Effects Of YAP In Vascular Calcification And The Underlying Mechanisms Exploration

BackgroundThe incidence rate of calcification is rising continuously with the change of the living style and the aging of the mouth.It is a common pathological condition of atherosclerosis,diabetes,end-stage renal disease and aging,and exacerbates cardiovascular related death.Current researches confirm that the prevalence of vascular calcification and the incidence of cardiovascular events are two important characteristics of chronic kidney disease(CKD).Most observational studies have found a positive correlation between CKD and vascular calcification.Dysregulation of mineral metabolism and elevated levels of circulating calcium(Ca)and phosphate(Pi)are key factors in the occurrence and progression of vascular calcification in CKD.Therefore,active prevention and treatment of calcification is of great importance in reducing the incidence of adverse events in patients with chronic kidney disease and diabetes.However,active dialysis can not delay the development of canal calcification.Drugs for the treatment of chronic renal failure and cardiovascular diseases have not been found to effectively inhibit vascular calcification.Although there have been many clinical studies on the effect of vascular calcification on the prognosis of the disease,the basic research on the mechanism of cells and division in the process of the occurrence and progression of vascular calcification is only at the beginning,and there is an urgent need for further basic research.Studies have shown that the occurrence of vascular calcification is not a simple passive calcium deposition,but a complex regulatory process with multiple mechanisms and signal pathways.These include(1)Phenotypic transformation of vascular smooth muscle cells,which will lose the original contraction phenotypic markers,like Calponin,and express osteogenic phenotypic markers,like RUNX2,etc.(2)Apoptosis and release of matrix vesicles.;(3)Imbalance of protein balance between promoting calcification and inhibiting calcification;(4)Autophagy;(5)Oxidative stress,endoplasmic reticulum stress,etc.YAP is the direct downstream effector of Hippo pathway.It can be used as a transcriptional co-activator to interact with transcriptional factors such as TEAD and RUNX2 to regulate the expression of target genes.It is the main regulator of multiple organ development and volume control.Recent studies have found that Hippo-YAP signaling pathway may be involved in the pathological process of bone formation and osteogenic differentiation.YAP can act as a co-inhibitory factor of RUNX2 in response to Src stimulation and inhibit the osteogenic differentiation of mesenchymal cells.YAP can also participate in classical signaling pathways of osteogenic differentiation such as Wnt pathway and promote osteogenic differentiation.Since YAP is related to the process of osteogenic differentiation,and the occurrence of vascular calcification is similar to the process of bone formation,we speculate that YAP may be involved in the regulation of vascular calcification.Autophagy refers to the process that cells degrade misfolded cytoplasm,aging organelles and other intracellular wastes for energy reuse.Studies have shown that under the stimulation of high phosphorus,autophagy as a protective response will increase significantly,and the increased autophagy can inhibit the calcification of vascular smooth muscle cells induced by high phosphorus by inhibiting the release of matrix vesicles.At present,the research on the relationship between YAP and autophagy mainly focuses on the field of tumor.In human ovarian cancer cells,YAP can induce cisplatin resistance by enhancing autophagy flow,and then enhance the proliferation,invasion and migration of these cells;In glioblastoma,YAP can promote autophagy by promoting the up regulation of HMGB1 and promoting its translocation from nucleus to cytoplasm.The relationship between YAP and autophagy is less studied in vascular calcification,so it is of great significance to study the relationship between YAP and autophagy in vascular calcification.In conclusion,this experiment established a vascular calcification model in vivo and in vitro to explore the role of Yap in vascular calcification and its mechanism in vascular calcification,so as to provide basis for the treatment of vascular calcification in CKD.Purpose(1)To study the expression of YAP in vascular calcification model of rats with chronic renal failure and vascular smooth muscle cell calcification model,and its effect on calcium salt deposition and the expression of osteogenic phenotypic markers in vascular calcification.(2)To study the regulatory effect of YAP on autophagy:to determine whether YAP is involved in the regulation of vascular calcification through autophagy.(3)To study the molecular mechanism of YAP regulating autophagy:to clarify whether there is a direct effect between YAP,Bcl2 and Beclin1.Methods(1)To establish a rat model of vascular calcification in chronic renal failure:The rat model of chronic renal failure vascular calcification was established by intragastric administration of adenine for 8 weeks.YAP lentivirus vector was constructed,and 6-week-old SD male rats were injected with YAP lentivirus through caudal vein.Two weeks later,the virus was expressed and intragastric administration of adenine was used for 8 weeks.(2)Echocardiographic left ventricular ejection fraction(LVEF)and left ventricular short axis shortening(LVFS)were measured dynamically.(3)At the end of the experiment,the body weight was measured,and the blood was taken from the cardiac apex of anesthetized rats.The thoracic aorta and abdominal aorta were retained and sub packed for follow-up experiments.(4)Detect vascular calcification and vascular morphology:Alizarin red staining and von Kossa staining were used to detect calcium salt deposition,and VVG staining was used to observe the structure of vascular elastic plate.(5)Detection of VSMC phenotype conversion in vascular tissue:The expression levels of osteogenic phenotypic markers Opan,RUNX2 and contractile phenotypic marker Calponin were detected by Western blot.(6)Detect the autophagy of VSMC in vascular tissue:The expression levels of autophagy related proteins ATG5 and LC3 were detected by Western blot.(7)Establish vascular smooth muscle cell calcification model:A7r5 smooth muscle cell line was selected and the smooth muscle cells were stimulated with 3.25mmol/l Pi for 6 days to construct the calcification model of vascular smooth muscle cells in vitro.(8)Construction of adenovirus vector overexpressing YAP:The YAP overexpression adenovirus vector was constructed and transfected into rat vascular smooth muscle cell line(A7r5)with empty vector as control to verify the overexpression effect of YAP overexpression adenovirus.(9)Establishment of silent YAP model:(10)Si-YAP sequence was constructed and transfected into rat vascular smooth muscle cells(A7r5)with Si-NC as control to verify the silencing effect of YAP.(11)Detection of YAP content and subcellular localization in vascular smooth muscle cells:The content and subcellular localization of YAP in smooth muscle cells were detected by Western blot.(12)Detect calcification of vascular smooth muscle cells:Calcium deposition in vascular smooth muscle cells was observed by calcium ion detection and alizarin red staining.(13)Detection of phenotype conversion of vascular smooth muscle cells:The expression levels of osteogenic phenotypic markers(BMP2,RUNX2)and contractile phenotypic markers(Calponin)were detected by Western blot.(14)Detection of autophagy of vascular smooth muscle cells:The expression levels of autophagy related proteins ATG5 and LC3 were detected by Western blot.The expression level of LC3 was detected by immunofluorescence.The changes of osteogenic phenotypic marker RUNX2 of vascular smooth muscle cells were detected after using autophagy inhibitor 3-methyladenine(3-MA).(15)Detect the binding of YAP,Bcl2 and Beclinl and the ubiquitination level of Beclinl:The binding of YAP,Bcl2 and Beclinl and the ubiquitination level of Beclinl were detected by immunocoprecipitation.Results(1)In vivo experiments:1)The vascular calcification model of CKD rats was established.CKD rats had significant media membrane calcification,calcium salt deposition in vascular media tissue and the expression of VSMC osteogenic phenotypic markers RUNX2 and OPN increased.2)The expression of YAP decreased in vascular media tissue and heart tissue of CKD rats.3)A lentiviral vector(LV-YAP)overexpressing YAP was constructed.LV-YAP increased the expression level of YAP in rats.4)Overexpression of YAP inhibited calcium deposition in vascular media tissue of CKD rats and the expression of osteogenic phenotypic markers RUNX2 and OPN of VSMC,and promoted the expression of contractile phenotypic marker Calponin.5)In CKD rats,the expression of VSMC apoptosis marker Cleaved-caspase3 increased,and overexpression of YAP significantly decreased the expression of apoptosis marker Cleaved-caspase3.6)The expression of VSMC autophagy markers ATG5 and LC3 Ⅱ increased in CKD rats.Overexpression of YAP further increased the expression of autophagy markers LC3 Ⅱ.(2)In vitro experiment:1)Calcification model of VSMC was constructed.When stimulated by high phosphorus,the expression of osteogenic phenotypic markers RUNX2 and BMP2 of VSMC increased,and the expression of contractile phenotypic marker Calponin decreased.2)Adenovirus vector(Ad-YAP)overexpressing YAP was constructed.Ad-YAP increased the expression level of YAP in VSMC.3)Overexpression of YAP inhibited the calcium deposition of VSMC and the expression of osteogenic phenotypic markers RUNX2 and BMP2,and promoted the expression of contractile phenotypic marker Calponin.4)A silent YAP model was constructed.Silent YAP promoted the calcium deposition of VSMC and the expression of osteogenic phenotypic markers BMP2.5)After high phosphorus stimulation,the expression of apoptosis markers Cleaved-caspase 3 and Bcl2/Bax in VSMC increased,and overexpression of YAP significantly decreased the expression of apoptosis markers cleaved caspase 3 and Bcl2/Bax.6)After high phosphorus stimulation,the expression of autophagy markers ATG5 and LC3 Ⅱ in VSMC increased,and overexpression of YAP further increased the expression of autophagy markers ATG5 and LC3 Ⅱ.7)After stimulation of YAP overexpressing VSMC with autophagy inhibitor 3-methyladenine(3-MA),the expression level of osteogenic phenotypic marker RUNX2 increased compared with YAP overexpression group.8)After high phosphorus stimulated VSMC,the binding of Beclin1 to YAP increased,and the binding of Beclinl to Bcl2 decreased.After overexpression of YAP,the binding of Beclinl to Bcl2 further decreased.9)After high phosphorus stimulated VSMC,the ubiquitination level of Beclinl increased.Overexpression of YAP further increased the ubiquitination level of Beclin1..Conclusion(1)The expression of YAP was down regulated in the CKD rats calcification model of heart and vascular media tissues and the vascular smooth muscle cells calcification model.(2)Overexpression of YAP can inhibit calcification of vascular media tissue and vascular smooth muscle cells.(3)YAP can affect the combination of Beclinl and Bcl2 by affecting the ubiquitination of Beclinl,then affect autophagy,finally affect vascular calcification.