The Regulative Study Of Human Vascular Smooth Muscle Cell Proliferation Was Induced By Endothelin-1

Molecular biology and cell biology methods were employed to study vascular smooth muscle cell proliferation induced by endothelin-1. Five parts were included in this paper.Part I Effects of endothelin-1 on human vascular smooth muscle cell proliferation Endothelin-1(ET-1) is a potent vasoconstrictor. Recent studies have revealed that ET-1 may promote cell proliferation. We investigated that ET-1 affect on human vascular smooth muscle cell (HVSMC) proliferation. The effects of ET-1 and PD98059 on HVSMC were evaluated by MTT assay. The content of DNA was defined by [3H]-TdR assay and cell cycle was analyzed by flow cytomerty. Firstly, ET-1 (100 nmol/L) stimulates HVSMC proliferation compared with the group without ET-1 (P<0.05) and PD98059 group (P<0.05). Secondly, ET-1 stimulated DNA synthesis of HVSMC compared with the group without ET-1 (P<0.05). Thirdly, ET-1 promoted the cell cycle transition from G0/G1 phase to S phase. Cell percentage of G0/G1 phase has obviously decreased compared with the group without ET-1 (P<0.05). Cell percentage of S phase was increased compared with the group without ET-1 (P<0.05). ET-1 promotes HVSMC proliferation.Part II Effects of endothelin-1 on phenotype transtion of human vascular smooth muscle cell The vascular smooth muscle cell may transit their phenotype from contractile to a synthetic state, and make HVSMC proliferative state. We investigated the relation which ET-1 affects on phenotype and proliferation of cultured human vascular smooth muscle cells (HVSMC). l-CaD (Low-Caldesmon) appears syntheticphenotype of HVSMC . h-CaD (High-Caldesmon) appears contractile phenotype of HVSMC. RT-PCR method was used. The expression of h-CaD appeared on the first day, decreased on the second day, and disappeared at third day. ET-1(100 nmol/L) faintly triggered the expression of l-CaD at second day, gradually increased following dependent-time manner, and reach the maximum on the seventh day (P<0.05). Our research indicated that ET-1 inhibits h-CaD expression of HVSMC, but induced l-CaD expression of HVSMC.. ET-1 could stimulate phenotype change of HVSMC transition from contractile phenotype to synthetic phenotype. ET-1 accelerates HVSMC phenotype transition.Part III Effects of endothelin-1 on cell cycle progression of human vascular smooth muscle cellAlthough it is well established that ET-1 has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this part, we examined the effects of ET-1 on the cell-cycle regulatory machanism, including cyclins, and cdk inhibitors in HVSMC. Western blot analysis was employed. The protein expression of cyclin D1 appeared at the sixteenth hour after ET-1 stimulation, reach the maximum at the thirty-second hour, and descended at the fortieth hour (P<0.05). The protein expression of cyclin A appeared at the twenty-four hour after ET-1 stimulation, and reach the maximum at the fortieth hour (P<0.05). The protein expression of p27kip1 decreased at the eighth hour after ET-1 stimulation, and reach the minimum at the fortieth hour (P<0.05). Under the absence of ET-1 stimulation, the expression of p27kip1 remained unchanged (P>0.05).These results point out that ET-1 enhanced the protein expression of cyclin D1 and cyclin A in dependent-time manner, but ET-1 decreased the protein expression of p27kip1 in dependent-time manner. The research indicates that ET-1 depend on cell cycle progression in proliferative HVSMC.Part IV Effects of endothelin-1 MEK/ERK pathway and ETA receptor expression of human vascular smooth muscle cell Many studies suggest that endothelin A receptor and mitogen-activated proteinkinase (MEK)/extracellular signal-regulated kinases 1 and 2(ERK1 and ERK2, respectively) play a key role in the signal transduction pathway leading to cell proliferation. In this study, activation of MEK/ERK signaling pathway...