Objective Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species (ROS) production in vascular smooth muscle cells (VSMCs) and induces vascular dysfunction by promoting oxidative stress. Methods and Results Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O2•-) levels in VSMCs at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C‐terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O2•− in vitro and ex vivo. TSP1-treatment of VSMCs activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase (Nox) organizer subunit p47phox and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and Nox1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow following ischemia reperfusion. Conclusion Our data suggest a highly-regulated process of ROS stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report to our knowledge of a matricellular protein acting as a ligand for Nox activation and through specific engagement of integrin-associated protein CD47.

Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Matricellular proteins play diverse roles in modulating cell behavior by engaging specific cell surface receptors and interacting with extracellular matrix proteins, secreted enzymes, and growth factors. Studies of such interactions involving thrombospondin-1 have revealed several physiological functions and roles in the pathogenesis of injury responses and cancer, but the relatively mild phenotypes of mice lacking thrombospondin-1 suggested that thrombospondin-1 would not be a central player that could be exploited therapeutically. Recent research focusing on signaling through its receptor CD47, however, has uncovered more critical roles for thrombospondin-1 in acute regulation of cardiovascular dynamics, hemostasis, immunity, and mitochondrial homeostasis. Several of these functions are mediated by potent and redundant inhibition of the canonical nitric oxide pathway. Conversely, elevated tissue thrombospondin-1 levels in major chronic diseases of aging may account for the deficient nitric oxide signaling that characterizes these diseases, and experimental therapeutics targeting CD47 show promise for treating such chronic diseases as well as acute stress conditions that are associated with elevated thrombospondin-1 expression.

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-a (SIRP-a) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-a in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-a on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-a. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-a and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47 phox (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-a. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxidemediated vasodilation in a SIRP-a-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-a signaling axis and was associated with increased superoxide production and cell death. A SIRP-a antibody that blocks thrombospondin-1 activation of SIRP-a mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-a activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-a on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.

The objective of this study was to evaluate the long term performance of cell-free vascular grafts made from a fast-degrading elastic polymer. We fabricated small arterial grafts from microporous tubes of poly(glycerol sebacate) (PGS) reinforced with polycaprolactone (PCL) nanofibers on the outer surface. Grafts were interpositioned in rat abdominal aortas and characterized at 1 year post-implant. Grafts remodeled into “neoarteries” (regenerated arteries) with similar gross appearance to native rat aortas. Neoarteries mimic arterial tissue architecture with a confluent endothelium and media and adventita-like layers. Patent vessels (80%) showed no significant stenosis, dilation, or calcification. Neoarteries contain nerves and have the same amount of mature elastin as native arteries. Despite some differences in matrix organization, regenerated arteries had similar dynamic mechanical compliance to native arteries in vivo. Neoarteries responded to vasomotor agents, albeit with different magnitude than native aortas. These data suggest that an elastic vascular graft that resorbs quickly has potential to improve the performance of vascular grafts used in small arteries. This design may also promote constructive remodeling in other soft tissues.