Comparative Proteomics Study Of Carotid Atherosclerotic Stable And Unstable Plaques

Objective: Atherosclerosis is a progressive, mutifactorialartery diseasewhich histological is complex and diverse. The pathological manifestationsinclude intimal thickening, artery stenosis, even cause fatal coronary occlusion,myocardial infarction or cerebral infarction. Carotid atherosclerosis is a riskfactor for ischemic cerebrovascular disease, Carotid artery stenosis caused byplaque, hemodynamic changes and emboli formation of plaque off are themain mechanism for cerebral ischemia. Comparative proteomics focuses onthe changes and differences of proteomics under different conditions aiming toprovide the basis for revealing certain biological phenomena and discipline.By comparative proteomics studies, we can have a complete and clearunderstanding of the protein regulatory networks for the occurrence anddevelopment of disease, even provide strong evidences for furthercomprehension of the mechanism. Past studies have demonstrated thatatherosclerotic plaque instability is mainly related to inflammation and metalprotease secretion. The physiopathologic mechanism is not clear. Therefore,understanding the differences between stable plaque and unstable plaque onthe protein expression level is essential. The results can help us comprehendthe pathophysiology of atherosclerosis and the mechanism of unstable plaquegenesis and development. At the same time, it can provide reliable basis foridentifying early biomarkers of atherosclerosis and looking for new drug target.In this study, by applying comparative proteomics to atheroscleroticplaque.We have isolated and identified a panel of proteins that are differentlyrepresented in stable and unstable plaque so as to deeply understandatherosclerosis pathogenesis and provide a new direction for the treatment ofatherosclerosis.Methods:1Specimen and clinical data collecting： We collectedatherosclerotic plaques undergoing carotid endarterectomy at department of Vascular Surgery, Second Hospital of Hebei Medical University fromMar.2010to Nov.2012.We collected clinical data, including gender, age, pasthistory, results of carotid artery ultrasound (intimal thickness, plaque echointensity, artery stenosis, etc.) Selected cases must meet the following criteria:the same gender, age from60to70years old, no underlying disease, rate ofcarotid artery stenosis is over70%, the past six months there have beenmanifestations of cerebral ischemia.2According to the standard ofAmerican Heart Association (AHA), the plaques were distinguished as stableplaque group and unstable plaque group.3Extraction of protein fromspecimen, FASP digestion, extraction of peptides, desalination, The proteinswere identified using Lable-Free quantitative combined with LC-MS/MS.4Western blot to verify the expression level of protein DJ-1.Results:1All of the cases were divided into2groups,3cases in stable plaquegroup,3cases in unstable plaque group.2A total of63proteins were identified by Lable-Free quantitativecombined with LC-MS/MS. Compared to unstable plaques,stable onesshowed reduced abundance of: Procollagen-lysine, Collagen alpha-1(XXI)chain, Protein Z-dependent protease inhibitor, Growth arrest-specific protein6,Sulfhydryl oxidase1,Coagulation factor X, Collagen alpha-1(I) chain,Complement C1q subcomponent subunit A, Glia-derived nexin, Collagenalpha-2(I) chain,Alpha-2-antiplasmin,Collagen alpha-1(VI) chain, Collagenalpha-2(VI) chain, Collagen alpha-3(VI) chain, Kallistatin, Ribonuclease4,Fibrillin-1,Metalloproteinase inhibitor3,Insulin-like growth factor-bindingprotein complex acid labile subunit,Hemoglobin subunitgamma-2,Carboxypeptidase B2,N-acetylmuramoyl-L-alanine amidase, PTBdomain-containing engulfment adapter protein1,Macrophage metalloelastase,Lysozyme C.In stable plaques the more abundant proteins were: Proactivatorpolypeptide, Calnexin,Aquaporin-1, Protein disulfide-isomerase A6,Alpha-parvin,40S ribosomal protein SA, CD44antigen, Alpha-crystallin Bchain, Chloride intracellular channel protein1, Retinal dehydrogenase1NADH-cytochrome b5reductase3, Coagulation factor XIII A chain, Myosin light chain1/3,Galectin-1Ribonuclease inhibitor Alcohol dehydrogenase[NADP(+)]Protein S100-A4,Cytosol aminopeptidase Coronin-1A,Rab GDPdissociation inhibitor alpha,14-3-3protein beta/alpha, Transaldolase,Macrophage-capping protein, Rho GDP-dissociation inhibitor2,ProteinS100-A10,Actin-related protein2,14-3-3protein gamma,14-3-3proteinepsilon, Tropomyosin alpha-4chain, Clathrin heavy chain1,14-3-3protein eta,Neutral alpha-glucosidase AB, Phosphoglucomutase-like protein5,Plectin,Lipoma-preferred partner, Cytosolic non-specific dipeptidase,Protein DJ-1,PDZ and LIM domain protein7. All of the proteins wereanalyzed by GeneCodis3.The63proteins are related to apoptotic,celladhesion,proteolysis,blood coagulation,aging, carbohydrate metabolic process,signal transduction etc.3For protein DJ-1,the increased levels stable versus unstable plaques wasconfirmed by Western blot analysis.Conclusions:1By applying proteomics to human carotid artery plaque extract, wehave identified a panel of proteins that are differently represented in stable andunstable plaques. They are referring to apoptotic, cell adhesion, proteolysis,blood coagulation, aging, carbohydrate metabolic process, signal transductionetc. These proteins may be involved in the pathogenesis of AS and plaquesinstability, laying the foundation for further scanning of exploring thedisease-specific markers and intervention targets.2Western blotting was used to confirm different levels of protein DJ-1, inorder to lay the foundation for studying the proteins participation in functionnetwork.