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• W2087929503 abstract "Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPα (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2. Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPα (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2. Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction. NO preserves vascular health by minimizing the adhesion of inflammatory cells to the vessel wall, limiting platelet activation, and increasing blood vessel diameter and blood flow by relaxing vascular smooth muscle cells (VSMC). 3The abbreviations used are: VSMC, vascular smooth muscle cell(s); DEA/NO, diethylamine/NONOate; sGC, soluble guanylate cyclase; SIRP, signal-regulatory protein; TSP, thrombospondin; BSA, bovine serum albumin; PBS, phosphate-buffered saline.3The abbreviations used are: VSMC, vascular smooth muscle cell(s); DEA/NO, diethylamine/NONOate; sGC, soluble guanylate cyclase; SIRP, signal-regulatory protein; TSP, thrombospondin; BSA, bovine serum albumin; PBS, phosphate-buffered saline. These actions of NO are mediated by activating soluble isoforms of guanylate cyclase (sGC) to increase cGMP levels, resulting in downstream activation of cGMP-dependent protein kinases and ion channels (1Ignarro L.J. J. Physiol. Pharmacol. 2002; 53: 503-514PubMed Google Scholar). Physiological NO/cGMP signaling is limited by several phosphodiesterases that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein that is produced by vascular and inflammatory cells that regulates cellular behavior by engaging several cell surface receptors. Recently we reported that TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and VSMC (2Isenberg 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 (207) Google Scholar, 3Isenberg J.S. Wink D.A. Roberts D.D. Cardiovasc. Res. 2006; 71: 785-793Crossref PubMed Scopus (93) Google Scholar). TSP1 also plays a role in promoting platelet thrombus formation and hemostasis by antagonizing the antithrombotic activity of NO (4Isenberg J.S. Hyodo F. Matsumoto K. Romeo M.J. Abu-Asab M. Tsokos M. Kuppusamy P. Wink D.A. Krishna M.C. Roberts D.D. Blood. 2007; 109: 1945-1952Crossref PubMed Scopus (89) Google Scholar). In all of these vascular cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated fluxes in cGMP by engaging its receptor CD47 (5Isenberg J.S. Ridnour L.A. Dimitry J. Frazier W.A. Wink D.A. Roberts D.D. J. Biol. Chem. 2006; 281: 26069-26080Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Nanomolar concentrations of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated uptake of myristate into vascular cells (6Isenberg J.S. Jia Y. Fukuyama J. Switzer C.H. Wink D.A. Roberts D.D. J. Biol. Chem. 2007; 282: 15404-15415Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In vivo, mice lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater increases in regional blood flow in response to a NO challenge than wild type controls (4Isenberg J.S. Hyodo F. Matsumoto K. Romeo M.J. Abu-Asab M. Tsokos M. Kuppusamy P. Wink D.A. Krishna M.C. Roberts D.D. Blood. 2007; 109: 1945-1952Crossref PubMed Scopus (89) Google Scholar). After an ischemic insult, the absence of TSP1 or CD47 in transgenic mice is associated with better maintenance of tissue perfusion and enhanced tissue survival. Similarly, targeting TSP1 or CD47 using function blocking antibodies enhances ischemic tissue perfusion and survival in wild type mice and pigs (7Isenberg J.S. Romeo M.J. Abu-Asab M. Tsokos M. Oldenborg A. Pappan L. Wink D.A. Frazier W.A. Roberts D.D. Circ. Res. 2007; 100: 712-720Crossref PubMed Scopus (90) Google Scholar, 8Isenberg J.S. Romeo M.J. Maxhimer J.B. Smedley J. Frazier W.A. Roberts D.D. Ann. Surg. 2008; 247: 860-868Crossref PubMed Scopus (43) Google Scholar). TSP1 belongs to a family of five secreted glycoproteins that share an evolutionarily conserved C-terminal signature domain (9Carlson C.B. Lawler J. Mosher D.F. Cell. Mol. Life Sci. 2008; 65: 672-686Crossref PubMed Scopus (137) Google Scholar). TSP1 and TSP2 form a distinct subfamily of trimeric proteins that exhibit similar anti-angiogenic activities for endothelial cells in vitro and activities in vivo to block tumor growth. Despite their similarities in structure, TSP1 and TSP2 have markedly different expression patterns after tissue injury, with TSP1 being immediately expressed and maximal at day 3, whereas TSP2 was not expressed until day 7 and was maximal 10 days after injury (10Agah A. Kyriakides T.R. Lawler J. Bornstein P. Am. J. Pathol. 2002; 161: 831-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In addition, large amounts of TSP1 but not TSP2 are stored in platelet α-granules and released into the wound environment. Polymorphisms in TSP1 and TSP2 have been linked to altered risk of premature myocardial infarction (11Topol E.J. McCarthy J. Gabriel S. Moliterno D.J. Rogers W.J. Newby L.K. Freedman M. Metivier J. Cannata R. O'Donnell C.J. Kottke-Marchant K. Murugesan G. Plow E.F. Stenina O. Daley G.Q. Circulation. 2001; 104: 2641-2644Crossref PubMed Scopus (252) Google Scholar, 12Boekholdt S.M. Trip M.D. Peters R.J. Engelen M. Boer J.M. Feskens E.J. Zwinderman A.H. Kastelein J.J. Reitsma P.H. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 24-27Crossref PubMed Google Scholar). A 3′-untranslated region polymorphism in TSP2 is also associated with type 2 diabetes in men (13Yamaguchi S. Yamada Y. Matsuo H. Segawa T. Watanabe S. Kato K. Yokoi K. Ichihara S. Metoki N. Yoshida H. Satoh K. Nozawa Y. Int. J. Mol. Med. 2007; 19: 631-637PubMed Google Scholar). The molecular basis for these associations is unclear. Less is known about the roles of the pentameric TSP3–5 in vascular cells. TSP3 and TSP5 (also known as cartilage oligomeric matrix protein) appear to serve their primary functions in bone development (14Hankenson K.D. Hormuzdi S.G. Meganck J.A. Bornstein P. Mol. Cell. Biol. 2005; 25: 5599-5606Crossref PubMed Scopus (48) Google Scholar, 15Posey K.L. Hayes E. Haynes R. Hecht J.T. Int. J. Biochem. Cell Biol. 2004; 36: 1005-1012Crossref PubMed Scopus (44) Google Scholar). However, a polymorphism in TSP4 is associated with premature myocardial infarcts in certain populations (11Topol E.J. McCarthy J. Gabriel S. Moliterno D.J. Rogers W.J. Newby L.K. Freedman M. Metivier J. Cannata R. O'Donnell C.J. Kottke-Marchant K. Murugesan G. Plow E.F. Stenina O. Daley G.Q. Circulation. 2001; 104: 2641-2644Crossref PubMed Scopus (252) Google Scholar, 16Wessel J. Topol E.J. Ji M. Meyer J. McCarthy J.J. Am. Heart J. 2004; 147: 905-909Crossref PubMed Scopus (50) Google Scholar, 17Cui J. Randell E. Renouf J. Sun G. Green R. Han F.Y. Xie Y.G. Am. Heart J. 2006; 152: 543.e1-543.e5Crossref Scopus (22) Google Scholar). A proatherogenic activity for the A387P variant of TSP4 was proposed based on its differential ability to modulate proliferation of endothelial and VSMC (18Stenina O.I. Desai S.Y. Krukovets I. Kight K. Janigro D. Topol E.J. Plow E.F. Circulation. 2003; 108: 1514-1519Crossref PubMed Scopus (83) Google Scholar). Cardiovascular functions of TSP4 may also be linked to the high expression of TSP4 in heart (19Lawler J. Duquette M. Whittaker C.A. Adams J.C. McHenry K. DeSimone D.W. J. Cell Biol. 1993; 120: 1059-1067Crossref PubMed Scopus (117) Google Scholar) and its altered expression in that tissue during hypertensive heart failure (20Rysa J. Leskinen H. Ilves M. Ruskoaho H. Hypertension. 2005; 45: 927-933Crossref PubMed Scopus (95) Google Scholar). The C-terminal domain of TSP1 is sufficient to mediate CD47-dependent inhibition of cGMP signaling (5Isenberg J.S. Ridnour L.A. Dimitry J. Frazier W.A. Wink D.A. Roberts D.D. J. Biol. Chem. 2006; 281: 26069-26080Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Of the two CD47 binding VVM motifs identified in this domain of TSP1, the first is conserved among all five TSPs, suggesting that CD47 binding could be a universal attribute of this family (21Gao 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 (327) Google Scholar). Based on structural evidence that the VVM motifs may not be accessible (22Adams J.C. Bentley A.A. Kvansakul M. Hatherley D. Hohenester E. J. Cell Sci. 2008; 121: 784-795Crossref PubMed Scopus (34) Google Scholar, 23Kvansakul M. Adams J.C. Hohenester E. EMBO J. 2004; 23: 1223-1233Crossref PubMed Scopus (123) Google Scholar), however, conservation of VVM motifs may not be sufficient to predict CD47 binding. Uncertainty regarding the location of the CD47 binding site in the G domain of TSP1 therefore limits interpretation of the known sequence homology to predict CD47 binding to other TSP family members. Although CD47 recognition of other TSPs has not been demonstrated experimentally, a local deficiency of inflammation-associated T cell apoptosis shared by TSP1-, CD47-, and TSP2-null mice is consistent with this hypothesis (24Ticchioni M. Raimondi V. Lamy L. Wijdenes J. Lindberg F.P. Brown E.J. Bernard A. FASEB J. 2001; 15: 341-350Crossref PubMed Scopus (47) Google Scholar). Furthermore, a 21-residue peptide from the C-terminal domain of TSP4 was found to decrease human umbilical vein endothelial cell proliferation similar to the CD47 binding peptides from TSP1, although it lacks the VVM motif and no interaction with CD47 was demonstrated (25Congote L.F. Temmel N. FEBS Lett. 2004; 576: 343-347Crossref PubMed Scopus (23) Google Scholar). To directly address whether other TSP family members can inhibit NO responses and signaling in vascular cells, we now compare binding of recombinant signature domains of TSP1, TSP2, and TSP4 to cell surface CD47 and inhibition of NO-stimulated cell responses and cGMP signaling by these domains. We also compared acute tissue blood flow and perfusion responses to ischemic challenge in TSP1 and TSP2-null mice and cGMP responses in primary cultures of vascular cells isolated from these mice. These studies clearly demonstrate that CD47 selectively interacts with TSP1 and that the signature domains of TSP2 and TSP4 are less potent inhibitors of NO signaling in vascular cells in vitro. Furthermore, we show that the role of TSP1 to acutely limit recovery from ischemic injury in vivo is not shared by TSP2. Cells and Reagents—Human aortic VSMC were obtained from Lonza (Switzerland) and maintained in the appropriate growth medium provided by the manufacturer. Wild type and CD47 negative (clone JinB8) Jurkat cells were obtained from Drs. Kevin Gardner and Eric Brown, respectively, and maintained in RPMI 1640 containing 10% fetal calf serum. The nitric oxide donors diethylamine/NONOate (DEA/NO) and diethylenetriamine/NONOate were provided kindly by Dr. Larry Keefer (NCI-Frederick, Maryland). Type I collagen was obtained from Inamed Biomaterials (Fremont, CA). TSP1 was prepared as previously described from fresh platelets obtained under an approved protocol from the transfusion service of the National Institutes of Health (26Roberts D.D. Cashel J. Guo N. J. Tissue Cult. Methods. 1994; 16: 217-222Crossref Scopus (44) Google Scholar). Recombinant proteins derived from TSP4 were expressed in insect cells using baculovirus (27Misenheimer T.M. Mosher D.F. J. Biol. Chem. 2005; 280: 41229-41235Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recombinant regions of TSP1 and TSP2 as summarized in Fig. 1A were prepared as described (28Annis D.S. Gunderson K.A. Mosher D.F. J. Biol. Chem. 2007; 282: 27067-27075Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Recombinant extracellular domain of human SIRPα including all three Ig domains fused to a modified human Fc domain was prepared as described (29Piccio L. Vermi W. Boles K.S. Fuchs A. Strader C.A. Facchetti F. Cella M. Colonna M. Blood. 2005; 105: 2421-2427Crossref PubMed Scopus (77) Google Scholar). SIRPα and E123CaG1 were labeled using Na125I by the iodogen method as previously described for TSP1 (30Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (140) Google Scholar). A CD47 monoclonal antibody (B6H12) was purified from conditioned medium of the corresponding hybridoma (American Type Culture Collection). An isotype-matched control IgG1 was obtained from Santa Cruz Biotechnology. TSP1 antibody A6.1 was obtained from Thermo Scientific (Cheshire, UK). An antisense morpholino oligonucleotide complementary to murine TSP2 mRNA (5′-GGCCAGTGCCCAGAGCATCTTGTCT-3′) and a 5-base mismatched control morpholino were obtained from Gene Tools (Philomath, OR). Animals—C57BL/6 wild type and TSP1-null mice were housed in a pathogen-free environment with ad libitum access to water and rat chow on a 12-h light-dark cycle. TSP2-null male mice 12 weeks of age and matched wild type mice in a B6129sf1/J background were obtained from The Jackson Laboratory (Bar Harbor, ME). All animal studies conformed to the guidelines of the Animal Care and Use Committee of the National Cancer Institute of the NIH. Primary Murine Vascular Cell Isolation—Using sterile technique, whole lungs were excised from 12-week-old wild type, TSP1, and TSP2-null male mice, minced into 1–2-mm fragments, and incubated in basal medium with collagenase type II (Worthington, MA). The cell suspension was then plated in standard culture flasks (Nunc) in the presence of endothelial cell growth medium (Lonza) and used at the first passage. Under such conditions staining for CD31 has demonstrated 85–95% of cells to be endothelial cells (2Isenberg 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 (207) Google Scholar). Cell Adhesion Assay—Human aortic VSMC were plated at 10,000 cells/well onto 96-well plates (Nunc Maxisorb) precoated with type I collagen (3 μg/ml). Cells were incubated for 1 h in basal medium with 0.1% BSA and no growth factors and the indicated concentrations of recombinant TSP2, -3, and -4 domains ± DEA/NO. Plates were then washed with PBS, fixed with glutaraldehyde, stained with crystal violet, and washed. Absorbed stain was solubilized with acetic acid from fixed cells and quantified using a Micro580 Elisa plate reader (Dynatech Laboratories, Alexandria, VA) at 450 nm. Cell Proliferation Assay—Primary wild type and TSP2-null lung-derived endothelial cells were harvested as described (2Isenberg 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 (207) Google Scholar) and plated at a density of 10,000 cells/well in standard endothelial cell growth medium ± NO (10 μm diethylenetriamine/NONOate) and incubated for 72 h at 37 °C and 5% CO2. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent (Promega, Madison, WI) was then added, and the product was quantified at 450 nm. Intracellular Cyclic GMP Measurement—Yucatan white hairless pig femoral artery VSMC (8Isenberg J.S. Romeo M.J. Maxhimer J.B. Smedley J. Frazier W.A. Roberts D.D. Ann. Surg. 2008; 247: 860-868Crossref PubMed Scopus (43) Google Scholar) or wild type and TSP1- and TSP2-null murine endothelial cells (105 cells/well) grown overnight in 6- or 12-well culture plates (Nunc) were pretreated for 24 h with medium containing 2% fetal calf serum and weaned off serum over 48 h before treatment with DEA/NO with or without the indicated treatment agents in serum-free medium containing 0.1% BSA. Intracellular cGMP levels were determined using an enzyme immunoassay (Amersham Biosciences) as per the manufacturer's instructions. Hindlimb Ischemia Model—Wild type and TSP2-null 12-week-old male mice underwent inhalation general anesthesia with 1.5% isoflurane. Using sterile techniques, the femoral artery at the level of the inguinal ligament was identified and under operative magnification ligated with a 5–0 nylon suture, and the skin incision was closed. Limb perfusion was then measured at 5-min intervals for the first hour postoperatively and at 72 h post-operatively via laser Doppler (Moor Instruments, Devon England). Also at 72 h post-operatively, vascular remodeling was assessed in the medial thigh musculature above and below the point of femoral artery ligation. Laser Doppler Analysis—Dorsal cutaneous flap or hindlimb perfusion was determined in wild type and TSP2-null male mice under light general anesthesia via inhalation of 1.5% isoflurane. Core body temperature was constantly monitored and maintained at 35 °C. Pre-operative and post-operative perfusion flux levels were obtained at 5-min intervals with the following machine parameters: scan area, 1.6 × 2.5 cm; scan speed, 4 ms/pixel; scan time, 1 min 54 s; override distance, 25 cm. The override distance was 20 cm. In hindlimb studies the opposite unoperated limb served as an internal control. Ischemic Soft Tissue Flap Model—Under inhalation isoflurane (1.5%), animals underwent creation of a random myocutaneous (McFarlane) flap as previously described (4Isenberg J.S. Hyodo F. Matsumoto K. Romeo M.J. Abu-Asab M. Tsokos M. Kuppusamy P. Wink D.A. Krishna M.C. Roberts D.D. Blood. 2007; 109: 1945-1952Crossref PubMed Scopus (89) Google Scholar). Flap viability was assessed 72 h post-operatively. Determination of Flap Survival—Clinical assessment of flap perfusion was performed with notation of color, capillary refill, and bleeding to needle-stick being recorded. Flap dimensions were then traced onto a clear plastic sheet with demarcation of viable and non-viable areas made. Weights of segments of sheeting corresponding to viable versus non-viable portions of flaps were then determined, and % survival was expressed as a percentage versus total as previously described (4Isenberg J.S. Hyodo F. Matsumoto K. Romeo M.J. Abu-Asab M. Tsokos M. Kuppusamy P. Wink D.A. Krishna M.C. Roberts D.D. Blood. 2007; 109: 1945-1952Crossref PubMed Scopus (89) Google Scholar). CD47 Binding Studies—Jurkat T lymphoma cells (1 × 106 cells/well) were suspended in PBS containing divalent cations and 0.1% BSA with the indicated treatment reagents for 30 min at 37 °C. 125I-SIRPα or 125I-E123CaG1 was then added, and the cells were incubated at room temperature on a plate shaker for 1 h. Cells were separated by centrifugation through silicone oil (Nye Co, New Bedford, MA), and cell-bound radioactivity was quantified using a PerkinElmer Life Sciences gamma counter. Background counts in the absence of cells were determined for each experiment and subtracted to determine net binding. Detection of TSP2 Secretion—A heparin-BSA capture assay was used to detect TSP2 levels in the conditioned media. A 50-μl volume of heparin-BSA conjugate diluted to 100 ng/ml was adsorbed onto 96-well plate wells overnight at 4 °C. The next day the wells were blocked with 250 μl/well 5 mm Tris, 1% BSA, 0.02 mm phenylmethylsulfonyl fluoride, 150 mm NaCl, 1 mm CaCl2, pH 7.8, for 30 min. Samples were dispensed at 50 μl per well and allowed to bind to the immobilized heparin-BSA for 2 h at 37 °C. The wells were aspirated, and 50 μl of polyclonal anti-mouse TSP2 diluted 1:1000 in 50 mm Tris and 1% BSA was added to each well. The antibody was allowed to bind to the immobilized TSP2 for 2 h at 37 °C. After aspiration, the wells were washed 3 times with 50 μl per well of Dulbecco's phosphate-buffered saline with 0.02% BSA, 0.02 mm phenylmethylsulfonyl fluoride, and 0.05% Tween 20. Peroxidase-conjugated goat anti-rabbit IgG (Kirkegaard and Perry) was diluted to 1:1000 in Dulbecco's phosphate-buffered saline with 0.05% Tween 20 and allowed to bind for 1 h at room temperature. The wells were washed 3 times with Dulbecco's phosphate-buffered saline with 0.05% Tween 20. A 50-μl volume of o-phenylenediamine dihydrochloride was dissolved in phosphate-citrate buffer with sodium perborate (Sigma #P4922), and 30 μl of hydrogen peroxide was added to each well just before use. After 10 min, 3 m sulfuric acid was added to stop the color development. Absorbance values were read at 490 nm. Statistics—Results of vascular cell in vitro data are presented as the mean ± S.D. of at least three experiments. Significance was calculated with Student's t test or, where appropriate, one-way and two-way analysis of variance using a standard software package (Origin) with p < 0.05. In vivo tissue survival and perfusion study results represent the mean ± S.D. of five pairs of wild type and TSP2-null animals. Selective Binding of the Signature Domain of TSP1 to CD47-expressing Cells—Because others have failed to detect TSP1 binding to a recombinant extracellular domain of CD47 (22Adams J.C. Bentley A.A. Kvansakul M. Hatherley D. Hohenester E. J. Cell Sci. 2008; 121: 784-795Crossref PubMed Scopus (34) Google Scholar), we chose to examine TSP1 binding to native CD47 using a cell binding assay. TSP1 engages multiple cell surface receptors, and high affinity binding via the N domain to heparan sulfate proteoglycans is typically dominant in such cell binding studies (31Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To detect CD47 binding, therefore, we used a recombinant signature domain of TSP1 that is known to mediate CD47-dependent signaling but lacks the high affinity heparin-binding site (Fig. 1A). The only other cell surface receptors known to interact with this domain are αvβ3 integrin, which is not expressed in Jurkat T lymphoma cells (32Li Z. Calzada M.J. Sipes J.M. Cashel J.A. Krutzsch H.C. Annis D.S. Mosher D.F. Roberts D.D. J. Cell Biol. 2002; 157: 509-519Crossref PubMed Scopus (117) Google Scholar), and β1 integrins, which interact with low affinity with the E123 domain (33Calzada M.J. Annis D.S. Zeng B. Marcinkiewicz C. Banas B. Lawler J. Mosher D.F. Roberts D.D. J. Biol. Chem. 2004; 279: 41734-41743Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In Jurkat cells, β1 integrin interaction with this domain of TSP1 requires activating signals that were not present in these studies. Jurkat cells were also chosen for the binding studies based on their well characterized CD47-mediated responses to TSP1 (32Li Z. Calzada M.J. Sipes J.M. Cashel J.A. Krutzsch H.C. Annis D.S. Mosher D.F. Roberts D.D. J. Cell Biol. 2002; 157: 509-519Crossref PubMed Scopus (117) Google Scholar) and the availability of a somatic mutant of the Jurkat line lacking CD47 (JinB8) (24Ticchioni M. Raimondi V. Lamy L. Wijdenes J. Lindberg F.P. Brown E.J. Bernard A. FASEB J. 2001; 15: 341-350Crossref PubMed Scopus (47) Google Scholar). 125I-Labeled E123CaG1 bound at much higher levels to wild type Jurkat cells than to CD47-negative JinB8 cells (Fig. 1B). Furthermore, binding to Jurkat cells expressing CD47 was inhibited to background levels in a dose-dependent manner by the function-blocking CD47 antibody B6H12 (Fig. 1C). An isotype-matched control IgG1 did not significantly inhibit binding of E123CaG1 at the same concentrations. Binding was also significantly inhibited by the TSP1-derived CD47 binding peptide 1102FIRVVMYEGKK1112 (7N3) at 1 μm (p = 0.02) but not by the respective control peptide FIRGGMYEGKK (604, Fig. 1D). Specific binding of 125I-E123CaG1 to Jurkat cells was inhibited in the presence of 22 pm TSP1 (Fig. 1B), consistent with the known potency of TSP1 for inhibiting NO/cGMP signaling via CD47 (5Isenberg J.S. Ridnour L.A. Dimitry J. Frazier W.A. Wink D.A. Roberts D.D. J. Biol. Chem. 2006; 281: 26069-26080Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Unlabeled E123CaG1 was similarly active for self-inhibition of labeled ligand binding to Jurkat cells (Fig. 1E). In contrast, the corresponding signature domain of TSP2 (E123CaG2) was inactive over the same concentration range, and although the signature domain of TSP4 (E1234CaG4) produced consistent inhibition, the changes did not achieve significance (Fig. 1E). Therefore, high affinity interaction with CD47 appears to be relatively specific for the signature domain of TSP1. Selective Inhibition by TSP1 of SIRPα Binding to Cell Surface CD47—We were concerned that the relatively high non-displaceable fraction of E123CaG1 binding in Fig. 1 could reflect residual interactions with other TSP1 receptors. Therefore, competition with the well characterized CD47 ligand SIRPα (34Brown E.J. Frazier W.A. Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar) was used to further assess TSP1 binding to cell surface CD47. TSP1 potently and dose-dependently inhibited 125I-SIRPα binding to Jurkat cells (Fig. 2A). Analysis of the binding data using Scafit (Version 2.4 of Ligand software (35Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7759) Google Scholar)) gave an apparent dissociation constant for TSP1 of 12 pm with a Bmax of ∼5000 sites/cell (Fig. 2A, inset). The latter value is consistent with the known copy numbers for CD47 on other cell lines (36Subramanian S. Parthasarathy R. Sen S. Boder E.T. Discher D.E. Blood. 2006; 107: 2548-2556Crossref PubMed Scopus (114) Google Scholar). The apparent Kd is consistent with the known potency of TSP1 for inhibiting cGMP signaling, but we caution that this number is dependent on assumptions used in the analysis and does not represent an intrinsic dissociation constant. The requirement of CD47 for this interaction was confirmed by complete inhibition of 125I-SIRPα binding to Jurkat cells in the presence of the CD47 blocking antibody B6H12 and the absence of 125I-SIRPα binding to CD47-negative JinB8 cells (Fig. 2B). The dose dependence for B6H12 inhibition of SIRPα binding paralleled that for inhibiting E123CaG1 binding (compare Figs. 2C and 1C), and an isotype-matched control IgG1 did not significantly inhibit binding of SIRPα to wild type Jurkat cells (Fig. 2C). E123CaG1 showed a similar dose dependence for inhibiting SIRPα binding to Jurkat cells as intact TSP1, but the corresponding signature domains of TSP2 (E123CaG2) and TSP4 (E1234CaG4) were essentially inactive (Fig. 2D). This confirmed the relative specificity of CD47 for TSP1 and also established that TSP1 binding to CD47 can prevent interaction with its counter-receptor SIRPα. This has been hypothesized to occur in smooth muscle cells based on reduced co-immunoprecipitation of SIRPα with CD47 in the presence of TSP1 (37Maile L.A. Clemmons D.R. Circ. Res. 2003; 93: 925-931Crossref PubMed Scopus (38) Google Scholar), but here is the first direct proof that such inhibition occurs at the level of SIRPα-CD47 binding. Differential Effects of TSPs on NO-driven cGMP in Porcine VSMC—As previously reported for human VSMC (5Isenberg J.S. Ridnour L.A. Dimitry J. Frazier W.A. Wink D.A. Roberts D.D. J. Biol. Chem. 2006; 281: 26069-26080Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), pre" @default.
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• W2087929503 title "Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses" @default.
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