The Molecular Mechanism And Effect Of DNA Double-strand Breaks On The Activity Of Vascular Smooth Muscle Cells In Intimal Hyperplasia

ObjectiveTo establish the model of intima hyperplasia via mechanical balloon injury of carotid artery,and verify DNA damage and repair in the process of intima hyperplasia.Further to observe effect of H₂O₂ induced DNA damage on vascular smooth muscle cells?VSMC?,and understand the relationship between DNA-damage related poly?ADP-ribose?ylation?PARylation?and VSMC proliferation and death.We aim to expound the mechanisms underlying VSMC proliferation and death in intima hyperplasia,and in order to provide the new biomarker on artery injury or the potential therapeutic targets of intima hyperplasia or atherosclerosis.Methods1.Seven-week-old male SD rats were randomly divided into two groups:normal control group?Ctrl group,n=10?,balloon-injured group?HE group,n=50?.We established the carotid balloon-injured rat model to examine the remodeling process of neointima.Using HE and immune-staining,we examined VSMC proliferation and intima hyperplasia in the injured artery.Moreover,we performed immune-staining and Western blotting assays to study DNA damage and homologous recombination repair in the VSMC.We also examined the expression of Rad51 and Brca2 by RT-PCR and analysis the relationship between DNA damage and homologous recombination repair during intima hyperplasia.2.Using comet assays and immune-staining,we examined oxidative damage repair in murine aorta smooth muscle cells?MOVAS?.We use laser microirradiation and immune-staining to examine poly?ADP-ribose??PAR?recruitment.Importantly,we performed Q-TOF mass spectrometry analysis and dot blot assays to study and compare PAR metabolism by different cell lines including MOVAS and murine embryo fibroblasts?MEF?.We also examined the expression of PARP-1,PAR and PARG by western blot and immunostaining,and used PARG inhibitor to facilitate PARylation to observe the change of PARylation-dependent DNA damage repair and cell survival.Results1.Our findings demonstrated that the neointima became dramatical hyperplasia till 28days following injury,and became steady to 40 days.DNA double strand breaks marker?H2AX first occurred in VSMC at the sites of lesion,then the DNA damge appeared in the VSMC of the neointima,associated with proliferative marker PCNA.DNA repair protein Rad51 and?H2AX were co-located in the nucleus.Following DNA damage,PAR was synthesis in 30 min.Rad51 and Brca2,two major DNA repair factors,were up-regulated in neointima,which activated DNA damage repair process,and then decreased with DNA damage repaired.This DNA repair process could make a role in the VSMCs proliferation and intima hyperplasia.2.We analyzed oxidative damage repair in MOVAS and found that compared to MEFs,VSMCs are hypersensitive to oxidative damage to induce DNA double strand breaks.Further analysis shows that oxidative damage repair in VSMCs is suppressed by low level of PARylation.The low level of PARylation in response to oxidative damage is not caused by lacking PARP-1,the major poly?ADP-ribose?polymerase activated by oxidative damage.We found that the expression level of PARG,the enzyme hydrolyzing PAR,is significantly higher than that in MEFs and other cell line.Using PARG inhibitor to suppress the enzymatic activity of PARG facilitates the oxidative damage-induced PARylation as well as DNA damage repair.Conclusions1.Thus,the balloon injury induced rat model was successfully made in this study,which is mature and can be used in the basic study of vascular surgery.Our study provides a novel molecular mechanism to explain the VSMC proliferation and death during intima hyperplasia.DNA damage related protein could be used as the new bio-marker on testing intima hyperplasia.Activating DNA repair pathway could be a potential approach for the clinical treatment of intima hyperplasia.2.Our study demonstrates a novel molecular mechanism of oxidative damage-induced VSMCs proliferation and death.Moreover,this study reveals the important role of PARylation in DNA repair,and PARylation could be as a defense mechanism against oxidative injury of vascular smooth muscle cells to maintain normal function.Importantly,PARG inhibitors facilitates the oxidative damage-induced PARylation as well as DNA damage repair,which could be as potential drugs for improve intima hyperplasia and atherosclerosis.