Endogenous developmental endothelial locus-1 limits ischaemia- related angiogenesis by blocking inflammationFinancial support: This work was supported by the Else Kröner-Fresenius-Stiftung (2013_A2 to E. C.). E. C. and S. D. are members of the Excellence Cluster Cardiopulmonary System (DFG; Exc147–1), the German Centre for Cardiovascular Research (BMBF) and the LOEWE Center for Gene and Cell Therapy (Hessen, Germany). S. C. is also supported by a grant from the LOEWE Center for Gene and Cell Therapy. T.C. was supported by the ERC (ENDHOMRET), the DFG (INST 515/11–1) and DE026152 from the NIH. M.E. was supported by the Else Kröner-Fresenius-Stiftung. G.H. was supported by DE026152, DE024716 and DE015254 from the NIH. S.K. was supported by a Grant of DAAD (Deutscher Akademischer Austauschdienst).
12 May 2016
Accepted after major revision: 05 March 2017
28 November 2017 (online)
We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.
Supplementary Material to this article is available online at www.thrombosis-online.com.
- 1 Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell 2011; 146: 873-887.
- 2 Sapieha P, Joyal JS, Rivera JC. et al. Retinopathy of prematurity: understanding ischaemic retinal vasculopathies at an extreme of life. J Clin Invest 2010; 120: 3022-3032.
- 3 Kopp HG, Ramos CA, Rafii S. Contribution of endothelial progenitors and proangiogenic haematopoietic cells to vascularisation of tumor and ischaemic tissue. Curr Opin Haematol 2006; 13: 175-181.
- 4 Chambers SE, O’Neill CL, O’Doherty TM. et al. The role of immune-related myeloid cells in angiogenesis. Immunobiology 2013; 218: 1370-1375.
- 5 Chavakis E, Dimmeler S. Homing of progenitor cells to ischaemic tissues. Anti-oxidants Redox Signal 2011; 15: 967-980.
- 6 Chavakis E, Koyanagi M, Dimmeler S. Enhancing the outcome of cell therapy for cardiac repair: progress from bench to bedside and back. Circulation 2010; 121: 325-335.
- 7 Murdoch C, Muthana M, Coffelt SB. et al. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008; 8: 618-631.
- 8 Chavakis E, Aicher A, Heeschen C. et al. Role of beta2-integrins for homing and neovascularisation capacity of endothelial progenitor cells. J Exp Med 2005; 201: 63-72.
- 9 Jin H, Su J, Garmy-Susini B. et al. Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors. Cancer Res 2006; 66: 2146-2152.
- 10 Mitroulis I, Alexaki VI, Kourtzelis I. et al. Leukocyte integrins: role in leukocyte recruitment and as therapeutic targets in inflammatory disease. Pharmacol Ther 2015; 147: 123-135.
- 11 Choi EY. et al. Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science 2008; 322: 1101-1104.
- 12 Eskan MA, Jotwani R, Abe T. et al. The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss. Nat Immunol 2012; 13: 465-473.
- 13 Choi EY, Lim JH, Neuwirth A. et al. Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination. Mol Psychiatry 2015; 20: 880-888.
- 14 Maekawa T, Hosur K, Abe T. et al. Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3beta-C/EBPbeta pathway. Nat Commun 2015; 6: 8272.
- 15 Chavakis E, Choi EY, Chavakis T. Novel aspects in the regulation of the leukocyte adhesion cascade. Thromb Haemost 2009; 102: 191-197.
- 16 Hajishengallis G, Chavakis T. Endogenous modulators of inflammatory cell recruitment. Trends Immunol 2013; 34: 1-6.
- 17 Hidai C, Zupancic T, Penta K. et al. Cloning and characterisation of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the alphavbeta3 integrin receptor. Genes Dev 1998; 12: 21-33.
- 18 Dasgupta SK, Le A, Chavakis T. et al. Developmental endothelial locus-1 (Del-1) mediates clearance of platelet microparticles by the endothelium. Circulation 2012; 125: 1664-1672.
- 19 Shin J, Maekawa T, Abe T. et al. DEL-1 restrains osteoclastogenesis and inhibits inflammatory bone loss in nonhuman primates. Sci Transl Med 2015; 7: 307ra155.
- 20 Mitroulis I, Kang YY, Gahmberg CG. et al. Developmental endothelial locus-1 attenuates complement-dependent phagocytosis through inhibition of Mac-1-integrin. Thromb Haemost 2014; 111: 1004-1006.
- 21 Kanczkowski W, Chatzigeorgiou A, Grossklaus S. et al. Role of the endothelial-derived endogenous anti-inflammatory factor Del-1 in inflammation-mediated adrenal gland dysfunction. Endocrinology 2013; 154: 1181-1189.
- 22 Kourtzelis I, Kotlabova K, Lim JH. et al. Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation. Thromb Haemost 2016; 115: 781-788.
- 23 Penta K, Varner JA, Liaw L. et al. Del1 induces integrin signaling and angiogenesis by ligation of alphaVbeta3. J Biol Chem 1999; 274: 11101-1119.
- 24 Zhong J, Eliceiri B, Stupack D. et al. Neovascularisation of ischaemic tissues by gene delivery of the extracellular matrix protein Del-1. J Clin Invest 2003; 112: 30-41.
- 25 Ho HK, Jang JJ, Kaji S. et al. Developmental endothelial locus-1 (Del-1), a novel angiogenic protein: its role in ischaemia. Circulation 2004; 109: 1314-1319.
- 26 Fan Y, Zhu W, Yang M. et al. Del-1 gene transfer induces cerebral angiogenesis in mice. Brain Res 2008; 1219: 1-7.
- 27 Ciucurel EC, Sefton MV. Del-1 overexpression in endothelial cells increases vascular density in tissue-engineered implants containing endothelial cells and adi-pose-derived mesenchymal stromal cells. Tissue Eng Part A 2014; 20: 1235-1252.
- 28 Kown MH, Suzuki T, Koransky ML. et al. Comparison of developmental endothelial locus-1 angiogenic factor with vascular endothelial growth factor in a porcine model of cardiac ischaemia. Ann Thorac Surg 2003; 76: 1246-1251.
- 29 Hsu GP, Mathy JA, Wang Z. et al. Increased rate of hair regrowth in mice with constitutive overexpression of Del1. J Surg Res 2008; 146: 73-80.
- 30 Connor KM, Krah NM, Dennison RJ. et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protocols 2009; 4: 1565-1573.
- 31 Limbourg A, Korff T, Napp LC. et al. Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischaemia. Nat Protoc 2009; 4: 1737-1746.
- 32 Chavakis E, Carmona G, Urbich C. et al. Phosphatidylinositol-3-kinase-gamma is integral to homing functions of progenitor cells. Circ Res 2008; 102: 942-949.
- 33 Choi EY, Orlova VV, Fagerholm SC. et al. Regulation of LFA-1-dependent inflammatory cell recruitment by Cbl-b and 14–3–3 proteins. Blood 2008; 111: 3607-3614.
- 34 Carmona G, Chavakis E, Koehl U. et al. Activation of Epac stimulates integrin-dependent homing of progenitor cells. Blood 2008; 111: 2640-2646.
- 35 Chavakis E, Hain A, Vinci M. et al. High-mobility group box 1 activates integrin-dependent homing of endothelial progenitor cells. Circ Res 2007; 100: 204-212.
- 36 Carmona G, Gottig S, Orlandi A. et al. Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood 2009; 113: 488-497.
- 37 Manavski Y, Carmona G, Bennewitz K. et al. Brag2 differentially regulates beta1– and beta3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis. Basic Res Cardiol 2014; 109: 404.
- 38 Athanasopoulos AN, Schneider D, Keiper T. et al. Vascular endothelial growth factor (VEGF)-induced up-regulation of CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing. J Biol Chem 2007; 282: 26746-26753.
- 39 Athanasopoulos AN, Economopoulou M, Orlova VV. et al. The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms. Blood 2006; 107: 2720-2727.
- 40 Economopoulou M, Bdeir K, Cines DB. et al. Inhibition of pathologic retinal neovascularisation by alpha-defensins. Blood 2005; 106: 3831-3838.
- 41 Economopoulou M, Langer HF, Celeste A. et al. Histone H2AX is integral to hypoxia-driven neovascularisation. Nat Med 2009; 15: 553-558.
- 42 Langer HF, Chung KJ, Orlova VV. et al. Complement-mediated inhibition of neovascularisation reveals a point of convergence between innate immunity and angiogenesis. Blood 2010; 116: 4395-4403.
- 43 Economopoulou M, Avramovic N, Klotzsche-von Ameln A. et al. Endothelial-specific deficiency of Junctional Adhesion Molecule-C promotes vessel normal-isation in proliferative retinopathy. Thromb Haemost 2015; 114: 1241-1249.
- 44 Baker M, Robinson SD, Lechertier T. et al. Use of the mouse aortic ring assay to study angiogenesis. Nat Protoc 2012; 7: 89-104.
- 45 Pitulescu ME, Schmidt I, Benedito R. et al. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protocols 2010; 5: 1518-1534.
- 46 Chavakis E, Urbich C, Dimmeler S. Homing and engraftment of progenitor cells: a prerequisite for cell therapy. J Mol Cell Cardiol 2008; 45: 514-522.
- 47 Grossman PM, Mendelsohn F, Henry TD. et al. Results from a phase II multi-center, double-blind placebo-controlled study of Del-1 (VLTS-589) for intermittent claudication in subjects with peripheral arterial disease. Am Heart J 2007; 153: 874-880.
- 48 Soloviev DA, Hazen SL, Szpak D. et al. Dual Role of the Leukocyte Integrin alphaMbeta2 in Angiogenesis. J Immunol 2014; 193: 4712-4721.
- 49 Wu Y, Ip JE, Huang J. et al. Essential role of ICAM-1/CD18 in mediating EPC recruitment, angiogenesis, and repair to the infarcted myocardium. Circ Res 2006; 99: 315-322.
- 50 Numasaki M, Fukushi J, Ono M. et al. Interleukin-17 promotes angiogenesis and tumor growth. Blood 2003; 101: 2620-2627.