A Proteomic Focus on the Alterations Occurring at the Human Atherosclerotic Coronary Intima

Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries. Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries. Coronary heart disease remains the major cause of mortality in developed countries. In particular, coronary atherosclerosis is the responsible for the majority of the acute coronary syndromes. In the recent years, the understanding of atherosclerosis has experienced a drastic shift, because advances in basic research have pointed out the role of inflammation and the underlying cellular and molecular mechanisms that contribute to atherogenesis (1Libby P. Inflammation in atherosclerosis.Nature. 2002; 420: 868-874Crossref PubMed Scopus (6972) Google Scholar, 2Ross R. Atherosclerosis–an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19274) Google Scholar). Intimal thickening produced by the migration of vascular smooth muscle cells (VSMCs) 1The abbreviations used are:VSMCvascular smooth muscle cell2-DEtwo-dimensional electrophoresisACBatherosclerotic coronary biopsyANXA4annexin A4CANatherosclerotic coronary necropsyECendothelial cellECMextracellular matrixFLCferritin light chainH 8: 1249-1256Crossref PubMed Scopus (720) Google Scholar), in response to minimal endothelium injury often produced by a disturbance in the pattern of blood flow at bending points and near bifurcations of the arterial tree (4Fuster V. Fayad Z.A. Badimon J.J. Acute coronary syndromes: biology.Lancet. 1999; 353: S115-S119Google Scholar). Atherosclerosis initiates at this locations with circulating leukocytes recruitment because of vascular endothelium alteration, which triggers the expression of adhesion molecules (VCAM) (3Dzau V.J. Braun-Dullaeus R.C. Sedding D.G. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.Nat. Med. 2002; 8: 1249-1256Crossref PubMed Scopus (720) Google Scholar). Monocytes differentiate into macrophages within the arterial intima where they phagocyte lipids, finally turning into foam cells (5Virmani R. Kolodgie F.D. Burke A.P. Farb A. Schwartz S.M. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3244) Google Scholar). Lipid accumulation within the intima leads to the formation of a central lipid core that is surrounded by a fibrous cap generated by migrated VSMCs (6Libby P. Aikawa M. Stabilization of atherosclerotic plaques: new mechanisms and clinical targets.Nat. Med. 2002; 8: 1257-1262Crossref PubMed Scopus (498) Google Scholar). vascular smooth muscle cell two-dimensional electrophoresis atherosclerotic coronary biopsy annexin A4 atherosclerotic coronary necropsy endothelial cell extracellular matrix ferritin light chain hemtoxilin/eosin staining immunohistochemistry laser microdissection laser microdissection and pressure catapulting microfibril-associated glycoprotein 4 myosin regulatory light 2, smooth muscle isoform osteoglycin preatherosclerotic coronary necropsy preatherosclerotic radial biopsy serum amyloid protein. Proteomic analysis of plasma, circulating cells or atherome plaque from patients affected by atherosclerosis has lead to a better understanding of the initiation and development of this pathology, because proteins are the final effectors of all events triggered by lipid deposition onto the thickened intima. Although arterial tissue protein extracts have allowed characterizing many proteins involved in the pathogenesis of atherosclerosis (7de la Cuesta F. Alvarez-Llamas G. Gil-Dones F. Martin-Rojas T. Zubiri I. Pastor F. in atherosclerosis: the molecular mechanisms of PubMed Scopus Google Scholar), these have the of protein in all locations a proteomic analysis of of isolated by laser of this is that may to of protein. this has been by the of with a on of proteins in which by two-dimensional electrophoresis using of protein R. R. J. P. proteomic analysis using with laser microdissection and PubMed Scopus Google Scholar). In many of acute coronary not For this reason, is still to novel that events in atherosclerosis initiates within the intima, we aimed to study atherosclerotic coronary intima proteome compared with intima in the for of the disease at the where expression to the lead to the they may be finally into plasma, they may constitute in or For this purpose, intima from human atherosclerotic coronary and from preatherosclerotic and radial isolated by analysis by in the by of 13 proteins up-regulated and expression of a of these proteins validated by immunohistochemistry on an independent of of the of the study be in and were from and were from and from from and from and from and cap were from for and were from for and were from used for by annexin ferritin light myosin regulatory light and from hemoglobin from and from used for these which with and for two-dimensional and for were from biopsy and necropsy from coronary and radial from patients to coronary at the in and from heart at the la Coronary were in a of at the la in of sample in were by the were in in with and at In particular, of of the used in the an atherosclerotic coronary necropsy and a radial were in and used to a tissue for protein to those used for were to staining and to cells and VSMCs using and the of of plaque to the of the classification by (5Virmani R. Kolodgie F.D. Burke A.P. Farb A. Schwartz S.M. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3244) Google Scholar). were coronary from and fibrous cap which a lipid of an inflammatory and of coronary and radial for the study were those from necropsy were from not from events and with of atherosclerotic in of the affected arteries. not at at a they were preatherosclerotic they an intimal thickening with or (5Virmani R. Kolodgie F.D. Burke A.P. Farb A. Schwartz S.M. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3244) Google Scholar). radial during were biopsy because of the to coronary tissue from biopsy In histological analysis intima layer and by for laser microdissection has been previously in la Cuesta F. Alvarez-Llamas G. A. R. F. for two-dimensional analysis of human arterial intima and layers isolated by laser PubMed Scopus Google Scholar). from were and on were with this, a staining with in on and with all to or staining were with were on were not with tissue layer from to analysis isolated by in a For this purpose, the in an of for and for tissue Although microdissection of of in the laser were and for were and using the with microdissection which and catapulting in a of all and those that were not were by using a laser with In of intimal tissue and to a cap because that 4 an for analysis la Cuesta F. Alvarez-Llamas G. A. R. 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V. analysis of human coronary atherosclerotic a study of tissue by and PubMed Scopus Google and of hemoglobin and of with and protein by in these proteins by be in from annexin and of atherosclerotic preatherosclerotic atherosclerotic preatherosclerotic and atherosclerotic preatherosclerotic in the atherosclerotic coronary intima, by validated by in an independent cohort of atherosclerotic and six radial arteries, from biopsy of from these are in the together with the with the with ferritin light chain in atherome plaque extracellular of ferritin light chain were in the intima from mainly at the lipid many cells, that may have those in the lipid core and in the fibrous In radial macrophages within the intima and in the lipid of hemoglobin within atherosclerotic coronary intima by two-dimensional by analysis a to within atherosclerotic coronary intima. cells were in the lipid core and the fibrous cap of affected arteries, macrophages were in radial of on biopsy atherome by be by of were by a cells in arterial were mainly Although macrophages and were of in the atherosclerotic coronary intima, in the and were to this alteration, because the a cells in the intima from atherosclerotic and were VSMCs and VSMCs were at the fibrous cap and to the within the not in lipid In radial were VSMCs in arteries. identified were in the layer from VSMCs and in in intima be within the fibrous analysis of protein in atherosclerotic coronary intima, a of the protein be the tissue In analysis of of the for and within the atherosclerotic coronary intima Proteomic to in atherosclerosis F. la Cuesta F. Alvarez-Llamas G. Proteomic of PubMed Google Scholar), circulating cells J. la Cuesta F. J.J. J. F. human in the acute coronary a proteomic PubMed Scopus Google Scholar, J. la Cuesta F. J.J. J. F. the protein of circulating human an acute coronary PubMed Scopus Google Scholar), and atherome tissue analysis (7de la Cuesta F. Alvarez-Llamas G. Gil-Dones F. Martin-Rojas T. Zubiri I. Pastor F. in atherosclerosis: the molecular mechanisms of PubMed Scopus Google Scholar). Proteomic analysis of atherome tissue has been out by the tissue a may be a approach because of the great complexity of a sample by three layers and cell that have in layer. In and proteome may protein at the intima, where major changes during atherosclerosis initiation and development are a for the of the arterial in to study from the Coronary layers were by analysis together with tissue which the of coronary proteome to J. 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In proteomic within the atherosclerotic coronary intima by with two-dimensional has the of 13 proteins implicated in the migrative capacity of composition, coagulation, and heat shock response, and intraplaque hemorrhage deposition. proteins validated by ferritin vimentin, and may be novel tissue of analysis in a of patients is to approach is to the of tissue which are to better the pathology, has the that on the that they are to blood in a to be which to be in to and from Proteomic for and with protein from Proteomic for with and from the la for with