RESUMO
Obesity is a major risk factor for liver and cardiovascular diseases. However, obesity-driven mechanisms that contribute to the pathogenesis of multiple organ diseases are still obscure and treatment is inadequate. We hypothesized that increased , glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme in the pentose shunt, is critical in evoking metabolic reprogramming in multiple organs and is a significant contributor to the pathogenesis of liver and cardiovascular diseases. G6PD is induced by a carbohydrate-rich diet and insulin. Long-term (8 months) high-fat diet (HFD) feeding increased body weight and elicited metabolic reprogramming in visceral fat, liver, and aorta, of the wild-type rats. In addition, HFD increased inflammatory chemokines in visceral fat. Interestingly, CRISPR-edited loss-of-function Mediterranean G6PD variant (G6PDS188F) rats, which mimic human polymorphism, moderated HFD-induced weight gain and metabolic reprogramming in visceral fat, liver, and aorta. The G6PDS188F variant prevented HFD-induced CCL7 and adipocyte hypertrophy. Furthermore, the G6PDS188F variant increased Magel2 - a gene encoding circadian clock-related protein that suppresses obesity associated with Prader-Willi syndrome - and reduced HFD-induced non-alcoholic fatty liver. Additionally, the G6PDS188F variant reduced aging-induced aortic stiffening. Our findings suggest G6PD is a regulator of HFD-induced obesity, adipocyte hypertrophy, and fatty liver.
Assuntos
Adipócitos , Dieta Hiperlipídica , Fígado Gorduroso , Glucosefosfato Desidrogenase , Hipertrofia , Obesidade , Animais , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Masculino , Ratos , Obesidade/metabolismo , Obesidade/genética , Obesidade/patologia , Obesidade/etiologia , Dieta Hiperlipídica/efeitos adversos , Adipócitos/metabolismo , Adipócitos/patologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/metabolismo , Fígado/patologia , Ratos Sprague-Dawley , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologiaRESUMO
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.
Assuntos
Metilases de Modificação do DNA , Regulação Enzimológica da Expressão Gênica , Glucosefosfato Desidrogenase , Hipertensão Pulmonar , Animais , Ratos , Metilação de DNA , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Hipertensão Pulmonar/genética , Oxigênio , Hipóxia Celular , Metilases de Modificação do DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Modelos Animais de DoençasRESUMO
Recent advances have revealed the importance of epigenetic modifications to gene regulation and transcriptional activity. DNA methylation, a determinant of genetic imprinting and the de novo silencing of genes genome-wide, is known to be controlled by DNA methyltransferases (DNMT) and demethylases (TET) under disease conditions. However, the mechanism(s)/factor(s) influencing the expression and activity of epigenetic writers and erasers, and thus DNA methylation, in healthy vascular tissue is incompletely understood. Based on our recent studies, we hypothesized that glucose-6-phosphate dehydrogenase (G6PD) is a modifier of DNMT and TET expression and activity and an enabler of gene expression. In the aorta of CRISPR-edited rats with the Mediterranean G6PD variant, we determined DNA methylation by whole-genome bisulfite sequencing, gene expression by RNA sequencing, and large artery stiffness by echocardiography. Here, we documented higher expression of Dnmt1, Dnmt3a, Tet2, and Tet3 in aortas from Mediterranean G6PDS188F variant (a loss-of-function single nucleotide polymorphism) rats than their wild-type littermates. Concomitantly, we identified 17,618 differentially methylated loci genome-wide (5787 hypermethylated loci, including down-regulated genes encoding inflammation- and vasoconstriction-causing proteins, and 11,827 hypomethylated loci, including up-regulated genes encoding smooth muscle cell differentiation- and fatty acid metabolism-promoting proteins) in aortas from G6PDS188F as compared to wild-type rats. Our results demonstrated that nitric oxide, which is generated in a G6PD-derived NADPH-dependent manner, increases TET and decreases DNMT activity. Further, we observed less large artery (aorta) stiffness in G6PDS188F as compared to wild-type rats. These results establish a noncanonical function of the wild-type G6PD and G6PDS188F variant in the regulation of DNA methylation and gene expression in healthy vascular tissue and reveal that the G6PDS188F variant contributes to reducing large artery stiffness.
Assuntos
Metilação de DNA , Glucosefosfato Desidrogenase , Animais , Ratos , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilases de Modificação do DNA/genética , Expressão Gênica , Impressão Genômica , Glucosefosfato Desidrogenase/genéticaRESUMO
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.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Histonas/metabolismo , Síndrome Metabólica/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Hemodinâmica , Humanos , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Camundongos Transgênicos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Mutação , Miócitos de Músculo Liso/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratos Sprague-Dawley , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Remodelação VascularRESUMO
Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP)-a fundamental glucose metabolism pathway-is vital for cell growth. Because treatment of PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg-1] during exposure to hypoxia (Hx + SU). A novel G6PD inhibitor (N-[(3ß,5α)-17-oxoandrostan-3-yl]sulfamide; 1.5 mg kg-1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle pressure-volume relationships and gene expression in lungs of normoxic, Hx, and Hx + SU and G6PD inhibitor-treated mice. RV systolic and end-diastolic pressures were higher in Hx and Hx + SU than normoxic control mice. Hx and Hx + SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, and decreased right ventricle pressures compared with untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx + SU mice and preclinical models of PH and appears to be a safe pharmacotherapeutic strategy. SIGNIFICANCE STATEMENT: The results of this study demonstrated that inhibition of a metabolic enzyme efficaciously reduces pulmonary hypertension. For the first time, this study shows that a novel inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the fundamental pentose phosphate pathway, modulates DNA methylation and alleviates pulmonary artery remodeling and dilates pulmonary artery to reduce pulmonary hypertension.
Assuntos
Cardiomegalia/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glucosefosfato Desidrogenase/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Animais , Pressão Sanguínea , Cardiomegalia/etiologia , Hipóxia Celular , Células Cultivadas , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipertensão Pulmonar/etiologia , Indóis/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Pirróis/toxicidade , Função Ventricular DireitaRESUMO
Pulmonary hypertension (PH) is a multicellular and progressive disease with a high mortality rate. Among many cell types, hematopoietic stem cells (HSCs) are incriminated in the pathogenesis of PH. However, our understanding of the mechanisms that increase HSCs in blood and lungs of hypertensive animals or patients and the role played by HSCs in the pathogenesis of PH remains elusive. Studies suggest that glycolysis is critical for the survival and growth of HSCs. In various cell types from hypertensive lungs of animals and patients, glycolysis and the glucose-6-phosphate dehydrogenase (G6PD) activity are increased. Herein, we demonstrated in mice that chronic hypoxia increased HSCs (CD34+, CD117+, CD133+, CD34+/CD117+, and CD34+/CD133+) in bone marrow and blood and around hypertensive pulmonary arteries in a time-dependent manner. Intriguingly, we found fewer CD133+ cells in the bone marrow of C57BL/6 mice compared with Sv129J mice, and C57BL mice developed less severe chronic hypoxia-elicited PH and heart failure than Sv129J mice. Similarly, the numbers of CD34+ and CD117+ cells in blood of patients with pulmonary arterial hypertension (PAH) were higher (>3-fold) compared with healthy individuals. By allogeneic bone marrow transplantation, we found that GFP+ bone marrow cells infiltrated the lungs and accumulated around the pulmonary arteries in lungs of hypoxic mice, and these cells contributed to increased α-adrenergic receptor-mediated contraction of the pulmonary artery cultured in hypoxia. Inhibition of G6PD activity with (3ß,5α)-3,21-dihydroxypregnan-20-one, a novel and potent G6PD inhibitor, decreased HSCs in bone marrow, blood, and lungs of hypoxic mice and reduced α-agonist-induced contraction of the pulmonary artery and established hypoxia-induced PH. We did not observe CD133+ cells around the pulmonary arteries in the lungs of chronically hypoxic G6PD-deficient mice. Furthermore, knockdown of G6PD and inhibition of G6PD activity: 1) downregulated canonical and noncanonical Wnt and Fzd receptors genes; 2) upregulated Bmpr1a; 3) decreased Cxcl12, and 4) reduced HSC (CD117+ and CD133+) numbers. In all, our findings demonstrate unexpected function for bone marrow-derived HSCs in augmenting α-adrenergic receptor-mediated contraction of pulmonary arteries and remodeling of pulmonary arteries that contribute to increase pulmonary vascular resistance in PAH patients and hypoxic mice and suggest that G6PD, by regulating expression of genes in the WNT and BMPR signaling, contributed to increase and release of HSCs from the bone marrow in response to hypoxic stimuli.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Hipertensão Pulmonar/fisiopatologia , Células-Tronco Pluripotentes/metabolismo , Artéria Pulmonar/fisiopatologia , Receptores Adrenérgicos alfa/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Contagem de Células , Células Cultivadas , Quimiocina CXCL12/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Coração/fisiopatologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hipertensão Pulmonar/etiologia , Hipóxia/sangue , Hipóxia/complicações , Hipóxia/genética , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44-/- mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N'-[(3ß,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 (Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.
Assuntos
Metilação de DNA/genética , Glucosefosfato Desidrogenase/genética , Hipertensão Pulmonar/genética , Hipóxia/genética , Animais , Proliferação de Células/genética , Família 2 do Citocromo P450/genética , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Pulmão/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Artéria Pulmonar/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Artérias/metabolismo , Canais de Cálcio Tipo L/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Potenciais de Ação , Androsterona/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Pressão Sanguínea , Bovinos , Cavéolas/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.
Assuntos
Sistemas CRISPR-Cas , Mapeamento de Epitopos/métodos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Transativadores/análise , Transativadores/metabolismo , Animais , Embrião de Mamíferos , Epitopos/análise , Camundongos , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/químicaRESUMO
Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182⯱â¯3â¯mmHg JCR, 145⯱â¯3â¯mmHg JCRâ¯+â¯20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.
Assuntos
Pressão Sanguínea , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Metaloproteinase 12 da Matriz/metabolismo , Síndrome Metabólica/enzimologia , Síndrome Metabólica/fisiopatologia , Rigidez Vascular , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Complacência (Medida de Distensibilidade) , Citocromo P-450 CYP4A/metabolismo , Família 4 do Citocromo P450/metabolismo , Diástole/efeitos dos fármacos , Elastina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hipertensão/complicações , Losartan/farmacologia , Masculino , Síndrome Metabólica/complicações , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Rigidez Vascular/efeitos dos fármacosRESUMO
Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone, which has the highest serum concentration among steroid hormones with DHEA sulfate (DHEAS). DHEA possesses an inhibitory action on glucose-6-phosphate dehydrogenase (G6PD), the first pentose-phosphate pathway enzyme that reduces NADP+ to NADPH. DHEA induced relaxation of high K+-induced contraction in rat arterial strips, whereas DHEAS barely induced it. We studied the effects of DHEA on L-type Ca2+ current ( ICa,L) of A7r5 arterial smooth muscle cells and compared the mechanism of inhibition with that produced by the 6-aminonicotinamide (6-AN) competitive inhibitor of G6PD. DHEA moderately inhibited ICa,L that was elicited from a holding potential (HP) of -80 mV [voltage-independent inhibition (VIDI)] and accelerated decay of ICa,L during the depolarization pulse [voltage-dependent inhibition (VDI)]. DHEA-induced VDI decreased peak ICa,L at depolarized HPs. By applying repetitive depolarization pulses from multiple HPs, novel HP-dependent steady-state inactivation curves ( f∞-HP) were constructed. DHEA shifted f∞-HP to the left and inhibited the window current, which was recorded at depolarized HPs and obtained as a product of current-voltage relationship and f∞-HP. The IC50 value of ICa,L inhibition was much higher than serum concentration. DHEA-induced VDI was downregulated by the dialysis of guanosine 5'- O-(2-thiodiphosphate), which shifted f∞-voltage to the right before the application of DHEA. 6-AN gradually and irreversibly inhibited ICa,L by VIDI, suggesting that the inhibition of G6PD is involved in DHEA-induced VIDI. In 6-AN-pretreated cells, DHEA induced additional inhibition by increasing VIDI and generating VDI. The inhibition of G6PD underlies DHEA-induced VIDI, and DHEA additionally induces VDI as described for Ca2+ channel blockers. NEW & NOTEWORTHY Dehydroepiandrosterone, the most abundantly released adrenal steroid hormone with dehydroepiandrosterone sulfate, inhibited L-type Ca2+ current and its window current in aortic smooth muscle cells. The IC50 value of inhibition decreased with the depolarization of holding potential to 15 µM at -20 mV. The inhibition occurred in a voltage-dependent manner as described for Ca2+ channel blockers and in a voltage-independent manner because of the inhibition of glucose-6-phosphate dehydrogenase.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Desidroepiandrosterona/farmacologia , Hormônios/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Artérias/citologia , Artérias/metabolismo , Células Cultivadas , Feminino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos WistarRESUMO
We have recently demonstrated that disruption of the murine cytochrome P-450 2c44 gene (Cyp2c44) exacerbates chronic hypoxia-induced pulmonary artery remodeling and hypertension in mice. Subsequently, we serendipitously found that Cyp2c44 gene disruption also increases hematopoietic stem cell (HSC) numbers in bone marrow and blood. Therefore, the objective of the present study was to investigate whether CYP2C44-derived eicosanoids regulate HSC proliferation/cell growth and whether increased HSCs contribute to chronic hypoxia-induced remodeling of pulmonary arteries in Cyp2c44 knockout mice. Our findings demonstrated that lack of CYP2C44 epoxygenase, which catalyzed the oxidation of arachidonic acid to epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic (HETE) acids, increases the numbers of 1) HSCs (CD34+, CD117+, and CD133+), 2) proangiogenic (CD34+CD133+ and CD34+CD117+CD133+) cells, and 3) immunogenic/inflammatory (CD34+CD11b+, CD133+CD11b+, F4/80+, CD11b+, and F4/80+CD11b+) macrophages in bone marrow and blood compared with wild-type mice. Among the various CYP2C44-derived arachidonic acids, only 15-HETE decreased CD117+ cell numbers when applied to bone marrow cell cultures. Interestingly, CD133+ and von Willebrand factor-positive cells, which are derived from proangiogenic stem cells, are increased in the bone marrow, blood, and lungs of mice exposed to chronic hypoxia and in remodeled and occluded pulmonary arteries of CYP2C44-deficient mice. In conclusion, our results demonstrate that CYP2C44-derived 15-HETE plays a critical role in downregulating HSC proliferation and growth, because disruption of the Cyp2c44 gene increased HSCs that potentially contribute to chronic hypoxia-induced pulmonary arterial remodeling and occlusion.NEW & NOTEWORTHY This study demonstrates that cytochrome P-450 2C44 plays a critical role in controlling the phenotype of hematopoietic stem cells and that when this enzyme is knocked out, stem cells are differentiated. These stem cells give rise to increased circulating monocytes and macrophages and contribute to the pathogenesis of chronic hypoxia-induced pulmonary artery remodeling and hypertension.
Assuntos
Proliferação de Células , Família 2 do Citocromo P450/deficiência , Células-Tronco Hematopoéticas/enzimologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensão Pulmonar/enzimologia , Hipóxia/complicações , Artéria Pulmonar/enzimologia , Remodelação Vascular , Antígeno AC133/metabolismo , Animais , Antígenos CD34/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Células Cultivadas , Doença Crônica , Família 2 do Citocromo P450/genética , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Macrófagos/enzimologia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Monócitos/enzimologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transdução de SinaisRESUMO
In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18ß-glycyrrhetinic acid (18ß-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18ß-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18ß-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18ß-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.
Assuntos
Junções Comunicantes/fisiologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Miócitos de Músculo Liso/fisiologia , Vasoconstrição , Animais , Bovinos , Hipóxia Celular , Células Cultivadas , Células Endoteliais , Junções Comunicantes/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Artéria Pulmonar/citologiaRESUMO
Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2 We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 µM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 µM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 µM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L.
Assuntos
Aorta Torácica/efeitos dos fármacos , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/toxicidade , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Rotenona/toxicidade , Superóxidos/metabolismo , Animais , Antioxidantes/farmacologia , Aorta Torácica/enzimologia , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Potenciais da Membrana , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Óxido Nítrico/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Fatores de TempoRESUMO
Heart failure, a major cause of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), is an outcome of complex biochemical processes. In this study, we determined changes in microRNAs (miRs) in the right and left ventricles of normal and PAH rats. Using an unbiased quantitative miR microarray analysis, we found 1) miR-21-5p, miR-31-5 and 3p, miR-140-5 and 3p, miR-208b-3p, miR-221-3p, miR-222-3p, miR-702-3p, and miR-1298 were upregulated (>2-fold; P < 0.05) in the right ventricle (RV) of PAH compared with normal rats; 2) miR-31-5 and 3p, and miR-208b-3p were upregulated (>2-fold; P < 0.05) in the left ventricle plus septum (LV+S) of PAH compared with normal rats; 3) miR-187-5p, miR-208a-3p, and miR-877 were downregulated (>2-fold; P < 0.05) in the RV of PAH compared with normal rats; and 4) no miRs were up- or downregulated with >2-fold in LV+S compared with RV of PAH and normal. Upregulation of miR-140 and miR-31 in the hypertrophic RV was further confirmed by quantitative PCR. Interestingly, compared with control rats, expression of mitofusin-1 (MFN1), a mitochondrial fusion protein that regulates apoptosis, and which is a direct target of miR-140, was reduced in the RV relative to LV+S of PAH rats. We found a correlation between increased miR-140 and decreased MFN1 expression in the hypertrophic RV. Our results also demonstrated that upregulation of miR-140 and downregulation of MFN1 correlated with increased RV systolic pressure and hypertrophy. These results suggest that miR-140 and MFN1 play a role in the pathogenesis of PAH-associated RV dysfunction.
Assuntos
Ventrículos do Coração/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertrofia Ventricular Direita/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Mitocondriais/metabolismo , Disfunção Ventricular Direita/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Potencial da Membrana Mitocondrial , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 µM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide.
Assuntos
Angiotensina II/farmacologia , Vasos Coronários/efeitos dos fármacos , Ferroquelatase/metabolismo , Heme/metabolismo , Mitocôndrias/efeitos dos fármacos , Guanilil Ciclase Solúvel/metabolismo , Superóxidos/metabolismo , Animais , Bovinos , Vasos Coronários/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ativadores de Enzimas/farmacologia , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/enzimologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Organoides , Superóxido Dismutase/metabolismo , Técnicas de Cultura de Tecidos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inflamação/prevenção & controle , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Bovinos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocromo P-450 CYP4A/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genótipo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Inflamação/enzimologia , Inflamação/genética , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Mutantes , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias Musculares/enzimologia , Músculo Liso Vascular/enzimologia , Mutação , Miócitos de Músculo Liso/enzimologia , Fenótipo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
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.
Assuntos
Aorta/metabolismo , Proteínas Contráteis/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/efeitos dos fármacos , Western Blotting , Bovinos , Cromatografia Líquida , Proteínas Contráteis/efeitos dos fármacos , Proteínas Contráteis/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Técnicas de Silenciamento de Genes , Glucosefosfato Desidrogenase/genética , Imunoprecipitação , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Ratos , Fator de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Espectrometria de Massas em Tandem , Transativadores/efeitos dos fármacos , Transativadores/genética , Transativadores/metabolismoRESUMO
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.
Assuntos
Glucosefosfato Desidrogenase/genética , Hipertensão Pulmonar/enzimologia , Miócitos de Músculo Liso/enzimologia , Artéria Pulmonar/patologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular , Proliferação de Células , Indução Enzimática , Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Ratos , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/metabolismo , Regulação para CimaRESUMO
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.