The Role And Mechanism Of PCSK9 In Graft Vascular Disease

Part Ⅰ Construction of a mouse model of graft vascular disease and analysis of PCSK9 expressionObjective: In this part,we compared 3 surgical techniques of vascular anastomoses widely used in the mouse model of graft vascular disease(GVD)and determined the technique with the most appreciable outcomes.And then,we also investigated the expression of PCSK9 in the mouse model of GVD.Method: 1.The abdominal aorta of male C57BL/6(H-2b)and BALB/c(H-2d)mice were used as donor vessel,and the abdominal aorta of C57BL/6(H-2b)mice were anastomosed in situ by end-to-end anastomosis,sleeve and cuff suture in sterile environment.2.Operative Parameters were recorded: the weight of mouse,the diameter of aorta,the time of preparation of receptor artery,the time of artery anastomosis and the time of total operation.The limb movements of mice were observed continuously for 8 weeks after operation.3.The grafts were collected at the 8th week postoperatively and stained with HE and Masson.4.The abdominal aorta of male C57BL/6(H-2b)and BALB/c(H-2d)mice were used as donor’s vessel,and the best way was chosen to anastomose the abdominal aorta of recipient C57BL/6(H-2b)male mice in situ to construct the model of GVD in mice.5.At the 8th week after transplantation,the serum of mice was collected for PCSK9 ELISA and investigated the expression of PCSK9 in a mouse model of GVD.And the expression of PCSK9 in transplanted vessels,liver,kidney and small intestine was detected by WB and IHC techniques.Results: 1.Three methods of vascular anastomosis were successfully used to construct the model of GVD in mice,and there was no significantly difference in pathological severity between different methods of vascular anastomosis.Sleeve suture had a shorter vascular anastomosis time and total operation time than end-to-end anastomosis and cuff technique.Sleeve suture and cuff technique had significantly fewer amount of bleeding from the site of vascular anastomosis than end-to-end anastomosis.Moreover,sleeve suture had the highest success rate among these 3 techniques.So,we think that sleeve suture is superior to end-to-end anastomosis and cuff technique with regard to vascular grafting in the murine model.2.The mouse model of GVD was established by sleeve method.Serum PCSK9 was detected at 8 weeks after transplantation.The content of PCSK9 in allograft mice was higher than that in isograft mice.At the same time,the transplanted vessels in recipient mice were collected.The PCSK9 in liver,kidney and small intestine were also detected by WB and IHC.The expression of PCSK9 was increased in the transplanted vessels and liver in allografts mice.These results suggest that PCSK9 may play an important role in vascular stenosis in the mouse model of GVD.Conclusion: 1.Sleeve suture is superior to the other 2 anastomosis techniques and should be more used to study graft vessels in the mouse of GVD.2.The level of PCSK9 in serum was significantly increased in allografts mice,and the expression of PCSK9 in transplanted vessels and liver were also increased in allografts mice.Part Ⅱ The role and mechanism of PCSK9 participate in the mouse model of graft vascular diseaseObjective: In this part,we explored that the role and mechanism of PCSK9 participated in the mouse model of GVD.Method: 1.The abdominal aorta of BALB/C(H-2d)mice was used as a donor vessel and transplanted into the PCSK9 knockout mice or the wild-type male C57BL/6(H-2b)mice in the male.At the 2nd week after transplantation,the infiltration of macrophages and T cells was detected by IHC,and the expression of local inflammatory genes was detected by RT-q PCR.2.HE,EVG and Masson staining were performed at the 8th week postoperatively to determine the effect of PCSK9 knock-out on the pathological of the transplanted vessels.3.The BMDMS and VSMCs of WT and PCSK9-/-mice were stimulated by the serum in the mouse model of graft vascular disease.The migration of cells was detected by Transwell assay,and the proliferation of cells was detected by Edu and WB assay.4.VSMCs from WT and PCSK9-/-mice were stimulated with serum from the mouse model of graft vascular disease,and the alteration of MEK,TGF-β/SMAD3,NLRP3 inflammatory corpuscles were detected by WB.The change of signal pathway in vivo was verified by WB and immunohistochemistry.Results: 1.The infiltration of F4/80+ macrophages in the PCSK9-/-group was significantly less than that in the WT group,while the infiltration of CD3+ T cells was not significantly different between the two groups.The m RNA expression of Il1b、Il6、Il18、Ifng、Tgfb、Ccl2、Vcam1 in PCSK9-/-group was lower than that in WT group.2.The ratio of I/M,the area of neointima and the deposition of collagen fibers in PCSK9-/-group were significantly lower than those in WT group.3.PCSK9-/-BMDMs and WT BMDMs had no difference in the number of cell migration and the number of Edu positive cells,but PCSK9-/-VSMCs and WT VSMCs had lower number of cell migration and Edu positive cells than WT mice.4.There was no difference in the activation of TGF-b/SMAD3 signaling pathway between PCSK9-/-VSMCs and WT VSMCs,but the activation of NLRP3 in PCSK9-/-VSMCs was lower than that in WT VSMCs.We also analyzed the changes of signal pathways in the grafts by WB and IHC.The results showed that the expression of NLRP3 and IL-1β in PCSK9-/-mice was significantly lower than that in WT mice.Conclusion: 1.PCSK9 knock-out inhibits graft stenosis,macrophage recruitment and inflammatory factor expression during GVD.2.PCSK9 knock-out inhibits the proliferation and migration of vascular smooth muscle cells.3.PCSK9 knock-out inhibits the activation of NLRP3 inflammatory corpuscles in vascular smooth muscle cells.Part Ⅲ Effects of PCSK9 inhibitors on graft vascular disease in humanized PCSK9 miceObjective: In this part,we mainly studied the effect of PCSK9 inhibitor(Evolocumab)on the humanized PCSK9 mouse model of GVD.Method: 1.The abdominal aorta of BALB/C(H-2d)mice was used as a donor vessel and transplanted into the humanized PCSK9 transgenic mice of C57BL/6(H-2b)background.After operation,the recipient mice were subcutaneously injected with Evolocumab(10mg/kg)or Ig G every 2 weeks.At the 8th week after operation,liver tissues were obtained and LDLR expression was analyzed by WB and IF.2.HE,EVG and Masson staining were performed at the 8th week after operation to determine the effect of PCSK9 inhibitor on the pathology of the transplanted vessels in the humanized PCSK9 mice.Results: 1.The expression of LDLR in liver was higher in PCSK9 inhibitor(Evolocumab)intervention group.2.The ratio of I/M,the area of neointima and the deposition of collagen fibers in PCSK9 inhibitor(Evolocumab)intervention group were significantly lower than those in Ig G group.Conclusion: PCSK9 inhibitor(Evolocumab)can inhibit the degradation of LDLR in the liver of humanized PCSK9 mice and reduce the stenosis of transplanted vessels.