The Mechanisms Underlying Inhibitory Effects Of Atorvastatin And Hydrogen Peroxide On Vascular Smooth Muscle Contractility

The mechanisms underlying inhibitory effects of atorvastatin andhydrogen peroxide on vascular smooth muscle contractilityBackground: Hypertension is one of high risky factors contributing to thedevelopment of atherosclerosis, a vascular lesion characterized by hardening andthickening of the blood vessel wall. Under certain pathological conditions, such asoxidative stress, vascular smooth muscle （SM） loses its contractility or smoothmuscle cells （SMCs） do not contract properly in response to various vasoactivefactors. Prolonged loss of contractility results in wall stiffness of the blood vessel andmay lead to subsequent hypertension. In many other cases, however, vascular SM hasincreased contractile reactivity than normal subjects, leading to persistentvasoconstriction and hypertension. In the later, one of the most efficient treatments isto relax the blood vessel or to inhibit vascular hyper-contractility. Our preliminarystudies have shown that atorvastatin （ATV）, an inhibitor of3-hydroxy-3-methylglutaryl （HMG）-coenzyme A （CoA） reductase and a widelyprescribed lipid-lowering drug, has potential inhibitory effects on the expression ofSM contractile proteins and contractility. In addition, our preliminary studies havedemonstrated that treatment with hydrogen peroxide （H₂O₂）, an oxidative stressinducer, resulted in an irreversible loss of vascular SM contractility. Therefore, thisthesis focuses on the mechanisms that regulate SM contraction from two directions:（1） ATV regulates myocardin expression and the involvement of the RhoA-ROCKpathway;（2） H₂O₂inhibits vascular SM contractility through a rapamycin-sensitivepathway.Part-I ATV inhibits vascular contractility through suppression ofmyocardin expressionIn addition to lipid-lowing effects, ATV inhibits the RhoA-Rho-associatedkinase （ROCK） pathway in vascular SMCs and critically inhibits SM function.Myocardin is a co-activator of serum response factor （SRF）, which up-regulates smooth muscle contractile proteins. The RhoA-ROCK pathway, which directlytriggers SM contraction, also increases myocardin gene expression. Therefore weinvestigated whether ATV inhibits myocardin gene expression in SMCs.In mice injected with ATV （i.p.20μg/g/d） for5天, myocardin gene expressionwas significantly down-regulated in aortic and carotid arterial tissues with decreasedexpression of myocardin target genes, SM α-actin and SM22. Correspondingly, thecontractility of aortic rings in mice treated with ATV or the ROCK inhibitor Y-27632was reduced in response to treatment with either KCl or phenylephrine. In culturedmouse and human aortic SMCs, KCl treatment stimulated the expression ofmyocardin, SM α-actin and SM22. These stimulatory effects were prevented by ATVtreatment. ATV-induced inhibition of myocardin expression was prevented bypre-treatment with either mevalonate or geranylgeranyl pyrophosphate, but notfarnesyl pyrophosphate. Treatment with Y-27632mimicked ATV-effects on the geneexpression of myocardin, SM α-actin and SM22, further suggesting a role for theRhoA-ROCK pathway in ATV effects. Furthermore, ATV treatment inhibited RhoAmembrane translocation and activation; these effects were prevented by pre-treatmentwith mevalonate. We conclude that ATV inhibits myocardin gene expression in vivoand in vitro, suggesting a novel mechanism for ATV inhibition of vascularcontraction.Part-II H₂O₂induces loss of vascular SM contractility through aRapamycin-sensitive mechanismRapamycin, an inhibitor of the mammalian target of Rapamycin （mTOR）pathway, has been shown to extend the lifespan of mice, and oxidative stress playscritical roles in vascular aging involving loss of compliance of arteries. We examined,therefore, whether Rapamycin has protective effects on the inhibition of vascular SMcontractility by H₂O₂.Prolonged （3h） exposure to H₂O₂induced complete loss of contraction of mouseaortic rings to either KCl or phenylephrine, which was prevented by pre-treatmentwith Rapamycin. H₂O₂-induced loss of contractility was unaffected by treatment withactinomycin D or cycloheximide, inhibitors of gene transcription and protein synthesis, respectively. Western blot analysis showed that there was no increase inphosphorylation of S6K or4EBP1in response to H₂O₂treatment, suggestinginvolvement of the mTOR complex-2（mTORC2）. H₂O₂treatment inhibitedphosphorylation of the20kDa regulatory light chains of myosin （MLC20）, whichwas partially blocked by Rapamycin treatment. Interestingly, the calcineurininhibitors cyclosporine A and FK506were found to mimic the Rapamycin effect, andRapamycin inhibited calcineurin activation induced by H₂O₂. We conclude thatRapamycin inhibits H₂O₂-induced loss of vascular contractility, likely through anmTORC2-calcineurin pathway.Conclusions: Based on above results, we conclude that （1） ATV inhibition ofvascular SM contractility may be beneficial to its treatment in hypertension;（2）H₂O₂-induced loss of vascular SM contractility may result in vascular dysfunctionand subsequent vascular disease.