Hypertension promotes atherosclerosis and is a major source of morbidity and mortality. We show that mice lacking T and B cells (RAG-1−/− mice) have blunted hypertension and do not develop abnormalities of vascular function during angiotensin II infusion or desoxycorticosterone acetate (DOCA)–salt. Adoptive transfer of T, but not B, cells restored these abnormalities. Angiotensin II is known to stimulate reactive oxygen species production via the nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase in several cells, including some immune cells. Accordingly, adoptive transfer of T cells lacking the angiotensin type I receptor or a functional NADPH oxidase resulted in blunted angiotensin II–dependent hypertension and decreased aortic superoxide production. Angiotensin II increased T cell markers of activation and tissue homing in wild-type, but not NADPH oxidase–deficient, mice. Angiotensin II markedly increased T cells in the perivascular adipose tissue (periadventitial fat) and, to a lesser extent the adventitia. These cells expressed high levels of CC chemokine receptor 5 and were commonly double negative (CD3+CD4−CD8−). This infiltration was associated with an increase in intercellular adhesion molecule-1 and RANTES in the aorta. Hypertension also increased T lymphocyte production of tumor necrosis factor (TNF) α, and treatment with the TNFα antagonist etanercept prevented the hypertension and increase in vascular superoxide caused by angiotensin II. These studies identify a previously undefined role for T cells in the genesis of hypertension and support a role of inflammation in the basis of this prevalent disease. T cells might represent a novel therapeutic target for the treatment of high blood pressure.

Objectives To examine the expression and activity of the calcium dependent NADPH oxidase in human atherosclerotic coronary arteries. Background The Nox based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several Nox homologs have been identified but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear. Nox5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels. Methods Coronary arteries from patients undergoing cardiac transplant with CAD or without CAD were studied. Nox5 was quantified and visualized using Western blotting, immunofluorescence and quantitative real-time PCR. Calcium dependent NADPH oxidase activity, corresponding greatly to Nox5 activity was measured by electron paramagnetic resonance. Results Both western blotting and quantitative real time PCR indicated a marked increase in Nox5 protein and mRNA in CAD vs non CAD vessels. Calcium dependent NADPH driven production of reactive oxygen species in vascular membranes, reflecting Nox5 activity was increased 7 fold in CAD and correlated significantly with Nox5 mRNA levels among subjects. Immunofluorescence demonstrated that Nox5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced in coronary lesions. Conclusions These studies identify Nox5 as a novel, calcium dependent source of reactive oxygen species in atherosclerosis.

Background— Atrial fibrillation (AF) is associated with an increased risk of stroke due almost exclusively to emboli from left atrial appendage (LAA) thrombi. Recently, we reported that AF was associated with endocardial dysfunction, limited to the left atrium (LA) and LAA and manifest as reduced nitric oxide (NO · ) production and increased expression of plasminogen activator inhibitor-1. We hypothesized that reduced LAA NO · levels observed in AF may be associated with increased superoxide (O 2 ·− ) production. Methods and Results— After a week of AF induced by rapid atrial pacing in pigs, O 2 ·− production from acutely isolated heart tissue was measured by 2 independent techniques, electron spin resonance and superoxide dismutase–inhibitable cytochrome C reduction assays. Compared with control animals with equivalent ventricular heart rates, basal O 2 ·− production was increased 2.7-fold ( P <0.01) and 3.0-fold ( P <0.02) in the LA and LAA, respectively. A similar 3.0-fold ( P <0.01) increase in LAA O 2 ·− production was observed using a cytochrome C reduction assay. The increases could not be explained by changes in atrial total superoxide dismutase activity. Addition of either apocyanin or oxypurinol reduced LAA O 2 ·− , implying that NADPH and xanthine oxidases both contributed to increased O 2 ·− production in AF. Enzyme assays of atrial tissue homogenates confirmed increases in LAA NAD(P)H oxidase ( P =0.04) and xanthine oxidase ( P =0.01) activities. Although there were no changes in expression of the NADPH oxidase subunits, the increase in superoxide production was accompanied by an increase in GTP-loaded Rac1, an activator of the NADPH oxidase. Conclusions— AF increased O 2 ·− production in both the LA and LAA. Increased NAD(P)H oxidase and xanthine oxidase activities contributed to the observed increase in LAA O 2 ·− production. This increase in O 2 ·− and its reactive metabolites may contribute to the pathological consequences of AF such as thrombosis, inflammation, and tissue remodeling.

The adaptive immune response and, in particular, T cells have been shown to be important in the genesis of hypertension. In the present study, we sought to determine how the interplay between ANG II, NADPH oxidase, and reactive oxygen species modulates T cell activation and ultimately causes hypertension. We determined that T cells express angiotensinogen, the angiotensin I-converting enzyme, and renin and produce physiological levels of ANG II. AT1 receptors were primarily expressed intracellularly, and endogenously produced ANG II increased T-cell activation, expression of tissue homing markers, and production of the cytokine TNF-alpha. Inhibition of T-cell ACE reduced TNF-alpha production, indicating endogenously produced ANG II has a regulatory role in this process. Studies with specific antagonists and T cells from AT1R and AT2R-deficient mice indicated that both receptor subtypes contribute to TNF-alpha production. We found that superoxide was a critical mediator of T-cell TNF-alpha production, as this was significantly inhibited by polyethylene glycol (PEG)-SOD, but not PEG-catalase. Thus, T cells contain an endogenous renin-angiotensin system that modulates T-cell function, NADPH oxidase activity, and production of superoxide that, in turn, modulates TNF-alpha production. These findings contribute to our understanding of how ANG II and T cells enhance inflammation in cardiovascular disease.

Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.

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