RESUMO
Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.
Assuntos
Biopterinas/análogos & derivados , Óxido Nítrico Sintase Tipo III/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antioxidantes/metabolismo , Biopterinas/metabolismo , Linhagem Celular , Células Cultivadas , Doxiciclina/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Metotrexato/metabolismo , Camundongos , Células NIH 3T3 , Óxido Nítrico Sintase Tipo III/genética , Oxirredução , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Superóxidos/metabolismo , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
BACKGROUND: Vascular injury results in loss of endothelial nitric oxide (NO), production of reactive oxygen species (ROS), and the initiation of an inflammatory response. Both NO and ROS modulate inflammation through redox-sensitive pathways. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) that regulates enzymatic synthesis of either nitric oxide or ROS. We hypothesized that endothelial BH4 is an important regulator of inflammation and vascular remodeling. METHODS AND RESULTS: Endothelium-targeted overexpression of GTP cyclohydrolase 1 (GCH), the rate limiting enzyme in BH4 synthesis, increased levels of tetrahydrobiopterin (BH4), reduced endothelial superoxide, improved eNOS coupling, and reduced vein graft atherosclerosis in transgenic GCH/ApoE-KO mice compared to ApoE-KO controls. Immunohistochemistry using anti-MAC-3 and MAC-1 antibody staining revealed a marked reduction in vein graft macrophage content, as did RT-PCR expression of macrophage marker CD68 mRNA levels in GCH/ApoE-KO mice. When we investigated the potential mediators of this reduction, we discovered that mRNA and protein levels of MCP-1 (CCL2) but not RANTES (CCL5) were significantly reduced in GCH/ApoE-KO aortic tissue. Consistent with this finding we found a decrease in CCR2-mediated, but not CCR5-mediated, chemotaxis in vascular tissue and plasma samples from GCH/ApoE-KO animals. CONCLUSIONS: Increased endothelial BH4 reduces vein graft neointimal hyperplasia and atherosclerosis through a reduction in vascular inflammation. These findings highlight the importance of MCP-1/CCR2 signaling in the response to vascular injury and identify novel pathways linking endothelial BH4 to inflammation and vascular remodeling.
Assuntos
Aterosclerose/prevenção & controle , Biopterinas/análogos & derivados , Vasos Sanguíneos/lesões , Quimiocina CCL2/metabolismo , Endotélio Vascular/metabolismo , Receptores CCR2/metabolismo , Vasculite/prevenção & controle , Animais , Aorta/metabolismo , Apolipoproteínas E/deficiência , Aterosclerose/etiologia , Biopterinas/metabolismo , Artérias Carótidas/cirurgia , Quimiotaxia , Feminino , GTP Cicloidrolase/metabolismo , Humanos , Hiperplasia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo , Superóxidos/metabolismo , Túnica Íntima/patologia , Regulação para Cima , Vasculite/complicações , Veias Cavas/metabolismo , Veias Cavas/patologia , Veias Cavas/transplante , Ferimentos e Lesões/complicaçõesRESUMO
Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species (ROS) derived from vascular NADPH oxidases in both vascular smooth muscle cells (VSMCs) and endothelial cells. Recent evidence suggests an important role for VSMC NADPH oxidases in vascular ROS production. However, it is unclear whether increased NADPH oxidase activity in endothelial cells alone is sufficient to alter overall vascular ROS production and hemodynamics. We sought to address these questions using transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NADPH oxidase, Nox2. Aortas of Nox2 transgenic (Nox2-Tg) mice had increased total Nox2 mRNA and protein levels compared with wild-type littermates. Both p22phox mRNA and protein levels were also significantly elevated in Nox2-Tg aortas. Aortic superoxide production was significantly increased in Nox2-Tg mice compared with wild-type, but this difference was abolished by endothelial removal. Superoxide dismutase inhibition increased superoxide release and levels of Mn superoxide dismutase protein were significantly elevated in aortas from Nox2-Tg mice compared with wild type. Increased ROS production from endothelial Nox2 overexpression led to increased endothelial nitric oxide synthase protein and extracellular signal-regulated kinase 1/2 phosphorylation in transgenic aortas. Basal blood pressure was similar, however the pressor responses to both acute and chronic angiotensin II administration were significantly increased in Nox2-Tg mice compared with wild type. These results demonstrate that endothelial-targeted Nox2 overexpression is sufficient to increase vascular NADPH oxidase activity, activate downstream signaling pathways, and potentiate the hemodynamic response to angiotensin II, despite compensatory increases in vascular antioxidant enzymes. Endothelial cell Nox2-containing NADPH oxidase plays an important functional role in vascular redox signaling.
Assuntos
Angiotensina II/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Endotélio Vascular/enzimologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Animais , Ativação Enzimática , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredutases/metabolismo , RNA Mensageiro/metabolismo , Superóxidos/metabolismo , Sistema Vasomotor/fisiologiaRESUMO
AIMS: Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species derived from NADPH oxidases. However, it remains unclear whether a primary increase in superoxide production specifically in the endothelium alters the initiation or progression of atherosclerosis. METHODS AND RESULTS: Mice overexpressing Nox2 specifically in the endothelium (Nox2-Tg) were crossed with ApoE(-/-) mice to produce Nox2-Tg ApoE(-/-) mice and ApoE(-/-) littermates. Endothelial overexpression of Nox2 in ApoE(-/-) mice did not alter blood pressure, but significantly increased vascular superoxide production compared with ApoE(-/-) littermates, measured using both lucigenin chemiluminescence and 2-hydroxyethidium production (ApoE(-/-), 19.9 ± 6.3 vs. Nox2-Tg ApoE(-/-), 47.0 ± 7.0 nmol 2-hydroxyethidium/aorta, P< 0.05). Increased endothelial superoxide production increased endothelial levels of vascular cell adhesion protein 1 and enhanced macrophage recruitment in early lesions in the aortic roots of 9-week-old mice, indicating increased atherosclerotic plaque initiation. However, endothelial-specific Nox2 overexpression did not alter native or angiotensin II-driven atherosclerosis in either the aortic root or the descending aorta. CONCLUSION: Endothelial-targeted Nox2 overexpression in ApoE(-/-) mice is sufficient to increase vascular superoxide production and increase macrophage recruitment possible via activation of endothelial cells. However, this initial increase in macrophage recruitment did not alter the progression of atherosclerosis. These results indicate that Nox-mediated reactive oxygen species signalling has important cell-specific and distinct temporal roles in the initiation and progression of atherosclerosis.
Assuntos
Doenças da Aorta/enzimologia , Apolipoproteínas E/deficiência , Aterosclerose/enzimologia , Quimiotaxia , Células Endoteliais/enzimologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Superóxidos/metabolismo , Angiotensina II , Animais , Doenças da Aorta/induzido quimicamente , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Apolipoproteínas E/genética , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Pressão Sanguínea , Modelos Animais de Doenças , Progressão da Doença , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NADPH Oxidase 2 , NADPH Oxidases/genética , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Epigenetic processes play a key regulatory role in cancer. Hypermethylation in the CpG islands of the promoter regions of many tumour suppressor genes leads to the recruitment of co-repressors, altered chromatin structure, and ultimately transcriptional silencing. Key components in the regulation of DNA methylation are DNA methyltransferases (DNMT1, 2, 3A and 3B) and methyl CpG-binding proteins, which recognize methyl cytosine residues and recruit transcriptional repressor complexes, including histone deacetylases (HDAC). DNMT1 is responsible for the maintenance of DNA methylation patterns during replication. Inhibitors of this enzyme may potentially lead to DNA hypomethylation, and re-expression of tumour suppressor genes. Several DNMT inhibitors are currently being evaluated in preclinical and clinical studies, include various analogues of adenosine, cytidine or deoxycytidine. However, such drugs have had limited clinical success, perhaps because of cytotoxicity associated with their incorporation into DNA. Non-nucleoside small molecule inhibitors of DNMTs can directly block DNMT activity, and may be able to circumvent this cytotoxicity. Post-translational modifications of histones play a key role, not only in regulating chromatin structure and gene expression, but also in genomic stability. Histone acetylation (HAT) and histone deacetylation (HDAC) affect chromatin condensation, with concomitant effects on gene transcription. A further range of compounds is being evaluated for clinical use as HDAC inhibitors, including hydroxamic acids such as Trichostatin A (TSA) and Suberoyl anilide bishydroxamide (SAHA). MicroRNAs are also found to play a key role in cancer development, and novel approaches to their regulation may provide a susceptible anticancer drug target. Because of the interdependence of epigenetic processes, combinations of these approaches may have maximum clinical efficacy.
Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Humanos , MicroRNAs/metabolismo , Nucleosídeos/química , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêuticoRESUMO
GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.
Assuntos
Biopterinas/análogos & derivados , Proteínas de Transporte/metabolismo , Células Endoteliais/enzimologia , GTP Cicloidrolase/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Antibacterianos/farmacologia , Biopterinas/biossíntese , Biopterinas/genética , Proteínas de Transporte/genética , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , GTP Cicloidrolase/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células NIH 3T3RESUMO
Tetrahydrobiopterin (BH4) is a critical determinant of endothelial nitric-oxide synthase (eNOS) activity. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxide rather than NO. However, the stoichiometry of intracellular BH4/eNOS interactions is not well defined, and it is unclear whether intracellular BH4 deficiency alone is sufficient to induce eNOS uncoupling. To address these questions, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in BH4 synthesis, to selectively induce intracellular BH4 deficiency by incubation with doxycycline. These cells were stably co-transfected to express a human eNOS-green fluorescent protein fusion protein, selecting clones expressing either low (GCH/eNOS-LOW) or high (GCH/eNOS-HIGH) levels. Doxycycline abolished GTPCH mRNA expression and GTPCH protein, leading to markedly diminished total biopterin levels and a decreased ratio of BH4 to oxidized biopterins in cells expressing eNOS. Intracellular BH4 deficiency induced superoxide generation from eNOS, as assessed by N-nitro-L-arginine methyl ester inhibitable 2-hydroxyethidium generation, and attenuated NO production. Quantitative analysis of cellular BH4 versus superoxide production between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a striking linear relationship between eNOS protein and cellular BH4 stoichiometry, with eNOS uncoupling at eNOS:BH4 molar ratio >1. Furthermore, increasing the intracellular BH2 concentration in the presence of a constant eNOS:BH4 ratio was sufficient to induce eNOS-dependent superoxide production. This specific, reductionist approach in a cell-based system reveals that eNOS:BH4 reaction stoichiometry together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling, even in the absence of exogenous oxidative stress.