Tamara M. Paravicini

NADPH Oxidases, Reactive Oxygen Species, and Hypertension Clinical implications and therapeutic possibilities

Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS production (termed “oxidative stress”) has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and chronic kidney disease. A major source for vascular and renal ROS is a family of nonphagocytic NAD(P)H oxidases, including the prototypic Nox2 homolog-based NAD(P)H oxidase, as well as other NAD(P)H oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase. NAD(P)H oxidase-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important processes underlying cardiovascular and renal remodeling in hypertension and diabetes. These findings have evoked considerable interest because of the possibilities that therapies against nonphagocytic NAD(P)H oxidase to decrease ROS generation and/or strategies to increase nitric oxide (NO) availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress target organ damage associated with hypertension and diabetes. Here we highlight current developments in the field of reactive oxygen species and cardiovascular disease, focusing specifically on the recently identified novel Nox family of NAD(P)H oxidases in hypertension. We also discuss the potential role of targeting ROS as a therapeutic possibility in the management of hypertension and cardiovascular disease.

Increased NADPH-Oxidase Activity and Nox4 Expression During Chronic Hypertension Is Associated With Enhanced Cerebral Vasodilatation to NADPH In Vivo

Background and Purpose— We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels. Methods— Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats. Results— NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu2+/Zn2+–superoxide dismutase inhibitor), catalase (H2O2 scavenger), or tetraethylammonium (BKCa channel inhibitor). Conclusions— Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu2+/Zn2+–superoxide dismutase to H2O2. H2O2 then elicits vasodilatation via activation of BKCa channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.

Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 5 (Nox5) Regulation by Angiotensin II and Endothelin-1 Is Mediated via Calcium/Calmodulin-Dependent, Rac-1-Independent Pathways in Human Endothelial Cells

Rationale: Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives: We sought to test whether vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+-sensitive Nox5, associated with Rac-1, generates superoxide (O2·−) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results: Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O2·− production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II–induced, but not ET-1–induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions: Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca2+/calmodulin-dependent, Rac-1–independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Increased NADPH Oxidase Activity, gp91phox Expression, and Endothelium-Dependent Vasorelaxation During Neointima Formation in Rabbits

Reactive oxygen species including superoxide and hydrogen peroxide are important mediators in atherogenesis. We investigated the enzymatic source of vascular superoxide and its role in endothelium-dependent vasorelaxation during neointima formation. Silastic collars positioned around carotid arteries of rabbits for 14 days induced neointimal thickening. Using lucigenin-enhanced chemiluminescence, superoxide production was detectable in collared artery sections, but not in controls, only after inactivation of endogenous Cu2+/Zn2+-superoxide dismutase (Cu2+/Zn2+-SOD) with diethyldithiocarbamate (DETCA). Dihydroethidium staining indicated that endothelium and adventitia were the major sites of superoxide generation. Superoxide production in DETCA-treated collared arteries was enhanced further by NADPH and was inhibited by diphenyleneiodonium, suggesting NADPH oxidase was the source of the radical in collared arteries. Moreover, real-time PCR demonstrated 11-fold higher expression of the gp91phox subunit of NADPH oxidase in collared arteries than in controls. In vascular reactivity studies, endothelium-dependent vasorelaxation to acetylcholine did not differ between collared and control sections. However, treatment with DETCA reduced relaxations to acetylcholine in collared rings, but not in controls. NADPH further reduced relaxations to acetylcholine in DETCA-treated collared sections, but not in controls. In DETCA/NADPH-treated collared rings, sensitivity to nitroprusside, in contrast to acetylcholine, exceeded that of controls. Moreover, further treatment of such rings with exogenous Cu2+/Zn2+-SOD restored acetylcholine relaxations without altering nitroprusside responses. Thus, early neointimal lesions induced by periarterial collars are associated with elevated gp91phox expression and increased NAPDH-oxidase-dependent superoxide production in endothelium and adventitia. However, endothelium-dependent vasorelaxation is largely preserved due to the actions of Cu2+/Zn2+-SOD and increased smooth muscle sensitivity to nitric oxide.

Downregulation of Renal TRPM7 and Increased Inflammation and Fibrosis in Aldosterone-Infused Mice. Effects of Magnesium

Hyperaldosteronism is associated with hypertension, cardiovascular fibrosis, and electrolyte disturbances, including hypomagnesemia. Mechanisms underlying aldosterone-mediated Mg2+ changes are unclear, but the novel Mg2+ transporters TRPM6 and TRPM7 may be important. We examined whether aldosterone influences renal TRPM6/7 and the TRPM7 downstream target annexin-1 and tested the hypothesis that Mg2+ administration ameliorates aldosterone-induced cardiovascular and renal injury and prevents aldosterone-associated hypertension. C57B6 mice were studied (12 weeks, n=8 to 9/group); (1) control group (0.2% dietary Mg2+), (2) Mg2+ group (0.75% dietary Mg2+), (3) aldosterone group (Aldo, 400 &mgr;g/kg/min and 0.9% NaCl drinking water), and (4) Aldo+Mg2+ group. Blood pressure was unaltered by aldosterone and was similar in all groups throughout the experiment. Serum Na+ was increased and serum K+ and Mg2+ decreased in the Aldo group. Aldo mice had hypomagnesuria and proteinuria, and renal, cardiac, and aortic fibrosis, which were normalized by Mg2+ supplementation. Renal and cardiovascular expression of interleukin-6, VCAM1 and COX2 was increased in the Aldo group. Magnesium attenuated renal and cardiac interleukin-6 content and decreased renal VCAM1 and cardiac COX2 expression (P<0.05). Aldosterone decreased expression of renal TRPM7 and the downstream target annexin-1 (P<0.05) without effect on TRPM6. Whereas Mg2+ increased mRNA expression of TRPM6 and TRPM7, it had no effect on TRPM7 and annexin-1 protein content. Our data demonstrate that aldosterone mediates blood pressure-independent renal and cardiovascular fibrosis and inflammation through Mg2+-sensitive pathways. We suggest that altered Mg2+ metabolism in hyperaldosteronism may relate to TRPM7 downregulation and that Mg2+ protects against cardiovascular and renal damaging actions of aldosterone.

Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase

Reactive oxygen species (ROS) such as superoxide (O•−2) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Activation of NADPH-oxidase leads to dilatation of the basilar artery in vivo via production of H2O2, but the endogenous stimuli for this unique vasodilator mechanism are unknown. Shear stress is known to activate both NADPH-oxidase and phosphatidylinositol-3 kinase (PI3-K) in cultured cells. Hence, this study used a cranial window preparation in anesthetized rats to investigate whether increased intraluminal blood flow could induce cerebral vasodilatation via the activation of NADPH-oxidase and/or PI3-K. Bilateral occlusion of the common carotid arteries to increase basilar artery blood flow caused reproducible, reversible vasodilatation. Topical treatment of the basilar artery with the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) (0.5 and 5 μmol/L) inhibited flow-induced dilatation by up to 50% without affecting dilator responses to acetylcholine. Treatment with the H2O2 scavenger, catalase similarly attenuated flow-induced dilatation, suggesting a role for NADPH-oxidase-derived H2O2 in this response. The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) partially reduced flow-induced dilatation, and combined treatment with a ROS inhibitor (DPI or catalase) and l-NAME caused a greater reduction in flow-induced dilatation than that seen with any of these inhibitors alone. Flow-induced dilatation was also markedly inhibited by the PI3-K inhibitor, wortmannin. Increased O•−2 production in the endothelium of the basilar artery during acute increases in blood flow was confirmed using dihydroethidium. Thus, flow-induced cerebral vasodilatation in vivo involves production of ROS and nitric oxide, and is dependent on PI3-K activation.

Dysregulation of Vascular TRPM7 and Annexin-1 Is Associated With Endothelial Dysfunction in Inherited Hypomagnesemia

Inadequate magnesium intake and hypomagnesemia may contribute to chronic diseases, such as hypertension. The novel magnesium transporter TRPM7 is a critical regulator of magnesium homeostasis in vascular cells, but its role in pathophysiology is unclear. In a model of hypomagnesemia, we examined microvascular structure and function, TRPM7 expression, and vascular inflammatory status using inbred mice selected for normal-high intracellular magnesium levels or low intracellular magnesium levels (MgLs). Blood pressure was significantly increased in MgLs compared with normal-high intracellular magnesium levels. Pressurized myography of mesenteric resistance arteries showed that MgLs had significantly impaired endothelial function together with decreased plasma nitrate levels and endothelial NO synthase expression when compared with normal-high intracellular magnesium levels. Significant differences in vascular structure were also evident in both mesenteric arteries and aortas from MgLs. Aortas from MgLs had increased medial cross-sectional areas, whereas mesenteric arteries from MgLs had increased lumen diameters with increased medial cross-sectional areas, indicating outward hypertrophic remodeling. Expression of the magnesium transporter TRPM7 was significantly elevated in the vasculature of MgLs, whereas expression of a TRPM7 downstream target, the anti-inflammatory molecule annexin-1, was reduced. MgLs had increased expression of vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1, indicating vascular inflammation. Taken together, these data demonstrate that the inherited magnesium status of MgLs and normal-high intracellular magnesium levels mice affects magnesium transporter expression, endothelial function, vascular structure, and inflammation. Our findings suggest a potential regulatory role for TRPM7 signaling in the maintenance of vascular integrity. Alterations in magnesium status and/or TRPM7 signaling may contribute to vascular injury in conditions associated with hypomagnesemia.

TRPM7: A unique channel involved in magnesium homeostasis

TRPM7 is a ubiquitously expressed cation channel with a fused alpha kinase domain. It is highly permeable to magnesium and calcium, and is negatively gated by intracellular Mg(2+) and Mg-ATP. Substrates for the TRPM7 kinase domain include annexinA1 and myosin IIA heavy chain, and there is evidence to suggest a functional interaction between the channel and kinase domains. Alterations in the expression and activity of TRPM7 have profound effects on cell proliferation and differentiation. Genetic deletion of TRPM7 in model systems demonstrates that this channel is critical for cellular growth and embryonic development. Here, we provide a brief overview of the activity of TRPM7 and the associated regulatory mechanisms. We will then discuss the biological functions of TRPM7, emphasizing its role in development and the potential pathophysiological significance of TRPM7 in neurological and cardiovascular disease.

Cerebral vascular effects of reactive oxygen species: recent evidence for a role of NADPH-oxidase.

1. Reactive oxygen species (ROS) are a diverse family of molecules that are produced throughout the vascular wall. Many ROS, such as the superoxide anion (·O2–) and hydrogen peroxide (H2O2), are now known to act as cellular signalling molecules within blood vessels. In particular, these molecules can exert powerful effects on vascular tone.

Prenatal exposure to dexamethasone in the mouse alters cardiac growth patterns and increases pulse pressure in aged male offspring.

Exposure to synthetic glucocorticoids during development can result in later cardiovascular and renal disease in sheep and rats. Although prenatal glucocorticoid exposure is associated with impaired renal development, less is known about effects on the developing heart. This study aimed to examine the effects of a short-term exposure to dexamethasone (60 hours from embryonic day 12.5) on the developing mouse heart, and cardiovascular function in adult male offspring. Dexamethasone (DEX) exposed fetuses were growth restricted compared to saline treated controls (SAL) at E14.5, but there was no difference between groups at E17.5. Heart weights of the DEX fetuses also tended to be smaller at E14.5, but not different at E17.5. Cardiac AT1aR, Bax, and IGF-1 mRNA expression was significantly increased by DEX compared to SAL at E17.5. In 12-month-old offspring DEX exposure caused an increase in basal blood pressure of ∼3 mmHg. In addition, DEX exposed mice had a widened pulse pressure compared to SAL. DEX exposed males at 12 months had an approximate 25% reduction in nephron number compared to SAL, but no difference in cardiomyocyte number. Exposure to DEX in utero appears to adversely impact on nephrogenesis and heart growth but is not associated with a cardiomyocyte deficit in male mice in adulthood, possibly due to compensatory growth of the myocardium following the initial insult. However, the widened pulse pressure may be indicative of altered vascular compliance.