscle cells proliferation possibly via blocking signal transduction of MEK-ERK1/2

To elucidate the effect of hydroxysafflor yellow A ( HYSA) on the proliferation of vascular smooth muscle cells (VSMCs) and the related mechanism. VSMCs derived from SD rats were treated with DMEC culture medium (Control), 10 ng/ml PDGF (PDGF group), pretreatment with HYSA at different doses (1, 5, 10, 20, 40, 60 µmol/L) for 24 h then cotreatment with PDGF. After 24 h, MTT assay, Western blot and immunohistochemical staining were performed to evaluate the inhibitory effects of HYSA on VSMCs proliferation. HYSA inhibited PDGF induced VSMCs proliferation in a dose-dependent manner, dowregulated proliferating cell nuclear antigen (PCNA) expression and blocked PDGF activated PDGFR-MEK-ERK1/2 signaling pathway. HYSA inhibits VSMCs proliferation possibly via downregulating the expression of PCNA and blocking MEK-ERK1/2 signal transduction in VSMCs.

MicroRNA-136 Promotes Vascular Muscle Cell Proliferation Through the ERK1/2 Pathway by Targeting PPP2R2A in Atherosclerosis

Aberrant proliferation of vascular smooth muscle cells [VSMCs] is implicated in the pathogenesis of vascular pathologies such as atherosclerosis and restenosis. Accumulating evidences have revealed that microRNAs are involved in cell proliferation in various pathological conditions. In the present study, we showed that miR-136 was up regulated in human coronary atherosclerotic plaques when compared with normal coronary artery tissues. Moreover, miR-136 levels were up regulated in proliferative vascular smooth muscle cells induced by platelet-derived growth factor [PDGF] or serum. In cultured VSMCs, over expression of miR-136 stimulated cell proliferation. PPP2R2A was proved to be the direct target gene of miR-136 and knockdown of PPP2R2A had a proliferative effect on VSMCs. miR-136-induced PPP2R2A down-regulation was accompanied by increased expression of ERK1/2 phosphorylation. Inhibition of ERK1/2 abolished the effect of miR-136 and knockdown of PPP2R2A on VSMCs proliferation. In summary, aberrant miR-136 up regulation in atherosclerosis contributes to abnormal VSMC proliferation through suppressing the ERK1/2 pathway by targeting PPP2R2A. Our study also suggested that specific modulation of miR-136 in human VSMCs may provide a potential approach for the treatment of atherosclerosis.

Role of NF-κB/survivin signal pathway on intima hyperplasia of rat carotid balloon injury restenosis model

To investigate the role of NF-κB/survivin signal pathway in the intima hyperplasia of rat carotid balloon injury restenosis model. NF-κB siRNA lentivirus vector (titer was 1 × 10⁸ TU/ml) was established. Carotid balloon injury restenosis model was made in 33 SD rats. The rats were divided into 4 groups according to different processing methods, including negative control (NC) group (n = 11), NF-κB siRNA group (n =11), NF-κB siRNA+YM155 (survivin inhibitor) (n = 11), the uninjured carotid artery served as the normal control group (n = 11). After 7 days, the carotid sample (n = 5 each group) were harvested to detect the NF-κB and survivin mRNA expression by RT-PCR.The carotid sample were harvested on 28 days (n = 6 each group) for HE staining and measuring intima hyperplasia. Immunohistochemical method was also used to detect the expression of intima proliferation cell nuclear antigen (PCNA) and media α-SM-actin. (1) After 7 days, NF-κB and survivin mRNA expression was significant higher in NC group than in normal control group (P<0.05), the NF-κB mRNA expression was significantly lower in NF-κB siRNA group than in NC group (P<0.05) and similar between NF-κB siRNA group and NF-κB siRNA+YM155 group. The survivin mRNA expression was significantly lower in NF-κB siRNA group compared to NC group (P<0.05) and significantly higher in NF-κB siRNA group than in NF-κB siRNA+YM155 group (P<0.05). (2) After 28 days, intima hyperplasia was observed in NC (0.13 ± 0.01), NF-κB siRNA (0.11 ± 0.01) and NF-κB siRNA+YM155 group (0.09 ± 0.01) mm² (P<0.05). Media area was similar among NC group, NF-κB siRNA group and NF-κB siRNA+YM155 group (P>0.05). I/M ratio was gradually reduced among NC group (1.55 ± 0.07), NF-κB siRNA group (0.92 ± 0.08), NF-κB siRNA+YM155 group (0.76 ± 0.06, all P<0.05). Similar results were found in the residual restenosis rate: NC group (58.71 ± 0.02) %, NF-κB siRNA group (32.13 ± 0.05) %, NF-κB siRNA+YM155 group (26.42 ± 0.03) % (all P<0.05) and expression of vascular smooth muscle cell PCNA: NC group (45.32 ± 7.21) %, NF-κB siRNA group (36.54 ± 6.42) %, NF-κB siRNA+YM155 group (28.57 ± 6.31) % (all P<0.05). On the contrary, the IOD of α-SM-actin in media increased gradually: NC group (0.055 ± 0.006), NF-κB siRNA group (0.072 ± 0.011), NF-κB siRNA+YM155 group (0.084 ± 0.008, all P<0.05). Inhibiting NF-κB expression can significant decrease intima hyperplasia in this model, and this effect may be mediated by inhibiting survivin and reducing the proliferation of vascular smooth muscle cells.

Atherosclerosis and beneficial effects of parnaparin

In recent years atherosclerosis has become a serious health challenge given its causal association with cardiovascular disease and stroke, which predictably leads to most of the morbidity and mortality worldwide [1]. The mechanism by which in this pathology there is an extensive proliferation of vascular smooth muscle cells (VSMCs) and their migration to the intima remain poorly understood. Recently, low molecular weight heparin (LMWH) has been shown to inhibit the proliferation of VSMCs and, in particular, a kind of these heparins, parnaparin (PNP), acts on the development and clinical course of atherosclerosis in animal models [2]. We hypothesized that PNP might modulate VSMCs migration and thereby decrease vascular remodelling in atherosclerosis. Vascular remodelling was evalu- ated in aortas of an animal model of atherosclerosis, (ApoE)-deficient mice (ApoE -/-) with normal or western diet. We treated animals with PNP to evaluate its pro- tective effect. Thereafter, we evaluated morphological changes and the expression of proteins linked to atherogenesis and atheroprogression in particular an enzyme involved in oxidative stress, iNOS, some inflammatory mediators, such as TNF-α, IL-1 and IL-6, and markers of VSMCs alterations (PAI-1 and TSP1). Our results demonstrated that PNP downregulates VSMCs proliferation and migration, mediated by PAI-1 and TSP1, and reduces inflammation and oxidative stress in vessels. These data suggested that LMWH, in particular PNP, could be a theoretically practical tool in the prevention of atherosclerotic vascular modification.

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