CTRP6 Inhibits PDGF-BB-induced Vascular Smooth Muscle Cell Proliferation And Migration And Its Mechanism

Background : Atherosclerosis is the main cause of cardiovascular disease and the leading cause of morbidity and mortality in the world.Atherosclerosis is a complex pathology that involves several processes including endothelial dysfunction,lipid infiltration,oxidative stress,inflammation and cellular proliferation and migration.As advancing atherosclerosis culminates into lumen-narrowing stenotic plaques causing ischemia,surgical interventions are needed to restore proper tissue perfusion.Such interventions rely on mechanical opening and widening of the afflicted vessel area and can be performed by percutaneous transluminal angioplasty(PTA),utilizing either balloon dilation or vascular stent placement.Although these procedures allow successful revascularization,it is well known that in a substantial number of cases the treated vessel might,over time,develop restenosis.Restenosis limits the long-term outcome of PTA.Though a main goal of the surgical intervention accomplished by PTA is to create and preserve an open lumen,it is also important to achieve reconstitution of a functional intimal endothelial coverage.However,the surgical treatment involving mechanical dilation might cause further injury to the vessel wall already afflicted by plaque formation,stimulating the inflammatory response and subsequent tissue remodeling and repair.The overshooting response will limit the success of therapeutic intervention.In the case of balloon angioplasty,both inward vessel remodeling(the loss of inner vessel diameter)as well as excessive VSMC proliferation invading the luminal space,form abnormal hyperplasia of the new intima and contribute to lumen loss.In the situation of stent placement,only VSMC proliferation remains a major treatment-limiting factor.Vascular smooth muscle cells(VSMCs)are involved in the control of vascular tone anddiameter through the mechanism of contraction.Mature VSMC is quiescent and has a‘contractile’ phenotype associated with the stable production of smooth muscle(SM)contractile proteins.Those include SM α-actin,SM22α,SM myosin heavy chain(MHC),H1-calponin,and smoothelin,all are recognized as selective VSMC markers.In steady state,VSMCs very rarely proliferate and exhibit a low synthetic activity.VSMCs are not terminally differentiated and hence exhibit phenotypic plasticity.This feature discriminates VSMCs from skeletal muscle cells and cardiomyocytes that are terminally differentiated.The capacity of VSMCs to switch the phenotype is regulated by external signals and accompanied by induction of expression of VSMC markers related to proliferation and motility and decrease in expression of selective VSMC markers.Generally,phenotypic changes in VSMCs are required during embryonic angiogenesis,neocascularization,vascular remodeling,and repair of vessel injury.In response to vascular injury or local environmental changes,differentiated vascular smooth muscle cells can be dedifferentiated,manifested as cell proliferation,migration and synthesis of extracellular matrix,and reduced expression of selective VSMC markers.As a large body of in vitro data suggests,VSMC plasticity is remarkable and can lead to acquisition of many functionally distinct phenotypes(synthetic SMC,macrophage-,MSC-chondrogenic-or osteogenic-like cells),with very different outcomes in disease progression.Moreover,VSMC phenotype transition is an environmental cue-guided,rather than spontaneous.When stimulated by extracellular cytokines,shear forces,matrix components and other factors,vascular smooth muscle cells can significantly reduce the expression of selective VSMC markers,improve the ability of proliferation,migrated and synthesized extracellular matrix,and participate in the formation of new intima.The migratory and proliferative abilities of VSMCs are regulated by a multitude of factors,including platelet-derived growth factor-BB(PDGF-BB).platelet-derived growth factor(PDGF)as a serum-derived growth factor,has been found to promote the growth of fibroblasts,VSMCs and neural glial cells,and is a potent mitotic promoter.It plays an important role in the phenotypic transformation,proliferation and migration ofvascular smooth muscle cells,and is also an important regulator of angiogenesis.PDGFs,mainly produced by endothelial cells,activated macrophages and smooth muscle cells,are stored and released by platelets upon activation.PDGFs consists of four ligands(PDGF-A,PDGF-B,PDGF-C,and PDGF-D),which form five kinds of functional dimmers.PDGF-BB homologous dimer is the most effective stimulant to the proliferation of vascular smooth muscle cells.PDGF-BB was reported to initiate various biological effects via the activation of intracellular signal transduction pathways that play important roles in modulating VSMC proliferation and migration.Therefore,inhibiting PDGF-BB-mediated VSMC proliferation and migration may be a good way to prevent the development and progression of atherosclerosis and restenosis.C1q/tumor necrosis factor-related proteins(CTRPs)are a highly conserved family of adiponectin paralogs.CTRP6,a member of CTRPs,is made up of four separate domains including an N-terminal signal peptide,a short variable domain,a collagen-like domain,and a C-terminal C1q-like globular domain.AdipoR1 was identified as a receptor of CTRP6 in intramuscular adipocytes.Increasing evidences have reported that CTRP6 was involved in a variety of physiological processes,such as cell proliferation,lipid metabolism,insulin sensitivity,energy balance and cardiac-protection.A recent study showed that CTRP6 overexpression significantly inhibited Angiotensin II(AngII)-mediated vascular endothelial dysfunction and apoptosis.However,the effects of CTRP6 on VMSC proliferation and migration remain unknown.This study aimed to investigate its role in VSMCs and explore the possible mechanism.Objective: 1.Identify the expression of CTRP6 at both mRNA and protein levels in atherosclerotic plaques and normal arteries.2.Identify the changes in the expression levels of CTRP6 after the stimulation of human arterial smooth muscle cells with PDGF-BB.3.Observe the effects of CTRP6 overexpression on proliferation and migration of VSMCs exposed to PDGF-BB,and explore the possible mechanism.Materials and Methods: 1.qRT-PCR and Western Blot were used to detect thedifferences in gene and protein expression levels of CTRP6 in the intimal tissue of atherosclerosis and normal artery tissue,so as to preliminarily determine whether CTRP6 is related to atherosclerosis.Then,PDGF-BB was used to stimulate human arterial smooth muscle cells,promoting the ability of proliferation and migration.qRT-PCR and Western Blot were used again to detect the changes in the expression levels of CTRP6 in human arterial smooth muscle cells after PDGF-BB stimulated.Eukaryotic expression vector pcDNA3.1-CTRP6 was constructed using gene recombination technology.For in vitro transfection,HASMCs were transfected with pcDNA3.1-CTRP6 or empty vector using LipofectamineTM 2000.The expression of CTRP6 in human arterial smooth muscle cells transfected with pcDNA3.1-CTRP6 was detected by western blot.2.MTT assay was used to detect the effect of CTRP6 on the proliferation of PDGF-BB stimulated human arterial smooth muscle cells.Scratch wound assay,Transwell assay and Transwell invasion assay were performed to observe the changes in the cellular motility of CTRP6 in PDGF-BB stimulated human arterial smooth muscle cells.Then detect the influence of CTRP6 on the expression of MMP-2,MMP-9 in human arterial smooth muscle cells.Then,we examined the effects of exogenous CTRP6 on functional changes in HASMCs.3.The expression of CTRP6 in PDGF-BB-stimulated human arterial smooth muscle cells was enhanced by transfection with pcDNA3.1-CTRP6.Then detect the influence of CTRP6 on the expression of VSMC contractile makers(α-SMA and SM22α)in human arterial smooth muscle cells,and investigated the effect of CTRP6 on the activation of PI3K/Akt/mTOR in HASMCs exposed PDGF-BB.RESULTS: In part one,CTRP6 expression was dramatically down-regulated in human atherosclerotic tissues and in cultured VSMCs stimulated by PDGF-BB.The mRNA expression levels of CTRP6 were remarkably down-regulated in human atherosclerotic tissues,as compared with the normal tissues.The results of western blot indicated that the protein expression levels of CTRP6 were lower in human atherosclerotic tissues than that of in the normal tissues.Furthermore,we found that the expression of CTRP6 at both mRNA and protein levels was dramatically reduced in HASMCs exposed to PDGF-BB.HASMCs were incubated with pcDNA3.1-CTRP6 or pcDNA3.1 for 48 h,and the transfection efficiency was confirmed using western blot analysis.Overexpression of CTRP6 significantly increased the protein expression of CTRP6 in HASMCs,as compared with the vector group.In addition,we found that pcDNA3.1-CTRP6 could recover the decrease of CTRP6 expression by PDGF-BB in HASMCs.In part two,CTRP6 overexpression inhibits the proliferation and migration of VSMCs exposed to PDGF-BB.In order to examine the effect of CTRP6 on HASMCs proliferation,CTRP6 was overexpressed in HASMCs using the pcDNA3.1-CTRP6.The results of MTT assay indicated that as compared with the untreated cells,PDGF-BB treatment markedly induced the proliferation of HASMCs.However,CTRP6 overexpression significantly inhibited PDGF-BB-induced HASMCs proliferation.Then,we evaluated the effect of CTRP6 on HASMCs migration using the Transwell assay(Transwell migration assay and Transwell invasion assay)and Scratch wound assay.The number of migrated VSMCs was greatly increased in PDGF-BB-stimulated VSMCs as compared with the control group in the both Transwell assay and PDGF-BB also enhanced VSMC migration in vitro scratch-wound assay.This effect was potently reversed by CTRP6 overexpression.CTRP6 overexpression alone did not affect HASMCs proliferation and migration.We examined the effects of exogenous CTRP6 on functional in HASMCs found that exogenous CTRP6 significantly suppressed PDGF-BB-induced HASMCs proliferation and migration,in a concentration-dependent manner.We observed that PDGF-BB treatment significantly increased the protein expression of MMP-2 and MMP-9 in HASMCs,but overexpression of CTRP6 efficiently suppressed PDGF-BB-induced the expression of MMP-2 and MMP-9 in HASMCs.CTRP6 overexpression alone did not affect the expression of MMP-2 and MMP-9.In part three,CTRP6 increases expression of VSMC markers in PDGF-BB-stimulated VSMCs.Phenotypic switch of VSMCs is an important step for VSMC proliferation and migration during the development and progression of atherosclerosis.Therefore,we examined the effect of CTRP6 on VSMCs markersexpression in PDGF-BB-stimulated HASMCs.PDGF-BB treatment significantly decreased the expression of VSMC contractile makers(α-SMA and SM22α)in HASMCs as compared with the control group.However,the decreased α-SMA and SM22α levels in PDGF-BB-stimulated HASMCs were prevented by CTRP6 overexpression.CTRP6 overexpression alone did not affect the expression of α-SMA and SM22α.We examined the effects of exogenous CTRP6 on phenotypic changes in HASMCs.Exogenous CTRP6 also markedly reversed PDGF-BB-reduced the protein expression of α-SMA and SM22 in HASMCs.To further explore the mechanism of CTRP6-inhibited HASMCs proliferation and migration induced by PDGF-BB,we investigated the effect of CTRP6 on the activation of PI3K/Akt/mTOR in HASMCs exposed PDGF-BB.The results of western blot analysis demonstrated that as compared with the control group,PDGF-BB treatment significantly increased the phosphorylation of PI3 K,Akt and mTOR in HASMCs.However,overexpression of CTRP6 sharply prevented the activation of PI3K/Akt/mTOR pathway in HASMCs in response to PDGF-BB.Additionally,we found that overexpression of CTRP6 inhibited phosphorylation of PI3 K,Akt and mTOR in a time-dependent manner in PDGF-BB-stimulated HASMCs.Conclusion: 1.CTRP6 expression was dramatically down-regulated in human atherosclerotic tissues.2.CTRP6 inhibited PDGF-BB-induced VSMC proliferation and migration,as well as increased expression of α-SMA and SM22α and suppressed expression of MMP2 and MMP9 in PDGF-BB-stimulated VSMCs.3.In conclusion,these findings showed that CTRP6 inhibits PDGF-BB-induced VSMC proliferation and migration,at least in part,through suppressing the PI3K/Akt/mTOR signaling pathway.Therefore,CTRP6 may be a potential target for the treatment of atherosclerosis.