| Background and ObjectivesAtherosclerosis (AS) and coronary heart disease are the common diseases in the cardiovascular system of which causes are still to be identified. Although percutaneous transluminal coronary angioplasty (PTCA) is a highly effective procedure to reduce the severity of coronary artery stenosis, it's long term success is significantly limited by the high rate of restenosis.The pathogenesis of AS involve abnormal lipid metabolism, injury of vascular endothelium, infiltration of macrophages, proliferation of vascular smooth muscle cells (VSMCs), adhesion and aggregation of platelet. The proliferation and apoptosis of VSMC may play important roles. In the normal condition, the proliferation and apoptosis of cells are in dynamic balance, and which maybe protect means to keep the cellular dynamic stability. Recent evidence suggest that the imbalance of prolifeation and apoptosis lead to abnormal proliferation of VSMC and the AS conformation. The apoptosis is more common in the restenotic lesion than in the primary AS.The rennin-angiotensin-aldosterone system (RAS)is a main liquid regulation system in the body, and play an important role in the VSMC proliferation. Researches show that Ang Ⅱ can promote the synthesis of DNA and proteins in myocardial cells, fibroblasts and VSMCs. This effect of Ang Ⅱ is weak in early phase of cell cycle, but it can significantly promote the synthesis of DNA in later phase and increase the cell numbers. A research found that Ang Ⅱ could induce the neointimal hyperplasia after injury of the carotid artery endothelium in a rat model. Other researches showed that Ang Ⅱ can promote the VSMC hypertrophy , but can't increase the cell numbers. The recent resarches focus on the antagonist of RAS including ACEI and Ang Ⅱ receptor antagonist, which can inhibit the effect of Ang Ⅱ on VSMC.In this study, we used method of cell culture of the rat aortic smooth muscle in vitro,observed the effect of Ang Ⅱ, ACEI captopril and Ang Ⅱ receptor antagonist losatan on the proliferation of the VSMC, the proliferation rules and the apoptosis of the VSMC, probed into the mechanism of the antiatherosclerosis and restenosis prevention of captopril and losartan on cytological and molecular biological levels.MethodsMale Wistar rats were killed and the thoratic aorta was taken out for primary culture; Vascular smooth muscle cells were used for the experiment at passage numbers 5-10. Cultured VSMCs were divided into control group, Ang II group, CP group, LT group and CP+LT group according to stimuli used. Each group was subdivided according to different Ang II concentration (10~8, 10'7, 10"6, 10"5and 10~4mol/L) and different action time (12, 24, 48 and 72h). The proliferation of cells, cell cycle and apoptosis ratio, cell phenotype changes and the ultramicrostructure of the cultured cells were determined by the methods of MTT, cell number counting, 3H-TdR incorporation, flow cytometry, and transmission electron microscope.Results1. Angll stimulated VSMC proliferation in a concentration-depending and time-depending style. Compared with the control group, the OD value, cell number counting, and 3H-TdR were increased to 111.88%, 108.03% and 203.89% respectively at the Ang II concentration of 10"6mol/L, 24 hours. There are significant difference(PO.Ol). At the concentration of 10"8mol/L and 10"4mol/L, the effect of Ang II was not significant. The OD value, cell number counting, and 3H-TdR were increased toll 1.88%, 108.03% and 203.89% at the action time of 24h; 246.11%, 122.7% and 192.63% at 48h respectively. There are significant difference(P<0.01) compared with the control group. Angll had no effect on VSMC proliferation at 12h (P>0.05), and the proliferation promoting effect was significant decreased at 72h (P>0.05).2. CP or LT could inhibit the effect of Ang II on the proliferation of VSMCs. Compared with Ang II group, at the Ang II action time of 24h, 48h, and 72h, the MTT OD value was decreased to 34.74%, 56.08%, 57.54%(P<0.05), the cellnumber counting was decreased to 38.78%, 45.41% and 44.37% (PO.05); and the 3H-TdR was decreased to 55.38%, 50.38% and 51.19% (PO.05) respectively in the CP group. There was a similarity in the LT group (PO.05). Combination of CP and LT had more significant inhibition effect on the proliferation of VSMCs(PO.Ol).3. Angll promoted the transition of the VSMC from Go/Gi phase to S/G2-M phase. With serum starvation treatment for 72h, 89.27% of VSMCs located in the G0/G1 phase. After stimulation of Ang II at the concentration of 10"7mol/L, 10"6mol/L ,10"5mol/L and 10"4mol/L respectively for 24h about 11.61%, 43.35%, 55.87% and 45.64% of VSMCs transmitted into S/G2 -M phase. There were significant difference(PO.05) compared with the control group, except when the concentration was 10"8mol/L(P>0.05). The effect of Ang II was increased with the action time prolonging. The percentage of VSMCs in thes/G2 -M phase were 23.97%, 55.87%, 57.54% and 51.11% at 12h, 24h, 48h and 72h. There were singnificant increase compared with the control group(P<0.05) except for the action time of 12h(P>0.05).4. CP or LT significantly inhibited the proliferating activity of VSMCs stimulated by Ang II . At the Ang II concentration of 10"7mol/L, 10"6mol/L and 10'5mol/L, the percentage of VSMCs in the S/G2 -M phase were decreased to 21.17%, 22.48%, and 19.35% in CP group (PO.05), 26.95%, 25.62% and 16.77% in LT group (PO.05); 21.23%, 19.87% and 11.61% in CP+LT group (PO.05) respectively compared with the control group. The inhibition effect intensity of CP or LT was also related to the action time.5.Ang II could induce apoptosis at high concentration 10"4mol/L). Compared with the control group, the apoptosis ratio was significantly increased(PO.05). CP or LT could induce apoptosis of the proliferation VSMCs stimulated by Ang II. Compared with the Ang II group, at the concentration of 10"7, 10"6, 10"5 and 10"4mol/L, the apoptosis ratio was increased to 10.33 ±0.23(P<0.05), 22.65 ± 1. 97(P<0.05), 15. 08+1. 04(P<0.05) and 18. 80 ±1. 11 (PO.05) in CP group; 10.34+1. 05(P<0.05), 28. 46±3. 31(P<0.01), 25. 53±3. 57(P<0.01) and 27. 33... |
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