Pitavastatin Activates Mitophagy To Protect Epc Proliferation Through A Calcium-Dependent CAMK1-PINK1-Pathway In Atherosclerotic Mice

BackgroundAtherosclerosis（AS）is one of cardiovascular diseases with high incidence,which is also considered as the main cause of the cardio cerebral thrombotic diseases and vascular wall disorders.With the improvement of people’s living standards,AS has become the leading cause of death worldwide.Accumulating exist evidences suggested endothelial dysfunction is an early marker for AS which could be detected prior to the structural changes of vessel wall.It is not only the initial event of AS and subsequently contributes to the development of AS.Therefore,the early prevention of endothelial cell dysfunction and timely repairment of the damaged vascular endothelium have become the potential targets of the therapeutic approach for AS.Plenty of studies revealed that endothelial progenitor cells（EPCs）have the potential to promote neovascularization by repairing the damaged blood vessels.Compared with the proliferation and migration of adjacent endothelial cells,the targeted homing effect of circulating EPCs on AS plaque is more important when the blood vessel is under severe stress.However,the drugs to improve the biological function of EPCs are lack of specificity and may have other side effects on the body.At the same time,due to the susceptibility of EPCs to various circulation risk factors,the mobilization and survival time of the transplanted EPCs have not been properly improved,and further limits the application of targeted EPCs treatment to the clinical practice.Pitavastatin（PTV）is a new-generation lipophilic statin,indicating for the treatment of dyslipidemia and clinical prevention of cardiovascular diseases.In addition to its lipid-lowering effect,PTV also has other biological function such as anti-inflammatory,anti-oxidation,anti-apoptosis and vascular endothelial protection.However,whether PTV can improve the biological function of EPCs under AS condition and the underlying mechanism are still unclear.ObjectiveThis study intends to explore the effect of PTV on the biological function of EPCs in AS condition.And we further explored whether PTV could activate mitophagy via CAMK1-PINK1pathway,thereby promoting the repairment of damaged blood vessels.MethodsIn the integral experiment,after Apo E^-/-mice being fed with high fat diet,the establishment of AS mice model was verified by aortic oil red staining and immunohistochemistry.In vitro,bone marrow mononuclear cells were collected by density gradient centrifugation and cultured with differential adhesion assay subsequently.Purified EPCs were identified by phagocytosis test,immunofluorescence（IF）and flow cytometry.PTV was served as the intervention reagent to treat EPCs,and the expression of CAMK1,PINK1,PARK2 and other autophagy-related genes were knocked down by sh RNA respectively to explore the specific mechanism.CCK-8and RTCA assays were used to detect the proliferation of EPCs.Immunoblotting（WB）and IF were performed to investigate the expression and distribution of protein in EPCs.The morphology of mitochondria was observed using transmission electron microscope.WB,fluorescent protein reporter assay system and mt-Keima were applied to evaluate the level of mitophagy.Cytoplasmic/mitochondrial calcium uptake was detected by the Fluo-3/Rhod-2probe respectively using confocal imaging.WB,fluorescence colocalization were used to determine the key molecules contributed to the PTV-induced mitophagy.In vivo,the homing of EPCs was identified by autologous EPCs transplantation in combination with the fluorescent tracer technique,and the reendothelialization of damaged vessels was evaluated by Evans blue staining.Results1.PTV restored the proliferation ability and mitophagy of EPCs in AS miceAortic oil red staining and immunohistochemistry confirmed the plaque formation and vascular wall inflammatory infiltration phenotype of AS mice.IF and flow cytometry identified the bone marrow-derived EPCs by phagocytic characteristics（UEA-I and Dil-Ac-LDL）and the expression of classical markers（CD34 and CD133）.The morphology and functional abnormality of EPCs was aggravated with development of AS.CCK-8 and RTCA results showed decreased proliferation of EPCs in AS mice,which can be reversed by PTV treatment in a concentration-dependent manner.WB and immunofluorescence showed inhibited mitophagy flux of EPCs in AS mice,and the treatment of PTV also significantly recovered the levels of mitophagy,which was attributed to the activation of mitophagy formation rather than the degradation inhibition of autophagy-lysosome.2.PTV improved EPC proliferation dependent on PINK1-PARK2-mediated mitophagyAs the development of AS,the expression levels of PINK1 and PARK2,as well as mitophagy flux were decreased in EPCs,both of which can be recovered with the treatment of PTV.It is the process that Atg7 involved and PINK1-PARK2 dependent pathway to participant in the PTV-induced mitophagy.In vitro,the protective effect of PTV on mitophagy and EPCs proliferation can be blocked by knocking down of Pink or Park2.PINK1 is the key protein during endothelial injury repairment through recruiting its downstream PARK2 to maintain a moderate mitophagy level.3.PTV activated EPCs mitophagy via CAMK1-PINK1 pathway to promote the repairment of the damaged vesselsIn vitro experiments,PTV-activated mitophagy was accompanied by mitochondrial calcium release and the activation of CAMK1,while Camk1 knockdown downregulated the expression of PINK1 and PARK2,which further inhibited PARK2-mediated mitophagy and EPCs proliferation.When blocking the CAMK1-PINK1 dependent mitophagy by sh RNA targeting the key nodes,we found that the phosphorylation level of CAMK1 is essential for the protective effect of PTV on mitophagy and EPCs proliferation.Finally,the autologous EPCs transplantation experiment showed that PTV pretreatment was conducive to the recruitment and homing of EPCs to the site of damaged vessels,which depended on the activation of mitophagy mediated by CAMK1-PINK1.Conclusions1.PTV promotes the proliferation of EPCs effectively and protects the vascular endothelium damaged by AS.2.PTV-activated mitophagy is essential for the improvement EPC proliferation3.CAMK1-PINK1 is a novel mitophagy activation pathway,which maintains EPCs proliferation under atherosclerotic condition.