Thrombospondin-1 Activation of Signal-Regulatory Protein-α Stimulates Reactive Oxygen Species Production and Promotes Renal Ischemia Reperfusion Injury

Abstract

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.

RNAi-Mediated Down-Regulation of CD47 Protects against Ischemia/Reperfusion-Induced Myocardial Damage via Activation of eNOS in a Rat Model

“…Sharifi-Sanjani et al found that lack of CD47 provided protection from cardiac stress and alleviated heart failure [34]. TSP1 can not only regulate the CD47, but also can activate the Nox-1 receptor to increases superoxide in vascular and epithelial cells [35,36] Previous research shows that TSP-1 has an inflammatory-associated role in myocardial infarction and MIRI [37] The basic mechanism underlying the effect of CD47 blockade in alleviating MIRI was determined by several in vivo experiments demonstrating that limitation of the CD47/TSP1 signaling pathway can alleviate IRI by increasing eNOS-derived NO signaling. In liver and kidney I/R models, CD47 knockout mice were found to suffer less damage compared to control mice after periods of vascular occlusion followed by recanalization [13,17].…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Similarly, TSP1-mediated increases in superoxide were abated in VSMCs treated with NADPH oxidase 1 (Nox1) siRNA or in aortic rings from Nox1 -/-mice but not in cells treated with control siRNA or in aortic rings from WT mice. In renal tubular epithelial cells, TSP1 also increased Nox1-derived superoxide levels and this involved interaction with SIRPa (307). Superoxide levels represent a balance between enzymatic production and catabolism by SOD.…”

“…TSP1 (2.2 nM) decreased NOmediated vasodilation of aortic rings from WT and Thbs1 -/-mice but not Cd47 -/-mice (11). Treatment of WT murine vessels with the membrane-permeable superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) partially reversed the inhibitory actions of TSP1 (307). Tempol can inhibit the Fenton reaction, a source of hydroxyl radical that limits arterial vasodilation (263).…”

“…In vivo, studies in Thbs1 -/-and Cd47 -/-mice indicate that the TSP1-CD47 axis limits blood flow and tissue survival under conditions of fixed ischemia (85, 100a) as well as ischemia-reperfusion (36,271,307), situations characterized by significant oxidative stress. Conversely, suppressing CD47 expression using an adenoviral siRNA vector protected myocardial tissue from ischemia-reperfusion injury in rats while elevating eNOS phosphorylation and NO production, decreasing gp91 phox and superoxide, decreasing tissue malondialdehyde, and increasing SOD activity (288).…”

“…Although this is an acceptable starting point, methods exist that enable precise characterization of ROS. Detailed chemical and biophysical characterization of ROS has been reported for some TSP1 studies (36,307), but this remains to be addressed for other matricellular proteins.…”