| Objective: In recent years, with the development of endovascularintervention techniques and the improvement of the safety and efficacy ofstent techniques, stent implantation has become the primary treatment ofcoronary artery stenosis. Not only be able to significantly improve thenarrow-related symptoms and prognosis, but also to prevent the occurrence ofischemic events. Although the use of stents has dramatically increased ininterventional cardiology, in-stent restenosis continues to be a serious problemand is more troublesome when it occurs. Furthermore, pharmacologicalapproaches have generally been unsuccessful in suppressing in-stent restenosis.Although the use of drug-eluting stents significantly reduce the incidence ofrestenosis, however, the long-term restenosis rate has no significant differencecompared with bare-metal stents. Late neointimal proliferation remains apotential adverse outcome with the stent-based drug delivery. Recently,vascular smooth muscle cells(VSMCs) proliferation, migration, extracellularmatrix accumulation has been recognized as very important components ofin-stent restenosis after stent implantation. Since drug-eluting stents haveseveral limitations and are expensive, there may become a need for systemictherapies to maintain neointimal inhibition.Rho-kinase plays an important role in adhesion, migration, proliferation,and cytokinesis of VSMCs and other vascular wall cells. Rho-kinase issubstantially involved in the signal transduction initiated by angiotensinII(ANG II), plateletderived growth factor (PDGF), thrombin, and endothelin-1,all of which may play an important role in the pathogenesis of restenosis,especially in that of in-stent restenosis. In addition, Rho-kinase plays a crucialrole in the reactive oxygen species (ROS) augmentation, ROS have beenimplicated in the pathogenesis of neointima formation in part by promoting VSMC growth and by stimulating proinflammatory events. Therebyaccelerating vascular restenosis. In our research, the inhibitory effect offasudil on VSMCs proliferation in vitro was explored and the potentialmolecular mechnisms associated with the effect was examined. In order tofurther understand the pharmacological effects of fasudil on vascularrestenosis. And expect it to become a new drug for the prevention andtreatment of in-stent restenosis.Method: VSMCs of Sprague-Dawley(SD) rat aortic were isolated andcultured. Cells were made quiescent with serum-free medium for24h afterreach70%-80%confluence. Fetal bovine serum(FBS) were then used anddifferent concention of1,10,100umol/l of fasudil were plused. The culturedcells were divided into5groups randomly: group A (serum-free group);group B (fetal bovine serum(FBS) group); group C (FBS+1μmol/L fasudilintervention group); group D (FBS+10μmol/L intervention group); group E(FBS+100μmol/L intervention group). The proliferation and migration ofVSMC were assayed; The cell cycle and apoptosis were examined by flowcytometric analysis; Western blot were performed to detect the activation ofRas,MEK1/2, ERK1/2and Akt of VSMC.Results:(1) Fasudil inhibited rat VSMCs proliferation and migrationfollowing stimulation of VSMC cultures with fetal bovine serum(FBS) in adose-dependent manner.MTT assay showed that VSMC proliferation in the fasudil group wassignificantly lower than in the control group in a dose-dependentmanner(P<0.05). Within a range of1,10,100μmol/L, the concentration offasudil increased, the VSMC proliferation activity decreased. Compared withcontrol group, decreased by2.04%,8.98%,30.39%(24h);6.17%,23.36%,59.82%(48h), respectively.BrdU assay showed that VSMC proliferation in the fasudil group wassignificantly inhibited in a dose-dependent manner(P<0.05). Within a range of1,10,100μmol/L, the concentration of fasudil increased, the VSMCproliferation activity decreased. Compared with control group, decreased by 9.94%39.36%49.37%(24h);7.38%,57.26%,70.74%(48h), respectively.(2) Fasudil blocked FBS-induced progression from the G0/G1phase to Sphase in a dose-dependent manner.Treatment with1,10,100μmol/L fasudil for24h inhibited FBS-inducedG1-S progression, as demonstrated by the increase in G0/G1cells (5.1%,14.2%,22.4%compared with control group), accompanied by concurrentdecrease in S-phase cells (3.9%，14.8%，19.1%compared with control group).These results suggest that hydrogen may prevent FBS-induced S-phase entryin VSMCs (P<0.05).(3) Fasudil promoted early and late apoptosis of VSMC in adose-dependent manner.Proportion of early apoptotic cells increased from0.51%(serum-freegroup),1.16%(FBS group),1.96%(FBS+1μmol/L fasudil interventiongroup),2.1%(FBS+10μmol/L intervention group) to4.09%(FBS+100μmol/L intervention group); Proportion of late apoptotic cells increasedfrom4.03%(serum-free group),6.64%(FBS group),8.21%(FBS+1μmol/Lfasudil intervention group),13.15%(FBS+10μmol/L intervention group) to24.73%(FBS+100μmol/L intervention group). These results suggested thatfasudil could enhance VSMCs apoptosis (P<0.05, fasudil intervention groupversus serum group).(4) Fasudil significantly inhibited FBS-stimulated intracellular Ras,MEK1/2, ERK1/2activation, but did not in Akt activation.Ras-MEK1/2-ERK1/2pathway and Akt phosphorylation are involved inthe effect of fasudil on FBS-stimulated VSMCs. Stimulation of cells with10%FBS induced Ras activation. Cells treated with fasudil exhibited a obviousinhibition of FBS-induced Ras activation in a dosedependent manner(P<0.05).Treatment of FBS-stimulated VSMCs with fasudil markedly decreasedphosphorylated MEK1/2and ERK1/2levels， also in a dosedependentmanner(P<0.05). FBS induced a profound increase in Akt activation. The levelof Akt phosphorylation following FBS stimulation was not significantlyinhibited by treatment with fasudil(P>0.05). Conclusion: Our results suggest that fasudil could attenuate VMSCsproliferation and Ras-MEK1/2-ERK1/2pathway blockage and cell apoptosisincreasing may play a critical role. |
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