Mechanisms Of Smooth Muscle Cell-Specific Knockout Of CD38 Promoting Atherosclerotic Calcification

Background and Objects:Atherosclerosis is a chronic inflammatory vascular disease and an important pathological basis of coronary heart disease.The mortality rate of cardiovascular disease caused by atherosclerosis still ranks first among Chinese residents and the disease is one of the leading killers of human health.Thrombus formation from rupture of unstable atherosclerotic plaques may lead to adverse cardiovascular events（such as myocardial infarction etc.）.Factors that affect plaque stability include the size of the necrotic core,the composition and thickness of the fibrous cap,and others,of which,the presence of microcalcification in the plaque seriously reduces the stability of the plaque.However,there is currently no effective drug to treat vascular calcification and the underlying molecular and cellular mechanisms of regulating vascular calcification are not fully understood.At present,it is generally believed that the differentiation of vascular cells,mainly smooth muscle cell（SMC）,into osteoblast-like cells leading to calcium salt deposition is the root cause of vascular calcification.CD38 is a single-chain type II transmembrane glycoprotein with a molecular weight of 45 k Da,which can participate in cell adhesion,signal transduction and calcium signaling.Previous studies in our group found that macrophage CD38 deletion increased inflammation and aggravated atherosclerosis by promoting macrophage polarization to M1 type.However,the role of CD38,especially smooth muscle cell CD38,in vascular calcification is not clear.Therefore,this project firstly intended to use smooth muscle cell-specific CD38 knockout mice to prepare atherosclerotic calcification model,evaluate the effect of smooth muscle cell CD38 on high-fat diet-induced atherosclerotic calcification and clarify the molecular mechanisms in vivo.Secondly,the aortic ring and smooth muscle cell calcification model would be used in vitro to further reveal the role and molecular mechanisms of SMC CD38 in the development of calcification.Our study will reveal the new function of CD38 in the field of vascular calcification,and provide a new theoretical basis for the prevention and treatment of clinical vascular calcification diseases.Experimental Methods:1.Preparations and analysis of atherosclerotic calcification model in mice:1)Preparation and identification of smooth muscle cell-specific CD38 knockout mice:The CD38^f/f mice and SM22α^cre mice were used to mate to obtain smooth muscle cell-specific CD38 knockout mice and the genotypes of the mice were identified by PCR technology.CD38^f/f SM22α^cre mice were crossed with Apo E^-/-mice to obtain the Apo E^-/-CD38^f/f mice and Apo E^-/-CD38^f/fSM22α^cre mice from littermates,which were used in this study.2)Preparation and analysis of atherosclerotic calcification mouse model:Eight-week-old Apo E^-/-CD38^f/f mice and Apo E^-/-CD38^f/fSM22α^cre mice were fed with a high-fat diet for 16 weeks,and then aortas were collected from the mice.Oil red O staining was performed with the whole aortas and aortic roots to detect plaque formation;Alizarin red S,Picrosirius red and VVG staining were performed with the aortic roots to detect the degree of vascular calcification and plaque stability;and the calcium content was examined with the whole aortas.2.Establishments and analysis of smooth muscle cells and aortic ring calcification model:1)Establishment and analysis of calcification model of smooth muscle cells with overexpressed CD38:Human smooth muscle cells were infected with lentivirus containing CD38 expression vector to construct stable human smooth muscle cell lines with overexpressed CD38.The cell lines were cultured with calcification medium for5 days to establish the calcification model,and the effect of CD38 on calcification was analyzed by Alizarin red S staining and calcium content determination.2)Establishment and analysis of calcification model in smooth muscle cells by inhibiting CD38 activity:Primary mouse smooth muscle cells and human smooth muscle cell lines were cultured in calcification medium for 5 days to establish calcification models under presence of CD38 enzyme activity inhibitor 78-C,and the effect of CD38 on calcification was analyzed by Alizarin red S staining and calcium content determination.3)Establishment and analysis of smooth muscle cell calcification and aortic ring calcification model with smooth muscle-specific knockout of CD38:The thoracic aortas of eight-week-old CD38^f/f mice and CD38^f/fSM22α^cre mice or Apo E^-/-CD38^f/fmice and Apo E^-/-CD38^f/fSM22α^cre mice were isolated to extract smooth muscle cells,or cut into～5 mm long pieces.Then cells were cultured with calcification medium for5 days and the aortic rings were cultured with calcification medium for 14 days to establish calcification model.The effect of CD38 on calcification was analyzed by Alizarin red S staining and calcium content determination.3.Analysis of the mechanisms of CD38 deficiency in smooth muscle cells promoting vascular calcification:The protein levels of RUNX2,BMP2,ALP,OPN,p-Smad1/5/8,LRP6,DKK1 andβ-catenin,and so on,which is the related signaling molecules of calcification-related signaling pathways BMP2-（p-Smad1/5/8）and Wnt/β-catenin were detected in aortic roots or SMCs by Western Blot and immunofluorescence staining to explore the mechanisms of CD38 regulating vascular calcification.Results:1.Smooth muscle cell-specific knockout of CD38 significantly promoted vascular calcification in high-fat diet-induced atherosclerotic calcification model mice.2.Overexpression of CD38 improved smooth muscle cell calcification,while inhibition of CD38 activity promoted smooth muscle cell calcification,and smooth muscle cell-specific knockout of CD38 promoted SMC and aortic ring calcification.3.CD38 in smooth muscle cells inhibited BMP2-（p-Smad1/5/8）and Wnt/β-catenin signaling pathways,reduced the differentiation of smooth muscle cells into osteoblast-like cells and inhibited vascular calcification.4.The expressions of CD38 were decreased in calcification plaques of patients with atherosclerosis.Conclusions:Smooth muscle cell-specific deletion of CD38 significantly promoted the occurrence and development of atherosclerotic calcification in vivo and in vitro.The mechanisms may be related to the inhibition of BMP2-（p-Smad1/5/8）and Wnt/β-catenin signaling pathways,in which CD38 reduced the differentiation of smooth muscle cells into osteoblast-like cells.In addition,the expressions of CD38 were decreased in calcification plaques of patients with atherosclerosis,further confirming that CD38 plays an important role in the development of vascular calcification.