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Objective To investigate the effects of ghrelin on proliferation of vascular smooth muscle cells(VSMC)and the expression of mitochondrial fusion 2(Mfn-2)in cultured human aortic smooth muscle cells(HASMCs).Methods HASMCs were cultured in vitro,treated with different concentrations(10-9,10-8,10-7,10-6,10-5 mol/L)ghrelin or 10-6 mol/L ghrelin for different time(0,6,12,18,24 h).Subconfluent HASMCs at passage 4-6 were used in experiments.MTT essay was used to investigate the effect on proliferation of HASMCs.RT-PCR and Western blot were used to analyse the expression of Mfn-2.Results 10-7-10-5 mol/L ghrelin inhibited the proliferation of HASMCs,and the inhibitory effect of concentration of 10-6 mol/L was the most obvious(P<0.01).Ghrelin inhibited the proliferation of HASMCs in 6-24 h,and it reached the peak at 24 h(P<0.01).10-6 mol/L ghrelin significantly increased the expression of Mfn-2 mRNA and protein(P<0.01).The up-regulation of 10-6 mol/L ghrelin on Mfn-2 mRNA and protein expression reached the peak at 18 h(P<0.01).Conclusion Ghrelin might inhibit the proliferation of HASMC by up-regulating the expression of Mfn-2.
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AIM:To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells ( SMCs) .METH-ODS:Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 ( containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1 ×105 units/L of penicillin and streptomycin, respectively).EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding.Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats.Vascular SMCs were cultured by tissue explant method and identified byα-SM-actin immunofluorescence.In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower cham-ber.The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE).The protein expression ofα-SM-actin and osteopontin was detected by Western blotting.[3H]-TdR incor-poration assay was used to determine the proliferation.SMC migration was analyzed by scratch wound healing assay.RE-SULTS:Compared with P3 group,α-SM-actin expression in P4 group significantly decreased and osteopontin protein ex-pression obviously increased, whereas no significant change was found in P4YE group.Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs.Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phe-notype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION:Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs pro-liferation and migration.Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.
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Objective To study the effects of endothelial nitric oxide synthase (eNOS) gene transfection on endothelial progenitor cells (EPCs) transplantation in the process of injured vascular endothelium repair. Methods EPCs were cultured and expanded in vitro. EPCs were transduced with pseudotyped retroviral vectors expressing eNOS gene (pMCV-eNOS-EPCs) or green fluorescent protein gene (pMCV-GFP-EPCs). EPCs with expressing eNOS, GFP or saline were injected respectively into rat injured artery model by tail vein injection after balloon injury and again 24 hours. 14 days after transplantation. eNOS expression in injured artery was detected by RT-PCR, western blot and immunohistochemical methods. The morphology of arterial intima and media was studied by optical microscopy and image analysis system. Results Compared with GFP-EPCs group and control group, the mRNA and protein of eNOS were obviously high expressed in eNOS-EPCs group. EPCs transplantation reduce lumen stenosis and inhibit neointimalhyperplasia (eNOS-EPCs group vs.control group, 0.58±0.05 vs. 1.56±0.21, P < 0.01;GFP-EPCs group vs. control group, 0.84±0.09 vs.1.56±0.21, P < 0.05). eNOS gene transfection could further enhance this anti-proliferative effects (eNOS-EPCs group vs. GFP-EPCsgroup,0.58±0.05 vs. 0.84±0.09, P < 0.05). Furthermore, eNOS modified EPCs could improve the endothelial function of injured vascular endothelium. Conclusions eNOS gene transfection could increase the anti-proliferative effect of EPCs transplantation on injured artery and obviously ameliorate endothelial function.
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Objective To investigate the different influences of simvastatin on proliferation of rat smooth muscle progenitor cells(SPCs) versus endothelial progenitor cells (EPCs) and identify the compounds that differentially inhibit SPCs and EPCs proliferation for clinical usefulness. Methods Total mononuclear cells (MNCs) were isolated from bone marrow of rats by Fieoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. SPCs outgrew from the culture of MNCs in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas EPCs were obtained in the presence of vascular endothelial growth factor. SPCs were identified as adherent cells positive for α-smooth muscle actin (α-SMA) by indirect immunofluoreseent staining. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining. SPCs and EPCs were stimulated by simvastatin (0.01～10.00 μmol/L) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). SPCs and EPCs proliferation were assayed with 3H-TdR incorporation and manual counting respectively. Results Simvastatin obviously inhibited SPCs proliferation. At the concentration of 0. 01 μmol/L for 12 h,simvastatin significantly reduced the number of SPCs by (5.8±3.1)% compared with control group (P<0.05). Simvastatin significantly stimulated EPCs proliferation, which was dose- and time dependent and reached maximum at 1 μmol/L after 24 hours (2.0±0.1 fold increase, P<0.01).Conclusions Simvastatin displays different effects on SPCs (inhibited) and EPCs (promoted)proliferation. Local application of simvastatin may inhibit arterial restenosis and promote reendothelialization of injured vessels.
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AIM: To investigate the feasibility of reendothelialization of the injured arterial wall by autologous endothelial cell transplantation and their influences on neointima proliferation. METHODS: New Zealand white rabbits (n=30) were subjected to bilateral iliofemoral artery balloon injury. Cultured, autologous venous endothelial cells were immediately transplanted into one vessel(transplantation group), whereas the contralateral artery received medium only(control group). Reendothelialization of the injured arterial wall was analysed 4 hours or 4 days after cell transplantation by fluorescent tracing、scanning electron microscope(SEM) and Evans blue staining. Pathology analysis was employed 28 days after cell transplantation to evaluate neointima proliferation. RESULTS: The transplanted endothelial cells had adhered into the aterial wall 4 hours after transplantation and began to attach and spread 4 days later. A number of fluorescent labeling endothelial cells were observed in the endothelial injured arterial wall. The vessels in control group were stained nearly completely by Evans blue, whereas about 60% area was not stained in transplantation group. Pathological examination demostrated that neointimal area and maximal intima thickness in transplantation group significant decreased than those in control. CONCLUSION: Autologus endothelial cells were effectively transplanted into the injured arterial wall by balloon catheter, and it can relieve neointima proliferation in the long time.