Dan Sorescu

NAD(P)H Oxidase: Role in Cardiovascular Biology and Disease

Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent work has shown that NAD(P)H oxidases are major sources of superoxide in vascular cells and myocytes. The biochemical characterization, activation paradigms, structure, and function of this enzyme are now partly understood. Vascular NAD(P)H oxidases share some, but not all, characteristics of the neutrophil enzyme. In response to growth factors and cytokines, they produce superoxide, which is metabolized to hydrogen peroxide, and both of these reactive oxygen species serve as second messengers to activate multiple intracellular signaling pathways. The vascular NAD(P)H oxidases have been found to be essential in the physiological response of vascular cells, including growth, migration, and modification of the extracellular matrix. They have also been linked to hypertension and to pathological states associated with uncontrolled growth and inflammation, such as atherosclerosis.

Cell transformation by the superoxide-generating oxidase Mox1

Reactive oxygen species (ROS) generated in some non-phagocytic cells are implicated in mitogenic signalling and cancer. Many cancer cells show increased production of ROS, and normal cells exposed to hydrogen peroxide or superoxide show increased proliferation and express growth-related genes. ROS are generated in response to growth factors, and may affect cell growth, for example in vascular smooth-muscle cells. Increased ROS in Ras-transformed fibroblasts correlates with increased mitogenic rate. Here we describe the cloning of mox1, which encodes a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase of phagocytes, gp91phox. mox1 messenger RNA is expressed in colon, prostate, uterus and vascular smooth muscle, but not in peripheral blood leukocytes. In smooth-muscle cells, platelet-derived growth factor induces mox1 mRNA production, while antisense mox1 mRNA decreases superoxide generation and serum-stimulated growth. Overexpression of mox1 in NIH3T3 cells increases superoxide generation and cell growth. Cells expressing mox1 have a transformed appearance, show anchorage-independent growth and produce tumours in athymic mice. These data link ROS production by Mox1 to growth control in non-phagocytic cells.

Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role in Vascular Physiology and Pathophysiology

Emerging evidence indicates that reactive oxygen species, especially superoxide and hydrogen peroxide, are important signaling molecules in cardiovascular cells. Their production is regulated by hormone-sensitive enzymes such as the vascular NAD(P)H oxidases, and their metabolism is coordinated by antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Both of these reactive oxygen species serve as second messengers to activate multiple intracellular proteins and enzymes, including the epidermal growth factor receptor, c-Src, p38 mitogen-activated protein kinase, Ras, and Akt/protein kinase B. Activation of these signaling cascades and redox-sensitive transcription factors leads to induction of many genes with important functional roles in the physiology and pathophysiology of vascular cells. Thus, reactive oxygen species participate in vascular smooth muscle cell growth and migration; modulation of endothelial function, including endothelium-dependent relaxation and expression of a proinflammatory phenotype; and modification of the extracellular matrix. All of these events play important roles in vascular diseases such as hypertension and atherosclerosis, suggesting that the sources of reactive oxygen species and the signaling pathways that they modify may represent important therapeutic targets.

NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-β1–Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts

Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-β1 (TGF-β1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle α-actin (SM α-actin) de novo and produce extracellular matrix. We hypothesized that TGF-β1–stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-β1 potently upregulates the contractile marker SM α-actin mRNA (7.5±0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-β1, Nox4 mRNA is dramatically upregulated by 16.2±0.8-fold (n=3, P<0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80±5%, inhibits NADPH-driven superoxide production in response to TGF-β1 by 65±7%, and reduces TGF-β1–induced expression of SM α-actin by 95±2% (n=6, P<0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-β1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-β1 stimulation of Smad 2/3 phosphorylation by 75±5% and 68±3%, respectively (n=7, P<0.0001). We conclude that Nox 4 mediates TGF-β1–induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure.

Nox4 Is Required for Maintenance of the Differentiated Vascular Smooth Muscle Cell Phenotype

Objective—The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process. Methods and Results—Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle α-actin (SM α-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM α-actin–based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-α actin, SM-MHC, and calponin, as well as SM-α actin stress fibers. Nox1 depletion did not decrease these parameters. Conclusion—Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.

Hemodynamic Shear Stresses in Mouse Aortas: Implications for Atherogenesis

Objective—The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is known about local variations in wall shear stress (WSS) in the mouse and whether the levels of WSS are comparable to those in humans. The objective of this study was to determine WSS values in the mouse aorta and to relate these to expression of gene products associated with atherosclerosis. Methods and Results—Using micro-CT and ultrasound methodologies we developed a computational fluid dynamics model of the mouse aorta and found values of WSS to be much larger than those for humans. We also used a quantum dot-based approach to study vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression on the aortic intima and demonstrated that increased expression for these molecules occurs where WSS was relatively low for the mouse. Conclusions—Despite large differences in WSS in the two species, the spatial distributions of atherogenic molecules in the mouse aorta are similar to atherosclerotic plaque localization found in human aortas. These results suggest that relative differences in WSS or in the direction of WSS, as opposed to the absolute magnitude, may be relevant determinants of flow-mediated inflammatory responses.

Angiotensin II and atherosclerosis.

Numerous clinical and laboratory data are now available supporting the hypothesis that the renin-angiotensin system is mechanistically relevant in the pathogenesis of atherosclerosis. The traditional role of the renin-angiotensin system in the context of blood pressure regulation has been modified to incorporate the concept that angiotensin II (Ang II) is a potent proinflammatory agent. In vascular cells, Ang II is a potent stimulus for the generation of reactive oxygen species. As a result, Ang II upregulates the expression of many redox-sensitive cytokines, chemokines, and growth factors that have been implicated in the pathogenesis of atherosclerosis. Extensive data now confirm that inhibition of the renin-angiotensin system inhibits atherosclerosis in animal models as well as in humans. These studies provide mechanistic insights into the precise role of Ang II in atherosclerosis and suggest that pharmacologic interventions involving the renin-angiotensin system may be of fundamental importance in the treatment and prevention of atherosclerosis.

Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet

The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase β-oxidation.

Ranolazine Improves Cardiac Diastolic Dysfunction Through Modulation of Myofilament Calcium Sensitivity

Rationale: Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (INa), reducing the net cytosolic Ca2+ efflux. Objective: Oxidative stress in the DOCA-salt model may increase late INa, resulting in diastolic dysfunction amenable to treatment with ranolazine. Methods and Results: Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E′:sham, 31.9±2.8, sham+ranolazine, 30.2±1.9, DOCA-salt, 41.8±2.6, and DOCA-salt+ranolazine, 31.9±2.6; P=0.018). The end-diastolic pressure-volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham, 0.16±0.01 versus sham+ranolazine, 0.18±0.01 versus DOCA-salt, 0.23±0.2 versus DOCA-salt+ranolazine, 0.17±0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, &tgr;, improving with ranolazine (DOCA-salt, 0.18±0.02, DOCA-salt+ranolazine, 0.13±0.01, sham, 0.11±0.01, sham+ranolazine, 0.09±0.02 seconds; P=0.0004). Neither late INa nor the Ca2+ transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca2+ with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca2+ response and cross-bridge kinetics. Conclusions: Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells.

Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2*- production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2*-. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2*- with DEPMPO. We confirmed that the main source of O2*- from VSMC membranes is an NAD(P)H oxidase and that the O2*- formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2*- when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC(50)s ( approximately 10-50 microM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O2*- (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2*-. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2*- production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme.