Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

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).³ 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 (1).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 (2, 3). TSP1 also plays a role in promoting platelet thrombus formation and hemostasis by antagonizing the antithrombotic activity of NO (4). In all of these vascular cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated fluxes in cGMP by engaging its receptor CD47 (5). Nanomolar concentrations of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated uptake of myristate into vascular cells (6). 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 (4). 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 (7, 8).TSP1 belongs to a family of five secreted glycoproteins that share an evolutionarily conserved C-terminal signature domain (9). 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 (10). 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 (11, 12). A 3′-untranslated region polymorphism in TSP2 is also associated with type 2 diabetes in men (13). 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 (14, 15). However, a polymorphism in TSP4 is associated with premature myocardial infarcts in certain populations (11, 16, 17). A proatherogenic activity for the A387P variant of TSP4 was proposed based on its differential ability to modulate proliferation of endothelial and VSMC (18). Cardiovascular functions of TSP4 may also be linked to the high expression of TSP4 in heart (19) and its altered expression in that tissue during hypertensive heart failure (20).The C-terminal domain of TSP1 is sufficient to mediate CD47-dependent inhibition of cGMP signaling (5). 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 (21). Based on structural evidence that the VVM motifs may not be accessible (22, 23), 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 (24). 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 (25).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.