The Effects Of CAPE On The Growth Of VSMCs In Vitro And Its Inhibiting Effects On The Restenosis Induced By Intima Injury After Balloon Lesions In Rats And The Mechanisms

PARTⅠThe Effects of on the growth Dynamics of Vascular Smooth Muscle Cells in vitroObjective: To observe the effects of caffeic acid phenethyl ester (CAPE) ranged from 5 mg﹒L-1 to 80 mg﹒L-1 on the growth dynamics of vascular smooth muscle cells (VSMCs) stimulated by lipopolysaccharide (LPS)Methods:LPS or CAPE was added into the medium to treat VSMCs. MTT assay method was used to descript the effects of LPS and CAPE on the growth of the VSMCs in 72h or 120h. Cells were stimulated by 1 mg﹒L-1 LPS when cells were treated by CAPE; Cell clones were counted in plates after cultured for 3 week. Immunocytochemistry(ICC)was utilized to detect the effect of CAPE on the exprssion of proliferating cell nuclear antigen (PCNA) in VSMCs when cells had been cultured for 72h.Results: 0.1-10 mg﹒L-1 LPS showed a significantly inducible effect on the growth of VSMCs. And CAPE showed an inhibitory effect on the proliferation of VSMCs activated by 1 mg﹒L-1 LPS. The inhibitive curve showed CAPE excerted a significantly inhibitory effect in a dose- and time-dependent manner, especially when the cells had been exposed in culture medium with CAPE for over 72h.LPS had an opposite effect on the generation of cell clones to CAPE. 0.1-10 mg﹒L-1 LPS can promote VSMCs to emerge clones but CAPE play a part in inhibiting the generation of cell clones. The difference was more remarkable in the higher concentration groups.While VSMCs were activated by LPS of 0.1-10 mg﹒L-1, the rates of the cells positive for PCNA increased compared with the control. After treated with different concentrations of CAPE, the positive rate for PCNA was decreased from 91.8%±1.7% in the control down to 40.0%±1.8% in the 80 mg﹒L-1 CAPE treated group. Compared with the control, the rates of the cells positive treated with LPS or CAPE were significantly different (p<0.05 or p<0.01). The results also showed that LPS and CAPE both affected the expression of PCNA in a dose-dependent manner.DSMO had no remarkable effect on the growthof VSMCsConclusions: MTT assay, cell clones experiment and ICC for PCNA showed 0.1-10 mg﹒L-1 LPS significantly stimulated the proliferation of VSMCs in a dose-dependent manner. But CAPE ranged from 5-80 mg﹒L-1 inhibited the proliferation of VSMCs stimulated by 1 mg﹒L-1 LPS in a dose- and time- dependent manner. Meanwhile, CAPE of different concentrations decreased the positive rate of PCNA which indicated that it was one of the mechanisms that CAPE inhibited the expression of protein related to cell cycle.PARTⅡThe Mechanisms of CAPE Inhibiting the Growth of VSMCs Cultured in vitro Objective:To elucodate the primary mechanisms of CAPE ranged from 5 mg﹒L-1 to 80 mg﹒L-1 which inhibited the growthof VSMCs.Methods:The changes of IL-6 and TNF-αin the supernatant liquid were determined by Enzyme-linked Immunosorbnent Assay (ELISA). The effect of CAPE on the cell cycle of VSMCs was measured by flow cytometry (FCM) assay and the same method was utilized to detect the apoptosis of VSMCs. Electrophoretic mobility shift assays (EMSA) was used to examine nuclear factor of kappa B (NF-κB) activation in VSMCs. Real-time quantitative PCR method was usded to show the exprssion levels of Sur, Bcl-2 and Bax mRNA.Results: LPS ranged from 0.1 to10 mg﹒L-1 stimulated VSMCs to generate IL-6 and TNF-αin the supernatant liquid and CAPE ranged from 5 to 80 mg﹒L-1 lowered the levels of IL-6 and TNF-αin a dose- and time-dependent manner.After cells had been exposed to CAPE (5.10.20 mg﹒L-1) for 24 h, FCM analysis displayed that the rates of cells in G0/G1 stage in the treated groups significantly increased compared with the control and the percentage of S stage were down-regulated(p<0.05). FCM analysis also determined the rate of the apoptosis of VSMCs in the control group(4.2%±0.3%) was significantly less than the treated groups (10.1%±0.7%,18.9%±1.3%,27.3%±2.4%) (p<0.05 or p<0.01).LPS can up-regulated the expression of Sur mRNA in VSMCs when cells were treated with LPS ranged from 0.1 mg﹒L-1 to 10 mg﹒L-1 ( p<0.05 ). The experiment results also showed that CAPE down-regulated significantly the expression levels of Sur mRNA (p<0.05 or p<0.01). The changing tendency of the expression of Bcl-2 mRNA was similar to Sur but Bax mRNA had the contrary tendency.LPS enhanced NF-κB avtivity in VSMCs but CAPE inhibited the activation in VSMCs stimulated by 1 LPS mg﹒L-1Conclusions: Both ELISA and EMSA experiments showed that CAPE lowered VSMCs to serect cytokines and inhibited NF-κB activation in cells. CAPE also regulated the cell cycle, urged the apoptosis of cells and changed the expression levels of Sur, Bcl-2 and Bax mRNA. These indicated that the mechanisms of CAPE impacting VSMCs may be relative to its regulating the cell cycle and the expression of genes of apoptosis. And its inhibitory effect on the serection of cytokine and NF-κB activation also played an important role in inhibiting the growthof VSMCs. So we conclued rhat CAPE had the potential as an effective anti-restenosis therapy after PCI.PARTⅢPreventive Effects and its Mechanisms of CAPE on Vascular Restennosis Induced by intima Injury after Balloon LesionsObjective: To study the mechanism of restenosis for the prevention and treatment following percutaneous coronary intervention (PCI), we replicated the dynamic models of cellular proliferation and vascular remodeling after intimal denudation of rats carotid common arteries at different time points and to investigate the effects of CAPE on the proliferation of VSMCs induced by intimal denudation after balloon lesions.Methods: SD rats (weighing 300~350g) were used to replicate the dynamic models of cellular proliferation and vascular remodeling after intimal denudation of rats carotid common arteries and the rats were randomly divided into sham-injured group (S group), injured group (I group) and injured+20 mg kg-1 CAPE-treated group (CAPE group). Animals were respectively killed after baloonlesions at different time points (3d, 7d, 14d or 28d). The injured sections were taken out and made into specimens for HE staining and elastic fiber staining. Neointimal thickness (H), neointimal area (NIA), media area (MA), internal elastic membrane (IEM) cross section area, external elastic membrane (EEM) cross section area and lumen area (LA) were measured by computer image analysis system, then the neointimal/media area ratio (NIA/MA) and lumen stenosis index (NIA/IEM) were calculated. The mean optical density values were employed to show the protein expression changes of Sur, Bax and Bcl-2 protein in the carotid wall cells by computer image analysis system. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nucle and labeling(TUNEL)determined the apoptosis in vessel wall. The changes of IL-6 and TNF-αin the serum were determined by ELISA. EMSA was used to examine nuclear factor of kappa B (NF-κB) activation in vessel wall. The expression levels of Sur, Bax and Bcl-2 mRNA in the tissues of carotid wall cells were detected with real-time quantitative PCR method.Results: Pathomorphometrical analysis:Single layer endothelium was only showed in non-injury arteries(S group)at different time points. The proliferation of VSMCs was spotted on the surface of lumen at 3 d after balloon injury. The neointima had been formed and continuously thicken at 7 d after injury. The neointimal thickness and area as well as extracellular matrix were gradually increased after 14 d. The thickness and area of the media were gradually increased during 3 ~ 14 d,in which the medial area significantly increased at 14 d compared with non-injured vessel. Lumen area initially decreased at 3~ 7 d after injury. Lumen area after 14 d was significantly less than that of non-injured vessel. NIA/MA and NIA/IEM gradually slightly increased at 3 ~ 7 d after injury,it was maximal at 14 d. The indexes in CAPE group had contrary changes compared with I group.Sur and Bcl-2 protein be detected mainly in VSMCs of arteries media. Their levels were very low, and they increased in I groups, CAPE can improve the states. Sur and Bcl-2 protein increased in I group and lowered after rats were administered CAPE. Bax protein were mainly detected in VSMCs of media of carotid wall and increases slightly in I group, after rats treated with CAPE, the level of Bax protein increased significantly in a time-dependent manner.No cells TUNEL positive in S group. The cells positive increased in I group in early time but decreased later. CAPE increased the cells positiveThe concentrations of IL-6 and TNF-αin S group in the serum increased slightly.Those in Igroup increased more remarkbly compared with S group. And CAPE inhibited the serection of L-6 and TNF-α.NF -κB activation in balloon - injured arteries was examined by electrophoretic mobility shift assay. No detectable NF -κB activation was found in normal arteries, which was detected immediately following injury, peaked at 12 hours, lasted 48 h, returned to baseline at 72h.Real-time quantitative PCR showed that the expression levels of Sur and Bcl-2 mRNA increased in I group compared with S group and they decreased in CAPE group compared with I group. And that of Bax showed a contrary change. Conclusion: CAPE produced a significant inhibition of neointimal hyperplasia in rats carotid arteries after balloon dilation, the inhibition effect had an obviously time-dependent relationship and may partly be raleted to inhibit the expression of genes such as Sur and Bcl-2 which can spur the growthof cells and promote the expression of Bax gene which show an effect of inducing cell apotosis. Maybe it is one of the mechanisms of CAPE inhibiting neointimal hyperplasia after PCI.