| Objective:This study was designed to explore the mechanisms under exercise modulated VSMC phenotype switching in hypertension and to figure out the targeting effects of miR-143/145 on the signaling pathways in the process.We first observed the characteristics of VSMC phenotype during aging and hypertension,and how miR-143/145 were influenced.On the base of that,rat age was selected to be subjected to long-term exercise training to examine the effect on VSMC phenotype.miR-143/145 expression and Akt activation were also determined.At last,we wanted to discuss the regulatory role of miR-145 on Akt feed back loop during phenotype switching in isolated primary VSMC.Methods:In experiment 1,mesenteric arteries from 1-,3-,9-,and 16-month-old WKY and SHR were isolated,artery histology were evaluated by staining with HE.VSMC phenotype marker including a-actin,calponin,and OPN were measured.Total Akt,p38,and ERK and their phosphorylated forms were also determined by Western blot.miR-143/145 were quantified by real-time PCR.In experiment 2,3-month old WKY and SHR were subjected to 8wk moderate treadmill training with blood pressure being monitored.After the last bout of exercise,mesenteric arteries were isolate to determine VSMC phenotypic marker,miR143/145 expression and Akt phosphorylation.In experiment 3,miR-145 mimic and miR-145 inhibitor were transfected into primary cultured VSMC,there were 4 observing time points(12h,24h,48h,and 72h)for determining the VSMC morphology stained by a-actin,VSMC phenotype marker,Akt phosphorylation,and IGF-1R、IRS-1,p70S6K mRNA expressions.Results:(1)VSMC contractile marker a-actin and calponin expression both reached the peak at 3M and then decreased,while OPN increased with age in SHR with no obvious changes in WKY.miR-143/145 firstly increased with age and then decreased in both WKY and SHR,and miR-143/145 in WKY-3M was significantly higher than the age-matched SHR(P<0.05).Akt phosphorylation was sensitive to both age and hypertension:Akt activation was inhibited with aging;phosphorylation of Akt in SHR-3M was significantly lower than WKY-3M(P<0.05).p38 phosphorylation differed significantly after being adult(P<0.05)but did not change with age.ERK phosphorylation was inhibited with age but did not differ between WKY and SHR.(2)Exercise significantly reduced SBP and DBP in SHR(P<0.05),and hypertensive arteriole thickness in SHR was significantly reduced(P<0.05).VSMC contractile marker calponin in SHR-E was significantly increased compared with SHR-C(P<0.05),while synthesis marker OPN in SHR-E was reduced significantly than SHR-C(P<0.05).Exercise was effective to normalize miR-145 but there was no obvious effect on miR-143.Akt was also activated by exercise with significance than the control one(P<0.05).(3)miR-145 overexpression by transfecting miR-145 mimic into VSMC increased a-actin significantly after 12h and 24h(P<0.05),while miR-145 inhibitor made a-actin decrease at 24h(P<0.05),calponin decrease at 12h,24h,and 48h(P<0.05),and OPN increase at 24h(P<0.05)with statistical significance.Akt phosphorylation was inversely regulated by miR-145:Akt activation was significantly inhibited by miR-145 mimic at 12h,24h,and 48h(P<0.01)and enhanced by miR-145 inhibitor at 12h,24h,and 48h(P<0.01).miR-145 targeted IRS-1 and IGF-1R mRNA,the upstream signals,but its targeting effects were not obvious on p70S6K mRNA.Conclusions:(1)Aging and hypertension accelerate the VSMC phenotype switching in arterioles,promoting the synthesis phenotype;miR-143/145 is inversely regulated with contractile VSMC during aging hypertension;Akt phosphorylation was also inhibited during this process.(2)Exercise ameliorates the high blood pressure and remodels arterioles,which may rely on its regulatory role on VSMC switching from proliferative to contractile phenotype.miR-143/145 could be involved in the exercise induced VSMC switching.(3)In isolated cultured VSMC,miR-145 inversely regulates Akt activation by targeting its upstream signals. |
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