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

Abstract

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47. Pathological angiogenesis or the lack thereof underlies a number of major diseases (1). Proangiogenic signals from vascular endothelial growth factors (VEGF)2 and fibroblast growth factors (FGF1 and FGF2) to induce blood vessel formation are opposed by signals from endogenous angiogenesis inhibitors, including two thrombospondins (TSP1 and TSP2) and proteolytic fragments of several extracellular matrix components (2, 3). Defining the mechanism of action of these inhibitors has been complicated by the finding that vascular cells express multiple receptors for several of these molecules. In the case of TSP1, endothelial cells express at least eight receptors, and some of these elicit pro-rather than anti-angiogenic responses (4, 5). The activities of some TSP1 receptors differ between large vessel and microvascular endothelial cells, and some are regulated by specific contextual signals (4 -6). CD36, a member of the scavenger receptor B family, is a TSP1 receptor that is selectively expressed in microvascular endothelium (7,8). CD36 was initially reported to recognize CSVTCG sequences in the type 1 repeats of TSP1 (9), but further studies identified higher affinity binding to the adjacent GVQXR sequences in the second and third type 1 repeats (10, 11). CD36 binding was markedly enhanced by epimerization of the first Ile in a peptide from the second type 1 repeat 434 GDGVITRIR 442 , where I (Ile) is the D isomer (11). TSP1, recombinant type 1 repeats of TSP1, and peptide mimetics of its CD36 binding sequences inhibit FGF2-stimulated endo...

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