Mechanistic And Translational Studies On The Role Of Sema7A In Atherosclerotic Plaque Formation

Atherosclerosis is a multifactorial chronic inflammatory vascular disease.Along with lipid deposition in the subendothelium of the blood vessels,vulnerable plaque is formed.When plaques rupture,thrombosis and arterial stenosis are formed,causing myocardial infarction(MI),stroke and other severe clinical events.Cardiovascular disease has become the number one cause of death in Chinese residents and atherosclerosis is the main cause account for cardiovascular disease.Therefore,in-depth understanding of the pathogenesis and mechanism of atherosclerosis to strengthen the prevention and treatment of atherosclerosis will contribute to reduction of the incidence of cardiovascular disease and improvement of the health and quality of life of the Chinese.Under normal circumstances,endothelial cells covering the vascular lumen sense the physical,chemical and biological stimuli of blood flow and respond to these stimuli to protect the integrity of blood vessels and maintain vascular homeostasis.When exposed to disturbed blood flow,endothelial cells undergo phenotypic changes,increasing permeability,release of cytokines and adhesion of leukocytes.Changes in these structures and functions of the vascular wall endothelial cells greatly increasing their susceptibility to atherogenesis.However,the underlying molecular mechanisms by which disturbed flow led to changes of intima function so far are not well understood.Semaphorin family,originally discovered as guidance of neural growth,were shown to be involved in many physiological and pathological functions.After we reported that Sema4 D was involved in atherosclerosis,we recently discovered that other family member,Sema7A participates in the development of atherosclerosis.However,the mechanism of Sema7A involvement in atherosclerosis remains unclear.In this study,we found that Sema7A responds to disturbed blood flow and promotes atherosclerosis through interaction with its corresponding receptor ?1?1.In our translational studies,we found that serum Sema7A level is related to acute atherothrombotic stroke,suggesting its potential application as a biomarker in the diagnosis and prevention of related diseases.Objective:Explore the mechanism of axon guidance molecule Sema7A regulating the pathogenesis of atherosclerosis and provide a theoretical basis for the clinical intervention of atherosclerosis.Methods: The project will use a variety of knockout mice,combined with cellular and molecular biology and protein technology to study the mechanism of Sema7A in the pathogenesis of atherosclerosis and explore the effect of axon guidance molecules on the endothelial cell phenotype changes and inflammatory cell infiltration during development of atherosclerosis as well as the role of Sema7A in the translational studies of prevention and treatment of atherosclerosis.1.Regulation of endothelial Sema7A expression,disturbed flow and cell function.(1)Expression pattern of Sema7A in vascular endothelial cells.Previous studies have shown that knockdown of Sema7A significantly reduces lipid deposition in the aorta and formation of atherosclerotic plaques and plaque reduction mainly concentrated in the aortic arch,where disturbed blood flow occurred.We examined Sema7A expression levels in the aortic arch,exposed to disturbed blood flow and in the descending aortic trunk,exposed to laminar blood flow of blood vessels to observe whether the expression of Sema7A has regional preference.To further confirm the regulation of Sema7A expression exactly caused by the flow,we will establish a more purified model of blood disturbance by ligation the carotid artery in vivo and the artificially oscillatory flow model of endothelial cells in vitro.And the demonstration of the relationship between blood disturbance and Sema7A expression pattern will pave the way for the next step in the experimental plan and design.(2)Endothelial Sema7A transcriptional regulation.We found that in the disturbed flow regions,Sema7A m RNA and protein levels in vascular endothelial cells were significantly up-regulated compared to laminar flow regions,suggesting that transcriptional regulation plays an important role in Sema7A expression regulation.Sema7A gene was analyzed by Qiagen database and the promoter region of Sema7A was found to contain many binding sites for transcriptional factors associated with shear flow in bloodstream.It is speculated that these transcription factors may mediate the up-regulation of Sema7A expression under disturbance of blood flow.Thus we will study the regulation of transcription factor changes and effects on Sema7A expression modulation.(3)The effect of Sema7A deletion or overexpression on the regulation of adhesion molecules under disturbed flow conditions.Selectins,adhesion molecules ICAM-1,VCAM-1 are the major adhesion molecules that mediate leukocyte rolling,adhesion and invasion.In the case of disturbed flow,Sema7A deletion or overexpression may affect the main adhesion molecule expression.Thus we will utilize proteomics experiments and ligand-receptor interaction assays for investigation.2.Effect of Sema7A knockout on leukocyte rolling,adhesion and invasion,and atherosclerotic plaque formation.(1)The effect of Sema7A cell-specific knockout on the leukocyte rolling and adhesion mediated by Sema7A and its receptor ?1?1.Cytokines induced inflammatory models were used to detect the rolling and adhesion of leukocytes in the endothelium under inflammatory conditions in vivo and in vitro.And ?1?1-specific blocking antibody and Sema7A knockout cells were used to further confirm that Sema7A and its receptor ?1?1 interaction are inseparable and indispensable during endothelial cell leukocyte rolling adhesion.(2)The bone marrow transplantation and ligation model were used to analyze the plaque development between different knockout mice.Using mice with high-fat diet to build atherosclerosis induction model combined with the partial carotid artery ligation model to observe difference between different sources of Sema7A and atherosclerosis plaque formation and demonstrate roles of different sources of Sema7A on atherosclerosis formation.3.The relationship between Sema7A and clinical acute atherothrombotic stroke(AAS)patients.Since Sema7A participates in the development of atherosclerosis through a series of pathological effects such as the response to disturbed flow and its involvement in leukocyte infiltration,it is necessary to study the application of Sema7A as a target for translational studies.In patients with cerebral or carotid atherosclerosis,rupture of vulnerable plaques may result in acute thrombus formation that blocks blood supply to the brain.This disease,known as AAS,is one of the most common and life-threatening cardiovascular disorders.In order to explore the relationship between Sema7A and clinical acute atherothrombotic stroke.Serum levels of soluble Sema7A were measured in healthy controls and patients with acute atherothrombotic stroke and adjusting for known cardiovascular risk factors and baseline covariates including body mass index,total cholesterol,triglycerides,low density lipoprotein cholesterol,high-density lipoprotein cholesterol,fasting blood glucose,smoking and drinking status to measure whether serum Sema7A level is an independent risk factor for acute atherothrombotic stroke,and the study will provide experimental and theoretical basis for Sema7A application in clinical atherosclerosis treatment.Results:1.CREB is a transcription factor known to respond to shear stress by analysis of in silico promoters and we have identifyed potential binding sites for c AMP response element binding protein(CREB)in the human and mouse Sema7A promoter regions.We found that the expression of endothelial Sema7A is regulated by PKA/CREB signaling and these data suggest that inhibition of PKA/CREB signaling may provide a potential mechanism to regulate flow-induced endothelial Sema7A upregulation.2.Sema7A expression upregulates in the aortic arch and ligation of the left carotid artery,exposed to disturbed flow,significantly up-regulated the expression of adhesion molecules ICAM-1 and VCAM-1.3.Using Sema7A overexpression endothelial cells,we showed that up-regulation of endothelial Sema7A activates focal adhesion kinase signaling pathway(FAK)and downstream NF?B signaling pathway and activate transcriptional transcription of ICAM-1/VCAM-1 through the receptor ?1?1,which promoting the rolling and adhesion of leucocytes.4.Endothelial Sema7A plays a more important role in the formation of atherosclerotic plaque.Using bone marrow transplantation combined with partial carotid artery ligation model,we found that specific knockout of Sema7A in the endothelium significantly reduce atherosclerotic plaque lipid deposition and the size of plaque formation.5.We measured serum levels of soluble Sema7A in AAS patients and in age-and sex-matched healthy controls.Compared with the control group,the serum Sema7A levels in AAS patients were significantly higher compared to controls.After adjusting for known cardiovascular risk factors and baseline covariates(including body mass index,total cholesterol,triglycerides,LDL cholesterol,HDL cholesterol,fasting blood glucose,smoking and alcohol consumption),serum Sema7A levels remained unchanged evident increase in AAS patients.These results suggest that Sema7A is an independent risk factor for acute atherothrombotic stroke and may be a novel diagnostic marker for atherosclerosis treatment.Conclusion:1.Sema7A is up-regulated by disturbed blood flow,mediating endothelial cell interactions through receptor integrin ?1?1,leading to up-regulation of adhesion molecules and endothelial dysfunction,to promote leukocyte rolling adhesion and development of atherosclerosis.2.The level of Sema7A in the serum of patients with clinical acute atherosclerotic thrombotic stroke(AAS)is significantly elevated and is an independent risk factor for AAS.3.Targeting Sema7A may provide an important theoretical basis and a powerful experimental basis for the diagnosis and treatment of atherosclerosis.