RSS-Feed abonnieren
![](/products/assets/desktop/img/oa-logo.png)
DOI: 10.1055/s-0041-1725042
Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis
Funding This research was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, SCHA 808/9–1 and 9–2 to K.S.).![](https://www.thieme-connect.de/media/10.1055-s-00033990/202102/lookinside/thumbnails/10-1055-s-0041-1725042_200089-1.jpg)
Abstract
Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied.
Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis (n = 46), atherosclerotic carotid artery plaques (n = 9), and abdominal aortic aneurysms (n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson–Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals.
Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.
Publikationsverlauf
Eingereicht: 29. Oktober 2020
Angenommen: 18. Januar 2021
Artikel online veröffentlicht:
14. April 2021
© 2021. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
-
References
- 1 Allison MA, Criqui MH, Wright CM. Patterns and risk factors for systemic calcified atherosclerosis. Arterioscler Thromb Vasc Biol 2004; 24 (02) 331-336
- 2 Rajamannan NM, Subramaniam M, Rickard D. et al. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation 2003; 107 (17) 2181-2184
- 3 Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol 2003; 23 (03) 489-494
- 4 Tanaka K, Sata M, Fukuda D. et al. Age-associated aortic stenosis in apolipoprotein E-deficient mice. J Am Coll Cardiol 2005; 46 (01) 134-141
- 5 Maldonado N, Kelly-Arnold A, Vengrenyuk Y. et al. A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture. Am J Physiol Heart Circ Physiol 2012; 303 (05) H619-H628
- 6 Shioi A, Ikari Y. Plaque calcification during atherosclerosis progression and regression. J Atheroscler Thromb 2018; 25 (04) 294-303
- 7 Sluimer JC, Kolodgie FD, Bijnens AP. et al. Thin-walled microvessels in human coronary atherosclerotic plaques show incomplete endothelial junctions relevance of compromised structural integrity for intraplaque microvascular leakage. J Am Coll Cardiol 2009; 53 (17) 1517-1527
- 8 Virmani R, Roberts WC. Extravasated erythrocytes, iron, and fibrin in atherosclerotic plaques of coronary arteries in fatal coronary heart disease and their relation to luminal thrombus: frequency and significance in 57 necropsy patients and in 2958 five mm segments of 224 major epicardial coronary arteries. Am Heart J 1983; 105 (05) 788-797
- 9 Kolodgie FD, Gold HK, Burke AP. et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med 2003; 349 (24) 2316-2325
- 10 Takaya N, Yuan C, Chu B. et al. Presence of intraplaque hemorrhage stimulates progression of carotid atherosclerotic plaques: a high-resolution magnetic resonance imaging study. Circulation 2005; 111 (21) 2768-2775
- 11 Akahori H, Tsujino T, Naito Y. et al. Intraleaflet haemorrhage is associated with rapid progression of degenerative aortic valve stenosis. Eur Heart J 2011; 32 (07) 888-896
- 12 Tziakas D, Chalikias G, Kapelouzou A. et al. Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin. Int J Cardiol 2013; 170 (02) 173-181
- 13 Tziakas DN, Chalikias GK, Stakos D. et al. Independent and additive predictive value of total cholesterol content of erythrocyte membranes with regard to coronary artery disease clinical presentation. Int J Cardiol 2011; 150 (01) 22-27
- 14 Yu MM, Xu Y, Zhang JH. et al. Total cholesterol content of erythrocyte membranes levels are associated with the presence of acute coronary syndrome and high sensitivity C-reactive protein. Int J Cardiol 2010; 145 (01) 57-58
- 15 Dawson TC, Lentsch AB, Wang Z. et al. Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC). Blood 2000; 96 (05) 1681-1684
- 16 Schnabel RB, Baumert J, Barbalic M. et al. Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators. Blood 2010; 115 (26) 5289-5299
- 17 Apostolakis S, Chalikias GK, Tziakas DN, Konstantinides S. Erythrocyte Duffy antigen receptor for chemokines (DARC): diagnostic and therapeutic implications in atherosclerotic cardiovascular disease. Acta Pharmacol Sin 2011; 32 (04) 417-424
- 18 Benson TW, Weintraub DS, Crowe M. et al. Deletion of the Duffy antigen receptor for chemokines (DARC) promotes insulin resistance and adipose tissue inflammation during high fat feeding. Mol Cell Endocrinol 2018; 473: 79-88
- 19 Tziakas DN, Chalikias G, Pavlaki M. et al. Lysed erythrocyte membranes promote vascular calcification. Circulation 2019; 139 (17) 2032-2048
- 20 Schaer DJ, Schaer CA, Buehler PW. et al. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 2006; 107 (01) 373-380
- 21 Buechler C, Ritter M, Orsó E, Langmann T, Klucken J, Schmitz G. Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli. J Leukoc Biol 2000; 67 (01) 97-103
- 22 Premont RT, Reynolds JD, Zhang R, Stamler JS. Role of nitric oxide carried by hemoglobin in cardiovascular physiology: developments on a three-gas respiratory cycle. Circ Res 2020; 126 (01) 129-158
- 23 Singel DJ, Stamler JS. Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin. Annu Rev Physiol 2005; 67: 99-145
- 24 Byrnes JR, Wolberg AS. Red blood cells in thrombosis. Blood 2017; 130 (16) 1795-1799
- 25 Pasterkamp G, Virmani R. The erythrocyte: a new player in atheromatous core formation. Heart 2002; 88 (02) 115-116
- 26 Virmani R, Kolodgie FD, Burke AP. et al. Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol 2005; 25 (10) 2054-2061
- 27 Kolodgie FD, Burke AP, Nakazawa G, Cheng Q, Xu X, Virmani R. Free cholesterol in atherosclerotic plaques: where does it come from?. Curr Opin Lipidol 2007; 18 (05) 500-507
- 28 Michel JB, Virmani R, Arbustini E, Pasterkamp G. Intraplaque haemorrhages as the trigger of plaque vulnerability. Eur Heart J 2011; 32 (16) 1977-1985 , 1985a, 1985b, 1985c
- 29 Kockx MM, Cromheeke KM, Knaapen MW. et al. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol 2003; 23 (03) 440-446
- 30 Matsumura M, Mintz GS, Kang SJ. et al. Intravascular ultrasound and near-infrared spectroscopic features of coronary lesions with intraplaque haemorrhage. Eur Heart J Cardiovasc Imaging 2017; 18 (11) 1222-1228
- 31 Sawada H, Hao H, Naito Y. et al. Aortic iron overload with oxidative stress and inflammation in human and murine abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 2015; 35 (06) 1507-1514
- 32 Martinez-Pinna R, Lindholt JS, Madrigal-Matute J. et al. From tissue iron retention to low systemic haemoglobin levels, new pathophysiological biomarkers of human abdominal aortic aneurysm. Thromb Haemost 2014; 112 (01) 87-95
- 33 Nordon IM, Hinchliffe RJ, Loftus IM, Thompson MM. Pathophysiology and epidemiology of abdominal aortic aneurysms. Nat Rev Cardiol 2011; 8 (02) 92-102
- 34 Cornelissen A, Guo L, Sakamoto A, Virmani R, Finn AV. New insights into the role of iron in inflammation and atherosclerosis. EBioMedicine 2019; 47: 598-606
- 35 Fontaine V, Jacob MP, Houard X. et al. Involvement of the mural thrombus as a site of protease release and activation in human aortic aneurysms. Am J Pathol 2002; 161 (05) 1701-1710
- 36 Pernow J, Mahdi A, Yang J, Zhou Z. Red blood cell dysfunction: a new player in cardiovascular disease. Cardiovasc Res 2019; 115 (11) 1596-1605
- 37 Paul A, Calleja L, Vilella E, Martínez R, Osada J, Joven J. Reduced progression of atherosclerosis in apolipoprotein E-deficient mice with phenylhydrazine-induced anemia. Atherosclerosis 1999; 147 (01) 61-68
- 38 de Winter RJ, Manten A, de Jong YP, Adams R, van Deventer SJ, Lie KI. Interleukin 8 released after acute myocardial infarction is mainly bound to erythrocytes. Heart 1997; 78 (06) 598-602
- 39 Tziakas DN, Chalikias GK, Tentes IK. et al. Interleukin-8 is increased in the membrane of circulating erythrocytes in patients with acute coronary syndrome. Eur Heart J 2008; 29 (22) 2713-2722
- 40 Sluimer JC, Gasc JM, van Wanroij JL. et al. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 2008; 51 (13) 1258-1265
- 41 da Silva Garrote-Filho M, Bernardino-Neto M, Penha-Silva N. Influence of erythrocyte membrane stability in atherosclerosis. Curr Atheroscler Rep 2017; 19 (04) 17
- 42 Schaer CA, Schoedon G, Imhof A, Kurrer MO, Schaer DJ. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circ Res 2006; 99 (09) 943-950
- 43 Yunoki K, Naruko T, Komatsu R. et al. Enhanced expression of haemoglobin scavenger receptor in accumulated macrophages of culprit lesions in acute coronary syndromes. Eur Heart J 2009; 30 (15) 1844-1852
- 44 Philippidis P, Mason JC, Evans BJ. et al. Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circ Res 2004; 94 (01) 119-126
- 45 Boyle JJ, Harrington HA, Piper E. et al. Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype. Am J Pathol 2009; 174 (03) 1097-1108
- 46 Guo L, Akahori H, Harari E. et al. CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis. J Clin Invest 2018; 128 (03) 1106-1124
- 47 Oba E, Aung NY, Ohe R, Sadahiro M, Yamakawa M. The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis. Am J Transl Res 2020; 12 (05) 1728-1740
- 48 Weidenbach H, Massmann J. Erythrocytophagocytosis by arterial smooth muscle cells in experimental atherosclerosis. Exp Pathol (Jena) 1978; 15 (02) 111-115
- 49 Ramakrishnan L, Pedersen SL, Toe QK. et al. The hepcidin/ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells. Sci Rep 2018; 8 (01) 12972
- 50 Levy AP, Levy JE, Kalet-Litman S. et al. Haptoglobin genotype is a determinant of iron, lipid peroxidation, and macrophage accumulation in the atherosclerotic plaque. Arterioscler Thromb Vasc Biol 2007; 27 (01) 134-140
- 51 Guo L, Harari E, Virmani R, Finn AV. Linking hemorrhage, angiogenesis, macrophages, and iron metabolism in atherosclerotic vascular diseases. Arterioscler Thromb Vasc Biol 2017; 37 (04) e33-e39
- 52 Vinchi F, Porto G, Simmelbauer A. et al. Atherosclerosis is aggravated by iron overload and ameliorated by dietary and pharmacological iron restriction. Eur Heart J 2020; 41 (28) 2681-2695
- 53 Lee TS, Shiao MS, Pan CC, Chau LY. Iron-deficient diet reduces atherosclerotic lesions in apoE-deficient mice. Circulation 1999; 99 (09) 1222-1229
- 54 Xie Y, Hou W, Song X. et al. Ferroptosis: process and function. Cell Death Differ 2016; 23 (03) 369-379
- 55 Mallat Z, Tedgui A. Apoptosis in the vasculature: mechanisms and functional importance. Br J Pharmacol 2000; 130 (05) 947-962
- 56 Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty. An observational study using intravascular ultrasound. Circulation 1992; 86 (01) 64-70
- 57 Vengrenyuk Y, Cardoso L, Weinbaum S. Micro-CT based analysis of a new paradigm for vulnerable plaque rupture: cellular microcalcifications in fibrous caps. Mol Cell Biomech 2008; 5 (01) 37-47
- 58 Lin R, Chen S, Liu G, Xue Y, Zhao X. Association between carotid atherosclerotic plaque calcification and intraplaque hemorrhage: a magnetic resonance imaging study. Arterioscler Thromb Vasc Biol 2017; 37 (06) 1228-1233
- 59 Rattazzi M, Bennett BJ, Bea F. et al. Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoE-deficient mice: potential role of chondrocyte-like cells. Arterioscler Thromb Vasc Biol 2005; 25 (07) 1420-1425
- 60 Morvan M, Arangalage D, Franck G. et al. Relationship of iron deposition to calcium deposition in human aortic valve leaflets. J Am Coll Cardiol 2019; 73 (09) 1043-1054
- 61 Clarke MC, Littlewood TD, Figg N. et al. Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration. Circ Res 2008; 102 (12) 1529-1538