We previously demonstrated a potent antagonism between TSP1 and pro-angiogenic signaling downstream of NO. Although ligation of CD36 by antibodies, recombinant type 1 repeats of TSP1, 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 TSP1 similarly inhibited NO signaling in vascular cells from wild type and CD36 null mice. Ligation of the TSP1 receptor CD47 by recombinant C-terminal regions of TSP1, TSP1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. TSP1 did not inhibit NO signaling in CD47 null vascular cells or NO-stimulated vascular outgrowth from CD47 null muscle explants in 3D cultures. Furthermore, the CD36-binding domain of TSP1 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 TSP1 at physiological concentrations.
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. An anti-angiogenic peptide derived from TSP1 type 1 repeats 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. Therefore, the fatty acid translocase activity of CD36 elicits pro-angiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.
The nitric oxide (NO)/cGMP pathway, by relaxing vascular smooth muscle cells, is a major physiological regulator of tissue perfusion. We now identify thrombospondin-1 as a potent antagonist of NO for regulating F-actin assembly and myosin light chain phosphorylation in vascular smooth muscle cells. Thrombospondin-1 prevents NO-mediated relaxation of pre-contracted vascular smooth muscle cells in a collagen matrix. Functional magnetic resonance imaging demonstrated that an NO-mediated increase in skeletal muscle perfusion was enhanced in thrombospondin-1 null relative to wild type mice, implicating endogenous thrombospondin-1 as a physiological antagonist of NO-mediated vasodilation. Using a random myocutaneous flap model for ischemic injury, tissue survival was significantly enhanced in thrombospondin-1 null mice. These findings demonstrate an important antagonistic relationship between NO/cGMP signaling and thrombospondin-1 in vascular smooth muscle cells to regulate vascular tone and tissue perfusion. Using mice lacking the TSP1 receptors CD36 or CD47, we showed that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. Blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.
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