Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.

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.

Vascular inflammation has traditionally been thought to be initiated at the luminal surface and progress through the media toward the adventitial layer. In recent years, however, evidence has emerged suggesting that the vascular adventitia is activated early in a variety of cardiovascular diseases and that it plays an important role in the initiation and progression of vascular inflammation. Adventitial fibroblasts have been shown to produce substantial amounts of NAD(P)H oxidase-derived reactive oxygen species (ROS) in response to vascular injury. Additionally, inflammatory cytokines, lipids and various hormones, implicated in fibroblast proliferation and migration, lead to recruitment of inflammatory cells to the adventitial layer and impairment of endothelium-dependent relaxation. Early in the development of vascular disease, there is clear evidence for progression toward a denser vasa vasorum which delivers oxygen and nutrients to an increasingly hypoxic and nutrient-deficient media. This expanded vascularization appears to provide enhanced delivery of inflammatory cells to the adventitia and outer media. A combined adventitial fibroblast and inflammatory cell-derived ROS therefore is expected to synergize their local effect on adventitial parenchymal cells leading to further cytokine release and a feed-forward propagation of adventitial ROS production. In fact, data from our laboratory and others suggest a broader paracrine positive feedback role for adventitia-derived ROS in medial smooth muscle cell hypertrophy and neointimal hyperplasia. A likely candidate responsible for the adventitia-derived paracrine signaling across the vessel wall is the superoxide anion metabolite hydrogen peroxide, which is highly stable, cell-permeant and capable of activating downstream signaling mechanisms in smooth muscle cells. This leads to phenotypic modulation of smooth muscle cells. This review addresses the role of adventitial NAD(P)H oxidase-derived ROS from a non-traditional, perivascular vantage of promoting vascular inflammation and will discuss how ROS derived from adventitial NAD(P)H oxidases may be a catalyst for vascular remodeling and dysfunction.

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2 and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In the present study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B loop of Nox2, would inhibit ROS production by Nox2-, but not by Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O2•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance (EPR) confirmed that Nox2ds inhibits O2•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production in either Nox1 or Nox4 oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2 oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.

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.

Our results identify imipramine as a new pharmacological tool to study macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving macropinocytosis.

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.