Manish Mittal
Publications
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6.08Impact points
Hypoxia induces K(v) channel current inhibition by increased NADPH oxidase-derived reactive oxygen species.
Free radical biology & medicine. 03/2012; 52(6):1033-42.
There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog ... [more] There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.
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12.92Impact points
Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration.
PLoS biology. 01/2010; 8(9).
Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thu... [more] Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.
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4.04Impact points
The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice.
American journal of physiology. Lung cellular and molecular physiology. 08/2009;
Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on... [more] Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose response curve was shifted leftward when investigating the effects of HMR1766 in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed pulmonary hypertension, right heart hypertrophy and pulmonary vascular remodeling. Treatment with HMR1766 (10mg/kg/day), after full establishment of pulmonary hypertension from day 21 to day 35, significantly reduced pulmonary hypertension, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH. Key words: Pulmonary hypertension, soluble guanylate cyclase (sGC), Hypoxia, Vascular remodeling.
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10.69Impact points
Heme Oxygenase-2 and Large-conductance Ca2+ activated K+ Channels: Lung Vascular Effects of Hypoxia.
American journal of respiratory and critical care medicine. 07/2009;
RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. Recently, a comple... [more] RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. Recently, a complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVE: We investigated, whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS and RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and co-immunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different between the wild-type, HO-2- and BKalpha-deficient mice. Exposure of mice to three weeks of chronic hypoxia resulted in a slight downregulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension which developed, quantified from right ventricular pressure, right heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different between the wild-type, HO-2- or BKalpha-deficient mice. CONCLUSION: It is demonstrated that neither deletion of HO-2 nor BK channels affects the acute, sustained and chronic vascular responses to alveolar hypoxia in the lung.
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9.21Impact points
Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.
Circulation research. 09/2007; 101(3):258-67.
Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen se... [more] Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O(2)) or normoxic (21% O(2)) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.
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7.69Impact points
Dietary electrolyte-driven responses in the renal WNK kinase pathway in vivo.
Journal of the American Society of Nephrology : JASN. 10/2006; 17(9):2402-13.
WNK1 and WNK4 are unusual serine/threonine kinases with atypical positioning of the catalytic active-site lysine (WNK: With-No-K[lysine]). Mutations in these WNK kinase genes can cause familial hyperkalemic hypertension (FHHt), an autosomal dominant, hypertensive, hyperkalemic disorder, implicating ... [more] WNK1 and WNK4 are unusual serine/threonine kinases with atypical positioning of the catalytic active-site lysine (WNK: With-No-K[lysine]). Mutations in these WNK kinase genes can cause familial hyperkalemic hypertension (FHHt), an autosomal dominant, hypertensive, hyperkalemic disorder, implicating this novel WNK pathway in normal regulation of BP and electrolyte balance. Full-length (WNK1-L) and short (WNK1-S) kinase-deficient WNK1 isoforms previously have been identified. Importantly, WNK1-S is overwhelmingly predominant in kidney. Recent Xenopus oocyte studies implicate WNK4 in inhibition of both thiazide-sensitive co-transporter-mediated Na+ reabsorption and K+ secretion via renal outer medullary K+ channel and now suggest that WNK4 is inhibited by WNK1-L, itself inhibited by WNK1-S. This study examined WNK pathway gene expression in mouse kidney and its regulation in vivo. Expression of WNK1-S and WNK4 is strongest in distal tubule, dropping sharply in collecting duct and with WNK4 also expressed in thick ascending limb and the macula densa. These nephron segments that express WNK1-S and WNK4 mRNA have major influence on long-term NaCl reabsorption, BP, K+, and acid-base balance, processes that all are disrupted in FHHt. In vivo, this novel WNK pathway responds with significant upregulation of WNK1-S and WNK4 with high K+ intake and reduction in WNK1-S on chronic lowering of K+ or Na+ intake. A two-compartment distal nephron model explains these in vivo findings and the pathophysiology of FHHt well, with WNK and classic aldosterone pathways responding to drivers from K+ balance, extracellular volume, and aldosterone and cross-talk through distal Na+ delivery regulating electrolyte balance and BP.
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Role of NADPH oxidases and KDR channels in the pathophysiology of hypoxia induced pulmonary hypertension
Circulation Research. 2007;101: 258-267.
The pulmonary vasculature has the unique ability to undergo vasoconstriction in response to acute hypoxia, a physiological mechanism known as hypoxic pulmonary vasoconstriction (HPV). Sustained or chronic hypoxia, however, leads to proliferation of vascular smooth muscle cells of pulmonary arteriole... [more] The pulmonary vasculature has the unique ability to undergo vasoconstriction in response to acute hypoxia, a physiological mechanism known as hypoxic pulmonary vasoconstriction (HPV). Sustained or chronic hypoxia, however, leads to proliferation of vascular smooth muscle cells of pulmonary arterioles, which causes a permanent increase in pulmonary vascular resistance, and may lead to right heart dysfunction. The underlying mechanisms of vascular proliferation under chronic hypoxia have not been fully defined. The NADPH oxidases are one family of recently discovered molecules which generate reactive oxygen species (ROS) and have been suggested to be important for cellular signaling under physiological conditions. However, NADPH oxidase generated oxidative stress can also lead to inflammation, vascular smooth muscle cell proliferation and endothelial damage under pathological conditions. Many homologs of NADPH oxidases exist, the classical homolog is gp91phox or NOX2, and the recently discovered homologs include NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2. Superoxide production by classical gp91phox is induced by assembly of the cytosolic subunits such as p40phox, p47phox and p67phox with membrane-bound gp91phox complex. A previous report from our laboratory has shown that the knockout mice of p47phox subunit exhibit reduced acute HPV as compared to wild type mice suggesting an essential role of NADPH oxidases in regulation of vascular tone in acute hypoxia. Against this background, the current thesis aimed to elucidate the role of NADPH oxidases in vascular remodeling in chronic hypoxia, and its possible downstream mediators. Screening of NADPH oxidase expression revealed that all subunits were expressed in the lung homogenate and that NOX4 was prominently up-regulated under chronic hypoxia. The NOX4 mRNA was also up-regulated in the microdissected vessels of mice exposed up to three weeks of chronic hypoxia. In addition, a functional interference with NOX4 using NOX4 siRNA resulted in reduced ROS production and reduced proliferation of pulmonary arterial smooth muscle cells (PASMC) revealing an important contribution of NOX4 in PASMC proliferation and particularly in hypoxia induced pulmonary hypertension. Intriguingly, a similar reflection was found in lungs of patients with idiopathic pulmonary hypertension that underwent lung transplantation. Further experiments demonstrated that NOX4 inhibited voltage-gated delayed rectifier K+ channels (KDR) under hypoxia. Pharmacological inhibition with apocynin and genetic ablation with NOX4siRNA resulted in increased KDR current under hypoxia. In addition, the current study demonstrated that NOX4 is essential for ET-1 mediated calcium influx in PASMC as NOX4 knockdown using NOX4 siRNA abolished the ET-1 mediated calcium influx under chronic hypoxia. Thus, the NOX4-ROS-KDR-[Ca2+] pathway may contribute to the development of pulmonary hypertension. Das pulmonale Gefäßsystem besitzt die besondere Eigenschaft, auf akute Hypoxie mit einer Vasokonstriktion zu reagieren. Dies ist ein physiologischer Mechanismus, der als hypoxische pulmonale Vasokonstriktion (HPV) bezeichnet wird. Anhaltende oder chronische Hypoxie führt darüberhinaus zu einer Proliferation der Media der Lungenarteriolen, das einen permanenten Anstieg des pulmonalen Gefäßwiderstandes auslöst, der im weiteren zu einem Cor pulmonale führen kann. Die der HPV zu Grunde liegenden Mechanismen sind bisher noch nicht umfassend geklärt worden. NADPH-Oxidasen sind eine vor kurzem entdeckte Proteinfamilie, die reaktive Sauerstoffspezies (ROS) generieren können und unter physiologischen Bedingungen für die zelluläre Signaltransduktion wichtig sind. Andererseits können die von NADPH-Oxidasen stammenden ROS unter pathologischen Bedingungen zu Entzündung und Proliferation von vaskulären glatten Muskelzellen und zur Schädigung des Endothels führen. In der Literatur sind mehrere Homologe von NADPH-Oxidasen beschrieben, wobei das klassische Homolog als gp91phox oder NOX2 bekannt ist. Weitere, erst kürzlich entdeckte Homologe umfassen NOX1, NOX3, NOX4, NOX5, DUOX1 und DUOX2. Die Superoxidproduktion der klassischen gp91phox wird durch die Anlagerung der zytosolischen Untereinheiten p40phox, p47phox und p67phox an gp91phox induziert. Unsere bisherigen Daten zeigen, dass Mäuse mit einer p47phox-Defizienz im Vergleich zu Wildtyp-Mäusen eine geringere akute HPV aufweisen, das auf eine essentielle Rolle der NADPH-Oxidasen für die Regulation des vaskulären Tonus unter akuter Hypoxie hinweist. Ziel der vorliegenden Arbeit war, die Rolle der NADPH-Oxidasen für den vaskulären Umbauprozess unter chronischer Hypoxie und deren Signaltransduktionsmechanismen aufzuklären. Untersuchungen der NADPH-Oxidasen Expression zeigten, dass alle Untereinheiten im Lungenhomogenat exprimiert waren und die Expression von NOX4 unter chronischer Hypoxie stark hochreguliert war. Darüberhinaus war die NOX4-mRNA auch in den durch Mikrodissektion gewonnenen Gefäßen der Mäuse, die 3 Wochen unter chronischer Hypoxie gehalten wurden, hochreguliert. Zusätzlich bewirkte eine funktionelle Interferenz von NOX4 mit NOX4-siRNA eine reduzierte ROS-Produktion und eine verringerte Proliferation der pulmonalarteriellen glatten Muskelzellen (PASMC). Dies weist auf eine bedeutungsvolle Rolle von NOX4 für die Proliferation von PASMC, insbesondere bei Hypoxia-induzierter pulmonaler Hypertonie, hin. Interessanterweise wurden ähnliche Ergebnisse in Lungen von Patienten mit idiopatischer pulmonaler Hypertonie nach Transplantation der Lunge gewonnen. Weitere Experimente zeigten, dass NOX4 unter chronischer Hypoxie die voltage gated delayed rectifier K+-Kanäle (KDR) hemmt. Eine pharmakologische Inhibition von NOX4 mittels Apocynin oder eine genetische Ablation durch siRNA erhöhten den KDR-Strom unter chronischer Hypoxie. Zusätzlich ist in der vorliegenden Arbeit gezeigt, dass NOX4 essentiell für den ET-1 vermittelten Kalziumeinstrom ist, da durch Reduzierung von NOX4 mittels NOX4 siRNA der ET-1 vermittelte Kalziumeinstrom unter chronischer Hypoxie nicht mehr vorhanden war. Zusammengefasst gesagt, könnte der NOX4-ROS-KDR-[Ca2+]-Signalweg somit zur Entstehung der pulmonalen Hypertonie beitragen.
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Heme oxygenase-2 and large-conductance Ca2+-activated K+ channels: lung vascular effects of hypoxia
Am J Respir Crit Care Med, v.180, 353-64 (2009).
RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme ... [more] RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVES: We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS: The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. CONCLUSIONS: It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.
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The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice
Am J Physiol Lung Cell Mol Physiol, v.297, L658-65 (2009).
Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on... [more] Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose-response curve was shifted leftward when the effects of HMR1766 were investigated in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed PH, right heart hypertrophy, and pulmonary vascular remodeling. Treatment with HMR1766 (10 mg x kg(-1) x day(-1)), after full establishment of PH from day 21 to day 35, significantly reduced PH, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH.
Following (1)
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Markus A. Queisser
Northwestern University
