Abstract 5642 Thrombospondin-1, via CD47, Blocks Endothelial-Dependent Arterial Relaxation by Limiting Endothelial Nitric Oxide Synthase Activation

Abstract

Introduction: The secreted protein thrombospondin-1 (TSP1) is an important negative regulator of nitric oxide/cGMP signaling in vascular cells. TSP1, signaling through its necessary receptor CD47, limits activation of soluble guanylate cyclase and cGMP-dependent protein kinase. Extending these discoveries we recently defined a role for TSP1 in the acute control of blood pressure. However, it is not clear if the inhibitory role TSP-CD47 signaling has on blood pressure is secondary to a direct effect on arterial relaxation. To investigate this we tested the hypothesis that TSP1 directly limits arterial vasorelaxation by limiting activation of endothelial nitric oxide synthase (eNOS). Methods: TSP1-CD47 regulation of eNOS activation was studied in endothelial cells and arteries. Vasorelaxation was assessed ex vivo in arteries from wild type, TSP1 and CD47 null mice. Endothelial dependent changes in blood pressure were studied in transgenic mice. Results: TSP1 and CD47 null arteries demonstrated significantly greater dilation in response to physiologic activation of eNOS by acetylcholine compared to wild type vessels. Treating the luminal endothelial compartment of both wild type and TSP1 null arteries (but not CD47 null) with TSP1 inhibited vessel relaxation to acetylcholine and potentiated vasoconstriction to phenylephrine. TSP1 and a recombinant fragment of the signature domain that specifically binds CD47 potently blocked both acetylcholine-stimulated eNOS activation and changes in intracellular calcium in cultured endothelial cells. In vivo, acetylcholine-stimulated hypotention in TSP1 null mice was blocked by intravenous replacement of TSP1. Conclusion: Circulating TSP1, through CD47, limits endothelial dependent arterial relaxation and demonstrates a role for circulating TSP1 as a hypertensive agent supporting blood pressure through continuous modulation of both endogenous NO production and signaling.