Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice

Abstract

Objectives The co-stimulatory CD40L–CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis.

Methods and Results Atherosclerotic plaques of apolipoprotein E-deficient (Apoe ^−/− ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCre^ERT2-driven) deficiency of CD40 in Apoe ^−/− mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs.

Conclusion Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Keywords

CD40 - CD40L - atherosclerosis - inflammation - plaque phenotype

• References

• 1 Stefanadis C, Antoniou CK, Tsiachris D, Pietri P. Coronary atherosclerotic vulnerable plaque: Current perspectives. J Am Heart Assoc 2017; 6 (03) e005543
• 2 van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994; 89 (01) 36-44
• 3 Wolf D, Stachon P, Bode C, Zirlik A. Inflammatory mechanisms in atherosclerosis. Hamostaseologie 2014; 34 (01) 63-71
• 4 Gimbrone Jr MA, García-Cardeña G. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 2016; 118 (04) 620-636
• 5 Tardif J-C, Kouz S, Waters DD. et al. Efficacy and safety of low-dose colchicine after myocardial infarction. N Engl J Med 2019; 381 (26) 2497-2505
• 6 Ridker PM, Everett BM, Thuren T. et al; CANTOS Trial Group. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med 2017; 377 (12) 1119-1131
• 7 Seijkens T, Kusters P, Chatzigeorgiou A, Chavakis T, Lutgens E. Immune cell crosstalk in obesity: A key role for costimulation?. Diabetes 2014; 63 (12) 3982-3991
• 8 Gerdes N, Zirlik A. Co-stimulatory molecules in and beyond co-stimulation - Tipping the balance in atherosclerosis?. Thromb Haemost 2011; 106 (05) 804-813
• 9 Lutgens E, Lievens D, Beckers L, Donners M, Daemen M. CD40 and its ligand in atherosclerosis. Trends Cardiovasc Med 2007; 17 (04) 118-123
• 10 Schönbeck U, Libby P. CD40 signaling and plaque instability. Circ Res 2001; 89 (12) 1092-1103
• 11 Schönbeck U, Mach F, Sukhova GK. et al. Expression of stromelysin-3 in atherosclerotic lesions: Regulation via CD40-CD40 ligand signaling in vitro and in vivo. J Exp Med 1999; 189 (05) 843-853
• 12 Schönbeck U, Mach F, Bonnefoy JY, Loppnow H, Flad HD, Libby P. Ligation of CD40 activates interleukin 1beta-converting enzyme (caspase-1) activity in vascular smooth muscle and endothelial cells and promotes elaboration of active interleukin 1beta. J Biol Chem 1997; 272 (31) 19569-19574
• 13 Schönbeck U, Mach F, Sukhova GK. et al. CD40 ligation induces tissue factor expression in human vascular smooth muscle cells. Am J Pathol 2000; 156 (01) 7-14
• 14 Mach F, Schönbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 1998; 394 (6689): 200-203
• 15 Schönbeck U, Sukhova GK, Shimizu K, Mach F, Libby P. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc Natl Acad Sci U S A 2000; 97 (13) 7458-7463
• 16 Lutgens E, Cleutjens KB, Heeneman S, Koteliansky VE, Burkly LC, Daemen MJ. Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype. Proc Natl Acad Sci U S A 2000; 97 (13) 7464-7469
• 17 Lutgens E, Gorelik L, Daemen MJ. et al. Requirement for CD154 in the progression of atherosclerosis. Nat Med 1999; 5 (11) 1313-1316
• 18 André P, Prasad KS, Denis CV. et al. CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism. Nat Med 2002; 8 (03) 247-252
• 19 Ahonen C, Manning E, Erickson LD. et al. The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells. Nat Immunol 2002; 3 (05) 451-456
• 20 Diehl L, Den Boer AT, van der Voort EI, Melief CJ, Offringa R, Toes RE. The role of CD40 in peripheral T cell tolerance and immunity. J Mol Med (Berl) 2000; 78 (07) 363-371
• 21 Kawai T, Andrews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 2000; 6 (02) 114
• 22 Kawabe T, Naka T, Yoshida K. et al. The immune responses in CD40-deficient mice: Impaired immunoglobulin class switching and germinal center formation. Immunity 1994; 1 (03) 167-178
• 23 Häkkinen T, Karkola K, Ylä-Herttuala S. Macrophages, smooth muscle cells, endothelial cells, and T-cells express CD40 and CD40L in fatty streaks and more advanced human atherosclerotic lesions. Colocalization with epitopes of oxidized low-density lipoprotein, scavenger receptor, and CD16 (Fc gammaRIII). Virchows Arch 2000; 437 (04) 396-405
• 24 Kotowicz K, Dixon GL, Klein NJ, Peters MJ, Callard RE. Biological function of CD40 on human endothelial cells: Costimulation with CD40 ligand and interleukin-4 selectively induces expression of vascular cell adhesion molecule-1 and P-selectin resulting in preferential adhesion of lymphocytes. Immunology 2000; 100 (04) 441-448
• 25 Hollenbaugh D, Mischel-Petty N, Edwards CP. et al. Expression of functional CD40 by vascular endothelial cells. J Exp Med 1995; 182 (01) 33-40
• 26 Popa M, Tahir S, Elrod J. et al. Role of CD40 and ADAMTS13 in von Willebrand factor-mediated endothelial cell-platelet-monocyte interaction. Proc Natl Acad Sci U S A 2018; 115 (24) E5556-E5565
• 27 Pluvinet R, Pétriz J, Torras J. et al. RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells. Blood 2004; 104 (12) 3642-3646
• 28 Aarts SABM, Reiche ME, den Toom M. et al. Macrophage CD40 plays a minor role in obesity-induced metabolic dysfunction. PLoS One 2018; 13 (08) e0202150
• 29 Ehling M, Adams S, Benedito R, Adams RH. Notch controls retinal blood vessel maturation and quiescence. Development 2013; 140 (14) 3051-3061
• 30 Hilgendorf I, Eisele S, Remer I. et al. The oral spleen tyrosine kinase inhibitor fostamatinib attenuates inflammation and atherogenesis in low-density lipoprotein receptor-deficient mice. Arterioscler Thromb Vasc Biol 2011; 31 (09) 1991-1999
• 31 Missiou A, Rudolf P, Stachon P. et al. TRAF5 deficiency accelerates atherogenesis in mice by increasing inflammatory cell recruitment and foam cell formation. Circ Res 2010; 107 (06) 757-766
• 32 Krohn JB, Hutcheson JD, Martínez-Martínez E. et al. Discoidin domain receptor-1 regulates calcific extracellular vesicle release in vascular smooth muscle cell fibrocalcific response via transforming growth factor-β signaling. Arterioscler Thromb Vasc Biol 2016; 36 (03) 525-533
• 33 Wolf D, Hohmann J-D, Wiedemann A. et al. Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice. Circ Res 2011; 109 (11) 1269-1279
• 34 Lindau A, Härdtner C, Hergeth SP. et al. Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression. Basic Res Cardiol 2016; 111 (02) 20
• 35 Mach F, Schönbeck U, Sukhova GK. et al. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: Implications for CD40-CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci U S A 1997; 94 (05) 1931-1936
• 36 Bentzon JF, Otsuka F, Virmani R, Falk E. Mechanisms of plaque formation and rupture. Circ Res 2014; 114 (12) 1852-1866
• 37 Boumpas DT, Furie R, Manzi S. et al; BG9588 Lupus Nephritis Trial Group. A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 2003; 48 (03) 719-727
• 38 Schuler W, Bigaud M, Brinkmann V. et al. Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation. Transplantation 2004; 77 (05) 717-726
• 39 Michel NA, Zirlik A, Wolf D. CD40L and its receptors in atherothrombosis-An update. Front Cardiovasc Med 2017; 4: 40
• 40 Tabas I, Bornfeldt KE. Macrophage phenotype and function in different stages of atherosclerosis. Circ Res 2016; 118 (04) 653-667
• 41 Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: A dynamic balance. Nat Rev Immunol 2013; 13 (10) 709-721
• 42 Karmann K, Hughes CC, Schechner J, Fanslow WC, Pober JS. CD40 on human endothelial cells: Inducibility by cytokines and functional regulation of adhesion molecule expression. Proc Natl Acad Sci U S A 1995; 92 (10) 4342-4346
• 43 Lutgens E, Lievens D, Beckers L. et al. Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile. J Exp Med 2010; 207 (02) 391-404
• 44 Wagner AH, Gebauer M, Pollok-Kopp B, Hecker M. Cytokine-inducible CD40 expression in human endothelial cells is mediated by interferon regulatory factor-1. Blood 2002; 99 (02) 520-525
• 45 Yellin MJ, Brett J, Baum D. et al. Functional interactions of T cells with endothelial cells: The role of CD40L-CD40-mediated signals. J Exp Med 1995; 182 (06) 1857-1864
• 46 Gerhardt T, Ley K. Monocyte trafficking across the vessel wall. Cardiovasc Res 2015; 107 (03) 321-330
• 47 Cybulsky MI, Iiyama K, Li H. et al. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest 2001; 107 (10) 1255-1262
• 48 Collins RG, Velji R, Guevara NV, Hicks MJ, Chan L, Beaudet AL. P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 2000; 191 (01) 189-194
• 49 Bourdillon MC, Poston RN, Covacho C, Chignier E, Bricca G, McGregor JL. ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet. Arterioscler Thromb Vasc Biol 2000; 20 (12) 2630-2635
• 50 Quillard T, Araújo HA, Franck G, Tesmenitsky Y, Libby P. Matrix metalloproteinase-13 predominates over matrix metalloproteinase-8 as the functional interstitial collagenase in mouse atheromata. Arterioscler Thromb Vasc Biol 2014; 34 (06) 1179-1186
• 51 Quillard T, Tesmenitsky Y, Croce K. et al. Selective inhibition of matrix metalloproteinase-13 increases collagen content of established mouse atherosclerosis. Arterioscler Thromb Vasc Biol 2011; 31 (11) 2464-2472
• 52 Sukhova GK, Schönbeck U, Rabkin E. et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 1999; 99 (19) 2503-2509
• 53 Allahverdian S, Chaabane C, Boukais K, Francis GA, Bochaton-Piallat ML. Smooth muscle cell fate and plasticity in atherosclerosis. Cardiovasc Res 2018; 114 (04) 540-550
• 54 Bennett MR, Sinha S, Owens GK. Vascular smooth muscle cells in atherosclerosis. Circ Res 2016; 118 (04) 692-702
• 55 Theoharis S, Krueger U, Tan PH, Haskard DO, Weber M, George AJ. Targeting gene delivery to activated vascular endothelium using anti E/P-selectin antibody linked to PAMAM dendrimers. J Immunol Methods 2009; 343 (02) 79-90
• 56 Melo LG, Gnecchi M, Pachori AS. et al. Endothelium-targeted gene and cell-based therapies for cardiovascular disease. Arterioscler Thromb Vasc Biol 2004; 24 (10) 1761-1774
• 57 Shuvaev VV, Brenner JS, Muzykantov VR. Targeted endothelial nanomedicine for common acute pathological conditions. J Control Release 2015; 219: 576-595
• 58 Kiseleva RY, Glassman PM, Greineder CF, Hood ED, Shuvaev VV, Muzykantov VR. Targeting therapeutics to endothelium: Are we there yet?. Drug Deliv Transl Res 2018; 8 (04) 883-902