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Angiogenesis In2 Β M Α Dual Role of the Leukocyte Integrin

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Angiogenesis In2 Β M Α Dual Role of the Leukocyte Integrin The Journal of Immunology Dual Role of the Leukocyte Integrin aMb2 in Angiogenesis Dmitry A. Soloviev,* Stanley L. Hazen,† Dorota Szpak,* Kamila M. Bledzka,* Christie M. Ballantyne,‡ Edward F. Plow,* and Elzbieta Pluskota* Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. aMb2(CD11b/CD18) and aLb2(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these b2 integrins in angiogenesis. 2/2 2/2 Angiogenesis was analyzed in wild-type (WT), aM-knockout (aM ), and aL-deficient (aL ) mice using B16F10 melanoma, 2/2 RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50–70% in aM mice, resulting in stunted tumor growth as compared with WT mice. In contrast, aL deficiency did not impair angiogenesis and tumor growth. The 2/2 neovessels in aM mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis 2/2 2/2 in the aM mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the aL 2/2 leukocytes, the aM myeloid cells showed impaired plasmin (Plm)-dependent extracellular matrix invasion, resulting from 50–75% decrease in plasminogen (Plg) binding and pericellular Plm activity. Surface plasmon resonance verified direct interaction of the aMI-domain, the major ligand binding site in the b2 integrins, with Plg. However, the aLI-domain failed to bind Plg. In addition, endothelial cells failed to form tubes in the presence of conditioned medium collected from TNF-a–stimulated PMNs derived from 2/2 the aM mice because of severely impaired degranulation and secretion of VEGF. Thus, aMb2 plays a dual role in angiogenesis, supporting not only Plm-dependent recruitment of myeloid cells to angiogenic niches, but also secretion of VEGF by these cells. The Journal of Immunology, 2014, 193: 4712–4721. one marrow–derived myeloid cells, particularly poly- degradation and remodeling and regulate the bioavailability of morphonuclear neutrophils (PMNs) and macrophages, various proangiogenic stimuli (5), all requisite events in angio- B are key regulators of tumor progression and metastasis. genesis (reviewed in Refs. 4, 6–8). In addition, PMNs and mac- One of the major tumor promoting functions of these cells is their rophages secrete urokinase-type plasminogen activator (uPA), facilitation of angiogenesis (reviewed in Refs. 1, 2). PMNs and which converts plasminogen (Plg) to plasmin (Plm). There are macrophages contribute to angiogenesis via a variety of well- diverse Plg receptors on leukocyte surface (reviewed in Ref. 9), established mechanisms. One example is their capacity to produce and bound Plm facilitates leukocyte migration/invasion by directly by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. and secrete a variety of proangiogenic factors such as vascular degrading ECM, and encourages leukocyte recruitment in a vari- endothelial growth factor A (VEGF-A), fibroblast growth factor ety of in vivo models of inflammation (10–12). (FGF), IL-8, IL-10, CXCL1/GRO, and COX-2 (3, 4). In addition, aMb2 (CD11b/CD18) and aLb2 (CD11a/CD18), the two most PMNs and macrophages are a rich source of numerous proteases, broadly studied members of the b2 integrin subfamily, are par- including neutrophil elastase, cathepsin G, and several metal- ticularly enriched in PMNs and macrophages, where they regulate loproteinases, which are crucial for extracellular matrix (ECM) diverse cell functions, including migration, adhesion, the respi- ratory burst, and cytokine production (13). In addition, we have previously demonstrated that aMb2 enhances uPA-dependent Plg *Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Learner Research Institute, Cleveland Clinic, Cleveland, OH 44195; activation on the PMN surface (14, 15), which has the potential to http://classic.jimmunol.org †Department of Molecular and Cellular Medicine, Cleveland Clinic, Cleveland, OH influence their recruitment to inflammatory or angiogenic sites ‡ 44195; and Baylor College of Medicine and Methodist DeBakey Heart and Vascular in vivo. Based on these observations, we hypothesized that a b Center, Houston, TX 77030 M 2 and aLb2, as key regulators of leukocyte functions, might be im- Received for publication January 24, 2014. Accepted for publication August 22, 2/2 2/2 2014. plicated in angiogenesis. In this study, we used aM and aL This work was supported by National Institute of Allergy and Infectious Diseases mice and three distinct in vivo angiogenesis models to show that Grant AIO 80596 (to D.A.S.), American Heart Association Grant SDG 0335088N (to aMb2, but not aLb2, is a critical contributor to angiogenesis. This Downloaded from E.P.), and National Institutes of Health–National Heart, Lung, and Blood Institute a b Grants R01 HL17964 and P01 HL07331 (to E.F.P.). function of M 2 is mediated by two distinct mechanisms: 1) support of Plm-dependent PMN and macrophage recruitment to Address correspondence and reprint requests to Dr. Elzbieta Pluskota, Department of Molecular Cardiology, NB50, Lerner Research Institute, Cleveland Clinic, 9500 angiogenic niches; and 2) enhancement of leukocyte production and Euclid Avenue, Cleveland, OH 44195. Email address: [email protected] secretion of the primary angiogenic growth factor, VEGF-A. The online version of this article contains supplemental material. Abbreviations used in this article: BM, bone marrow; BMT, BM transplantation; EC, Materials and Methods endothelial cell; ECM, extracellular matrix; FGF, fibroblast growth factor; GST, gluta- Materials thione S-transferase; KC, keratinocyte-derived cytokine; MAEC, mouse aortic endothe- lial cell; MOMA-2, macrophage–monocyte mAb; NG2, neural/glial Ag 2; Plg, Mouse VEGF165 and keratinocyte-derived cytokine (KC) were from plasminogen; Plm, plasmin; PMN, polymorphonuclear neutrophil; sc-uPA, single- Biosource International (Camarillo, CA); heparin was from Sigma- chain uPA; sFLT-1, soluble VEGF receptor-1; SMA, smooth muscle actin; SPR, surface plasmon resonance; uPA, urokinase-type plasminogen activator; VEGF-A, Aldrich (St. Louis, MO); biotin-conjugated anti-mouse CD31 mAb was vascular endothelial growth factor A. from BD Pharmingen (San Jose, CA); rabbit anti-smooth muscle actin (SMA; Abcam, Cambridge, MA); rabbit anti–neural/glial antigen 2 (NG2; Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Millipore, Temecula, CA); rabbit anti-mouse laminin (Serotec, Oxford, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400202 The Journal of Immunology 4713 UK), goat anti-Fibrin II (Accurate Chemical, Westbury, NY), purified, or transferred to 1.25% dextran T500 solution (1:9) to sediment erythrocytes FITC-labeled rat anti-Ly6G, clone 1A8, specific for mouse PMNs were for 30 min at room temperature (14, 18). Leukocyte-rich supernatants were from BD Pharmingen (San Jose, CA); anti-mouse macrophage–monocyte washed with PBS once, and PMNs were isolated by magnetic positive mAb (MOMA-2) was from Chemicon (Temecula, CA); and rat LEAF TM selection using mouse anti-Ly6G MicroBead Isolation Kit (Miltenyi Bio- purified anti-mouse aM integrin (clone M1/70) was from BioLegend (San tec, Auburn, CA) according to the manufacturer’s instructions. Eluted cells Diego, CA). Glu-Plg was isolated from normal human plasma by affinity were 98% granulocytes, of which more than 96% were neutrophils and chromatography on lysine-sepharose followed by gel filtration. Growth 1–2% were eosinophils. Contaminating lymphocytes and monocytes were factor–reduced Matrigel matrix was from BD Biosciences (San Diego, less than 2% as determined by Wright staining. PMN viability was .98% CA). Murine recombinant TNF-a was from R&D Systems. Cycloheximide as determined by trypan blue staining. The PMN yield was usually ∼0.5 3 6 and pentoxifylline were from Sigma-Aldrich. 10 per mouse, and blood pooled from 10–15 mice was used. For Plg activation assays, lymphocytes and monocytes were collected from buffy Mice coats and washed twice with the HBSS buffer. Leukocytes were obtained a 2/2 a 2/2 from blood pooled from 7–10 mice. The M mice were generated as described previously (16), and L For matrix invasion and Plg binding assays, macrophages and PMNs mice were purchased from the Jackson Laboratory. Both strains were were isolated from peritoneal lavages, PMNs at 6 h and macrophages at 72 h backcrossed for 12 generations into a C57BL/J6 background. The study was after i.p. thioglycolate injection, when their recruitment was at the highest conducted under protocols approved by the IACUC of the Cleveland Clinic. levels for each cell type (10). PMNs constitute 92% and macrophages Angiogenesis models in vivo constitute 90% of all cells in the 6- and 72-h peritoneal lavages, respec- tively. PMNs and macrophages harvested from lavages are referred to as Eight- to twelve-week-old mice were injected s.c. with 106 murine B16F10 “peritoneal PMN” or “peritoneal macrophages” in the manuscript to dis- melanoma or RM1 prostate cancer cells. Tumors were collected 8– tinguish them from blood cells. 14 d after injection and were weighed, photographed, and processed for immunohistochemical staining. Endothelial cells (ECs) were identified Matrix invasion assays ex vivo using a biotinylated mouse CD31 mAb, SMAs with anti-SMA Ab, peri- m cytes with anti-NG2 Ab, fibrin with anti-Fibrin II Ab, basement membrane Prechilled tissue culture inserts with porous (8- m pore size) polyester m with anti-laminin Ab, PMNs with rat anti-Ly6G (clone 1A8), and mono- membrane (Costar, Corning, NY) were coated with 50 l/insert Matrigel (0.5 mg/ml) overnight at 4˚C until dry. Next, matrices were rehydrated cytes–macrophages with MOMA-2 mAbs. Stained sections were analyzed m using fluorescent imaging microscopy (Leica, Wetzlar, Germany) and with 600 l DMEM F-12 for 1 h. Peritoneal PMNs or macrophages were suspended in serum-free DMEM F-12 medium (Life Technologies, ImagePro Plus Capture and Analysis software (Media Cybernetics).
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