The Role And Mechanism Of Shh In Vascular Smooth Muscle Cell Phenotype Switching And Skeletal Muscle Ischemia-reperfusion Injury

Background: Platelet-derived growth factor(PDGF)is known to induce phenotypic switching of vascular smooth muscle cells(VSMCs)from contractile to a pathological synthetic state,which played an essential role in proliferation of VSMCs.We have previously proved that Sonic hedgehog(Shh)can be up-regulated by PDGF in VSMC and abolishing Shh in vascular cells can prohibit the influence of PDGF on cell proliferation.Moreover,Krüppel-like factor 4(KLF4),as an important transcriptional regulate factor,was involve in PDGF induced VSMCs phenotypic switching as well,our prevous study indicate that KLF4 was closely related Shh function.These prompt us to speculate that Shh perhaps contribute to the phenotypic switching of VSMCs when induced by PDGF,and KLF4 perhaps involve in this process.Objective: In this study we investigate whether Shh participates in PDGF-induced phenotypic switching of VSMC.Further for examining the mechanism involved,we also investigate whether Shh controls phenotypic modulation of VSMC by mediating KLF4.This study will be an intensive complement and improvement for our previous results and provide some novel treatment idea for VSMC proliferation disease.Methods:(1)Firstly,PDGF-BB was used to stimulate VSMCs,the expressions of Shh signaling pathway and KLF4 signaling after PDGF-BB stimulation were observed by Western blot and RT-PCR.Subsequently,Imatinib o PD98059 w us d to inhibit d PDGFRβ or ERK1/2 signaling respectively.the roles of PDGFRβ and ERK1/2 signaling in PDGF induced Shh signaling pathway were investigated.(2)Secondly,α-actin and myocardin which were markers of differentiation of VSMCs,and tropomyosin 4(Tpm 4)and SMemb which were markers of dedifferentiation VSMCs were used to estimate the phenotypic state of VSMCs.Then whether Shh and KLF4 involve in PDGF-induced VSMCs phenotypic modulation was investigated.Subsequently,either Shh-si RNA or smoothened inhibitor cyclopamine was used to block Shh signaling,and either Shh-c DNA or recombinant N-Shh was used to facilitate Shh signaling.Then the role of Shh signaling in PDGF-induced VSMC phenotypic modulation was observed.And whether Shh alone could induced VSMC phenotypic modulation was investigated.(3)Lastly,a specific si RNA for KLF4 was used to knock down KLF4.Cells were stimulated by PDGF-BB or recombinant N-Shh protein to drive VSMC dedifferentiation.Then the effect of KLF4 on PDGF or Shh drived VSMC phenotypic modulation was investigated.Results: We found that PDGF stimulated Shh expression in VSMCs,which was mediated by activation of PDGFRβ/ER 1/2 c signa ing pathway.Further,we found PDGF-induced VSMC phenotypic modulation was accompanied by up-regulation of Shh/Gli family zinc finger 2(Gli2)signaling and KLF4.When inhibited Shh in the presence of PDGF,the expressions of KLF4 and VSMC dedifferentiation markers α-actin and myocardin were down-regulated and the effect of PDGF in inducing VSMC phenotypic switching was blocked.In the absence of PDGF,Shh signaling activation increased the expression of KLF4 and promoted VSMC dedifferentiation.The results indicate PDGF induced VSMC phenotypic switching through activation of Shh signaling.Finally,we found that KLF4 was closely involved in this process.On inhibition of KLF4,PDGF and Shh signaling induced VSMC phenotypic switching were both abrogated.Conclusion: This study proved that PDGF could stimulate Shh signaling pathway and KLF4 expressions in VSMCs;Shh signaling and KLF4 involved in PDGF induced VSMCs phenotypic switching;And PDGF induced VSMCs dedifferentiation though activation of Shh signaling;In addition,PDGF or Shh promoted VSMCs phenotypic switching dependent on KLF4.Taken together,the results provide critical insights into the newly discovered role of Shh in the pathogenesis of VSMCs proliferation,that is Shh mediates VSMCs phenotypic modulation through KLF4.Background: Limb ischemia/reperfusion(I/R)injury is a clinically common and severe disease.Skeletal muscle has high metabolic activity and thus sensitive to reperfusion injury after ischemia.Despite the blood flow is restored following severe ischemia,I/R injury causes continuous damage and necrosis of the skeletal muscle,which may lead to amputation and multisystem organ dysfunction syndrome.Sonic hedgehog(Shh)plays a critical role in post-natal skeletal muscle regeneration.And it is well established that Shh exerts a positive effect in skeletal muscle ischemia disease.However,the role of Shh in skeletal muscle I/R injury has not been investigated.Objective: In present study,we would construct a mice model of skeletal muscle I/R injury with tourniquet and then estimate the effect of Shh pathway in this pathological conditions.Subsequently,for examining the mechanism involved,we investigate whether Shh mediated I/R related skeletal muscular injury through the classical anabolic pathway AKT/m TOR/p70s6 k.Lastly,we would also investigate the role of Shh signaling in I/R related skeletal muscular apoptosis.We hope these could provide some novel therapeutic strategy for skeletal muscle I/R injury.Methods:(1)Young mice(10-14 weeks,male,C57BL/6)model of 3 h tourniquet induced hindlimb ischemia,and subsequent 1,3,5,7 or 14 d reperfusion were constructed.The skeletal muscle samples were harvested at different reperfusion time point,the Shh,Gli1 and Gli2 level was detected by Western blot.Meanwhile,the Shh expression in skeletal muscle tissue was also estimated by immunofluorescent assay.(2)To elucidate whether Shh pathway participated in regulation of skeletal muscle I/R injury,Shh signaling pathway was inhibited through intraperitoneal injection Smoothened inhibitor Cyclopamine.Conversely,Shh signaling pathway was over-expressed through intramuscular injection of plasmid encoding the human Shh gene(ph Shh).Subsequently,the skeletal muscle of I/R 7 d injury was harvested and evaluated.Then the Shh pathway protein expression was estimated by Western blot or immunofluorescent assay to verify the effiecency of Shh pathway inhibition and facilitation.Then the degree of skeletal muscle injury was evaluated by destruction scores based on H&E staining,and the degree of skeletal muscle fibrosis was quantified by Masson’s t ich om staining.(3)To inv stigat th tim-course of anabolic activity in skeletal muscle I/R injury model,the local expression of AKT,m TOR and p70s6 k and their phosphorylation protein in skeletal muscle in different reperfusion time were estimated by Western blot.To test whether AKT/m TOR/p70s6 k pathway was involved in the process of Shh triggered skeletal muscle protection following I/R injury,Shh pathway was over-expressed with ph Shh,and the expression of Akt/m TOR/p70s6 k pathway protein was detected at the time of 7 d reperfusion.To further investigate the underlying mechanism,ph Shh was used to over-express Shh pathway,and a dual PI3K-m TOR inhibitor NVP-BEZ235 was used to inhibit the AKT/m TOR/p70s6 k pathway.Then the expressions of Shh and AKT/m TOR/p70s6 k pathway,skeletal muscle destruction and intramuscular collagen deposition at the time of 7 d reperfusion were evaluated.(4)In present study,the cell apoptosis of skeletal muscle following I/R injury was evaluated.The time-course expressions of pro-apoptotic proteins Cleaved Caspase-3 and Bax,and anti-apoptotic protein Bcl2 in I/R injury skeletal muscle were detected by Western blot.To further investigate the role of Shh in skeletal muscle cells apoptosis following I/R injury,Shh pathway was over-expressed then the level of Cleaved Caspase-3 and the Bax/Bcl2 ratio in 7 d I/R injury skeletal muscle were examined by Western blot.In addition,skeletal muscle cell apoptotic level was estimated by TUNEL staining.Results:(1)Firstly,we found that Shh,Gli1 and Gli2 expressions were up-regulated due to I/R injury,all of them peaked at the time of 5 d reperfusion,and subsequently decreased.Immunofluorescent assay showed that Shh expression could be hardly examined in control and 1 d reperfusion skeletal muscle.Nevertheless,after 3 d and 5 d reperfusion,apparent Shh signaling expression was observed in the basement membrane of muscle which was co-localization with or adjacent to the myonuclei.While after 7 d and 14 d reperfusion,the Shh expression decreased.(2)In further research,we observed infarct areas in Cyclopamine treated skeletal muscle,and significant higher skeletal muscle destruction scores through H&E staining in Cyclopamine treatment group(P<0.05),indicated skeletal muscle damage caused by I/R injury was deteriorated after inhibition of Shh pathway.In addition,we found a higher percent of areas occupied by collagen in Cyc o amin t atm nt g ou th ough Masson’s t ich om staining(P<0.05).However,when Shh pathway was over-expressed through intramuscular injection of ph Shh,we obtained a reverse result.(3)Then we found from Western blot that accompanied with the rapid induction of Shh expression,the phosphorylation levels of AKT,m TOR and p70S6 K were all significantly increased in early phase but decreased in the late phase.Then we observed that the ratios of p-AKT/AKT,p-m TOR/m TOR and p-p70s6k/p70s6 k were significantly higher in the ph Shh treatment group,and the up-regulation of the phosphorylation of AKT,m TOR and p70S6 K induced by Shh was abolished with the treatment of NVP-BEZ235.(4)Finally,our data indicated that the level of Cleaved Caspase-3 and the Bax/Bcl2 ratio increased,and both of them peaked at the time of 7 d reperfusion and subsequently decreased.And facilitating Shh pathway could alleviate skeletal muscle cells apoptosis.Conclusion: Shh can be postnatally recruited in the setting of skeletal muscle I/R injury.In addition,Shh exerts protective effects against skeletal muscle I/R injury with respect to reducing skeletal muscle damage and fibrosis which depends on AKT/m TOR/p70S6 K pathway.Meanwhile,Shh exhibits an anti-apoptotic effect on skeletal muscle following I/R injury.