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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.

Objective As Ureaplasmas may be pathogens in preterm infants, this study was conducted to determine the incidence of invasive disease with Ureaplasma parvum and Ureaplasma urealyticum and the relationship with adverse outcomes in a prospective cohort of very low birth weight (VLBW) infants. Study Design DNA was extracted from the cord or venous blood and cerebrospinal fluid (CSF) samples obtained from 313 VLBW infants. PCR was performed using primers for the mba gene to detect all 14 serovars and then repeated for all positive samples using species-specific primers. Result Ureaplasma species were detected in serum and/or CSF samples from 74 of 313 (23.6%) infants. U. parvum was the predominant species (70%). Presence of Ureaplasma was significantly associated with elevated interleukin-1β in cord blood (odds ratio (OR) 2.6, 1.05 to 6.45, P = 0.039). Ureaplasma serum-positive infants had a 2.3-fold increased risk of intraventicular hemorrhage ≥ grade 3 (OR 2.50; 1.06 to 5.89, P = 0.036). Conclusion Invasive Ureaplasma occurs commonly in VLBW infants and may increase the risk for severe intraventricular hemorrhage.

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.

Objective Blood vessel hemodynamics have profound influences on function and structure of vascular cells. One of the main mechanical forces influencing vascular smooth muscle cells (VSMC) is cyclic stretch (CS). Increased CS stimulates reactive oxygen species (ROS) production in VSMC leading to their de-differentiation, yet the mechanisms involved are poorly understood. This study was designed to test the hypothesis that pathological CS stimulates Nox1-derived ROS via MEF2B, leading to VSMC dysfunction via a switch from a contractile to a synthetic phenotype. Approach and Results Using a newly developed isoform-specific Nox1 inhibitor and gene silencing technology, we demonstrate a novel pathway including MEF2B-Nox1-ROS is upregulated under pathological stretch conditions and this pathway promotes a VSMC phenotypic switch from a contractile to a synthetic phenotype. We observed that CS (10% at 1 Hz) mimicking systemic hypertension in humans increased Nox1 mRNA, protein levels, and enzymatic activity in a time-dependent manner and this upregulation was mediated by MEF2B. Furthermore, we show that stretch-induced Nox1-derived ROS upregulated a specific marker for synthetic phenotype (osteopontin), while downregulating classical markers for contractile phenotype (calponin1 and smoothelin B). In addition, our data demonstrated that stretch-induced Nox1 activation decreases actin fiber density and augments matrix metalloproteinase 9 activity, VSMC migration, and vectorial alignment. Conclusions These results suggest that CS initiates a signal through MEF2B that potentiates Nox1-mediated ROS production and causes VSMC to switch to a synthetic phenotype. The data also characterize a new Nox1 inhibitor as a potential therapy for treatment of vascular dysfunction in hypertension.