The Role And Mechanism Of TSPO And Its Ligands In Balloon Injury-induced Neointima Hyperplasia

BackgroundAbnormal growth of the intimal layer of blood vessels(neointima hyperplasia)is a prevalent severe pathophysiological process that contributes to the progression of atherosclerosis and in-stent restenosis.Vascular smooth muscle cells(VSMCs)are main constituent of the neointima in restenotic and atherosclerotic lesions.The molecular mechanisms underlying these complex series of changes are not completely understood.It is generally accepted that VSMCs transform phenotype,proliferate,migrate and secret e extracellular matrix,resulting in neointima forming in response to injury or other stimuli.During neointima formation,growth factors,such as transforming growth factor-?(TGF-?)and platelet-derived growth factor-BB(PDGF-BB),are produced locally.To date,the treatments aimed at suppressing neointima formation are extremely limited.Thus,elucidating the mechanisms governing VSMC growth modulation is necessary for understanding the pathogenesis and progression of many proliferative vascular diseases and ultimately identifying novel therapeutic targets to treat these diseases.The translocator protein(TSPO)is an 18 kDa protein distributing in the outer mitochondrial membrane.TSPO proteins from diverse organisms are highly conserved in sequence and also in function.TSPO expression alters readily in various cells exposed to physicochemical stimuli or pro-pathological factors.Roles for TSPO have been investigated in apoptosis,inflammation,HIV biosynthesis,cancer and Alzheimer's disease.In particular,TSPO is involved in several cardiovascular diseases,such as ischemia-reperfusion injury,cardiac arrhythmia,cardiac hypertrophy and atherosclerosis.PK11195 and Ro5-4864 are the two well studied TSPO ligands.In-vitro studies have shown PK11195 binding to macrophage-rich human atherosclerotic plaques.And radioactively labeled PK11195 has been tested for imaging atherosclerosis.Considering the role of TSPO in the regulation of cell proliferation has been shown and the abundance of TSPO in the inflammatory regions of atherosclerotic plaques,we inferred that there may be a link between TSPO and aberrant proliferation of VSMCs after angioplasty.Objectives1.Identify the expression of TSPO in VSMCs of balloon injured rat carotid artery and in PDGF-BB-induced A10 cells.2.Investigate the role of TSPO in PDGF-BB-induced VSMC proliferation and migration.3.Explore the effects and underlying mechanism of TSPO ligands on VSMC proliferation and migration in vitro and in vivo.MethodsBalloon injury model was established to induce neointima formation in the left carotid arteries in male SD rats(250±50 g body weight).The samples of carotid arteries were collected 1 day,7 days,14 days and 21 days after injury.The sections were immunostained to check the expressions of TSPO and a-SMA.Immunoblotting and PCR were applied to check the expression of TSPO protein/mRNA in homogenates of carotid artery tissue.In vitro experiments,A10 cells were treated with PDFG-BB to induce cell proliferation and migration.The expression of TSPO in the lysates was determined by immunoblotting.Cell proliferation was measured by cell number counting and MTT assays.Cell migration was measured by wound-healing and transwell assays.Transfection experiments were performed by using siRNA and GFP-TSPO to identify the role of TSPO in PDGF-BB-mediated proliferation and migration in A10 cells.PK11195 and Ro5-4864,the two best known TSPO specific ligands,were applied to check their effects on the PDGF-BB-mediated proliferation and migration in A10 cells,determined by MTT,wound-healing and transwell assays.The expressions of cyclin D1,cyclin E and PCNA were determined by immunoblotting.To uncover the mechanism of the effect of TSPO ligand on the aberrant growth of VSMCs,AMPK activity was detected by the ritio of phosphorylated AMPK to total AMPK.Compound C,a specific AMPK inhibitor,was applied to further investigate the role of AMPK in the inhibitory effect of PK11195 on PDGF-BB-mediated proliferation and migration in A10 cells.The therapeutic effect of PK11195 on balloon injury-induced neointima was explored in SD rats.Intraperitoneal injection of PK11195(3 mg/kg,per 3 days)was started with the balloon injury in rat carotid artery and continued for 2 weeks.Carotid artery sections were collected 14 days after injury and HE stained.The thickness ratio of intima to media was calculated to evaluate the neointima formation.VSMC proliferation was determined by immunostaining of PCNA.Expression of a-SMA was determined by immunoblotting.ResultsTSPO expression was significantly up-regulated in proliferative VSMCs in vivo and in vitro.PDGF-BB,via PKC/MAPK,increased TPSO expression in A10 cells.Over-expression of TSPO in A10 cells significantly promoted cell proliferation and migration.In contrast,down-regulation of TSPO expression by siRNA,as well as treatment with TSPO ligands PK11195 or Ro5-4864(10ng/ml),presented the opposite effects in A10 cells.Moreover,PK11195 induced AMPK activation.In particular,PK11195-mediated inhibitory effects on proliferation and migration of PDGF-BB-treated A10 cells were abolished by compound C(an AMPK specific inhibitor).Importantly,intraperitoneal injection of PK11195(3 mg/kg,per 3 days),starting from the initial balloon injury and lasting for 2 weeks,attenuated carotid neointima formation in rats by restoring balloon injury-induced phenotype switching of VSMCs.ConclusionTSPO is a key molecule that mediates VSMC proliferation and migration.TSPO ligand PK11195 reverses the balloon injury-induced VSMC phenotype switching by activating AMPK signaling,consequently inhibits the proliferative response of VSMC,and protects against balloon injury-induced neointima hyperplasia in rat carotid artery.