Ajay M. Shah

Plasma MicroRNA Profiling Reveals Loss of Endothelial MiR-126 and Other MicroRNAs in Type 2 Diabetes

Rationale: MicroRNAs (miRNAs) have been implicated in the epigenetic regulation of key metabolic, inflammatory, and antiangiogenic pathways in type 2 diabetes (DM) and may contribute to common disease complications. Objective: In this study, we explore plasma miRNA profiles in patients with DM. Methods and Results: Total RNA was extracted from plasma samples of the prospective population-based Bruneck study. A total of 13 candidate miRNAs identified by microarray screening and miRNA network inference were quantified by quantitative PCR in all diabetic patients of the Bruneck study and age- and sex-matched controls (1995 evaluation, n=80 each). Quantitative PCR assessment revealed lower plasma levels of miR-20b, miR-21, miR-24, miR-15a, miR-126, miR-191, miR-197, miR-223, miR-320, and miR-486 in prevalent DM, but a modest increase of miR-28-3p. Findings emerged as robust in multivariable analysis and were independent of the standardization procedure applied. For endothelial miR-126, results were confirmed in the entire Bruneck cohort (n=822) in univariate (odds ratio [95% confidence interval], 0.38 [0.26 to 0.55]; P=2.72×10−7) and multivariate analyses (0.57 [0.37 to 0.86]; P=0.0082). Importantly, reduced miR-15a, miR-29b, miR-126, miR-223, and elevated miR-28-3p levels antedated the manifestation of disease. Most differences in miRNA levels were replicated in plasma obtained from hyperglycemic Lepob mice. High glucose concentrations reduced the miR-126 content of endothelial apoptotic bodies. Similarly in patients with DM, the reduction of miR-126 was confined to circulating vesicles in plasma. Conclusions: We reveal a plasma miRNA signature for DM that includes loss of endothelial miR-126. These findings might explain the impaired peripheral angiogenic signaling in patients with DM.

Increased myocardial NADPH oxidase activity in human heart failure

OBJECTIVES This study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BACKGROUND A phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis. An increase in myocardial oxidative stress due to excessive production of ROS may be involved in the pathophysiology of congestive heart failure. Recent studies have suggested an important role for myocardial NADPH oxidase in experimental models of cardiac disease. However, it is unknown whether NADPH oxidase is expressed in the human myocardium or if it has any role in human heart failure. METHODS Myocardium of explanted nonfailing (n = 9) and end-stage failing (n = 13) hearts was studied for the expression of NADPH oxidase subunits and oxidase activity. RESULTS The NADPH oxidase subunits p22(phox), gp91(phox), p67(phox), and p47(phox) were all expressed at messenger ribonucleic acid and protein level in cardiomyocytes of both nonfailing and failing hearts. NADPH oxidase activity was significantly increased in end-stage failing versus nonfailing myocardium (5.86 +/- 0.41 vs. 3.72 +/- 0.39 arbitrary units; p < 0.01). The overall level of oxidase subunit expression was unaltered in failing compared with nonfailing hearts. However, there was increased translocation of the regulatory subunit, p47(phox), to myocyte membranes in failing myocardium. CONCLUSIONS This is the first report of the presence of NADPH oxidase in human myocardium. The increase in NADPH oxidase activity in the failing heart may be important in the pathophysiology of cardiac dysfunction by contributing to increased oxidative stress.

Activation of NADPH oxidase during progression of cardiac hypertrophy to failure

Abstract—Increased reactive oxygen species (ROS) production is implicated in the pathophysiology of left ventricular (LV) hypertrophy and heart failure. However, the enzymatic sources of myocardial ROS production are unclear. We examined the expression and activity of phagocyte-type NADPH oxidase in LV myocardium in an experimental guinea pig model of progressive pressure-overload LV hypertrophy. Concomitant with the development of LV hypertrophy, NADPH-dependent O2− production in LV homogenates, measured by lucigenin (5 &mgr;mol/L) chemiluminescence or cytochrome c reduction assays, significantly and progressively increased (by ≈40% at the stage of LV decompensation;P <0.05). O2− production was fully inhibited by diphenyleneiodonium (100 &mgr;mol/L). Immunoblotting revealed a progressive increase in expression of the NADPH oxidase subunits p22phox, gp91phox, p67phox, and p47phox in the LV hypertrophy group, whereas immunolabeling studies indicated the presence of oxidase subunits in cardiomyocytes and endothelial cells. In parallel with the increase in O2− production, there was a significant increase in activation of extracellular signal–regulated kinase 1/2, extracellular signal–regulated kinase 5, c-Jun NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinase. These data indicate that an NADPH oxidase expressed in cardiomyocytes is a major source of ROS generation in pressure overload LV hypertrophy and may contribute to pathophysiological changes such as the activation of redox-sensitive kinases and progression to heart failure.

Nox4 Is a Protective Reactive Oxygen Species Generating Vascular NADPH Oxidase

Rationale: The function of Nox4, a source of vascular H2O2, is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. Objective: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4−/− mice. Methods and Results: Nox4 was highly expressed in the endothelium and contributed to H2O2 formation. Nox4−/− mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4−/− lung endothelial cells (LECs) was attenuated and restored by low concentrations of H2O2, whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. Conclusion: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

Contrasting Roles of NADPH Oxidase Isoforms in Pressure-Overload Versus Angiotensin II–Induced Cardiac Hypertrophy

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)–induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox−/− mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II–induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox−/− mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox−/− and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox−/− mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload–induced LVH in both gp91phox−/− and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.

The E-loop Is Involved in Hydrogen Peroxide Formation by the NADPH Oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O2̇̄), Nox4 produces hydrogen peroxide (H2O2). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H2O2 versus L-012 and cytochrome c for O2̇̄. The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H2O2 to O2̇̄ generation while preserving expression and intracellular localization. In the presence of an NO donor, the O2̇̄-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O2̇̄. In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H2O2 but increased O2̇̄ formation by Nox4 without affecting Nox1-dependent O2̇̄ formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H2O2. Mutation of this but not of four other conserved histidines also switched Nox4 from H2O2 to O2̇̄ formation. Thus, H2O2 formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O2̇̄ egress and/or provide a source for protons, allowing dismutation to form H2O2.

Mechanism of endothelial cell NADPH oxidase activation by angiotensin II: Role of the p47phox subunit

Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952–19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47 phox knockout mice. Transfection of p47 phox cDNA into p47 phox−/− cells restored the angiotensin II response, whereas transfection of antisense p47 phox cDNA into wild-type cells depleted p47 phox and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47 phox -p22 phox complex formation but minimal detectable p47 phox phosphorylation. Angiotensin II induced rapid serine phosphorylation of p47 phox (within 1 min, peaking at ∼15 min), a 1.9 ± 0.1-fold increase in p47 phox -p22 phox complex formation and a 1.6 ± 0.2-fold increase in NADPH-dependent O 2 ⨪ production (p< 0.05). p47 phox was redistributed to “nuclear” and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47 phox and its enhanced binding to p22 phox .

Oxidative stress and redox signalling in cardiac hypertrophy and heart failure

Substantial evidence suggests the involvement of oxidative stress in the pathophysiology of congestive heart failure and its antecedent conditions such as cardiac hypertrophy and adverse remodelling after MI. Oxidative stress describes an imbalance between antioxidant defences and the production of reactive oxygen species (ROS), which at high levels cause cell damage but at lower levels induce subtle changes in intracellular signalling pathways (termed redox signalling). ROS are derived from many sources including mitochondria, xanthine oxidase, uncoupled nitric oxide synthases and NADPH oxidases. The latter enzymes are especially important in redox signalling, being implicated in the pathophysiology of hypertension and atherosclerosis, and activated by diverse pathologically relevant stimuli. We review the contribution of ROS to heart failure pathophysiology and discuss potential therapies that may specifically target detrimental redox signalling. Indeed, drugs such as ACE inhibitors and statins may act in part through such mechanisms. A better understanding of redox signalling mechanisms may enable the development of new targeted therapeutic strategies rather than the non-specific antioxidant approaches that have to date been disappointing in clinical trials.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

The identification of NOX4 as a major source of oxidative stress in stroke and demonstration of dramatic protection after stroke in mice by NOX4 deletion or NOX inhibition, opens up new avenues for treatment.