The severity of SARS-CoV-2 infection in K18-hACE2 mice is attenuated by a novel steroid-derivative in a gender-specific manner.

BACKGROUND AND PURPOSE: COVID-19 infections caused by SARS-CoV-2 disseminated through human-to-human transmission can evoke severe inflammation. Treatments to reduce the SARS-CoV-2-associated inflammation are needed and are the focus of much research. In this study, we investigated the effect of N-ethyl-N'-[(3ß,5α)-17-oxoandrostan-3-yl] urea (NEOU), a novel 17α-ketosteroid derivative, on the severity of COVID-19 infections. EXPERIMENTAL APPROACH: Studies were conducted in SARS-CoV-2-infected K18-hACE2 mice. KEY RESULTS: SARS-CoV-2-infected K18-hACE2 mice developed severe inflammatory crises and immune responses along with up-regulation of genes in associated signalling pathways in male more than female mice. Notably, SARS-CoV-2 infection down-regulated genes encoding drug metabolizing cytochrome P450 enzymes in male but not female mice. Treatment with NEOU (1 mg·kg-1 ·day-1 ) 24 or 72 h post-viral infection alleviated lung injury by decreasing expression of genes encoding inflammatory cytokines and chemokines while increasing expression of genes encoding immunoglobins. In situ hybridization using RNA scope™ probes and immunohistochemical assays revealed that NEOU increased resident CD169+ immunoregulatory macrophages and IBA-1 immunoreactive macrophage-dendritic cells within alveolar spaces in the lungs of infected mice. Consequentially, NEOU reduced morbidity more prominently in male than female mice. However, NEOU increased median survival time and accelerated recovery from infection by 6 days in both males and females. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that SARS-CoV-2 exhibits gender bias by differentially regulating genes encoding inflammatory cytokines, immunogenic factors and drug-metabolizing enzymes, in male versus female mice. Most importantly, we identified a novel 17α-ketosteroid that reduces the severity of COVID-19 infection and could be beneficial for reducing impact of COVID-19.

Mediterranean G6PD variant mitigates expression of DNA methyltransferases and right heart pressure in experimental model of pulmonary hypertension.

DNA methylation potentially contributes to the pathogenesis of pulmonary hypertension (PH). However, the role of DNA methyltransferases (DNMTs: 1, 3a, and 3b), the epigenetic writers, in modulating DNA methylation observed in PH remains elusive. Our objective was to determine DNMT activity and expression in the lungs of experimental rat models of PH. Because the activity of DNMTs is metabolically driven, another objective was to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in regulating DNMT expression and activity in the lungs of novel loss-of-function Mediterranean G6PD variant (G6PDS188F) rats. As outlined for modeling PH, rats injected with sugen5416 (SU) were placed in a hypoxia (Hx) chamber set at 10% oxygen for 3 weeks and then returned to normoxia (Nx) for 5 weeks (SU/Hx/Nx). Rats kept in atmospheric oxygen and treated with SU were used as controls. We assessed the activity and expression of DNMTs in the lungs of rats exposed to SU/Hx/Nx. WT rats exposed to SU/Hx/Nx developed hypertension and exhibited increased DNMT activity and Dnmt1 and Dnmt3b expression. In G6PDS188F rats, which developed less of a SU/Hx/Nx-induced increase in right ventricle pressure and hypertrophy than WT rats, we observed a diminished increase in expression and activity of DNMTs, DNA hypomethylation, increased histone acetylation and methylation, and increased expression of genes encoding NOS3 and SOD2-vascular-protective proteins. Collectively, increased DNMTs contribute to reduced expression of protective genes and to the pathogenesis of SU/Hx/Nx-induced experimental PH. Notably, G6PD regulates the expression of DNMTs and protective proteins in the lungs of hypertensive rats.

Mediterranean G6PD variant rats are protected from Angiotensin II-induced hypertension and kidney damage, but not from inflammation and arterial stiffness.

RATIONALE: Epidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model. OBJECTIVE: To determine the effect of Ang-II-induced hypertension (HTN) and stiffness in a rat model of the Mediterranean G6PD (G6PDS188F) variant and in wild-type (WT) rats. METHODS AND RESULTS: Our findings revealed that infusion of Ang-II (490 ng/kg/min) elicited less HTN and medial hypertrophy of carotid artery in G6PDS188F than in WT rats. Additionally, Ang-II induced less glomerular and tubular damage in the kidneys - a consequence of elevated pressure - in G6PDS188F than WT rats. However, Ang-II-induced arterial stiffness increased in G6PDS188F and WT rats, and there were no differences between the groups. Mechanistically, we found aorta of G6PDS188F as compared to WT rats produced less sustained contraction and less inositol-1,2,3-phosphate (IP3) and superoxide in response to Ang-II. Furthermore, aorta of G6PDS188F as compared to WT rats expressed lower levels of phosphorylated extracellular-signal regulated kinase (ERK). Interestingly, the aorta of G6PDS188F as compared to WT rats infused with Ang-II transcribed more (50-fold) myosin heavy chain-11 (MYH11) gene, which encodes contractile protein of smooth muscle cell (SMC), and less (2.3-fold) actin-binding Rho-activating gene, which encodes a protein that enhances SMC proliferation. A corresponding increase in MYH11 and Leiomodin-1 (LMOD1) staining was observed in arteries of Ang-II treated G6PDS188F rats. However, G6PD deficiency did not affect the accumulation of CD45+ cells and transcription of genes encoding interleukin-6 and collagen-1a1 by Ang-II. CONCLUSIONS: The G6PDS188F loss-of-function variant found in humans protected rats from Ang-II-induced HTN and kidney damage, but not from vascular inflammation and arterial stiffness.

G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases.

We aimed to determine 1) the mechanism(s) that enables glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)-driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs, and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC-restricted genes. G6PD knockdown or inhibition decreased (48.5%) histone deacetylase (HDAC) activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control Sprague-Dawley (SD) rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4 mo increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate that G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.NEW & NOTEWORTHY This study gives detailed mechanistic insight about the regulation of smooth muscle cell (SMC) phenotype by metabolic reprogramming and glucose-6-phosphate dehydrogenase (G6PD) in diabetes and metabolic syndrome. We demonstrate that G6PD controls the chromatin modifications by regulating histone deacetylase (HDAC) activity, which deacetylates histone 3-lysine 9 and 27. Notably, inhibition of G6PD decreases HDAC activity and enriches H3K27ac on myocardin gene promoter to enhance the expression of SMC-restricted genes. Also, we demonstrate for the first time that G6PD inhibitor treatment accentuates metabolic and transcriptomic reprogramming to reduce neointimal formation in coronary artery and large artery elastance in metabolic syndrome rats.

CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase (G6pd) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PDS188F) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced (P<0.05) in the arteries of G6PDS188F versus WT rats. Intriguingly, G6PDS188F mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-NG-nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PDS188F rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K+ channel proteins and (2) decreased voltage-gated Ca2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-NG-nitroarginine methyl ester and high-fat diet to G6PDS188F mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.

Glucose-6-phosphate dehydrogenase increases Ca2+ currents by interacting with Cav1.2 and reducing intrinsic inactivation of the L-type calcium channel.

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

Vascular smooth muscle cell contractile protein expression is increased through protein kinase G-dependent and -independent pathways by glucose-6-phosphate dehydrogenase inhibition and deficiency.

Homeostatic control of vascular smooth muscle cell (VSMC) differentiation is critical for contractile activity and regulation of blood flow. Recently, we reported that precontracted blood vessels are relaxed and the phenotype of VSMC is regulated from a synthetic to contractile state by glucose-6-phosphate dehydrogenase (G6PD) inhibition. In the current study, we investigated whether the increase in the expression of VSMC contractile proteins by inhibition and knockdown of G6PD is mediated through a protein kinase G (PKG)-dependent pathway and whether it regulates blood pressure. We found that the expression of VSMC-restricted contractile proteins, myocardin (MYOCD), and miR-1 and miR-143 are increased by G6PD inhibition or knockdown. Importantly, RNA-sequence analysis of aortic tissue from G6PD-deficient mice revealed uniform increases in VSMC-restricted genes, particularly those regulated by the MYOCD-serum response factor (SRF) switch. Conversely, expression of Krüppel-like factor 4 (KLF4) is decreased by G6PD inhibition. Interestingly, the G6PD inhibition-induced expression of miR-1 and contractile proteins was blocked by Rp-ß-phenyl-1,N2-etheno-8-bromo-guanosine-3',5'-cyclic monophosphorothioate, a PKG inhibitor. On the other hand, MYOCD and miR-143 levels are increased by G6PD inhibition through a PKG-independent manner. Furthermore, blood pressure was lower in the G6PD-deficient compared with wild-type mice. Therefore, our results suggest that the expression of VSMC contractile proteins induced by G6PD inhibition occurs via PKG1α-dependent and -independent pathways.

Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension.

Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension.

Increased reactive oxygen species, metabolic maladaptation, and autophagy contribute to pulmonary arterial hypertension-induced ventricular hypertrophy and diastolic heart failure.

Pulmonary arterial hypertension (PAH) is a debilitating and deadly disease with no known cure. Heart failure is a major comorbidity and a common cause of the premature death of patients with PAH. Increased asymmetrical right ventricular hypertrophy and septal wall thickening compress the left ventricular cavity and elicit diastolic heart failure. In this study, we used the Sugen5416/hypoxia/normoxia-induced PAH rat to determine whether altered pyridine nucleotide signaling in the failing heart contributes to 1) increased oxidative stress, 2) changes in metabolic phenotype, 3) autophagy, and 4) the PAH-induced failure. We found that increased reactive oxygen species, metabolic maladaptation, and autophagy contributed to the pathogenesis of right ventricular remodeling and hypertrophy that lead to left ventricular diastolic dysfunction. In addition, arterial elastance increased in PAH rats. Glucose-6-phosphate dehydrogenase is a major source of pyridine molecule (nicotinamide adenine dinucleotide phosphate), which is a substrate for nicotinamide adenine dinucleotide phosphate oxidases in the heart. Dehydroepiandrosterone, a 17-ketosteroid that reduces pulmonary hypertension and right ventricular hypertrophy, inhibited glucose-6-phosphate dehydrogenase, decreased oxidative stress, increased glucose oxidation and acetyl-coA, and reduced autophagy in the hearts of PAH rats. It also decreased arterial stiffness and improved left ventricular diastolic function. These findings demonstrate that pyridine nucleotide signaling, at least partly, mediates PAH-induced diastolic heart failure, and that reduction of glucose-6-phosphate dehydrogenase-derived nicotinamide adenine dinucleotide phosphate is beneficial to improve left ventricle diastolic function.

Glucose-6-phosphate dehydrogenase plays a critical role in hypoxia-induced CD133+ progenitor cells self-renewal and stimulates their accumulation in the lungs of pulmonary hypertensive rats.

Although hypoxia is detrimental to most cell types, it aids survival of progenitor cells and is associated with diseases like cancer and pulmonary hypertension in humans. Therefore, understanding the underlying mechanisms that promote survival of progenitor cells in hypoxia and then developing novel therapies to stop their growth in hypoxia-associated human diseases is important. Here we demonstrate that the proliferation and growth of human CD133(+) progenitor cells, which contribute to tumorigenesis and the development of pulmonary hypertension, are increased when cultured under hypoxic conditions. Furthermore, glucose-6-phosphate dehydrogenase (G6PD) activity was increased threefold in hypoxic CD133(+) cells. The increased G6PD activity was required for CD133(+) cell proliferation, and their growth was arrested by G6PD inhibition or knockdown. G6PD activity upregulated expression of HIF1α, cyclin A, and phospho-histone H3, thereby promoting CD133(+) cell dedifferentiation and self-renewal and altering cell cycle regulation. When CD133(+) cells were cocultured across a porous membrane from pulmonary artery smooth muscle cells (PASMCs), G6PD-dependent H2O2 production and release by PASMCs recruited CD133(+) cells to the membrane, where they attached and expressed smooth muscle markers (α-actin and SM22α). Inhibition of G6PD reduced smooth muscle marker expression in CD133(+) cells under normoxia but not hypoxia. In vivo, CD133(+) cells colocalized with G6PD(+) cells in the perivascular region of lungs from rats with hypoxia-induced pulmonary hypertension. Finally, inhibition of G6PD by dehydroepiandrosterone in pulmonary arterial hypertensive rats nearly abolished CD133(+) cell accumulation around pulmonary arteries and the formation of occlusive lesions. These observations suggest G6PD plays a key role in increasing hypoxia-induced CD133(+) cell survival in hypertensive lungs that differentiate to smooth muscle cells and contribute to pulmonary arterial remodeling during development of pulmonary hypertension.

Down-regulation of replication factor C-40 (RFC40) causes chromosomal missegregation in neonatal and hypertrophic adult rat cardiac myocytes.

BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 µM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 µM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 µM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Role of NAD(P)H oxidase in superoxide generation and endothelial dysfunction in Goto-Kakizaki (GK) rats as a model of nonobese NIDDM.

BACKGROUND: Cardiovascular disease is the leading cause of mortality in diabetics, and it has a complex etiology that operates on several levels. Endothelial dysfunction and increased generation of reactive oxygen species are believed to be an underlying cause of vascular dysfunction and coronary artery disease in diabetes. This impairment is likely the result of decreased bioavailability of nitric oxide (NO) within the vasculature. However, it is unclear whether hyperglycemia per se stimulates NADPH oxidase-derived superoxide generation in vascular tissue. METHODS AND RESULTS: This study focused on whether NADPH oxidase-derived superoxide is elevated in vasculature tissue evoking endothelial/smooth muscle dysfunction in the hyperglycemic (169+/-4 mg%) Goto-Kakizaki (GK) rat. By dihydroethidine fluorescence staining, we determined that aorta superoxide levels were significantly elevated in 9 month-old GK compared with age matched Wistar (GK; 195+/-6%, Wistar; 100+/-3.5%). Consistent with these findings, 10(-6) mol/L acetylcholine-induced relaxation of the carotid artery was significantly reduced in GK rats compared with age matched Wistar (GK; 41+/-7%, Wistar; 100+/-5%) and measurements in the aorta showed a similar trend (p = .08). In contrast, relaxation to the NO donor SNAP was unaltered in GK compared to Wistar. Endothelial dysfunction was reversed by lowering of superoxide with apocynin, a specific Nox inhibitor. CONCLUSIONS: The major findings from this study are that chronic hyperglycemia induces significant vascular dysfunction in both the aorta and small arteries. Hyperglycemic induced increases in NAD(P)H oxidase activity that did not come from an increase in the expression of the NAD(P)H oxidase subunits, but more likely as a result of chronic activation via intracellular signaling pathways.

Activation of glucose-6-phosphate dehydrogenase promotes acute hypoxic pulmonary artery contraction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O(2) tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N(2), 5% CO(2)) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD(+) and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca(2+) sensitivity to the myofilaments and diminished Ca(2+)-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca(2+)-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD(mut(-/-)) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP(+) ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.

Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH.

Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion (O(2)(-))] and the mechanisms underlying O(2)(-) production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O(2)(-) and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O(2)(-) generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9-11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P < 0.05). O(2)(-) levels were elevated (70-90%; P < 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91(ds-tat) (50 microM) or by the mitochondrial respiratory chain inhibitors antimycin (10 microM) and rotenone (50 microM). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 microM) also reduced (P < 0.05) O(2)(-) production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P < 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O(2)(-) generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O(2)(-) production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed.

Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver.

Glucose metabolism through the glycolysis and hexosamine pathway has been shown to be altered in type 2 diabetes. However, the fate of glucose through the pentose phosphate pathway (PPP) is currently unclear. In this study, we determined whether the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP, is modulated in the liver of Zucker obese fa/fa rats (9-11 weeks of age). We found that G6PD expression and activity, NADPH levels, and 6-phosphogluconate generation were significantly increased in the liver of fa/fa rats. Inhibition of PI3 kinase and Src kinases decreased (p < 0.05) G6PD activity in the fa/fa but not in the lean rat liver, suggesting that G6PD activity is regulated by PI3/Src kinase signaling pathways. G6PD-derived NADPH increased (p < 0.05) superoxide anion levels by 70-90% in fa/fa vs lean rat liver, which was inhibited by the NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and G6PD inhibitors 6-aminonicotinamide (1 mM) and dehydroepiandrosterone (100 microM), therefore indicating that elevated G6PD activity may be responsible for mediating superoxide generation. Interestingly, we also found a positive correlation between liver hypertrophy/increased G6PD activity (r2 = 0.77; p = 0.0009) and liver hypertrophy/superoxide production (r2 = 0.51; p = 0.0091) in fa/fa rats. Increased G6PD and NADPH oxidase expression and activity, in young hyperglycemic and hyperinsulinemic rats before the development of diabetes, seems to be a contributing factor in the induction of oxidative stress. Because inhibition of G6PD activity decreases oxidative stress, we conclude that G6PD behaves as a pro-oxidant in the fa/fa rat liver in type 2 diabetes.

Upregulation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase activity increases oxidative stress in failing human heart.

BACKGROUND: We previously found that higher NADPH levels produced by glucose-6-phosphate dehydrogenase (G6PD) can enhance myocardial superoxide generation by NAD(P)H oxidase in a dog model of dilated cardiomyopathy. Therefore, we tested whether G6PD activity is elevated and enhances NADPH level and increases NAD(P)H oxidase-derived superoxide production in the myocardium from patients with heart failure from ischemic cardiomyopathy. METHODS AND RESULTS: Surgical discards of left ventricle were collected from 8 congestive heart failure patients undergoing surgical ventricular restoration procedures, whereas control left ventricle tissue was obtained from 5 normal donor hearts deemed not suitable for transplantation. Biochemical assays were performed in tissue homogenates. We found that superoxide and hydrogen peroxide were elevated, respectively, by 9- and 3-fold in failing versus normal hearts (P < .05). The NAD(P)H oxidase inhibitors gp91(ds-tat), apocynin, and diphenyleneiodonium, significantly inhibited superoxide generation by approximately 75%, 89%, and 91%, respectively. Superoxide production by NAD(P)H oxidase increased 10- and 3-fold by adding NADPH (100 micromol/L) and NADH (100 micromol/L), respectively, in a DPI- and gp91(ds-tat)-inhibitable manner. Interestingly, chelerythrine, a PKC inhibitor, and PP2, a Src kinase family inhibitor, reduced G6PD activity (0.29 +/- 0.04 nM x min x mg protein) by 50% and 51% and these inhibitors also decreased myocardial superoxide by 99% and 79%, respectively. Furthermore, 6-aminonicotinamide, a G6PD inhibitor, decreased myocardial superoxide production by 71%. CONCLUSIONS: These data suggest that high NAD(P)H oxidase, fueled by G6PD-derived NADPH, generates most of the superoxide in failing hearts of patients with ischemic cardiomyopathy. In addition, PKC-Src kinase signaling pathways seem to coordinate the activation of both G6PD and NAD(P)H oxidase in human cardiac muscle.

Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart.

In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 +/- 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 microM) chemiluminescence was markedly increased in heart failure (1338 +/- 419 vs. 419 +/- 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 +/- 1.5 vs. 7.5 +/- 1.5 micromol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and the NO synthase inhibitor L-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 +/- 0.10 vs. 0.24 +/- 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure.

Phosphorylation of RIalpha by cyclin-dependent kinase CDK 2/cyclin E modulates the dissociation of the RIalpha-RFC40 complex.

We have previously demonstrated that the nuclear transport of the second subunit of the Replication Factor C complex, RFC40, by the regulatory subunit, RIalpha, of PKA is cell cycle specific and impairment in this transport results in G(1) arrest. In this study, we have investigated whether the cyclin-dependent kinases play a role in regulating the RIalpha-RFC40 complex formation. In this context, we have identified RIalpha as a novel substrate for the G(1)/S-Cyclin-dependent kinase, CDK2/Cyclin E, and found that RIalpha is specifically phosphorylated at the serine residue. Treatment of MCF7 cells with a CDK inhibitor, olomoucine, resulted in a significant accumulation in the RIalpha-RFC40 complex by 3.10 +/- 0.08 fold and a parallel decrease in the RFC40-37 complex formation by 73.73 +/- 11.81%. Furthermore, in vitro phosphorylation experiments suggest that, phosphorylation of RIalpha by CDK2/CyclinE kinase promotes the dissociation of the RIalpha-RFC40 complex and that once RIalpha is phosphorylated it cannot complex with RFC40. Inhibition of the serine-threonine phosphatase, PP1, by Calyculin A, significantly reduced the RIalpha-RFC40 complex formation, substantiating the in vitro phosphorylation data. Taken together, these findings suggest that CDK2/Cyclin E may function as downstream modulator that regulates the dissociation of the RIalpha-RFC40 complex and subsequently the association of the RFC40-RFC37 complex.

Cyclic AMP regulates the expression and nuclear translocation of RFC40 in MCF7 cells.

We have previously shown that the regulatory subunit of PKA, RIalpha, functions as a nuclear transport protein for the second subunit of the replication factor C complex, RFC40, and that this transport appears to be crucial for cell cycle progression from G1 to S phase. In this study, we found that N(6)-monobutyryl cAMP significantly up-regulates the expression of RFC40 mRNA by 1.8-fold and its endogenous protein by 2.3-fold with a subsequent increase in the RIalpha-RFC40 complex formation by 3.2-fold. Additionally, the nuclear to cytoplasmic ratio of RFC40 increased by 26% followed by a parallel increase in the percentage of S phase cells by 33%. However, there was reduction in the percentage of G1 cells by 16% and G2/M cells by 43% with a concurrent accumulation of cells in S phase. Interestingly, the higher percentage of S phase cells did not correlate with a parallel increase in DNA replication. Moreover, although cAMP did not affect the expression of the other RFC subunits, there was a significant decrease in the RFC40-37 complex formation by 81.3%, substantiating the decrease in DNA replication rate. Taken together, these findings suggest that cAMP functions as an upstream modulator that regulates the expression and nuclear translocation of RFC40.