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.

In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip. Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J ( n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure ( P = 0.013) and mean pulmonary artery pressure ( P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance ( P = 0.024) and RV effective arterial elastance ( P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.

Pulmonary hypertension is recognized as a leading cause of morbidity and mortality in patients with sickle cell disease (SCD). We now report benchtop phenotyping from the explanted lungs of the first successful lung transplant in SCD. Pulmonary artery smooth muscle cells (PASMCs) cultured from the explanted lungs were analyzed for proliferate capacity, superoxide (O 2 • − ) production, and changes in key pulmonary arterial hypertension (PAH)-associated molecules and compared with non-PAH PASMCs. Upregulation of several pathologic processes persisted in culture in SCD lung PASMCs in spite of cell passage. SCD lung PASMCs showed growth factor-and serum-independent proliferation, upregulation of matrix genes, and increased O 2• − production compared with control cells. Histologic analysis of SCDassociated PAH arteries demonstrated increased and ectopically located extracellular matrix deposition and degradation of elastin fibers. Biomechanical analysis of these vessels confirmed increased arterial stiffening and loss of elasticity. Functional analysis of distal fifth-order pulmonary arteries from these lungs demonstrated increased vasoconstriction to an α1-adrenergic receptor agonist and concurrent loss of both endothelial-dependent and endothelial-independent vasodilation compared with normal pulmonary arteries. This is the first study to evaluate the molecular, cellular, functional, and mechanical changes in endstage SCD-associated PAH.