Role And Mechanism Of Myotubularin-Related Protein 7 In Neointima Hyperplasiaafter Vascular Endothelial Injury

Objective:1.To explore the role of myotubularin-related protein 7（MTMR7）in the neointima hyperplasia after vascular endothelial injury;2.To investigate the role of MTMR7in proliferation and migration of mouse vascular smooth muscle cells（VSMC）;3.To explore the potential mechanism of which MTMR7 regulates proliferation and migration of mouse VSMCMethods:1.In vivo experiment,the mouse model of neointima hyperplasia after vascular endothelial injury was established by wire injury of the carotid artery endothelium.Mtmr7 was overexpressed via the carotid artery incubation and tail vein injection with adenovirus carrying Mtmr7.28 days after operation,the carotid artery morphology and hyperplastic neointima were analyzed by hematoxylin-eosin staining（HE）;the degree of carotid intimal hyperplasia was assessed by Ki-67 immunohistochemical staining;the protein level of MTMR7 in carotid artery was measured by Western blotting.2.In vitro experiments,mouse aortic smooth muscle cell line（MOVAS）was cultured.Platelet-derived growth factor BB（PDGF-BB）was used to induce proliferation and migration of VSMC.Adenovirus carrying Mtmr7 was uesd to overexpress Mtmr7.Insulin was uesd to restore the activity of mTORC1（Mammalian target of rapamycin complex 1）.The capacity of proliferation was analyzed by Cell counting kit-8 assay（CCK-8）and Ki-67immunofluorescence staining.The capacity of migration was assessed by scratch assay and transwell assay.The mRNA level of Mtmr7 was determined by qRT-PCR.Western blotting was used to measure the protein levels of MTMR7,4E binding protein 1（4EBP1）,phosphorylated（Phospho,p）-4EBP1^Thr37/46,S6 ribosomal protein（S6）,and p-S6^Ser235/236.Results:1.In vivo experiments,MTMR7 was expressed in mouse brain,blood vessel,liver,kidney,VSMC and other tissues.It was highly expressed brain.After carotid artery endothelial injury in mice,the ratios of carotid artery intima/media and Ki-67 positive VSMCs were increased（P<0.01）,and the protein level of MTMR7 was decreased（P<0.01）.Overexpression of Mtmr7 reduced the ratios of carotid artery intima/media and Ki-67positive VSMC after carotid artery endothelial injury in mice（P<0.01）.2.In vitro experiments,we found that the relative VSMC number（P<0.01）,the rate of Ki-67 positive VSMC（P<0.05）,the rate of scratch healing（P<0.01）and the migrated VSMC number were increased（P<0.01）after PDGF-BB treatment.Hower,we found decreased mRNA（P<0.01）and protein（P<0.05）level of MTMR7 in PDGF-BB-induced VSMC.Overexpression of Mtmr7 significantly reduced the relative VSMC number,the Ki-67 positive VSMC rate,the scratch healing rate,and the migrated VSMC number after PDGF-BB treatment（P<0.01）.The protein levels of p-4EBP1^Thr37/46and p-S6^Ser235/236were decreased in PDGF-BB-induced VSMC that achieved Mtmr7 Overexpression（P<0.05）.After Mtmr7 overexpression,insulin up-regulated the protein levels of p-S6^Ser235/236and p-4EBP1^Thr37/46in PDGF-BB-induced VSMC（P<0.05）.Insulin increased the relative VSMC number,the rate of Ki-67 positive VSMC,the rate of scratch healing and the migrated VSMC number at the same time（P<0.05 or P<0.01）.Conclusions:1.The expression of MTMR7 is decreased after vascular endothelial injury.Overexpression of Mtmr7 inhibits neointima hyperplasia after vascular endothelial injury.2.The expression of MTMR7 is decreased in proliferative VSMC.Overexpression of Mtmr7 inhibits the proliferation and migration of VSMC.3.MTMR7 inhibits the proliferation and migration of VSMC by reducing the activity of mTORC1,thereby improving neointima hyperplasia after vascular endothelial injury.