CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m605040200
ISSN1083-351X
AutoresJeff S. Isenberg, Lisa A. Ridnour, Julie Dimitry, William A. Frazier, David A. Wink, David D. Roberts,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoCD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations. CD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations. Thrombospondin-1 (TSP1) 2The abbreviations used are: TSP1, thrombospondin-1; CBD, thrombospondin-1 C-terminal binding domain; DEA/NO, diethylamine NONOate; DETA/NO, diethyltriamine NONOate; HASMC, human aortic vascular smooth muscle cell; HUVEC, human umbilical vein endothelial cell; MASMC, murine aortic smooth muscle cell; NoC1, trimeric thrombospondin-1 residues 1–356; 3TSR, thrombospondin type 1 repeats; VSMC, vascular smooth muscle cell; FGF2, fibroblast growth factor 2; FCS, fetal calf serum; BSA, bovine serum albumin. is a secreted glycoprotein that plays a relatively minor role in development of the murine vascular system (1Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Invest. 1998; 101: 982-992Crossref PubMed Scopus (388) Google Scholar, 2Wang S. Wu Z. Sorenson C.M. Lawler J. Sheibani N. Dev. Dyn. 2003; 228: 630-642Crossref PubMed Scopus (109) Google Scholar), but its regulated appearance in the extracellular matrix plays important roles in responses to acute injury and in several chronic disease states in the adult. TSP1 is a major component of platelet α-granules and is released from platelets upon activation, where it modulates platelet adhesion and the properties of fibrin clots formed following acute vascular injury (3Mosher D.F. Misenheimer T.M. Stenflo J. Hogg P.J. Ann. N. Y. Acad. Sci. 1992; 667: 64-69Crossref PubMed Scopus (21) Google Scholar, 4Bonnefoy A. Hantgan R. Legrand C. Frojmovic M.M. J. Biol. Chem. 2001; 276: 5605-5612Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). TSP1 released from platelets or produced locally in response to cytokines and growth factors also plays an important role in recruitment of mononuclear cells during the early phases of wound repair, and the absence of TSP1 delays excisional wound repair in mice (5Agah A. Kyriakides T.R. Lawler J. Bornstein P. Am. J. Pathol. 2002; 161: 831-839Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). TSP1 also accumulates in the neointima of atherosclerotic lesions (6Riessen R. Kearney M. Lawler J. Isner J.M. Am. Heart. J. 1998; 135: 357-364Crossref PubMed Scopus (94) Google Scholar), where it may stimulate vascular smooth muscle cell (VSMC) proliferation and migration by enhancing responsiveness to platelet-derived growth factor (7Isenberg J.S. Calzada M.J. Zhou L. Guo N. Lawler J. Wang X.Q. Frazier W.A. Roberts D.D. Matrix Biol. 2005; 24: 110-123Crossref PubMed Scopus (50) Google Scholar). Antibody blocking of TSP1 can reverse this response and enhance the re-endothelialization of an injured artery (8Chen D. Asahara T. Krasinski K. Witzenbichler B. Yang J. Magner M. Kearney M. Frazier W.A. Isner J.M. Andres V. Circulation. 1999; 100: 849-854Crossref PubMed Scopus (89) Google Scholar). An N700S coding sequence polymorphism in TSP1 that alters its conformation is associated with increased risk of premature familial myocardial infarction (reviewed in Ref. 9Stenina O.I. Byzova T.V. Adams J.C. McCarthy J.J. Topol E.J. Plow E.F. Int. J. Biochem. Cell Biol. 2004; 36: 1013-1030Crossref PubMed Scopus (41) Google Scholar). TSP1 is also a potent modulator of angiogenesis (10Lawler J. Detmar M. Int. J. Biochem. Cell Biol. 2004; 36: 1038-1045Crossref PubMed Scopus (184) Google Scholar). The N-terminal domain of TSP1 stimulates angiogenesis through its interactions with α3β1 and α4β1 integrins (11Calzada M.J. Zhou L. Sipes J.M. Zhang J. Krutzsch H.C. Iruela-Arispe M.L. Annis D.S. Mosher D.F. Roberts D.D. Circ. Res. 2004; 94: 462-470Crossref PubMed Scopus (86) Google Scholar, 12Chandrasekaran L. He C.Z. Al-Barazi H. Krutzsch H.C. Iruela-Arispe M.L. Roberts D.D. Mol. Biol. Cell. 2000; 11: 2885-2900Crossref PubMed Scopus (141) Google Scholar), but the central type 1 repeats contain sequences that potently inhibit angiogenesis via CD36 and/or heparan sulfate proteoglycan receptors (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar, 14Iruela-Arispe M.L. Lombardo M. Krutzsch H.C. Lawler J. Roberts D.D. Circulation. 1999; 100: 1423-1431Crossref PubMed Scopus (271) Google Scholar). Under most circumstances, the net activity of intact TSP1 is anti-angiogenic. Thus, the absence of TSP1 enhances experimental and tumor-induced angiogenic responses (15Lawler J. Miao W.M. Duquette M. Bouck N. Bronson R.T. Hynes R.O. Am. J. Pathol. 2001; 159: 1949-1956Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Bocci G. Francia G. Man S. Lawler J. Kerbel R.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12917-12922Crossref PubMed Scopus (372) Google Scholar, 17Yano K. Kajiya K. Ishiwata M. Hong Y.K. Miyakawa T. Detmar M. J. Invest. Dermatol. 2004; 122: 201-208Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Vascular effects of TSP1 are mediated by its binding to receptors on both endothelial and VSMCs. Endothelial cells express at least eight TSP1 receptors, and several of these are shared on VSMCs. Based on the activity of CD36 antibody FA6-152 to block the inhibitory effect of TSP1 on FGF2-induced microvascular endothelial cell motility and the failure of TSP1 to inhibit corneal angiogenesis induced by FGF2 in CD36–/– mice, CD36 is considered to be the primary TSP1 receptor that mediates this anti-angiogenic activity (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar, 18Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Crossref PubMed Scopus (860) Google Scholar). Some evidence indicates that TSP1 also inhibits angiogenesis through β1 integrins, CD47, LRP/calreticulin, and heparan sulfate proteoglycans (14Iruela-Arispe M.L. Lombardo M. Krutzsch H.C. Lawler J. Roberts D.D. Circulation. 1999; 100: 1423-1431Crossref PubMed Scopus (271) Google Scholar, 19Short S.M. Derrien A. Narsimhan R.P. Lawler J. Ingber D.E. Zetter B.R. J. Cell Biol. 2005; 168: 643-653Crossref PubMed Scopus (118) Google Scholar, 20Kanda S. Shono T. Tomasini-Johansson B. Klint P. Saito Y. Exp. Cell Res. 1999; 252: 262-272Crossref PubMed Scopus (65) Google Scholar, 21Elzie C.A. Murphy-Ullrich J.E. Int. J. Biochem. Cell Biol. 2004; 36: 1090-1101Crossref PubMed Scopus (67) Google Scholar), but the necessity of these receptors for TSP1 to inhibit angiogenesis has not been confirmed in the respective receptor-null mice. To develop effective angiogenesis inhibitors based on TSP1, it is important to establish whether additional signaling pathways may allow TSP1 to inhibit angiogenesis in a CD36-independent manner. We recently found that the anti-angiogenic activity of TSP1 is dramatically potentiated in the presence of low concentrations of nitric oxide (NO) donors (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar). This activity is mediated at least in part through inhibition by TSP1 of cGMP signaling via NO-mediated activation of soluble guanylyl cyclase in both endothelial cells (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar) and VSMCs (23Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; (in press)Google Scholar). Moreover, TSP1-null vascular cells exhibit elevated basal cGMP levels and enhanced cGMP and phenotypic responses to exogenous NO. The inhibitory activity of TSP1 on NO signaling was replicated by an agonist CD36 antibody and by a recombinant CD36-binding region of TSP1, suggesting that this activity is also mediated by CD36. However, our attempts to confirm this hypothesis led us to revise this model. We present here evidence that a second TSP1 receptor, CD47, plays the primary role in mediating the inhibitory activities of TSP1 for vascular cells. Remarkably, CD47 is also necessary for inhibition of NO signaling by CD36 ligands. Cells and Reagents—Human umbilical vein endothelial cells (HUVECs, Cambrex, Walkersville, MD) were maintained in endothelial cell growth medium (Cambrex) with 5% FCS in 5% CO2 at 37 °C. Cells were utilized at passages 4–8. Purity of cultures was monitored by immunochemical staining with monoclonal human anti-CD31 antibody and monoclonal anti-α smooth muscle actin from Sigma. Human aortic smooth muscle cells (HASMCs, Cambrex) were maintained in smooth muscle cell growth medium with the manufacturer's additives (SM-GM, Clonetics) and 5% FCS in 5% CO2 at 37 °C. Cells utilized were within passages 4–9. Purity of primary cultures was monitored by immunochemical staining with monoclonal human anti-CD31 antibody and α-smooth muscle actin (Sigma). DEA/NO and DETA/NO were kindly provided by Dr. Larry Keefer (NCI, National Institutes of Health (NIH), Frederick, MD). TSP1 was prepared from human platelets obtained from the NIH blood bank as previously described (24Roberts D.D. Cashel J. Guo N. J. Tissue Cult. Methods. 1994; 16: 217-222Crossref Scopus (44) Google Scholar). Recombinant proteins expressed in insect cells containing the N-terminal domains (NoC1), type 1 repeats (3TSR), or C-terminal regions of TSP1 (E3CaG1) were generously provided by Dr. Deane Mosher (University of Wisconsin) and Dr. Jack Lawler, Harvard Medical School (25Misenheimer T.M. Huwiler K.G. Annis D.S. Mosher D.F. J. Biol. Chem. 2000; 275: 40938-40945Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 26Tan K. Duquette M. Liu J.H. Dong Y. Zhang R. Joachimiak A. Lawler J. Wang J.H. J. Cell Biol. 2002; 159: 373-382Crossref PubMed Scopus (213) Google Scholar). The recombinant C-terminal cell-binding domain (CBD) was prepared as previously described (27McDonald J.F. Dimitry J.M. Frazier W.A. Biochemistry. 2003; 42: 10001-10011Crossref PubMed Scopus (30) Google Scholar). Murine anti-human CD36 antibody (clone SMΦ) was purchased from Chemicon International (Temecula, CA). CD36 antibody clone FA6-152 was purchased from Immunotech (Beckman Coulter). CD36 antibody clone 185-1G2 was purchased from Neomarkers (Fremont, CA). Anti-cyclin D1 (IgG1) and estrogen receptor-α (IgG2a) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HSP-60 monoclonal antibody (IgM) was purchased from Affinity Bioreagents Inc. (Golden, CO). Anti-CD47 antibody (clone CIKm1) was from ICN (Costa Mesa, CA). Type I collagen (Vitrogen) was from Cohesion Technologies (Palo Alto, CA). Peptides 246, 245, 7N3, 4N1-1, and 761 were prepared as described (28Barazi H.O. Li Z. Cashel J.A. Krutzsch H.C. Annis D.S. Mosher D.F. Roberts D.D. J. Biol. Chem. 2002; 277: 42859-42866Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Peptides 906 and 907 were prepared by Peptides International (Louisville, KY). B6H12 (anti-CD47) was purified by protein G affinity chromatography (Pierce) from conditioned media of the respective hybridoma (American Type Culture Collection). Murine Cell Cultures—Murine ASMCs were obtained from aortic segments harvested sterilely from C57B1/6 CD47-null or CD36-null mice as described (29Ray J.L. Leach R. Herbert J.M. Benson M. Methods Cell Sci. 2001; 23: 185-188Crossref PubMed Scopus (202) Google Scholar) and cultured in SM-GM (Cambrex) plus 20% FCS. In the case of CD36-null cells, culture flasks were pre-coated with 1% gelatin prior to cell plating. Wild-type C57B1/6 ASMC cultures were prepared as previously described (7Isenberg J.S. Calzada M.J. Zhou L. Guo N. Lawler J. Wang X.Q. Frazier W.A. Roberts D.D. Matrix Biol. 2005; 24: 110-123Crossref PubMed Scopus (50) Google Scholar). Cell culture purity was determined by immunohistochemistry staining for α-smooth muscle actin. Cells were used within passages 1–4 to minimize overgrowth of other cell types. Animals—C57B1/6 WT and TSP1-null (1Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Invest. 1998; 101: 982-992Crossref PubMed Scopus (388) Google Scholar) CD47-null (30Lindberg F.P. Bullard D.C. Caver T.E. Gresham H.D. Beaudet A.L. Brown E.J. Science. 1996; 274: 795-798Crossref PubMed Scopus (304) Google Scholar) and CD36-null mice (31Moore K.J. El Khoury J. Medeiros L.A. Terada K. Geula C. Luster A.D. Freeman M.W. J. Biol. Chem. 2002; 277: 47373-47379Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) were all extensively backcrossed on the C57Bl/6 background and were housed in a pathogen-free environment. Handling and care of animals was in compliance with the guidelines established by the Animal Care and Use Committees of the National Cancer Institute and of Washington University School of Medicine. Explant Invasion Assay—Muscle biopsies from the pectoralis major muscle of 8- to 12-week-old wild-type or transgenic mice were harvested and explanted into 100 μlof type I collagen gel in 96-well tissue culture plates as described (7Isenberg J.S. Calzada M.J. Zhou L. Guo N. Lawler J. Wang X.Q. Frazier W.A. Roberts D.D. Matrix Biol. 2005; 24: 110-123Crossref PubMed Scopus (50) Google Scholar). Following gelation, the embedded explants were overlaid with 75 μlof EGM plus 2% FCS in the presence or absence of a dose range of DETA/NO (0.01–1000 μm) and other indicated treatments and then incubated at 37 °C and 5% CO2 for 7 days, at which time maximum vascular cell migration through the matrix was measured. Cell Proliferation—Proliferation of vascular cells was measured with a non-radioactive colorimetric assay (CellTiter 96, Promega, Madison, WI). Briefly, to each well of a 96-well culture plate (Nunc, Denmark) 5 × 103 cells were suspended in 100 μl of culture medium with indicated treatments and incubated for 72 h at 37 °C in a 5% CO2 atmosphere. Following incubation 20 μl of tetrazolium compound/solubilization agent was added, and incubation continued for 4 h under the same conditions. The plate was then read on a MR580 Microelisa Auto Reader (Dynatech) at a wavelength of 490 nm. Appropriate zero time controls were run for all assays, and the optical density readings obtained were then subtracted from those obtained at 72 h. Cell Adhesion Assay—Cell adhesion was carried out in 96-well culture plates. After pre-coating wells with type I collagen (3 μg/ml in Dulbecco's phosphate-buffered saline) harvested cells were plated at a density of 1 × 104 cells/well in medium plus 0.1% BSA and treatment agents and incubated in 5% CO2 for 1 h. Wells were washed with phosphate-buffered saline, and cells were fixed with 1% glutaraldehyde for 10 min, washed, and stained with 1% crystal violet for 20 min. Excess stain was rinsed away, adherent cells were treated with 10% acetic acid, and plates were read at 570 nm. Intracellular cGMP Measurement—HUVECs (104 cells/well) were grown overnight in 96-well culture plates containing full growth medium with 2% FCS and weaned in growth medium without additives and 1% FCS over 24 h before treatment with NO donors and other agents in SM-GM without additives plus 0.1% BSA. Intracellular cGMP levels were determined according to the manufacturer's instructions using an enzyme immunoassay kit (Amersham Biosciences). In other experiments ASMCs from wild-type and CD36 or CD47-null mice were plated onto 96-well culture plates and incubated overnight in full growth medium. They were then weaned off serum as described to SM-GM plus 0.1% BSA and treated with NO donor and other agents as indicated. Intracellular cyclic nucleotides were determined via immunoassay. Statistics—All studies were repeated in triplicate, and results are presented as the mean ± S.D., with analysis of significance done using the Student's t test and a p < 0.05 taken as significant. Peptide Ligands of Three TSP1 Receptors Inhibit Explant Angiogenic Responses—As previously reported (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar), sustained exposure to exogenous NO released by the donor DETA/NO stimulated vascular outgrowth from muscle explants in three-dimensional collagen cultures to a greater extent in those from TSP1-null when compared with WT mice (Fig. 1A, controls). Consistent with their reported affects on endothelial cells in vitro and angiogenesis in vivo (14Iruela-Arispe M.L. Lombardo M. Krutzsch H.C. Lawler J. Roberts D.D. Circulation. 1999; 100: 1423-1431Crossref PubMed Scopus (271) Google Scholar, 20Kanda S. Shono T. Tomasini-Johansson B. Klint P. Saito Y. Exp. Cell Res. 1999; 252: 262-272Crossref PubMed Scopus (65) Google Scholar), a CD36-binding peptide from the third type 1 repeat of TSP1 (p245, VTCGGGVQKRSRL), a CD47-binding peptide from the C-terminal module of TSP1 (p7N3, FIRVVMYEGKK), and to a lesser extent a heparin- and transforming growth factor-β-binding peptide from the second type 1 repeat (p246, KRFKQDGGWSHWSPWSS) inhibited vascular cell outgrowth from both wild-type and TSP1-null explants stimulated by NO (Fig. 1). Conversely, the described pro-angiogenic activities of the N-terminal region of TSP1 (11Calzada M.J. Zhou L. Sipes J.M. Zhang J. Krutzsch H.C. Iruela-Arispe M.L. Annis D.S. Mosher D.F. Roberts D.D. Circ. Res. 2004; 94: 462-470Crossref PubMed Scopus (86) Google Scholar, 12Chandrasekaran L. He C.Z. Al-Barazi H. Krutzsch H.C. Iruela-Arispe M.L. Roberts D.D. Mol. Biol. Cell. 2000; 11: 2885-2900Crossref PubMed Scopus (141) Google Scholar) were reflected by enhanced vascular outgrowth from explants in the presence of recombinant NoC1. Assuming that these peptides act as agonists of their respective receptors, this indicates that ligating CD36, heparan sulfate proteoglycans, or CD47 is sufficient to inhibit NO-stimulated vascular outgrowth, whereas ligating β1 integrins or other TSP1 N-module receptors enhances vascular outgrowth under the same conditions. CD47 but Not CD36 Is Necessary for Inhibition by TSP1 of Explant Angiogenic Responses—Although the former data show that ligating CD36 or CD47 is sufficient to inhibit explant angiogenesis stimulated by NO, they do not prove that the respective receptors are necessary for activities of the peptide ligands or of intact TSP1. Furthermore, although these peptides clearly bind to the indicated receptors, structural studies have raised concerns that VVM peptides may not represent the true CD47 binding site in the C-terminal domain of TSP1 (32Kvansakul M. Adams J.C. Hohenester E. EMBO J. 2004; 23: 1223-1233Crossref PubMed Scopus (127) Google Scholar). To directly address the roles of CD36 and CD47 in the inhibitory activity of TSP1, muscle explants from mice lacking the respective receptors were placed into three-dimensional collagen cultures (Fig. 2). Similar to wild-type explants, NO dose-dependently stimulated vascular outgrowth in CD36- or CD47-null explants. Remarkably, the ability of exogenous TSP1 to antagonize this response was preserved in CD36-null explants (Fig. 2B) but lost in CD47-null explants (Fig. 2A). Thus, CD47 is necessary for the anti-angiogenic activity of TSP1 in this assay, but CD36 is not. This result was unexpected given that a recombinant CD36-binding domain of TSP1 (3TSR) and a CD36 antibody (SMΦ) described to be an agonist based on its ability to mimic TSP1 (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar) were shown previously to inhibit endothelial cell adhesion on type I collagen stimulated by acute NO exposure (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar). This prompted us to re-examine the role of CD36 in this activity of TSP1. CD36 Ligation Is Sufficient but Not Necessary for Inhibition of NO Responses by TSP1—A CD36-binding peptide derived from the third type 1 repeat (p906, VTAGGGVQKRSRL) and a derivative of the second type 1 repeat with enhanced CD36-binding and anti-angiogenic activity (p907, GDGV(D/I)TRIR (33Dawson D.W. Volpert O.V. Pearce S.F. Schneider A.J. Silverstein R.L. Henkin J. Bouck N.P. Mol. Pharmacol. 1999; 55: 332-338Crossref PubMed Scopus (135) Google Scholar)) similarly inhibited NO-stimulated endothelial cell adhesion (Fig. 3A). As previously reported, the CD36 agonist antibody SMΦ inhibited NO-stimulated cell adhesion, whereas a control IgM was inactive (Fig. 3C). However, two CD36 antibodies that were reported to antagonize inhibition by TSP1, FA6-152 (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar) and 185-1G2 (19Short S.M. Derrien A. Narsimhan R.P. Lawler J. Ingber D.E. Zetter B.R. J. Cell Biol. 2005; 168: 643-653Crossref PubMed Scopus (118) Google Scholar), also inhibited NO-stimulated adhesion (Fig. 3, B and D). Isotype-matched control antibodies did not inhibit NO-stimulated cell adhesion, demonstrating the specificity of these CD36 antibodies for blocking an NO response. Thus, various CD36 ligands are sufficient to inhibit NO-stimulated endothelial cell responses, but their mechanism of action may differ from the previously described TSP1 responses that were antagonized by the CD36 antibody FA6-152 (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar). Antagonism of NO signaling by TSP1 is conserved in VSMCs (23Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; (in press)Google Scholar). Consistent with the data for endothelial cells in Fig. 3, the CD36 antagonist antibodies FA6-152 and 185-1G2 but not isotype-matched control antibodies were dose-dependent inhibitors of NO-stimulated HASMC adhesion (Fig. 4A and results not shown). The FA6-152 antibody also prevented NO-induced accumulation of cGMP in HASMCs (Fig. 4B), consistent with its effects on NO-stimulated adhesion but not with its reported activity as a TSP1 antagonist (13Dawson D.W. Pearce S.F. Zhong R. Silverstein R.L. Frazier W.A. Bouck N.P. J. Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (552) Google Scholar, 18Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Crossref PubMed Scopus (860) Google Scholar). Therefore, inhibition of NO signaling by CD36 ligation is conserved in both types of vascular cells but is independent of the ability of CD36 ligands to mimic or inhibit TSP1 activity in other angiogenesis assays. To clarify the role of CD36 in inhibition by TSP1 of NO-stimulated responses, we used MASMCs derived from WT and CD36-null mice (Fig. 5). Low dose NO significantly stimulated adhesion of WT MASMCs on type I collagen, and TSP1 at 22 pm inhibited this response to control levels (Fig. 5A). Higher doses of TSP1 further inhibited adhesion below baseline. Remarkably, 22 pm TSP1 inhibited NO-stimulated adhesion of CD36-null MASMC to the same extent as in WT cells. Although higher concentrations of TSP1 further suppressed NO-induced adhesion of WT cells to levels below the basal level of the untreated controls, in the CD36-null cells further inhibition by TSP1 was only seen at 22 nm. These data established that CD36 is not necessary for picomolar concentrations of TSP1 to inhibit an NO-stimulated response, although a secondary inhibitory response at nanomolar concentrations of TSP1 does require CD36. We have shown that picomolar concentrations of TSP1 inhibit NO signaling at the level of cGMP (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar, 23Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; (in press)Google Scholar). To establish whether inhibition by TSP1 of NO signaling through cGMP requires CD36, cGMP levels were analyzed in the WT and CD36-null MASMCs (Fig. 5B). As shown previously (23Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; (in press)Google Scholar), NO-stimulated cGMP levels in WT cells were inhibited by exogenous TSP1. This activity of TSP1 does not require CD36, however, because the cGMP response was also completely inhibited by TSP1 in CD36-null MASMC (Fig. 5B). Notably, basal cGMP levels were similar in WT and CD36-null cells, suggesting that the previously reported effects of endogenous TSP1 on basal cGMP levels in vascular cells (22Isenberg J.S. Ridnour L.A. Perruccio E.M. Espey M.G. Wink D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13141-13146Crossref PubMed Scopus (222) Google Scholar, 23Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; (in press)Google Scholar) do not require CD36. CD47 Ligation Inhibits NO Responses—The explant data in Fig. 2 suggested that CD47 could mediate the CD36-independent regulation of NO signaling by TSP1. To further examine the role of CD47, we tested two CD47-binding sequences identified in the CBD of TSP1 (34Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Peptides containing the first (4N1-1, Fig. 6A) or second VVM motifs from this domain (7N3, Fig. 6B) inhibited NO-stimulated endothelial cell adhesion on type I collagen. Inhibition by peptide 7N3 was dose-dependent and maximal at 10 μm. Specificity was confirmed using a control peptide in which the first VVM motif was substituted by GGM (p4N1G, RFYGGMWK), which at 10 μm did not significantly inhibit NO-stimulated adhesion (data not shown). Although the VVM peptides clearly bind to CD47 (34Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 35Frazier W.A. Gao A.G. Dimitry J. Chung J. Brown E.J. Lindberg F.P. Linder M.E. J. Biol. Chem. 1999; 274: 8554-8560Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), crystal structures of recombinant C-terminal regions of TSP1 and TSP2 have raised doubts about the exposure of the VVM motifs in native TSP1 (32Kvansakul M. Adams J.C. Hohenester E. EMBO J. 2004; 23: 1223-1233Crossref PubMed Scopus (127) Google Scholar, 36Carlson C.B. Bernstein D.A. Annis D.S. Misenheimer T.M. Hannah B.L. Mosher D.F. Keck J.L. Nat. Struct. Mol. Biol. 2005; 12: 910-914Crossref PubMed Scopus (73) Google Scholar). Based on a crystal structure for this domain of the paralog TSP2 (36Carlson C.B. Bernstein D.A. Annis D.S. Misenheimer T.M. Hannah B.L. Mosher D.F. Keck J.L. Nat. Struct. Mol. Biol. 2005; 12: 910-914Crossref PubMed Scopus (73) Google Scholar), the third type 2 repeat, the Ca-binding repeats, and the G module fold together to form the C-terminal globular domain of TSP1. A recombinant construct containing these elements of TSP1 (E3CaG1, Fig. 1B) at ≥0.4 nm inhibited NO-driven but not basal HASMC adhesion to collagen (Fig. 7A). Recombinant G module (CBD), which is al
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