Cdc42 Regulates Vascular Calcification Via AKT Signal

Background:Currently,due to the advancement of human science and technology,the quality of life has been improved and human life has been extended,but this has also brought about a new problem of population aging.Cardiovascular disease has become one of the great factors threatening human health.One of the most fundamental cause of cardiovascular disease is vascular calcification,which is the direct cause of the deterioration of the primary disease and the increase of mortality.Diseases associated with vascular calcification include atherosclerosis,hypertension,and diabetes,especially in chronic kidney disease(CKD).According to statistics,up to 80%of CKD patients have cardiovascular disease due to vascular calcification.Therefore,vascular calcification has become a health concern in the world.Cell division cycle 42,Cdc42,which is a small GTPase belongs to the Rho family.In the past,the understanding of Rho proteins was mainly focused in tumor research field.Rho mediates a series of downstream phosphorylation-dephosphorylation reactions,thereby controlling the biological behaviors of cell migration,adhesion,chemotaxis and contraction,while the role of the Rho family in vascular calcification is not well understood.Objective:This study intends to investigate the role and changes of Cdc42 in vascular calcification and vascular smooth muscle cells(VSMCs)transformation into osteoblast-like cells in vitro and in vivo.Furthermore,it was further illustrated whether Cdc42 regulates vascular calcification and VSMCs to osteoblast-like cell phenotype transformation through activating AKT signaling pathway.This study proves Cdc42 an important target substance byinhibiting vascular calcification,and provides an effective experimental basis for clinically improving and reversing related diseases caused by vascular calcification.Methods:1.Rat aortic vascular smooth muscle cells were cultured and passaged after the cells were overgrown,and calcification was induced through calcifying medium(CM):DMEM containing 10 mM BGP and 3 mM CaC12.The mRNA and protein level Cdc42 was evaluated by qRT-PCR and Western blot,and the changes of Cdc42 in vascular calcification were established.The model of vascular calcification in vitro and in vivo was established.2.Cdc42 was overexpressed by adenovirus transfection technology.The final concentration of infectious virus was determined by pre-experiment.The rat aortic vascular smooth muscle cells were infected according to the pre-experimental results.The expression of Cdc42 was detected by Western blot.Alizarin red staining technique and calcium content were used to determine the role of Cdc42 in vascular calcification。3.Male 8-week-old SD rats(body weight 220g-250g)underwent right nephrectomy and 2/3 left renal artery ligation to construct CKD model,and was further divided into groups based on serum creatinine level(two weeks after operation,blood collection,plasma creatinine),so as sham operation group,surgical model group and Cdc42 inhibitor ML141 treated group.Immunohistochemical paraffin sections were used to observe changes in vascular calcification phenotype;calcification was confirmed by detecting relative calcium content;qRT-PCR and Western blot were used to detect the expression of bone-related transcription factors(BMP2,Runx2)in different groups,and further explain the relationship between Cdc42 and vascular calcification.Results:1.By constructing an in vitro and in vivo model of vascular calcification,it was observed that the expression level of Cdc42 in vascular smooth muscle cells was significantly up-regulated in the calcified medium(CM)group compared with the normal medium(GM)group(P<0.05).In vivo experiments also showed that the expression level of Cdc42 protein in the surgical model group also displayed an increasing trend compared with the Sham group(P<0.01).2.Adenovirus overexpressing Cdc42,was used to examine the role of Cdc42 in vascular calcification.Alizarin red staining and relative calcium content showed that mineral deposition was increased compared with the virus-negative control group(P<0.05);the expression levels of bone-related factors(BMP2,Runx2)were also increased(P<0.05).3.The Cdc42 inhibitor ML141 was used to determine the role of Cdc42 in vascular calcification.The calcification test group containing Cdc42 inhibitor ML141 was observed thatcompared with the calcification test group without Cdc42 inhibitor ML141,the alizarin red staining and relative calcium content test results were both decreased(P<0.01);the expression level of bone-related transcription factors(BMP2,Runx2)was lower in the calcification experimental group containing Cdc42 inhibitor ML141(P<0.05).The above results were further confirmed in the ex vivo experiment using vascular ring.4.The expression level of AKT was detected by different experimental models and experimental methods,indicating that the expression level of AKT was positively correlated with Cdc42,suggesting Cdc42 is to promote vascular calcification by activating AKT signaling pathway.5.Using AKT inhibitors to observe changes in vascular calcification phenotype successfully verified the crucial role of AKT signaling pathway in vascular calcification.Conclusion:1.High calcium and phosphate displayed a distinct ability in increasing the expression of Cdc42 in VSMCs and CKD rats.2.Overexpressed Cdc42 can promote calcification of VSMCs.3.inhibition of Cdc42 can reduce vascular calcification in VSMCs and CKD rats.4.AKT signaling mediates Cdc42-induced vascular calcification.