Translocator Protein 18 KDa Ligand Alleviates Neointimal Hyperplasia In The Diabetic Rat Artery Injury Model Via Activating PKG

Background:Diabetic patients undergoing surgical revascularization or percutaneous coronary intervention are highly prone to vascular restenosis.Neointimal hyperplasia after vascular injury plays a critical role in the process of vascular restenosis,but its mechanism has not been elucidated.It is commonly accepted that abnormal proliferation and migration of medial vascular smooth muscle cells（VSMCs）are the pathological causes of neointimal formation after tunica intima injury.Hyperglycemia or other conventional vascular risk factors,such as obesity and lipids,promote intimal hyperplasia via increased oxidative stress in lesions.Therefore,finding a novel molecule,that could be selectively targeted to inhibit neointimal formation and vascular remodeling,is a promising strategy for the treatment of cardiovascular complications after percutaneous coronary intervention in diabetes.Translocator protein（TSPO）is an 18 kDa ubiquitous protein that was previously described as peripheral benzodiazepines receptor.It is mainly localized in the outer mitochondrial membrane and has been shown to be involved in a wide range of important cellular processes,including proliferation,apoptosis,steroidogenesis,immunomodulation,gene expression and mitochondrial physiology.The interaction of TSPO with nicotinamide adenine dinucleotide phosphate（NADPH）oxidase links the generation of ROS to the induction of an antioxidant response to maintain redox homeostasis.TSPO is proposed as an outer mitochondrial membrane-based pathway to control intracellular Ca²⁺dynamics and redox transients in neuronal cytotoxicity.Of note,the TSPO component is considered to be a multifaceted molecule that could provide binding sites for synthetic ligands with high affinity.Large studies have been demonstrated that TSPO ligands can inhibit the proliferation of various cancer cell lines like melanomas,colon,esophagus,breast,astrocytes and mammary gland.Prototypical TSPO ligand Ro5-4864 has shown beneficial effects in experimental diabetic neuropathy.Recent studies have reported that TSPO function is modulated by selective ligands,leading to changes in glucose homoeostasis and cellular energy production.However,the role of TSPO drug ligands in vascular diseases,such as coronary artery restenosis and cardiovascular complications of type 2 diabetes mellitus remains elusive.Based on above mentioned views,we hypothesized that TSPO may serve as a potent target in the process of neointimal hyperplasia after angioplasty in diabetes.In the present study,we explored the protective effects of TSPO drug ligands PK 11195 and Ro5-4864 on VSMC proliferation and migration,and investigated whether PK 11195 could act as a potential reagent to prevent intimal hyperplasia after angioplasty in diabetes.Objective:1.To describe the role of TSPO in VSMC proliferation and migration induced by high glucose.2.To detect the effect of TSPO ligands on VSMC proliferation and migration induced by HG.3.To explore the underlying mechanism of TSPO ligands how to inhibit HG-induced VSMC proliferation and migration.4.To study effect of TSPO ligands on HG-induced neointimal hyperplasia in vivo.Methods:1.Hyperglycemia rat model was established by using STZ（60 mg/kg）injection.A10cells were treated with high glucose（25 mM）for different times or with indicated concentration glucose for 24 hours.TSPO expressions in rat aorta and A10 cells were detected by immunofluorescence staining,western blot（WB）and quantitative polymerase chain reaction（QT-PCR）.2.TSPO function of A10 cells was inhibited by siRNA or TSPO ligands（PK 11195and Ro5-4864）.A10 cells proliferation was detected by MTT assays and cell counting.A10cells migration was measured by Transwell?and scratch-wound assays.3.Role of cGMP/PKG on PK 11195-mediated inhibitory effect on A10 cell proliferation.A10 cells were treated with 25 mM HG for 24 hours in the presence of PK11195(10^-5 mol/L),ODQ（10 mM）,cAMP-Rp（10 mM）and/or KT5823（1 mM）.The cell proliferation was determined by MTT assay.cGMP activity was determined by ELISA via commercial cGMP activity assay kit.4.Using balloon injury to induce carotid neointima formation in type 2 diabetic rats,simultaneously injected with vehicle or PK 11195（3 mg/kg）twice per week for two weeks.After five times injection,carotid artery was harvested for neointima examination.hematoxylin-eosin（HE）and immunofluorescence staining were applied to evaluate effect of PK 11195 on neointima formation.Results:1.Both immunoblotting and immunofluorescence staining revealed that the expression of TSPO protein significantly upregulated in hyperglycemia rat aorta compared with vehicle group.Consistently,HG treatment leds to significant upregulation of TSPO expression in both concentration-and time-dependent manners in A10 cells.2.Knockdown of TSPO by using siRNA interference decreased A10 cell proliferation induced by HG evaluated by MTT analysis,and potently inhibited HG-mediated A10 cell migration which were determined by Transwell?and wound healing tests.Our findings also revealed that treatment with PK 11195 or Ro5-4864 dose-dependently reduced HG-induced proliferation of A10 cells determined by MTT and cell counting analysis,and suppressed HG-induced migration of A10 cells determined by Transwell?and wound healing studies.3.Our findings revealed that the inhibitory effects of PK 11195 on A10 cell proliferation was abolished by ODQ,the blocker of cGMP,but not by the cAMP blocker cAMP-Rp,as evaluated by MTT assay.Inhibitory effect of PK 11195 on HG-mediated proliferation in A10 cells was also blocked when PKG,which is the downstream mediator of cGMP signals,was inhibited by KT5823.Consistent with the effect of PK 11195 on cell proliferation,HG decreased cGMP production,which was reversed by PK 11195 treatment;while in presence of ODQ,the stimulatory effect of PK 11195 was blocked.4.PK 11195 was explored in the well-established balloon injury model in type 2diabetic rats.HE staining showed PK 11195 alleviated neointimal formation in the injured carotid arteries.The protective effect of PK 11195 on neointimal formation was correlated with its inhibitory effect on VSMC proliferation,as determined by PCNA immunofluorescence staining.Conclusion:This study revealed the crucial role of TSPO in balloon injury-induced neointimal hyperplasia in type 2 diabetic rats.TSPO ligand PK 11195 can attenuate neointimal hyperplasia through its anti-proliferative effects on VSMCs via cGMP/PKG pathway.Significance:This study suggests that TSPO inhibition suppresses the proliferation and migration of VSMCs induced by hyperglycemia,consequently,preventing atherosclerosis and restenosis after angioplasty in diabetic conditions.TSPO may be a potential therapeutic target to reduce arterial remodeling induced by angioplasty in diabetes.