Role And Mechanism Of Phosphatidylinositol 3-phosphate 5-kinase Type Ⅲ In Neointima Hyperplasia After Vascular Endothelial Injury

Objectives:To explore the role of phosphatidylinositol 3-phosphate 5-kinase type III（PIKfyve）in the neointima hyperplasia after vascular endothelial injury;To investigate whether PIKfyve plays a role in the proliferation and migration of vascular smooth muscle cell（VSMC）in mice,and to preliminarily explore the possible mechanism of its function.Methods:1.In vivo experiment:to explore the role of PIKfyve in neointima hyperplasia after vascular endothelial injury,the model of vascular endothelial injury was established,the male wild-type C57BL/6J mice were randomly divided into sham group,sham+YM201636group,carotid artery endothelial injury group,and carotid artery endothelial injury+YM201636 group.After modeling,they were intraperitoneally injected with saline or YM201636（2mg/kg/d）for 15 days,and the degree of hyperplastic neointima was detected by H&E staining 28 days after operation;cell proliferation rate in carotid neointima was detected by immunohistochemical staining;the expression level of PIKfyve in carotid artery was detected by q RT-PCR and Western blot;the levels of VSMC differentiation marker genes was determined by q RT-PCR.2.In vitro experiment:isolation and culture of primary vascular smooth muscle cells by enzymatic digestion method.Platelet-derived growth factor-BB（PDGF-BB）was used to induce proliferation and migration of VSMC.PIKfyve si RNA was used to silence PIKfyve’s expression,the inhibitor of PIKfyve was used to inhibit the activity of PIKfyve,and insulin was used to restore the activity of m TORC1.The proliferation rate of VSMC was detected by CCK-8 assay and Ki-67 immunofluorescence staining,the migration rate of VSMC was detected by scratch assay,and expression levels of PIKfyve,ribosomal protein S6（S6）,phospho（p）-S6^Ser235/236,4E binding protein 1（4EBP1）and p-4EBP1^Thr37/46 were detected by q RT-PCR and Western blot.Results:1.Compared with the sham group,the ratio of intima/media and positive cells of Ki-67were increased after vascular injury,and the expression of PIKfyve was increased after vascular injury.YM201636 inhibites PIKfyve activity,it could reduce the ratio of intima/media after carotid endothelial injury.The immunohistochemical results showed that YM201636 reduced the positive expression rate of Ki-67 in neointima after vascular endothelial injury;YM201636 reversed the decreased m RNA levels ofα-smooth muscle actin（α-SMA）,Calponin,and SM-myosin heavy chain（SM-MHC）after vascular injury.2.In vitro experiment,compared with the DMSO group,cell viability,migration and the rate of Ki-67 positive cells were increased in PDGF-BB group.The results of q RT-PCR and Western blot showed that the expression level of PIKfyve was increased in PDGF-BB group.The cell viability and the rate of Ki-67 positive cell induced by PDGF-BB were decreased when silence PIKfyve.In accordance with these results,inhibition of PIKfyve by YM201636 also reduced the increased cell viability and rate of Ki-67 positive cells after PDGF-BB intervention.Knocking down PIKfyve expression or decreasing its activity significantly inhibited the rate of VSMC migration induced by PDGF-BB.The phosphorylation of S6 and 4EBP1 was inhibited when knocking down PIKfyve expression or decreasing its activity,insulin restored the phosphorylation level of S6 and 4EBP1;When m TORC1 activity was recovered by insulin,the inhibitory effect of PIKfyve knockdown or inactivation on proliferation and migration rate of VSMC was reversed.Conclusions:1.The expression of PIKfyve increased after vascular endothelial injury,and the inhibition of PIKfyve activity reduced the neointima hyperplasia after vascular endothelial injury.2.PIKfyve may regulate the proliferation and migration of vascular smooth muscle cells via controlling m TORC1 activity,thus playing a role in the hyperplasia of neointima after vascular endothelial injury.