Ⅰ.Commutative Regulation Between Endothelial NO Synthase And Insulin Receptor Substrate 2 By MicroRNA Ⅱ.Cartilage Oligomeric Matrix Protein Inhibits EC Activation Through Interaction With Integrin α5

Part Ⅰ Commutative regulation between endothelial NO synthase and insulin receptor substrate 2 by miRNAssThe vascular endothelium,as a selectively permeable barrier between bloodstream and vessel wall,maintains the integrity of the blood vessel wall,responses to various mechanical and chemical stimuli from the circulating blood and produces a variety of cytokines,growth factors and other bioactive substances.It plays a key role in regulating cardiovascular function.Further,endothelium is the main site of inflammatory response.Endothelium dysfunction is an early stage of the development of various cardiovascular diseases such as atherosclerosis.Endothelial NO synthase(e NOS)which is expressed in endothelial cells(ECs)mediated nitric oxide(NO)releasing can expand blood vessels,regulate blood pressure,affect platelet aggregation,and inhibite smooth muscle cell proliferation.The lower bioavailability of e NOS-derived NO is one of the major indicators and mediators of endothelial dysfunction.Competitive endogenous RNAs(ce RNAs)reveal a new mechanism of the interaction of RNAs and represent a new mode for the regulation of gene expression introduced by Pier et al in 2011.RNA transcripts can crosstalk with and co-regulate each other using micro RNA response elements(MREs).mi RNAs are able to regulate the abundance of target genes,which can cross-talk with each other by competing for their shared mi RNAs.Ce RNA analysis tremendously expands functional information of coding and non-coding RNAs.Systemic ce RNA network analyse plays a role in biological and pathophysiological processes.eNOS expression is regulated by a number of transcriptional and posttranscriptional mechanisms,but the effects of competing endogenous RNAs on e NOS m RNA and the underlying mechanisms are still unknown.Our bioinformatic analysis revealed 3 highly-expressed e NOS-targeting mi RNAs(mi R-15 b,-16 and-30b)in human ECs.Among the 1103 m RNAs targets of these 3 mi RNAs,15 m RNAs share a common disease association with e NOS.Genes expression and correlation analysis in patients with cardiovascular diseases identified a significant correlation between e NOS and 7 of the predicted ce RNAs(insulin receptor substrate 2[IRS2],insulin receptor substrate 1[IRS1],beta-2 adrenergic receptor [ADRB2],C-reactive protein [CRP],neurogenic differentiation 1 [NEUROD1],solute carrier family 30 Member 8 [SLC30A8] and ubiquitin-conjugating enzyme E2 [UBE2E2]),among which insulin receptor substrate 2(IRS2)as the most correlated e NOS ce RNA.The expression of e NOS and IRS2 were coincidentally increased by application of laminar shear and reduced with e NOS or IRS2 si RNA transfection in human ECs,which was impeded by Dicer si RNA treatment.Moreover,luciferase reporter assay showed that the 3 mi RNAs directly target the 3’-UTR of e NOS and IRS2.Overexpression of the 3 mi RNAs decreases,while inhibition of them increases m RNA and protein levels of e NOS and IRS2.Functionally,silencing e NOS suppressed the AKT signal pathway,while IRS2 knockdown reduced NO production in ECs.In conclusion,we predicted the ce RNAs for e NOS and identified insulin receptor substrate 2(IRS2)as a major mi RNA-mediated ce RNA in human ECs and arteries.In ECs,the expression of e NOS and IRS2 regulating each other depended on mi RNAs via ce RNA crosstalk.Thus,our study provides a new regulation mode of e NOS and uncovers a new mechanism of the coincidence of complex cardiovascular diseases,such as hypertension and diabetes.Part Ⅱ Cartilage Oligomeric Matrix Protein inhibits EC activation through interaction with integrin α5Cardiovascular diseases(CVDs)such as atherosclerosis remain a leading cause of morbidity and mortality in the world.Endothelium is the main site of inflammatory response.Endothelium activation is an early stage of the development of various CVDs such as atherosclerosis.Increasing the understanding of the pathogenesis of endothelial cell(EC)activation may provide novel therapeutic targets to improve their prevention and treatment.Recently,many studies have proved cartilage oligomeric matrix protein(COMP)plays a crucial role in cardiovascular system.However,whether COMP plays a role in EC activation and the underlying mechanism is largely unknown.Therefore,our study aims to prove the function of COMP in EC activation.Cartilage oligomeric matrix protein(COMP),also known as thrombospondin-5(TSP-5),is a matricellular protein that is abundantly expressed in both cartilage and the cardiovascular system.Many studies have identified COMP as playing critical roles in maintaining cardiovascular homeostasis.COMP,expressed and produced by vascular smooth muscle cells(VSMCs),maintains VSMC contractile phenotypes.COMP deficiency enhances VSMC migration and aggravates VSMC calcification and atherosclerosis.Giving that the critical role of EC activation in CVDs and the protective role of COMP in cardiovascular system,we explore the role of COMP in EC activation and the underlying mechanism.Under normal physiological conditions,compared with WT mice,the expression of VCAM-1 and ICAM-1,markers for EC activation,was significantly increased in COMP-/-mice,especially in the inner curve of the vessel bow.Similarly,the protein levels of VCAM-1 and ICAM-1 enhanced in COMP knockout mice(COMP-/-)and decreased in smooth muscle cell specific COMP transgenic mice(SMC-COMP Tg).We also prove the phenomenon pathologically via left carotid artery partial ligation assay.In vitro,we confirmed that COMP can inhibit endothelial activation caused by oscillatory shear stress(OSS).Integrins,a large class of cell membrane receptor proteins,consists of 2 subunits,α and β that can form heterodimers through non-covalent bonds.Integrins can bind to extracellular matrix and mediate cell-cell cell-extracellular matrix interaction.Transmembrane signals are transmitted in both directions from “inside out” or “outside in”.Endothelial activation relevant integrins were α3β1,α6β1,α5β1,αvβ3,and αvβ5.In this study,we verified that COMP as an extracellular matrix can interact with integrin α5 through its TYP3 and C-terminal domains.Furthermore,in vivo experiments have demonstrated that the interaction of COMP and integrin α5 inhibited the activation of integrin α5 and the phosphorylation of downstream FAK and Src caused by fibronectin(FN)and OSS.Finally,we synthesized a short peptide containing 24 amino acids from the binding sequence of COMP based on the interaction of COMP and integrin α5.Both in vitro and in vivo experiments confirmed that this short peptide can act as an anti-atherosclerosis mediator by inhibiting EC activation induced by integrin α5.In summary,this study proved that the extracellular matrix protein COMP can inhibit the EC activation by interact with integrin α5 through its TYP3 and C-terminal domains.The synthesized short peptide from COMP sequence may act as an anti-atherosclerosis mediator by inhibiting EC activation.Thus,this study reveals a novel mechanism of the protective role of COMP in EC activation and provides a potential target for the treatment of atherosclerosis.