Multi-omics Based Research Of Molecular Mechanisms Underlying Endothelial Injury Under Oxidative Stress Induced By Oxidized Low-density Lipoprotein

Atherosclerosis causes cardiovascular and cerebrovascular diseases through the slow progressive lesion formation and arterial lumen narrowing.When plaque rupture and thrombosis formation,the common cardiovascular diseases present as acute coronary syndrome,myocardial infarction or stroke.Atherosclerosis is a chronic inflammation of the arterial wall,which is initialized by endothelial injury and dysfunction.The increased circulating cholesterol transported by low-density lipoprotein(LDL)-containing apolipoprotein promotes atherosclerosis and cardiovascular diseases.LDL particles remain in the intima after apolipoprotein binds to negative proteoglycans in the extracellular matrix,and is more easily subjected to oxidization by reactive oxygen species or enzymes released by inflammatory cells.Ox-LDL induces the expression of adhesion molecules and the secretion of endothelial chemokines,driving the infiltration of intima immune cells along with the deposition of platelet chemokines.The deposition of LDL on the arterial wall is a hallmark of atherosclerotic lesion.The elevated oxidative stress leads to LDL oxidation.Ox-LDL is involved in the atherosclerosis pathology through various mechanisms including impairing endothelial function,which is an early event of atherosclerosis.Nitric oxide(NO)plays a significantly role in vascular homostasis.One of the maj or factors for endothelial injury is the decreased NO synthesis and/or bioavailability Ox-LDL consists of various reactive aldehydes and lipid peroxides with strong oxidative ability.Its triggered oxidative stress seriously damages endothelial cells including abnormal NO synthesis and bioavailability.Protein S-nitrosylation is a ubiquitous and dynamic post-translational modification(PTM).This modification is not only involved in NO signal transduction,but also closely related to the cellular redox microenvironment.Ox-LDL was reported to impair the functional structures of biological macromolecules,particularly affecting the PTMs such as phosphorylation and ubiquitination,thus seriously interfering with cell signaling pathways.However,the effects of ox-LDL on some PTMs that are more directly related to the cellular redox state,for example,the S-nitrosylation,remain largely unknown.The glycocalyx coats healthy vascular endothelium and plays an important role in vascular homeostasis.The glycocalyx is mainly composed of glycoproteins and proteoglycans,which contains a core protein and covalently linked glycosaminoglycans(GAGs).GAGs are linear and negative charged polysaccharides consisted of repeating disaccharide units with different sulfated modes.The negative property allows GAGs to attract positive plasma proteins and growth factors,on the other hand,protect endothelial cells from being attacked by risk factors.Therefore,glycocalyx plays an important role as a barrier and molecular sieve.GAGs participate in cellular activities through regulating the structure and activity of their binding proteins.Distributed in the endothelial cell surface,the glycocalyx are sensitive to external stimulus.The leading GAG component of endothelia glycocalyx is heparan sulfate(HS),which accounts for 50%-90%.Though previous studies have demonstrated ox-LDL degraded the glycocalyx,no report is available for the structural and quantitative alterations of HS.Both carbohydrates and proteins are vital biomacromolecules in vivo.Proteins are responsible to perform biological functions.Carbohydrates serving as energy and structure substances are well involved in cell construction and signal transduction.The coupling of liquid chromatography and tandem mass spectrometry(LC-MS/MS)is featured by robust separation,excellent sensitivity and high resolution.LC-MS/MS is widely applied in sequencing,structure characterization,interaction detection,as well as qualitative and quantitative omics analysis.Bioinformatics is indispensable for data interpretation and mining in MS-based proteomics research,aiding in-depth understanding and efficient application.The "bottom-up" strategy for GAGs analysis refers to degrading intact polysaccharides into oligosaccharides by chemical or enzymatic methods,and characterizing oligosaccharide fingerprinting by LC-MS/MS.This study focused on elucidating molecular mechanisms underlying endothelial injury exerted by ox-LDL using human umbilical vein endothelial cell line EA.hy926.Relied on quantitative LC-MS/MS technique,this work was performed from the multi-omics perspective including proteome,S-nitrosoproteome,HS and heparan sulfate binding proteins(HSBPs),which have not been covered in previous research.Proteome study revealed the protein basis of endothelial dysfunction,and provided quantification information for S-nitrosylated proteins,making the attention on abnormal S-nitrosylation modification itself possible.On the other hand,proteome data was also the source of key HSBPs screening.This thesis enriches and extends the present understanding of endothelial injury under oxidative stress and the pro-atherogenic effect of ox-LDL,facilitating a promising direction and feasible workflow for future clinical samples in cardiovascular diseases.The main achievements of this thesis are as follows:1.Quantitative proteome analysis of endothelial cells under oxidative stress induced by ox-LDLThe combined iTRAQ approach and high-resolution mass spectrometry was applied and revealed a total of 260 differentially expressed proteins.With the help of bioinformatic tools,GO annotation and interaction network were performed to dig out the underlying significance.These significantly regulated proteins by ox-LDL were primarily distributed in extracellular,cell membrane and vesicular.With enzyme activity,receptor activity and glycoaminoglycan binding ability,the 260 abnormal expressed proteins participated in external stimulation response,wound healing,secretion,migration and some other important cellular activities,In the protein interaction network analysis,interaction involved in transcription,translation and platelet degranulation were sensitive to ox-LDL stimulus.This work studied the proteome basis behind endothelial injury.2.Effect of ox-LDL on S-nitrosylation related enzymes in endothelial cellsProtein S-nitrosylation is a reversible oxidative post-translational modification.This modification is not only an important NO signaling pathway but also closely related to cellular redox microenvironment.The physiological S-nitrosylation level is maintained by the balance between nitrosyaltion and denitrosylation.From western blot analysis,eNOS responsible for nitrosylation showed downregulated expression,and the expression of GSNOR responsible for denitrosylation were more slightly decreased.This imbalance explained why the whole S-nitrosylated protein level decreased under ox-LDL treatment.3.Quantitative S-nitrosoproteome in endothelial cells under oxidative stress induced by ox-LDLA quantitative S-nitrosoproteome workflow was established by "in solution"combination of iTRAQ and biotin switch technique.We discovered the reactivity of cysteines to iTRAQ reagents.The focus was the change of S-nitrosyaltion modification itself,realized by introducing a formula to exclude alteration caused by protein expression.Notably,the entire translation process was covered including initiation,elongation and termination.The 262 abnormal S-nitrosylated proteins were well involved in mRNA splicing and translation,suggesting their S-nitrosylation was susceptible to oxidative stress induced by ox-LDL.The interaction analysis and molecular docking simulation indicated S-nitrosylation was an important regulation mechanism for protein interaction.4.Structural characterization and quantification analysis of heparan sulfate in endothelial glycocalyx under oxidative stress induced by ox-LDLThe most abundant GAG of endothelial glycocalyx is HS.The negative charge reserves positive plasma proteins and growth factors,and also serves as a molecular sieve by repelling adverse molecules to protect endothelium.Located on the cellular surface,glycocalyx would be initially disturbed by external stimuli.The basis of this research section was fluorescent signal of HS on the surface of endothelial cells became much denser after ox-LDL treatment.In multi-reaction monitoring(MRM)MS analysis,the content of HS decreased by 50%compared with the control group.However,the number of sulfo groups per disaccharide increased regardless of N-sulfo and O-sulfo.PAGE was used to examine the molecular weight alteration,suggesting a wider molecular weight range and increased low molecular weight components under ox-LDL stress.In conclusion,ox-LDL indeed exerted adverse effect on the structure and content of HS in endothelia cells.5.Heparan sulfate binding proteins analysis in endothelial cells under oxidative stress induced by ox-LDLGAGs are generally involved in the cellular activities through regulating their protein partners.We screened potential HSBPs with important effects based on proteome data and fluorescent signal on proteome chip.Literature retrieval helped to find several consensus sequences for HSBPs.Through consideration of chip signal,consensus sequence and extracellular location,as well as BLI verification,a novel workflow was set up for candidate HSBPs discovery from quantitative proteome.According to the functional enrichment and interaction network,AHSG as a key HSBP was chosen for further investigation.Sulfate groups on various positions of HS differentially contributed to the binding with AHSG.From the immunocolocalization assay,HS facilitated the endocytosis of AHSG into endothelial cells,a possible explain for decreased serum AHSG during atherosclerosis.Efforts were made to investigate the non-anticoagulant heparin during endothelial injury by examining its influence on cell apoptosis induced by ox-LDL.The inhibited apoptosis implied non-anticoagulant components of heparin may be promising to intervene or alleviate atherosclerosis development caused by ox-LDL.