PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2008 September 25.
Published in final edited form as:
PMCID: PMC2553319
NIHMSID: NIHMS66094

CD47: A New Target in Cardiovascular Therapy

Abstract

CD47, originally named integrin-associated protein, is a receptor for thrombospondin-1. A number of important roles for CD47 have been defined in regulating the migration, proliferation and survival of vascular cells, and in regulation of innate and adaptive immunity. The recent discovery that thrombospondin-1 acts via CD47 to inhibit nitric oxide signaling throughout the vascular system has given new importance and perhaps a unifying mechanism of action to these enigmatic proteins. Here we trace the development of this exciting new paradigm for CD47 function in vascular physiology.

Integrin-associated protein (IAP) was discovered as a contaminant in preparations of αvβ3 integrin from human placenta. Certain monoclonal antibodies raised against these preparations had dramatic effects on integrin-dependent cell behaviors, but bound to a ca. 50 kDa protein rather than α or β integrin subunits 1. Cloning and sequencing IAP cDNA revealed a new member of the Ig superfamily with a single extracellular IgV domain, a unique 5 membrane-spanning domain and an alternatively spliced cytoplasmic tail 2 Figure 1A. Subsequent experiments found IAP to be identical to CD47, which is widely expressed on tissues, primary cells and nearly all cell lines, with prominent expression on leukocytes, platelets and erythrocytes 3, 4. At first, CD47 was an orphan receptor apart from its lateral membrane association in cis with αvβ3 and αIIbβ3.

FIGURE 1
Models of CD47 and TSP1

Thrombospondin-1 regulation of integrins requires CD47

Concurrent with the development of the CD47 knockout mouse, thrombospondin-1 (TSP1), a large, secreted glycoprotein, was identified as a potential trans ligand of CD47 5, and signal inhibitory receptor protein α (SIRPα) was recognized as a cell-bound counter-receptor for CD47 6. Also called SHPS-1 7, BIT 8 and p84 9 by different investigators in different species, SIRPα, is most highly expressed on myeloid lineage cells and functions with CD47 in regulating innate immunity and the transition to the adaptive immune response 1014.

TSP1 is a major component of platelet α-granules from which it is secreted upon platelet activation 15. While this localization suggested a role for TSP1 in thrombosis and hemostasis, identification of such a role has been elusive. In addition to blood-borne platelets, TSP1 is expressed at much lower levels in many if not all tissues and is a biosynthetic product of most cultured cells 16. The TSP family in mammals now has 5 members that consist of group A homotrimers (TSP-1 and -2) and group B homopentamers (TSPs 3–5). TSPs 1 and 2 have broad but distinct tissue expression patterns during development and through adulthood, and TSP1 is the only TSP found in platelets 1719. TSP 1 and 2 subunits have an identical domain organization (Figure 1B), but human TSP1 and 2 share only 54% amino acid sequence identity 20. The N domains attach TSP1 to the cell surface via several receptors (Figure 1B), while the G domains at the opposite end of each subunit interact functionally with CD47. The binding of TSP1 to so many receptors and matrix ligands serves to concentrate TSP1 at the cell surface and matrix, thereby dramatically enhancing its binding to what otherwise might be sites of rather low affinity. Among this plethora of receptors, none exclusively bind TSP1. This, along with contradictory and confusing reports of cell responses to TSP1, and the subtle phenotype initially described for the TSP1-null mouse21 have complicated efforts to understand the physiologic functions of TSP1.

While initial efforts to identify TSP1 receptors focused on integrins that might bind to the RGD sequence in the calcium-binding domain of TSP1, we found that cells could attach to a site contained in the C-terminal (now G) domain of TSP1 exclusive of the RGD sequence 22. Peptides having the cell binding activity were localized within the TSP1 G domain 23, 24, and one of these peptides was used to affinity label a ca. 50 kDa cell membrane receptor candidate 25. Concurrently, Eric Brown had discovered CD47 2 and suggested that it might be the TSP1 receptor. We confirmed that the 50 kDa protein was recognized by several CD47 antibodies and that the TSP1 G domain peptides augmented integrin functions such as chemotaxis and cell spreading in a CD47-dependent manner 5, 26. This activity of CD47 was dependent on functional heterotrimeric Gi 27, suggesting that CD47 might be an unconventional G protein coupled receptor (GPCR) with 5 rather than 7 transmembrane segments. CD47 signaling through Gi can access the “inside-out” pathway used by other GPCRs on platelets to activate αIIbβ3 28, 29, as well as αvβ3 and some β1 and β2 integrins 30, 31 (and our unpublished data). CD47 can also increase the avidity or clustering of integrins by associating with them in the plane of the membrane, an apparently signaling-independent process 32.

Biological Roles of CD47

The CD47-null mouse is lethally susceptible to bacterial infections that are of no consequence to wild type mice due to a delay in neutrophil recruitment and a weakened integrin-dependent oxidative burst response 33. In vitro data also support roles for CD47 in leukocyte adhesion to endothelium 12, leukocyte transmigration 34, and migration of dendritic cells 35. CD47 is important in the development and function of antigen presenting cells 11, 36, and has a role in immune cell apoptosis 37, 38. In the context of innate immunity, CD47 functions as a marker of self. When attempts were made to engraft CD47 null marrow cells into WT mice, the CD47 null cells were rapidly engulfed by splenic macrophages and dendritic cells 39. Experiments by Oldenborg and colleagues revealed that CD47 on circulating cells normally binds SIRPα on phagocytes and delivers a “don’t eat me” signal that prevents their phagocytosis 40, 41. This signal depends on the docking of SHP-1 phosphatase to phosphorylated ITIMS in the SIRPαcytoplasmic domain leading to SHP-1 activation and presumably dephosphorylation of one or more key components of the phagocytic machinery42, 43. A function of this mechanism is suggested by the report that phagocytosis of xenograft cells is augmented by the species incompatibility of donor CD47 with host SIRPα44.

TSP1 regulates vascular NO signaling through CD47

The role of CD47 in the regulation of NO signaling was found while focusing on another TSP1 receptor, CD36. A wealth of data both in vitro and in vivo has shown that TSP1, the first identified endogenous angiogenic inhibitor 45, has an important role in opposing tumor-driven angiogenesis and regulating episodes of angiogenesis in the adult 4649. Even so, the precise mechanism of this effect has remained elusive. Initial reports indicated that CD36 was the TSP1 receptor that mediates its anti-angiogenic effects 50. Receptors for several angiogenic factors such as VEGF can induce NO synthesis, and the NO/cGMP pathway is essential for angiogenesis 51, 52. Signaling through VEGFR2 activates Akt1 to phosphorylate endothelial nitric oxide synthase (eNOS), which renders it constitutively active, independent of its normal dynamic activation by calcium pulses and shear 53. This sustained activation of eNOS leads to increased endogenous NO production and a tonic cGMP increase which, through cGMP-dependent protein kinase (cGK) activation, coordinates the signaling pathways needed for endothelial proliferation, migration and survival 54. In contrast, higher levels of NO, such as that produced by iNOS during an inflammatory response, inhibit these responses or are cytotoxic 55.

Mouse muscle explant cultures provided the first evidence that the anti-angiogenic activity of TSP1 involves regulation of NO signaling 56. TSP1-null muscle explants produced a more robust vascular outgrowth than WT explants. Addition of NO donors exaggerated this difference, while the NOS inhibitor L-NAME blocked outgrowth. Further, NO-stimulated outgrowth was inhibited by exogenous TSP1. CD36-null muscle explants, though stimulated by exogenous NO, remained sensitive to inhibition by TSP1, demonstrating that CD36 is not necessary for TSP1 inhibition of NO-stimulated vascular cell responses. In contrast, NO-stimulated vascular outgrowth in CD47-null explants was not blocked by TSP1 57. Recombinant TSP1 domains and other specific ligands of CD36 or of CD47 inhibited NO responses in WT vascular cells. However, in cells lacking CD47, ligation of CD36 was unable to block NO signaling. Thus, while CD36 ligation is sufficient when CD47 is present, only CD47 ligation is necessary for inhibition of NO/cGMP signaling 57. These results clearly imply coupling of CD36 and CD47 either physically as “co-receptors” in the membrane or via convergent signaling pathways (Figure 2). Further studies in endothelial and vascular smooth muscle cells (VSMC) 57 and in platelets 58, revealed that not only does TSP1 ligation of CD47 block NO stimulation of cGMP production; it also inhibits the direct activation of cGK by cGMP analogs (Figure 2). Angiogenic signaling is only one of several physiologic roles of NO in cardiovascular homeostasis 59. Thus our data suggested that TSP1 signaling via CD47 might regulate NO responses in a much broader context.

Figure 2
Model of CD47-dependent regulation of NO signaling

CD47 inhibits NO action in vivo

NO is a major acute regulator of the cardiovascular system 60, 61. It plays a role in regulation of blood pressure and vascular tone and also adjusts regional tissue perfusion as the metabolic activity of tissues responds to changing demands. We therefore examined the role of TSP1 and CD47 in the physiological regulation of blood flow. VSMC contraction (and hence blood vessel diameter and flow) is controlled by kinases and phosphatases acting on the regulatory light chain-2 of myosin (MLC-2) 62. Pulsatile shear and calcium transients activate eNOS, producing NO, which then diffuses across membranes into VSMC, activating soluble guanylate cyclase (sGC). Cyclic GMP in turn, leads to a cGK-dependent activation of myosin light chain phosphatase, a rapid decrease in MLC-2 phosphorylation and VSMC relaxation. NO also activates sGC in the endothelial cell of origin leading to cGK-dependent, inhibitory phosphorylation of eNOS and termination of NO production, thus restoring VSMC contractility 63.

Blocking TSP1-CD47 alleviates tissue ischemia

TSP1, acting via CD47, inhibits the NO stimulation of sGC in both EC and VSMC 57. Exogenous NO donors inhibit the contraction of VSMC embedded in a collagen gel induced by serum or sphingosine-1-phosphate 64. Addition of TSP1 prevents NO-dependent relaxation of VSMC. CD47 null VSMC or WT VSMC in which CD47 expression has been knocked down with an antisense morpholino are insensitive to TSP1 inhibition of NO-stimulated relaxation 65.

These results were found to have implications in vivo in several models of tissue injury. Under ischemic challenge, tissue and blood vessels respond by increasing endogenous NO levels, leading to blood vessel dilation and increased tissue perfusion. A well-characterized skin flap model in WT mice routinely results in 40 to 60% necrosis of the ischemic flap within three days 64. However, skin flaps in TSP1- and CD47-null mice (but not CD36-nulls) experienced markedly enhanced perfusion and survival (Figure 3A) 65. Full thickness skin grafts (FTSG) are an even more stringent model of ischemia since they initially have no blood supply at all and must initiate neovascularization from the wound bed. Wild type FTSGs failed to survive on WT recipients, but survived and healed nearly completely on TSP1-null recipients (Figure 3B). TSP1-null grafts on WT recipients had intermediate survival66. Thus, TSP1/CD47 signaling limits tissue perfusion and survival following partial and complete ischemic challenges.

FIGURE 3
Improved healing of skin flaps and grafts in TSP1- and CD47-null mice

To explore the therapeutic potential of these discoveries, we targeted the TSP1/CD47 pathway using monoclonal antibodies to both TSP1 and CD47 and knockdown of CD47 expression using an antisense morpholino oligonucleotide. Each of these could be locally applied by injection into the ischemic soft tissues. Importantly, these therapeutics greatly enhanced ischemic tissue survival in WT animals to the level obtained in null animals 65, 66.

To determine if the improved tissue survival in these relatively simple ischemia models would be realized in more complex tissues, we examined a hindlimb ischemia model. Even under the dramatic ischemic challenge of complete femoral artery occlusion, TSP1- and CD47-null mice demonstrated restoration of vascular perfusion of the hindlimb at seven days after surgery to a level much superior to that of ischemic WT hindlimbs 65. These results further suggest that deleting either TSP1 or CD47 removes a barrier to vascular remodeling of ischemic tissues consistent with the potent inhibitory effects of TSP1 seen in angiogenic explant assays 56. However, real-time analysis of blood flow by laser Doppler flowmetry and blood oxygen level-dependent (BOLD)-MRI, revealed that increased tissue perfusion in TSP1 and CD47 knockout mice was achieved within minutes following an ischemic insult 65, 67, much too quickly for angiogenesis to occur. Such rapid reperfusion in the face of the permanently ligated femoral artery must require rapid remodeling of existing collateral vessels to bypass the ligation 68. This startling result indicated that TSP1 and CD47 acutely control blood flow under conditions of ischemic stress. Kopp et al. 69 report that platelet TSP1 is deposited in vessels downstream of femoral artery ligation, suggesting that the TSP1 responsible for the poor perfusion in ischemic WT limbs may be delivered by platelets. However, the extremely low (picomolar) concentrations at which TSP1 can suppress NO signaling in vascular cells in vitro 57, 70 suggests that the low levels of TSP1 present in the vascular wall may also contribute. The fact that both TSP1 and CD47 knockouts show the same rapid improvement in tissue perfusion strongly supports the functional relationship of these two proteins in this regulatory system.

There is a growing literature reporting sudden increases in TSP1 protein and mRNA in ischemic tissues, as much as 20 fold above normal tissue in the case of human legs amputated as a result of chronic ischemia 71 or myocardial infarction in rats 72 with more modest elevations seen in mouse kidney after ischemia/reperfusion 73, or in chronic ischemia due to systemic sclerosis in humans 74. In a rat middle cerebral artery stroke model, TSP1 increased in a biphasic manner with peaks at 1 and 3 days, while TSP2 appeared much later, peaking 2 weeks after the stroke 75. The extremely rapid appearance of TSP1 again suggests immediate delivery from platelets or its presence due to infiltrating inflammatory cells such as monocytes and macrophages. However, induction of TSP1 in endothelial, VSMCs, astroglia and kidney tubule cells was also seen. In view of our data above, it would seem that recruitment of TSP1-bearing cells to ischemic tissues or expression of TSP1 in resident cells could only exacerbate a bad situation.

Long-term effects of TSP1/CD47 signaling

Endothelial dysfunction, secondary to impaired NO bioavailability, is a hallmark of metabolic syndrome and of the aging cardiovascular system 76 61. We have begun to investigate the long-term consequences of the TSP1/CD47 inhibition of NO signaling. With advanced age (12–16 months) and atherosclerotic vasculopathy (ApoE−/− cross), TSP1- and CD47-null mice continued to demonstrate enhanced tissue perfusion and survival following ischemic challenge comparable that seen in young knockout animals 67. As expected, aged WT mice suffered much worse ischemia and necrosis and a corresponding drop in tissue cGMP levels compared to young WT animals. In contrast, tissue cGMP levels in aged TSP1- and CD47-null mice remained at levels comparable to those in young animals, suggesting that TSP1/CD47 contribute to the NO-insufficiency characteristic of aging.

Metabolic syndrome is a constellation of cardiovascular risk factors including obesity, hyperlipidemia, hypertension and insulin resistance 77. Interestingly, C57Bl/6 WT mice spontaneously develop metabolic syndrome 78, and eNOS-null mice develop metabolic syndrome more rapidly than WTs 79. CD47-null mice are leaner than matched WTs and appear to resist features of metabolic syndrome (our unpublished observations). A primary component of metabolic syndrome is diabetes. Murphy-Ullrich’s group reported that an NO donor could block the increase in TSP1 expression caused by high glucose 80, suggesting a mutually antagonistic link between TSP1 and NO signaling that could have important consequences for cardiovascular disease and its treatment.

TSP1 and the platelet enigma

The above studies indicate a pervasive role for TSP1 and CD47 in regulating NO signaling in blood vessels. Platelet function is also regulated by NO, the overall effect being to oppose activation and aggregation 81. This is consistent with the global role of NO to maintain a healthy, anti-thrombotic vascular system. However, local differences in the production and availability of NO can alter the fine balance between pro- and anti-thrombotic states.

One of the puzzling properties of the TSP1-null mouse was its apparent lack of a platelet phenotype even thought its platelets are completely devoid of TSP1 (or TSP2) 21. Earlier studies using TSP1 antibodies 82, 83 and peptides 29 suggested that TSP1 could facilitate platelet activation or aggregation in vitro, but this remained controversial 84, 85. In light of the newly discovered role for TSP1 as a regulator of NO signaling, we re-examined the function of TSP1 in platelets 58. The aggregation of human platelets activated with thrombin is delayed by fast acting NO donors, but this delay, and the NO-stimulated increase in platelet cGMP are abolished by adding exogenous TSP1. Freshly isolated TSP1-null mouse platelets have higher resting levels of cGMP, and addition of NO donors or supplementation of the traditional Tyrode’s buffer with L-arginine induces greater cGMP synthesis in TSP1 null platelets compared to WT. When small amounts of thrombin are added sequentially to stirred platelet suspensions, TSP1 null platelets require 2 to 3 times more thrombin for activation than WT platelets and are much more sensitive to inhibition by NO donors and cGMP analogs 58. A primary effect of NO in platelets is to prevent GTP loading of the small G protein Rap1b, which, upon binding GTP, activates αIIbβ3 81. TSP1 prevents the inhibition of Rap1b GTP loading by NO, thus facilitating αIIbβ3 activation, binding of fibrinogen, and aggregation. As in other vascular cell types, TSP1 binding to CD36 or CD47, both of which are highly expressed on platelets, is sufficient to inhibit NO signaling and thereby promote platelet activation and aggregation 58. Traditional methods for assessing platelet aggregation deplete platelets of both NO and the NOS substrate L-arginine, leading to progressive loss of endogenous NO and cGMP thereby lowering the barrier to activation. It is important to emphasize that NO does not prevent platelet activation, but only increases the level of agonist required to initiate the process. In a similar vein, TSP1 is not required for platelet activation, it simply lowers the threshold for platelet agonists. The massive amount of TSP1 discharged from activating platelets and the large number of CD47 receptors on the platelet surface combine to effectively abolish NO inhibition.

Why did “we” need TSP1 and CD47?

If NO is important in promoting cardiovascular health, why was the TSP1/CD47 inhibitory system selected during evolution and allowed to persist? What we see as maladaptive in the context of current longevity and lifestyle must have once provided an important selective advantage for our early vertebrate ancestors. We believe that the essential selective advantage of TSP1/CD47 antagonism of NO signaling was its ability to limit life-threatening hemorrhage. NO is an ancient signaling molecule, but the unique NO-responsive soluble GC of vertebrates may have evolved more recently86, perhaps coincident with the divergence of TSP1 from the ancestral TSP gene in lower animals 87 and the appearance of CD47 13. Closed, pressurized circulatory systems require finely-regulated hemostasis to control hemorrhage yet maintain flow. Packaging TSP1 in platelet α-granules allows a bandaid to be applied locally at a site of wounding, while permitting the rest of the circulatory system to continue functioning. Platelets secrete several compounds that further stimulate platelet activation, thus reinforcing the initial platelet layers at the blood-wound interface and recruiting more platelets from flowing blood. TSP1, unlike the other prothrombotic agents released from platelets, binds firmly to the platelet surface via a number of receptors (Fig. 1). TSP1 also binds to components of the clot matrix such as fibrinogen, fibronectin, and vWF. Thus, TSP1 is a tethered prothrombotic agent that is long-lived relative to compounds such as prostacyclins and ADP, which are rapidly degraded and/or diluted by blood flow. It could also be significant that TSP1 is a trimer with long and flexible subunits. In addition to acting on platelets, the released TSP1 can suppress NO signaling in cells exposed in the wounded vessel wall, promoting local vasoconstriction to further decrease bleeding. Local TSP1 binding to leukocyte CD47 will also attract and promote transmigration and oxidative bursting of neutophils 13, the first responders to infection.

A new target for cardiovascular therapeutics?

Hemostasis may have been the critical, evolutionarily selected function of TSP1 and/or CD47 in our ancestors. However, our present-day well-fed, sedentary, but nonetheless, long-lived contemporaries may have become unwitting victims of the TSP1/CD47 antagonism of NO signaling. The global role of NO regulation in the cardiovascular system is now well documented, but the beneficial effects of NO signaling are relatively easily overwhelmed by stressors such as hyperlipidemia, diabetes, smoking and others that disrupt this balance 61, 76, 88. Overcoming the beneficial effects of NO is made easier because TSP1 and CD47 continuously oppose NO signaling in circulating cells and blood vessels. The genetic knockout of TSP1 or CD47 relieves this antagonism, but has few discernable effects when mice are kept in a safe, pathogen-free environment. However, when stressors such as vessel injury, ischemia, hyperlipidemia and even old age are applied, we see that the knockouts fare substantially better than WT mice. Furthermore, therapeutic interventions that interrupt TSP1/CD47 signaling provide dramatic improvement in the WT response to ischemic stress. The previously unsuspected role for CD47 in limiting NO signaling provides a novel and accessible target to augment the beneficial effects of NO for the treatment of cardiovascular disease.

The data obtained so far present us with a new way of looking at the function and mechanism of TSP1 and CD47. However, many questions remain to be answered: What functions of TSP1 and CD47 are shared, and which are independent, i.e. due to TSP1’s binding to other receptors? What is the mechanism by which CD47 blocks NO signaling? Can CD47 regulate targets of NO that are independent of sGC and cGK? Answers to these questions will help to guide and define the role of CD47-targeted therapies, placing them in context with existing therapeutic approaches to improve the treatment of cardiovascular disease.

Acknowledgments

Source of Funding

Drs. Roberts and Isenberg received intramural funding from the National Cancer Institute and Dr. Frazier was funded by grants HL54390 and GM57573 from the National Institutes of Health.

Footnotes

Disclosures

Dr. Frazier is President of Vasculox, Inc.

Literature Cited

1. Brown EJ, Hopper L, Ho T, Gresham HD. Integrin associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins. J Cell Biol. 1990;111:2785–2794. [PMC free article] [PubMed]
2. Lindberg FP, Gresham HD, Schwarz E, Brown EJ. Molecular cloning of integrin associated protein: an immunoglobulin family member with multiple membrane spanning domains implicated in αvβ3-dependent ligand binding. J Cell Biol. 1993;123:485–496. [PMC free article] [PubMed]
3. Reinhold MI, Lindberg FP, Plas D, Reynolds S, Peters MG, Brown EJ. In vivo expression of alternatively spliced forms of integrin associated protein (CD47) J Cell Sci. 1995;108:3419–3425. [PubMed]
4. Lindberg FP, Lublin DM, Telen MJ, Veile RA, Miller YE, Donis-Keller H, Brown EJ. Rh-related antigen CD47 is the signal-transducer integrin associated protein. J Biol Chem. 1994;269:1567–1570. [PubMed]
5. Gao A-GFP, Lindberg MB, Finn SD, Blystone EJ, Brown Frazier WA. Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin. J Biol Chem. 1996b;271:21–24. [PubMed]
6. van Beek EM, Cochrane F, Barclay AN, van den Berg TK. Signal regulatory proteins in the immune system. J Immunol. 2005;175:7781–7787. [PubMed]
7. Babic I, Schallhorn A, Lindberg FP, Jirik FR. SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47 [published erratum appears in J Immunol (2000) 164, 5532] J Immunol. 2000;164:3652–3658. [PubMed]
8. Han X, Sterling H, Chen Y, Saginario C, Brown EJ, Frazier WA, Lindberg FP, Vignery A. CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation. J Biol Chem. 2000;275:37984–37992. [PubMed]
9. Jian P, Lagenaur CF, Narayanan V. Integrin-associated protein is a ligand for the P84 neural adhesion molecule. J Biol Chem. 1999;274:559–562. [PubMed]
10. Seiffert M, Cant C, Chen Z, Rappold I, Brugger W, Kanz L, Brown EJ, Ullrich A, Buhring HJ. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47. Blood. 1999;94:3633–3643. [PubMed]
11. Demeure CE, Tanaka H, Mateo V, Rubio M, Delespesse G, Sarfati M. CD47 engagement inhibits cytokine production and maturation of human dendritic cells. J Immunol. 2000;164:2193–2199. [PubMed]
12. Ticchioni M, Raimondi V, Lamy L, Wijdenes J, Lindberg FP, Brown EJ, Bernard A. Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow. Faseb J. 2001;15:341–350. [PubMed]
13. Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 2001;11:130–135. [PubMed]
14. Subramanian S, Boder ET, Discher DE. Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site. J Biol Chem. 2007;282:1805–1818. [PubMed]
15. Baenziger NL, Brodie GN, Majerus PW. Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem. 1972;247:2723–2731. [PubMed]
16. Adams JC, Lawler J. The thrombospondins. Int J Biochem Cell Biol. 2004;36:961–968. [PMC free article] [PubMed]
17. Varani J, Riser BL, Hughes LA, Carey TE, Fligiel SE, Dixit VM. Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells. Clin Exp Metastasis. 1989;7:265–276. [PubMed]
18. Reed MJ, Irueta-Arispe L, O’Brien ER, Truong T, Labell T, Bornstein P, Sage EH. Expression of thrombospondins by endothelial cells. Injury is correlated with TSP-1. Am J Pathol. 1995;147:1068–1080. [PubMed]
19. Hu CJ, Chen SD, Yang DI, Lin TN, Chen CM, Huang TH, Hsu CY. Promoter region methylation and reduced expression of thrombospondin-1 after oxygen-glucose deprivation in murine cerebral endothelial cells. J Cereb Blood Flow Metab. 2006;26:1519–1526. [PubMed]
20. LaBell TL, Byers PH. Sequence and characterization of the complete human thrombospondin 2 cDNA: potential regulatory role for the 3′ untranslated region. Genomics. 1993;17:225–229. [PubMed]
21. Lawler J, Sunday M, Thibert V, Duquette M, George EL, Rayburn H, Hynes RO. Thrombospondin-1 is required for normal murine pulmonary homeostatis and its absence causes pneumonia. J Clin Invest. 1998;101:982–992. [PMC free article] [PubMed]
22. Kosfeld MD, Pavlopoulos TV, Frazier WA. Cell attachment activity of the carboxyl-terminal domain of human thrombospondin expressed in escherichia coli. J Biol Chem. 1991;266:24257–24259. [PubMed]
23. Kosfeld MD, Frazier WA. Identification of a new cell adhesion motif in two homologous peptides from the COOH-terminal cell binding domain of human thrombospondin. J Biol Chem. 1993;268:8808–8814. [PubMed]
24. Kosfeld MD, Frazier WA. Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1. J Biol Chem. 1992;267:16230–16236. [PubMed]
25. Gao A-G, Frazier WA. Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins. J Biol Chem. 1994;269:29650–29657. [PubMed]
26. Gao A-G, Lindberg FP, Dimitry JM, Brown EJ, Frazier WA. Thrombospondin modulates αvβ3 function through integrin associated protein. J Cell Biol. 1996;135:533–544. [PMC free article] [PubMed]
27. Frazier WA, Gao A-G, Dimitry J, Chung J, Lindberg FP, Brown EJ, Linder ME. The thrombospondin receptor integrin-associated protein (CD47) functionally couples to heterotrimeric Gi. J Biol Chem. 1999;274:8554–8560. [PubMed]
28. Chung J, Gao A-G, Frazier WA. Thrombospondin acts via integrin-associated protein to activate the platelet integrin αIIbβ3. J Biol Chem. 1997;272:14740–14746. [PubMed]
29. Chung J, Wang XQ, Lindberg FP, Frazier WA. Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation. Blood. 1999;94:642–648. [PubMed]
30. Wang XQ, Frazier WA. The thrombospondin receptor CD47 (IAP) modulates and associates with alpha2 beta1 integrin in vascular smooth muscle cells. Mol Biol Cell. 1998;9:865–874. [PMC free article] [PubMed]
31. Barazi HO, Li Z, Cashel JA, Krutzsch HC, Annis DS, Mosher DF, Roberts DD. Regulation of integrin function by DC47 ligands. J Biol Chem. 2002;277:42859–42866. [PubMed]
32. McDonald JF, Zheleznyak A, Frazier WA. Cholesterol-independent interactions with CD47 enhance αvβ3 avidity. J Biol Chem. 2004;279:17301–17311. [PubMed]
33. Lindberg FP, Bullard DC, Caver TE, Gresham HD, Beaudet AL, Brown EJ. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science. 1996;274:795–798. [PubMed]
34. Cooper D, Lindberg FP, Gamble JR, Brown EJ, Vadas MA. The transendothelial migration of neutrophils involves integrin-associated protein (CD47) PNAS. 1995;92:3978–3982. [PubMed]
35. Hagnerud S, Manna PP, Cella M, Stenberg A, Frazier WA, Colonna M, Oldenborg P-A. Deficit of CD47 results in a defect of marginal zone DC, blunted immune response to particulate antigen and impairment of DC migration. J Immunol. 2006;176:5772–5778. [PubMed]
36. Avice M-N, Rubio M, Sergerie M, Delespesse G, Sarfati M. CD47 ligation selectively inhibits the development of human naïve T cells in to Th1 effectors. J Immunol. 2000;165:4624–4631. [PubMed]
37. Pettersen RDK, Hestdal K, Olafsen MK, Lie SO, Lindberg FP. CD47 signals T cell death. J Immunol. 1999;162:7031–7040. [PubMed]
38. Manna PP, Dimitry J, Oldenborg PA, Frazier WA. CD47 Augments Fas/CD95-mediated Apoptosis. Journal of Biological Chemistry. 2005;280:29637–29644. [PubMed]
39. Blazar BR, Lindberg FP, Ingulli E, Panoskaltsis-Mortari A, Oldenborg P-A, Iizuka K, Yokoyama WM, Taylor PA. CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells. J Exp Med. 2001;194:541–549. [PMC free article] [PubMed]
40. Oldenborg P-A, Zheleznyak A, Fang Y-F, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science. 2000;288:2051–2054. [PubMed]
41. Oldenborg PA, Gresham HD, Lindberg FP. CD47-signal regulatory protein alpha (SIRPalpha) regulates Fcgamma and complement receptor-mediated phagocytosis. J Exp Med. 2001;193:855–862. [PMC free article] [PubMed]
42. Ikeda H, Okazawa H, Ohnishi H, Murata Y, Oldenborg PA, Matozaki T. Mutational analysis of the mechanism of negative regulation by SRC homology 2 domain-containing protein tyrosine phosphatase substrate-1 of phagocytosis in macrophages. J Immunol. 2006;177:3123–3132. [PubMed]
43. Okazawa H, Motegi S-i, Ohyama N, Ohnishi H, Tomizawa T, Kaneko Y, Oldenborg P-A, Ishikawa O, Matozaki T. Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J Immunol. 2005;174:2004–2011. [PubMed]
44. Wang H, VerHalen J, Madariaga ML, Xiang S, Wang S, Lan P, Oldenborg PA, Sykes M, Yang YG. Attenuation of phagocytosis of xenogeneic cells by manipulating CD47. Blood. 2007;109:836–842. [PubMed]
45. Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, Bouck NP. A tumor supporessor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. PNAS. 1990;87:6624–6628. [PubMed]
46. Weinstat-Saslow DL, Zabrenetzky VS, VanHoutte K, Frazier WA, Roberts DD, Steeg PS. Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 1994;54:6504–6511. [PubMed]
47. Rodriguez-Manzaneque JC, Lane TF, Ortega MA, Hynes RO, Lawler J, Iruela-Arispe ML. Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A. 2001;98:12485–12490. [PubMed]
48. Ren B, Yee KO, Lawler J, Khosravi-Far R. Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta. 2006;1765:178–188. [PubMed]
49. Lawler J, Miao W-M, Duquette M, Bouck N, Bronson RT, Hynes RO. Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice. Am J Pathol. 2001;159:1949–1956. [PubMed]
50. Dawson DWFA, Pearce R, Zhong RL, Silverstein WA, Frazier Bouck NP. CD36 mediates the inhibitory effects of thrombospondin on endothelial cells. J Cell Biol. 1997;138:707–717. [PMC free article] [PubMed]
51. Cooke JP. NO and angiogenesis. Atheroscler Suppl. 2003;4:53–60. [PubMed]
52. Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A. 2001;98:2604–2609. [PubMed]
53. Ackah E, Yu J, Zoellner S, Iwakiri Y, Skurk C, Shibata R, Ouchi N, Easton RM, Galasso G, Birnbaum MJ, Walsh K, Sessa WC. Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis. J Clin Invest. 2005;115:2119–2127. [PMC free article] [PubMed]
54. Morbidelli L, Donnini S, Ziche M. Role of nitric oxide in the modulation of angiogenesis. Curr Pharm Des. 2003;9:521–530. [PubMed]
55. Ridnour LA, Thomas DD, Donzelli S, Espey MG, Roberts DD, Wink DA, Isenberg JS. The biphasic nature of nitric oxide responses in tumor biology. Antioxid Redox Signal. 2006;8:1329–1337. [PubMed]
56. Isenberg JS, Ridnour LA, Perruccio EM, Espey MG, Wink DA, Roberts DD. Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner. Proc Natl Acad Sci USA. 2005;102:13141–13146. [PubMed]
57. Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD. CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1. J Biol Chem. 2006;281:26069–26080. [PubMed]
58. Isenberg JS, Romeo MJ, Yu C, Yu CK, Nghiem K, Monsale J, Rick ME, Wink DA, Frazier WA, Roberts DD. Thrombospondin-1 stimulates platelet aggregation by blocking the anti-thrombotic activity of nitric oxide/cGMP signaling. Blood. 2007 in press. [PubMed]
59. Ignarro LJ, Cirino G, Casini A, Napoli C. Nitric oxide as a signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol. 1999;34:879–886. [PubMed]
60. Ignarro LJ. Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J Physiol Pharmacol. 2002;53:503–514. [PubMed]
61. Naseem KM. The role of nitric oxide in cardiovascular diseases. Mol Aspects Med. 2005;26:33–65. [PubMed]
62. Muto A, Fitzgerald TN, Pimiento JM, Maloney SP, Teso D, Paszkowiak JJ, Westvik TS, Kudo FA, Nishibe T, Dardik A. Smooth muscle cell signal transduction: Implications of vascular biology for vascular surgeons. J Vasc Surg. 2007;45(Suppl A):15–24. [PMC free article] [PubMed]
63. Haga JH, Li YS, Chien S. Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells. J Biomech. 2007;40:947–960. [PubMed]
64. Isenberg JS, Hyodo F, Matsumoto K, Romeo MJ, Abu-Asab M, Tsokos M, Kuppusamy P, Wink DA, Krishna MC, Roberts DD. Thrombospondin-1 limits ischemic tissue survival by inhibiting nitric oxide-mediated vascular smooth muscle relaxation. Blood. 2007;109:1945–1952. [PubMed]
65. Isenberg JS, Romeo MJ, Abu-Asab M, Tsokos M, Oldenborg A, Pappan L, Wink DA, Frazier WA, Roberts DD. Increasing survival of ischemic tissue by targeting CD47. Circ Res. 2007;100:712–720. [PubMed]
66. Isenberg JSPL, Romeo MJ, Abu-Asab M, Tsokos M, Wink DA, Frazier WA, Roberts DD. Blockade of thrombospndin-1-CD47 interactions prevents necrosis of full thickness skin grafts. Ann Surgery. 2007 in press. [PMC free article] [PubMed]
67. Isenberg JS, Hyodo F, Pappan LK, Abu-Asab M, Tsokos M, Krishna MC, Frazier WA, Roberts DD. Blocking thrombospondin-1/CD47 signaling alleviates deleterious effects of aging on tissue responses to ischemia. Arterioscler Thromb Vasc Biol. 2007;27:2582–2588. [PubMed]
68. Chalothorn D, Clayton JA, Zhang H, Pomp D, Faber JE. Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains. Physiol Genomics. 2007;30:179–191. [PubMed]
69. Kopp HG, Hooper AT, Broekman MJ, Avecilla ST, Petit I, Luo M, Milde T, Ramos CA, Zhang F, Kopp T, Bornstein P, Jin DK, Marcus AJ, Rafii S. Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization. J Clin Invest. 2006;116:3277–3291. [PMC free article] [PubMed]
70. Isenberg JS, Wink DA, Roberts DD. Thrombospondin-1 antagonizes nitric oxide-stimulated vascular smooth muscle cell responses. Cardiovasc Res. 2006;71:785–793. [PubMed]
71. Favier J, Germain S, Emmerich J, Corvol P, Gasc JM. Critical overexpression of thrombospondin 1 in chronic leg ischaemia. J Pathol. 2005;207:358–366. [PubMed]
72. Sezaki S, Hirohata S, Iwabu A, Nakamura K, Toeda K, Miyoshi T, Yamawaki H, Demircan K, Kusachi S, Shiratori Y, Ninomiya Y. Thrombospondin-1 is induced in rat myocardial infarction and its induction is accelerated by ischemia/reperfusion. Exp Biol Med (Maywood) 2005;230:621–630. [PubMed]
73. Thakar CV, Zahedi K, Revelo MP, Wang Z, Burnham CE, Barone S, Bevans S, Lentsch AB, Rabb H, Soleimani M. Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia. J Clin Invest. 2005;115:3451–3459. [PMC free article] [PubMed]
74. Distler JH, Jungel A, Pileckyte M, Zwerina J, Michel BA, Gay RE, Kowal-Bielecka O, Matucci-Cerinic M, Schett G, Marti HH, Gay S, Distler O. Hypoxia-induced increase in the production of extracellular matrix proteins in systemic sclerosis. Arthritis Rheum. 2007;56:4203–4215. [PubMed]
75. Lin TN, Kim GM, Chen JJ, Cheung WM, He YY, Hsu CY. Differential regulation of thrombospondin-1 and thrombospondin-2 after focal cerebral ischemia/reperfusion. Stroke. 2003;34:177–186. [PubMed]
76. Yetik-Anacak G, Catravas JD. Nitric oxide and the endothelium: history and impact on cardiovascular disease. Vascul Pharmacol. 2006;45:268–276. [PubMed]
77. Alberti KG, Zimmet P, Shaw J. Metabolic syndrome--a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet Med. 2006;23:469–480. [PubMed]
78. Biddinger SB, Almind K, Miyazaki M, Kokkotou E, Ntambi JM, Kahn CR. Effects of diet and genetic background on sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase 1, and the development of the metabolic syndrome. Diabetes. 2005;54:1314–1323. [PubMed]
79. Cook S, Hugli O, Egli M, Vollenweider P, Burcelin R, Nicod P, Thorens B, Scherrer U. Clustering of cardiovascular risk factors mimicking the human metabolic syndrome X in eNOS null mice. Swiss Med Wkly. 2003;133:360–363. [PubMed]
80. Wang S, Shiva S, Poczatek MH, Darley-Usmar V, Murphy-Ullrich JE. Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells. J Biol Chem. 2002;277:9880–9888. [PubMed]
81. Danielewski O, Schultess J, Smolenski A. The NO/cGMP pathway inhibits Rap 1 activation in human platelets via cGMP-dependent protein kinase I. Thromb Haemost. 2005;93:319–325. [PubMed]
82. Dixit VM, Haverstick DM, O’Rourke KM, Hennessy SW, Grant GA, Santoro SA, Frazier WA. A monoclonal antibody against human thrombospondin inhibits platelet aggregation. Proc Natl Acad Sci U S A. 1985;82:3472–3476. [PubMed]
83. Leung LL. Role of thrombospondin in platelet aggregation. J Clin Invest. 1984;74:1764–1772. [PMC free article] [PubMed]
84. Voit S, Udelhoven M, Lill G, Aktas B, Nieswandt B, Schror K, Weber AA. The C-terminal peptide of thrombospondin-1 stimulates distinct signaling pathways but induces an activation-independent agglutination of platelets and other cells. FEBS Lett. 2003;544:240–245. [PubMed]
85. Tulasne D, Judd BA, Johansen M, Asazuma N, Best D, Brown EJ, Kahn M, Koretzky GA, Watson SP. C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor gamma -chain-associated signaling pathway and by agglutination. Blood. 2001;98:3346–3352. [PubMed]
86. Fitzpatrick DLOHD, Burnell AM. Multiple lineage specific expansions within the guanylyl cyclase gene family. BMC Evolutionary Biology. 2006;6:26–43. [PMC free article] [PubMed]
87. McKenzie P, Chadalavada SC, Bohrer J, Adams JC. Phylogenomic analysis of vertebrate thrombospondins reveals fish-specific paralogues, ancestral gene relationships and a tetrapod innovation. BMC Evol Biol. 2006;6:33. [PMC free article] [PubMed]
88. Barbato JE, Tzeng E. Nitric oxide and arterial disease. J Vasc Surg. 2004;40:187–193. [PubMed]
89. Isenberg JSFW, Roberts DD. Thrombospondin is a central regulator of nitric oxide signaling in vascular pathology. Cell Mol Life Sci. 2007 in press.
90. Calzada MJ, Roberts DD. Novel integrin antagonists derived from thrombospondins. Curr Pharm Des. 2005;11:849–866. [PubMed]
91. Orr AW, Elzie CA, Kucik DF, Murphy-Ullrich JE. Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration. J Cell Sci. 2003;116:2917–2927. [PubMed]
92. Calzada MJ, Annis DS, Zeng B, Marcinkiewicz C, Banas B, Lawler J, Mosher DF, Roberts DD. Identification of novel β1 integrin binding sites in the type 1 and type 2 repeats of thrombospondin-1. J Biol Chem. 2004;279:41734–41743. [PubMed]
93. Dixit VM, Galvin NJ, O’Rourke KM, Frazier WA. Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes. J Biol Chem. 1986;261:1962–1968. [PubMed]
94. Kvansakul M, Adams JC, Hohenester E. Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats. EMBO J. 2004;23:1223–1233. [PubMed]
95. Carlson CB, Bernstein DA, Annis DS, Misenheimer TM, Hanna BA, Mosher DF, Keck JL. Structure of the calcium-rich signature domain of human thrombospondin-2. Nat Struct Biol Mol Biol. 2005;12:910–914. [PMC free article] [PubMed]
96. Isenberg JS, Jia Y, Fukuyama J, Switzer CH, Wink DA, Roberts DD. Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake. J Biol Chem. 2006;281:26069–26080. [PMC free article] [PubMed]