Gábor Csányi

NOX and Inflammation in the Vascular Adventitia

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. Combined adventitial fibroblast and inflammatory cell-derived ROS therefore are 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, leading to phenotypic modulation of smooth muscle cells. This review addresses the role of adventitial NAD(P)H oxidase-derived ROS from a nontraditional, 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.

Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation

AIMS Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation. METHODS AND RESULTS Acetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine(1177). TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice. CONCLUSION 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.

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2.

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 this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the 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 (O(2)(•-)) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O(2)(•-) production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by 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.

Platelet-derived HMGB1 is a critical mediator of thrombosis

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.

Thrombospondin-1 Regulates Blood Flow via CD47 Receptor–Mediated Activation of NADPH Oxidase 1

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 production in vascular smooth muscle cells 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 vascular smooth muscle cells 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 vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47phox and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. Conclusion—Our data suggest a highly regulated process of reactive oxygen species 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 NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.

NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS

NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns.

Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy.

AIMS Reactive oxygen species (ROS)-mediated intracellular signalling is well described in the vasculature, yet the precise roles of ROS in paracrine signalling are not known. Studies implicate interstitial ROS hydrogen peroxide (H(2)O(2)) in vascular disease, and plasma H(2)O(2) levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H(2)O(2) was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H(2)O(2) activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H(2)O(2) induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1. METHODS AND RESULTS Treatment of rat aortic SMCs (rASMC) with exogenous H(2)O(2) resulted in a concentration-dependent increase in Nox-derived superoxide (O(2)(•-)), determined by L-012 chemiluminescence, cytochrome c and electron paramagnetic resonance. Nox1 was verified as the source of O(2)(·-) by siRNA. Aqp1 siRNA attenuated H(2)O(2) cellular entry and H(2)O(2)-induced O(2)(•-) production. H(2)O(2) treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H(2)O(2)-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it. CONCLUSION Our results demonstrate for the first time that extracellular H(2)O(2), at pathophysiological concentrations, stimulates rASMC Nox1-derived O(2)(•-), subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H(2)O(2) enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.

Oxidative Stress in Cardiovascular Disease

In the special issue “Oxidative Stress in Cardiovascular Disease” authors were invited to submit papers that investigate key questions in the field of cardiovascular free radical biology. The original research articles included in this issue provide important information regarding novel aspects of reactive oxygen species (ROS)-mediated signaling, which have important implications in physiological and pathophysiological cardiovascular processes. The issue also included a number of review articles that highlight areas of intense research in the fields of free radical biology and cardiovascular medicine.

MEF2B-Nox1 Signaling Is Critical for Stretch-Induced Phenotypic Modulation of Vascular Smooth Muscle Cells

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 dedifferentiation, yet the mechanisms involved are poorly understood. This study was designed to test the hypothesis that pathological CS stimulates NADPH oxidase isoform 1 (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 that 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), whereas it downregulated 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.

Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease.

Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health.