The Role Of KPC1 In Neointima After Carotid Artery Balloon Injury In Rat

Objective To study the expression of kipl ubiquitylation-promoting complex 1(KPC1)in the neointima after carotid artery balloon injury in SD rats and to explore its biological function in neointimal formation process.Methods Primary vascular smooth muscle cells of rats were cultured using tissue adherent method.Vascular smooth muscle cell-specific marker a-SMA was analysed through immunofluorescence staining.Culturing vascular smooth muscle cells(VSMCs)proliferation model in vitro,qRT-PCR and western blot were detecting KPC1 mRNA,protein expression.We constructed Ad-shKPCl adenoviral vectors which downregulate KPC1 via RNAi technology.Transfecting it into AD293 cells for packaging,amplifying,and then vascular smooth muscle cells after being purified,western blot was detecting KPC1 protein expression.The adult male SD rats(450?550 g)were randomly divided into carotid artery injury model group and sham group.After 1,3,7,14 and 28 days of balloon injury,SD rats of the experimental group were sacrificed and harvested for histomorphology and molecular markers assay.Hematoxylin and eosin staining was used to assess the changes of neointimal formation in the carotid artery while western blot and qRT-PCR were used to evaluate the expression of KPC1.What's more,western blot was also used to assess proliferating cell nuclear antigen(PCNA)levels.Rats were randomly fell into three groups as to detecting impact of KPC1 overexpression on neointimal information,namely the sham group without balloon injury,Ad-Null group and Ad-KPCl group with which respectively suffering from 40 ?l Ad-Null,Ad-KPC1 incubation besides balloon injury.Neointimal formation was analysed with hematoxylin and eosin staining while immunohistochemistry was used to evaluate the expression of KPC1,CNN1 and CD31.Results We successfully achieved primary vascular smooth muscle cells via tissue adherent method.Immunofluorescence showed that a-SMA positive cells were more than 95%.After stimulation of VSMCs prolifleration by platelet-derived growm factor BB(PDGF-BB)in vitro,qRT-PCR and western blot data showed a gradual decreasing trend of KPC1 at mRNA and protein levels.Adenoviral vector containing the sequence of KPC1 interference was successfully constructed through RNAi technology,which can effectively downregulate expression of KPC1 protein.As the time of carotid arteries injury went,neointimal hyperplasia in carotid arteries revealed a gradually increasing trend.With VSMCs hyperplasia,neointimal and luminal stenosis were getting more and more serious,the thickness of intimal exceeded medial at 14 days.At 28 days,the irregular intimal hypelplasia was continued and the degree of vascular stenosis was more than 50%.Westen blot and qRT-PCR showed that the KPC1 expression levels of the experimental group were significalltly lower than that of the sham group(P<0.05),while the PCNA expression was significantly higher than that of sham group(P<0.05).KPC1 overexpression dramatically reduced neointimal formation(P<0.05),wheras promoted KPC1,CNN1 and CD31 expression levels(P<0.05).Conclusion KPC1 inhibits neointimal hyperplasia potentially by affecting vascular smooth muscle cell phenotypic transition.