Proteomic Analysis of Plasma-Purified VLDL, LDL, and HDL Fractions from Atherosclerotic Patients Undergoing Carotid Endarterectomy: Identification of Serum Amyloid A as a Potential Marker
Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects. 1. Introduction Cardiovascular diseases are the leading cause of death and illness in developed countries, with atherosclerosis being the most important contributor. Atherosclerosis is a chronic inflammatory condition that could turn into an acute clinical event due to plaque rupture and thrombosis [1]. Indeed, vascular inflammation not only plays a major role in the development of atherosclerosis but also contributes to the acute onset of thrombotic complications [2]. The selective retention of circulating apolipoprotein B100 containing lipoproteins in the subendothelial space, by means of specific interactions with artery wall proteoglycans, is currently thought to be the leading event in atherogenesis [3, 4]. Lipoproteins are supramolecular complexes that deliver insoluble lipids from the tissues where they are synthesized to those that metabolize or store them. They consist of hydrophobic molecules (core), particularly triacylglycerol and cholesteryl esters, stabilized by a coat of amphipathic compounds, namely, phospholipids, unesterified cholesterol, and proteins, with the latter referred to as
References
[1]
E. Lutgens, R.-J. van Suylen, B. C. Faber et al., “Atherosclerotic plaque rupture: local or systemic process?” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 23, no. 12, pp. 2123–2130, 2003.
[2]
P. Libby, “Inflammation in atherosclerosis,” Nature, vol. 420, no. 6917, pp. 868–874, 2002.
[3]
K. J. Williams and I. Tabas, “The response-to-retention hypothesis of early atherogenesis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 15, no. 5, pp. 551–562, 1995.
[4]
K. Sk？lén, M. Gustafsson, E. Knutsen Rydberg et al., “Subendothelial retention of atherogenic lipoproteins in early atherosclerosis,” Nature, vol. 417, no. 6890, pp. 750–754, 2002.
[5]
A. Jonas and M. C. Phillips, “Lipoprotein structure,” in Biochemistry of Lipids, Lipoprotein and Membranes, D. E. Vance and J. E. Vance, Eds., pp. 485–506, Elsevier, 5th edition, 2008.
[6]
V. M. Bolanos-Garcia and R. N. Miguel, “On the structure and function of apolipoproteins: more than a family of lipid-binding proteins,” Progress in Biophysics and Molecular Biology, vol. 83, no. 1, pp. 47–68, 2003.
[7]
G. Walldius, I. Jungner, I. Holme, A. H. Aastveit, W. Kolar, and E. Steiner, “High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study,” The Lancet, vol. 358, no. 9298, pp. 2026–2033, 2001.
[8]
A. J. Lepedda, E. Zinellu, and M. Formato, “Overview of current proteomic approaches for discovery of vascular biomarkers of atherosclerosis,” in Proteomics—Human Diseases and Protein Functions, T.-K. Man and J. R. Flores, Eds., pp. 3–32, InTech, 2012.
[9]
A. von Zychlinski, T. Kleffmann, M. J. A. Williams, and S. P. McCormick, “Proteomics of Lipoprotein(a) identifies a protein complement associated with response to wounding,” Journal of Proteomics, vol. 74, no. 12, pp. 2881–2891, 2011.
[10]
T. Vaisar, S. Pennathur, P. S. Green et al., “Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL,” Journal of Clinical Investigation, vol. 117, no. 3, pp. 746–756, 2007.
[11]
P. S. Green, T. Vaisar, S. Pennathur et al., “Combined statin and niacin therapy remodels the high-density lipoprotein proteome,” Circulation, vol. 118, no. 12, pp. 1259–1267, 2008.
[12]
T. Vaisar, P. Mayer, E. Nilsson, X.-Q. Zhao, R. Knopp, and B. J. Prazen, “HDL in humans with cardiovascular disease exhibits a proteomic signature,” Clinica Chimica Acta, vol. 411, no. 13-14, pp. 972–979, 2010.
[13]
K. Alwaili, D. Bailey, Z. Awan et al., “The HDL proteome in acute coronary syndromes shifts to an inflammatory profile,” Biochimica et Biophysica Acta, vol. 1821, no. 3, pp. 405–415, 2012.
[14]
J. Cubedo, T. Padró, R. Alonso, J. Cinca, P. Mata, and L. Badimon, “Differential proteomic distribution of TTR (pre-albumin) forms in serum and HDL of patients with high cardiovascular risk,” Atherosclerosis, vol. 222, no. 1, pp. 263–269, 2012.
[15]
M. Heller, E. Schlappritzi, D. Stalder, J.-M. Nuoffer, and A. Haeberli, “Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring,” Molecular and Cellular Proteomics, vol. 6, no. 6, pp. 1059–1072, 2007.
[16]
M. T. Mazura, H. L. Cardasis, D. S. Spellman, A. Liaw, N. A. Yates, and R. C. Hendrickson, “Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 17, pp. 7728–7733, 2010.
[17]
J. H. M. Levels, P. Geurts, H. Karlsson et al., “High-density lipoprotein proteome dynamics in human endotoxemia,” Proteome Science, vol. 9, article 34, 2011.
[18]
P. Davidsson, J. Hulthe, B. Fagerberg et al., “A proteomic study of the apolipoproteins in LDL subclasses in patients with the metabolic syndrome and type 2 diabetes,” Journal of Lipid Research, vol. 46, no. 9, pp. 1999–2006, 2005.
[19]
P. V. Bondarenko, S. L. Cockrill, L. K. Watkins, I. D. Cruzado, and R. D. Macfarlane, “Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein,” Journal of Lipid Research, vol. 40, no. 3, pp. 543–555, 1999.
[20]
A. J. Lepedda, A. Cigliano, G. M. Cherchi et al., “A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries,” Atherosclerosis, vol. 203, no. 1, pp. 112–118, 2009.
[21]
A. J. Lepedda, A. Zinellu, G. Nieddu et al., “Protein sulfhydryl group oxidation and mixed-disulfide modifications in stable and unstable human carotid plaques,” Oxidative Medicine and Cellular Longevity, vol. 2013, Article ID 403973, 8 pages, 2013.
[22]
J. Himber, E. Buhler, D. Moll, and U. K. Moser, “Low density lipoprotein for oxidation and metabolic studies. Isolation from small volumes of plasma using a tabletop ultracentrifuge,” International Journal for Vitamin and Nutrition Research, vol. 65, no. 2, pp. 137–142, 1995.
[23]
I. F. W. McDowell, J. McEneny, and E. R. Trimble, “A rapid method for measurement of the susceptibility to oxidation of low-density lipoprotein,” Annals of Clinical Biochemistry, vol. 32, no. 2, pp. 167–174, 1995.
[24]
R. Mastro and M. Hall, “Protein delipidation and precipitation by tri-n-butylphosphate, acetone, and methanol treatment for isoelectric focusing and two-dimensional gel electrophoresis,” Analytical Biochemistry, vol. 273, no. 2, pp. 313–315, 1999.
[25]
V. L. King, J. Thompson, and L. R. Tannock, “Serum amyloid A in atherosclerosis,” Current Opinion in Lipidology, vol. 22, no. 4, pp. 302–307, 2011.
[26]
S. P. Tam, A. Flexman, J. Hulme, and R. Kisilevsky, “Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1,” Journal of Lipid Research, vol. 43, no. 9, pp. 1410–1420, 2002.
[27]
J. A. Stonik, A. T. Remaley, S. J. Demosky, E. B. Neufeld, A. Bocharov, and H. B. Brewer, “Serum Amyloid a promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells,” Biochemical and Biophysical Research Communications, vol. 321, no. 4, pp. 936–941, 2004.
[28]
S. P. Tam, J. B. Ancsin, R. Tan, and R. Kisilevsky, “Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE？/？ mice,” Journal of Lipid Research, vol. 46, no. 10, pp. 2091–2101, 2005.
[29]
D. R. van der Westhuyzen, L. Cai, M. C. DeBeer, and F. C. DeBeer, “Serum amyloid A promotes cholesterol efflux mediated by scavenger receptor B-I,” Journal of Biological Chemistry, vol. 280, no. 43, pp. 35890–35895, 2005.
[30]
R. Kisilevsky, S. P. Tam, and J. B. Ancsin, “The anti-atherogenic potential of serum amyloid A peptides,” Current Opinion in Investigational Drugs, vol. 9, no. 3, pp. 265–273, 2008.
[31]
R. Badolato, J. M. Wang, W. J. Murphy et al., “Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes,” Journal of Experimental Medicine, vol. 180, no. 1, pp. 203–209, 1994.
[32]
Y. L. Ha, D. K. Sang, W. S. Jae et al., “Serum amyloid A induces CCL2 production via formyl peptide receptor-like 1-mediated signaling in human monocytes,” Journal of Immunology, vol. 181, no. 6, pp. 4332–4339, 2008.
[33]
Z. Dong, T. Wu, W. Qin et al., “Serum amyloid a directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice,” Molecular Medicine, vol. 17, no. 11, pp. 1357–1364, 2011.
[34]
H. Y. Lee, S. D. Kim, S. H. Baek et al., “Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation,” Biochemical and Biophysical Research Communications, vol. 433, no. 1, pp. 18–23, 2013.
[35]
C. J. Furlaneto and A. Campa, “A novel function of serum amyloid A: a potent stimulus for the release of tumor necrosis factor-α, interleukin-1β, and interleukin-8 by human blood neutrophil,” Biochemical and Biophysical Research Communications, vol. 268, no. 2, pp. 405–408, 2000.
[36]
C. Song, K. Hsu, E. Yamen et al., “Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes,” Atherosclerosis, vol. 207, no. 2, pp. 374–383, 2009.
[37]
S. Hua, C. Song, C. L. Geczy, S. B. Freedman, and P. K. Witting, “A role for acute-phase serum amyloid A and high-density lipoprotein in oxidative stress, endothelial dysfunction and atherosclerosis,” Redox Report, vol. 14, no. 5, pp. 187–196, 2009.
[38]
P. K. Witting, C. Song, K. Hsu et al., “The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein,” Free Radical Biology and Medicine, vol. 51, no. 7, pp. 1390–1398, 2011.
[39]
C. Song, Y. Shen, E. Yamen et al., “Serum amyloid A may potentiate prothrombotic and proinflammatory events in acute coronary syndromes,” Atherosclerosis, vol. 202, no. 2, pp. 596–604, 2009.
[40]
P. G. Wilson, J. C. Thompson, N. R. Webb, F. C. DeBeer, V. L. King, and L. R. Tannock, “Serum amyloid A, but not C-reactive protein, stimulates vascular proteoglycan synthesis in a pro-atherogenic manner,” American Journal of Pathology, vol. 173, no. 6, pp. 1902–1910, 2008.
[41]
J. B. Ancsin and R. Kisilevsky, “Serum amyloid A peptide interactions with glycosaminoglycans. Evaluation by affinity chromatography,” Methods in Molecular Biology, vol. 171, pp. 449–456, 2001.
[42]
K. D. O'Brien, T. O. McDonald, V. Kunjathoor et al., “Serum amyloid A and lipoprotein retention in murine models of atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25, no. 4, pp. 785–790, 2005.
[43]
T. Yamada, T. Kakihara, T. Kamishima, T. Fukuda, and T. Kawai, “Both acute phase and constitutive serum amyloid A are present in atherosclerotic lesions,” Pathology International, vol. 46, no. 10, pp. 797–800, 1996.
[44]
R. L. Meek, S. Urieli-Shoval, and E. P. Benditt, “Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 8, pp. 3186–3190, 1994.
[45]
G. Liuzzo, L. M. Biasucci, J. R. Gallimore et al., “The prognostic value of C-reactive protein and serum amyloid A protein in severe unstable angina,” The New England Journal of Medicine, vol. 331, no. 7, pp. 417–424, 1994.
[46]
A. I. Fyfe, L. S. Rothenberg, F. C. DeBeer, R. M. Cantor, J. I. Rotter, and A. J. Lusis, “Association between serum amyloid A proteins and coronary artery disease: evidence from two distinct arteriosclerotic processes,” Circulation, vol. 96, no. 9, pp. 2914–2919, 1997.
[47]
M. Erren, H. Reinecke, R. Junker et al., “Systemic inflammatory parameters in patients with atherosclerosis of the coronary and peripheral arteries,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 19, no. 10, pp. 2355–2363, 1999.
[48]
N. Rifai, R. Joubran, H. Yu, M. Asmi, and M. Jouma, “Inflammatory markers in men with angiographically documented coronary heart disease,” Clinical Chemistry, vol. 45, no. 11, pp. 1967–1973, 1999.
[49]
P. Jousilahti, V. Salomaa, V. Rasi, E. Vahtera, and T. Palosuo, “The association of c-reactive protein, serum amyloid a and fibrinogen with prevalent coronary heart disease—baseline findings of the PAIS project,” Atherosclerosis, vol. 156, no. 2, pp. 451–456, 2001.
[50]
J. R. Delanghe, M. R. Langlois, D. de Bacquer et al., “Discriminative value of serum amyloid A and other acute-phase proteins for coronary heart disease,” Atherosclerosis, vol. 160, no. 2, pp. 471–476, 2002.
[51]
K. Ogasawara, S. Mashiba, Y. Wada et al., “A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease,” Atherosclerosis, vol. 174, no. 2, pp. 349–356, 2004.
[52]
B. D. Johnson, K. E. Kip, O. C. Marroquin et al., “Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: The National Heart, Lung, and Blood Institute-Sponsored Women's Ischemia Syndrome Evaluation (WISE),” Circulation, vol. 109, no. 6, pp. 726–732, 2004.
[53]
K. W. J. Lee, J. S. Hill, K. R. Walley, and J. J. Frohlich, “Relative value of multiple plasma biomarkers as risk factors for coronary artery disease and death in an angiography cohort,” Canadian Medical Association Journal, vol. 174, no. 4, pp. 461–466, 2006.
