CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1

Abstract

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)2 is a secreted glycoprotein that plays a relatively minor role in development of the murine vascular system (1, 2), 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 ␣-granules and is released from platelets upon activation, where it modulates platelet adhesion and the properties of fibrin clots formed following acute vascular injury (3, 4). 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 (5).TSP1 also accumulates in the neointima of atherosclerotic lesions (6), where it may stimulate vascular smooth muscle cell (VSMC) proliferation and migration by enhancing responsiveness to platelet-derived growth factor (7). Antibody blocking of TSP1 can reverse this response and enhance the re-endothelialization of an injured artery (8). An N700S coding sequence polymorphism in TSP1 that alters its conformation is associated with increased risk of premature familial myocardial infarction (reviewed in Ref. 9).TSP1 is also a potent modulator of angiogenesis (10). The N-terminal domain of TSP1 stimulates angiogenesis through its interactions with ␣ 3 ␤ 1 and ␣ 4 ␤ 1 integrins (11, 12), but the central type 1 repeats contain sequences that potently inhibit angiogenesis via CD36 and/or heparan sulfate proteoglycan receptors (13,14). Under most circumstances, the net activity of intact TSP1 is ...

Thrombospondin-1 Inhibits Nitric Oxide Signaling via CD36 by Inhibiting Myristic Acid Uptake

“…Although engaging CD36 is sufficient to inhibit NO signaling as well as VEGF-induced cGMP formation, we subsequently found that CD36 is not necessary for the same activity of intact TSP1 (21,22). Rather, we found that the TSP1 receptor CD47 is necessary and sufficient for the anti-angiogenic signal elicited by picomolar concentrations of TSP1.…”

“…8A). This could be explained by the ability of CD47 signaling to inhibit NO signaling in vascular cells at the level of sGC (22). In this case some of the inhibition of myristate signaling by TSP1 seen in Fig.…”

“…Cells were utilized at passages 4 -8. CD47 null VSMC were prepared from aortic segments from knock out mice and grown in vascular smooth muscle growth medium as described (22). Cells were used between passages 2 through 8.…”

“…Rather, we found that the TSP1 receptor CD47 is necessary and sufficient for the anti-angiogenic signal elicited by picomolar concentrations of TSP1. However, a weaker inhibitory activity of TSP1, observed at 1-10 nM, was deficient in CD36 null vascular cells (22). Furthermore, synthetic derivatives of CD36-binding peptides, similar to those currently in phase II clinical trials as angiogenesis inhibitors (23,24), required CD36 for their ability to potently inhibit NO signaling.…”

Cellular, Pharmacological, and Biophysical Evaluation of Explanted Lungs from a Patient with Sickle Cell Disease and Severe Pulmonary Arterial Hypertension

“…19 Preclinical studies also suggest that an increased inflammatory response, cell adhesion, 20,21 and pathologic reactive oxygen species (ROS) production 22 contribute to SCD-associated vasculopathy. Interestingly, the proadhesive protein thrombospondin 1 (TSP1) was recently reported to inhibit NO signaling 23,24 while promoting PAH in preclinical models 25 and to be upregulated in SCD patient plasma. 26 Although studies have documented the hemodynamics and outcomes of SCD-associated PAH and interrogated the systemic vascular function of these patients, there have been no human studies directly evaluating the pulmonary vasculature.…”