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1.
Arterioscler Thromb Vasc Biol ; 41(2): 638-650, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267664

RESUMO

OBJECTIVE: Small GTPase Rap1 (Ras-association proximate 1) is a novel, positive regulator of NO release and endothelial function with a potentially key role in mechanosensing of atheroprotective, laminar flow. Our objective was to delineate the role of Rap1 in the progression of atherosclerosis and its specific functions in the presence and absence of laminar flow, to better define its role in endothelial mechanisms contributing to plaque formation and atherogenesis. Approach and Results: In a mouse atherosclerosis model, endothelial Rap1B deletion exacerbates atherosclerotic plaque formation. In the thoracic aorta, where laminar shear stress-induced NO is otherwise atheroprotective, plaque area is increased in Athero-Rap1BiΔEC (atherogenic endothelial cell-specific, tamoxifen-inducible Rap1A+Rap1B knockout) mice. Endothelial Rap1 deficiency also leads to increased plaque size, leukocyte accumulation, and increased CAM (cell adhesion molecule) expression in atheroprone areas, whereas vascular permeability is unchanged. In endothelial cells, in the absence of protective laminar flow, Rap1 deficiency leads to an increased proinflammatory TNF-α (tumor necrosis factor alpha) signaling and increased NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and elevated inflammatory receptor expression. Interestingly, this increased signaling to NF-κB activation is corrected by AKTVIII-an inhibitor of Akt (protein kinase B) translocation to the membrane. Together, these data implicate Rap1 in restricting Akt-dependent signaling, preventing excessive cytokine receptor signaling and proinflammatory NF-κB activation. CONCLUSIONS: Via 2 distinct mechanisms, endothelial Rap1 protects from the atherosclerosis progression in the presence and absence of laminar flow; Rap1-stimulated NO release predominates in laminar flow, and restriction of proinflammatory signaling predominates in the absence of laminar flow. Our studies provide novel insights into the mechanisms underlying endothelial homeostasis and reveal the importance of Rap1 signaling in cardiovascular disease.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/prevenção & controle , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Leucócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Placa Aterosclerótica , Transdução de Sinais , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
2.
Blood ; 129(21): 2917-2927, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28336528

RESUMO

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.


Assuntos
Plaquetas/imunologia , Antígenos CD36/imunologia , Hiperlipidemias/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 7 Ativada por Mitógeno/imunologia , Ativação Plaquetária/imunologia , Trombose/imunologia , Aloenxertos , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Plaquetas/patologia , Transplante de Medula Óssea , Antígenos CD36/genética , Humanos , Hiperlipidemias/genética , Hiperlipidemias/patologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Mutantes , Proteína Quinase 7 Ativada por Mitógeno/genética , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Ativação Plaquetária/genética , Trombose/genética , Trombose/patologia
3.
Chem Rev ; 117(15): 10043-10120, 2017 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-28654243

RESUMO

Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.


Assuntos
Antineoplásicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/tratamento farmacológico , Compostos Organofosforados/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Compostos Organofosforados/síntese química , Compostos Organofosforados/química
4.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845710

RESUMO

In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria.


Assuntos
Brônquios/química , Cromatos/efeitos adversos , Fígado/química , Mitocôndrias/química , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fígado/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Ratos , Xantina Desidrogenase/metabolismo
5.
Dev Neurosci ; 40(5-6): 534-546, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31163416

RESUMO

BACKGROUND: Chorioamnionitis from ascending bacterial infection through the endocervix is a potential risk factor for cerebral palsy. Tetrahydrobiopterin, an essential cofactor for nitric oxide synthase (NOS) and amino acid hydroxylases, when augmented in the fetal brain, prevents some of the cerebral palsy-like deficits in a rabbit hypoxia-ischemia model. OBJECTIVES: To study the effect of lipopolysaccharide (LPS)-induced intrauterine inflammation in preterm gestation on motor deficits in the newborn, and whether biosynthesis of tetrahydrobiopterin or inflammatory mediators is affected in the fetal brain. METHODS: Pregnant rabbits at 28 days gestation (89% term) were administered either saline or LPS into both endocervical openings. One group underwent spontaneous delivery, and neurobehavioral tests were performed at postnatal day (P) 1 and P11, with some kits being sacrificed at P1 for histological analysis. Another group underwent Cesarean section 24 h after LPS administration. Gene sequences for rabbit biosynthetic enzymes of tetra-hydrobiopterin pathways were determined and analyzed in addition to cytokines, using quantitative real-time polymerase chain reaction. RESULTS: Exposure to 200 µg/kg/mL LPS caused a locomotion deficit and mild hypertonia at P1. By P11, most animals turned into normal-appearing kits. There was no difference in neuronal cell death in the caudate between hypertonic and nonhypertonic kits at P1 (n = 3-5 in each group). Fetal brain GTP cyclohydrolase I was increased, whereas sepiapterin reductase and 6-pyruvoyltetrahydropterin synthase were decreased, 24 h after LPS administration. Neuronal NOS was also increased. Regardless of the position in the uterus or the brain region, expression of TNF-α and TGF-ß was decreased, whereas that of IL-1ß, IL-6, and IL-8 was increased (n = 3-4 in each group). CONCLUSIONS: This is the first study using an ascending LPS-induced intrauterine inflammation model in rabbits, showing mostly transient hypertonia and mainly locomotor deficits in the kits. Not all proinflammatory cytokines are increased in the fetal brain following LPS administration. Changes in key tetrahydro-biopterin biosynthetic enzymes possibly indicate different effects of the inflammatory insult.

6.
EMBO Rep ; 16(5): 628-37, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25807985

RESUMO

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.


Assuntos
Endotélio/metabolismo , Mecanotransdução Celular , Óxido Nítrico/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Pressão Sanguínea , Permeabilidade Capilar/genética , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Óxido Nítrico Sintase Tipo III/metabolismo , Especificidade de Órgãos/genética , Transdução de Sinais , Vasodilatação/genética , Proteínas rap1 de Ligação ao GTP/genética
7.
J Neurochem ; 132(4): 394-402, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25421613

RESUMO

We hypothesized that a deficiency in the descending serotonergic input to spinal cord may underlie postnatal muscle hypertonia after global antenatal hypoxic-ischemic injury in a rabbit model of cerebral palsy. Neurotransmitter content was determined by HPLC in the spinal cord of newborns with and without muscle hypertonia after fetal global hypoxic-ischemic brain injury and naïve controls. Contrary to our hypothesis, serotonin levels in both cervical and lumbar expansions and norepinephrine in cervical expansion were increased in hypertonic kits relative to non-hypertonic kits and controls, with unchanged number of serotonergic cells in caudal raphe by stereological count. Serotonergic fiber length per unit of volume was also increased in hypertonic kits' cervical and lumbar spinal cord, both in dorsal and ventral horns. Gene expression of serotonin transporter was increased and 5-HTR2 receptors were decreased in hypertonic kits relative to controls in cervical and lumbar cord. Intrathecal administration of non-selective serotonin receptor inhibitor methysergide decreased muscle tone in hypertonic kits only. Conversely, intrathecal administration of serotonin solution increased muscle tone only in non-hypertonic kits. We speculate that maturation of serotonergic system in spinal cord may be directly affected by decreased corticospinal connectivity after antenatal hypoxic-ischemic brain injury. Following prenatal hypoxia-ischemia, newborn rabbits exhibit elevated levels of serotonin in the spinal cord that were linked to muscle hypertonia. Serotonergic terminal density was also increased in hypertonic newborns' spinal cord. Intrathecal administration of the non-selective serotonin receptor inhibitor methysergide decreased muscle tone in hypertonic newborns only. Elevated spinal serotonin thus suggests a novel pathophysiological mechanism of hypertonia in cerebral palsy.


Assuntos
Paralisia Cerebral/metabolismo , Modelos Animais de Doenças , Hipóxia-Isquemia Encefálica/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/metabolismo , Medula Espinal/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Paralisia Cerebral/etiologia , Feminino , Hipóxia-Isquemia Encefálica/complicações , Dados de Sequência Molecular , Gravidez , Coelhos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
8.
Appl Magn Reson ; 46(8): 885-895, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26441482

RESUMO

This study validated the use of small unilamellar vesicles (SUVs) made of 1-palmitoyl-2-oleoylphosphatidylcholine with 1 mol% spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC) as an oxygen sensitive analyte to study cellular respiration. In the analyte the hydrocarbon environment surrounds the nitroxide moiety of 16-PC. This ensures high oxygen concentration and oxygen diffusion at the location of the nitroxide as well as isolation of the nitroxide moiety from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. The saturation-recovery EPR approach was applied in the analysis since this approach is the most direct method to carry out oximetric studies. It was shown that this display (spin-lattice relaxation rate) is linear in oxygen partial pressure up to 100% air (159 mmHg). Experiments using a neuronal cell line in suspension were carried out at X-band for closed chamber geometry. Oxygen consumption rates showed a linear dependence on the number of cells. Other significant benefits of the analyte are: the fast effective rotational diffusion and slow translational diffusion of the spin-probe is favorable for the measurements, and there is no cross reactivity between oxygen and paramagnetic ions in the lipid bilayer.

9.
Chem Res Toxicol ; 27(7): 1155-65, 2014 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-24890552

RESUMO

Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPPMPO, bearing a mitochondria-targeting triphenylphosphonium cationic moiety or guanidinium cationic group. All of the spin traps prepared have been observed to efficiently trap superoxide radical anions in a cell-free system. The superoxide spin adducts exhibited similar spectral properties, indicating no significant differences in the geometry of the cyclic nitroxide moieties of the spin adducts. The superoxide adduct stability was measured and observed to be highest (t1/2 = 73 min) for DIPPMPO nitrone linked to triphenylphosphonium moiety via a short carbon chain (Mito-DIPPMPO). The experimental results and DFT quantum chemical calculations indicate that the cationic property of the triphenylphosphonium group may be responsible for increased superoxide trapping efficiency and adduct stability of Mito-DIPPMPO, as compared to the DIPPMPO spin trap. The studies of uptake of the synthesized traps into isolated mitochondria indicated the importance of both cationic and lipophilic properties, with the DEPMPO nitrone linked to the triphenylphosphonium moiety via a long carbon chain (Mito10-DEPMPO) exhibiting the highest mitochondrial uptake. We conclude that, of the synthesized traps, Mito-DIPPMPO and Mito10-DEPMPO are the best candidates for potential mitochondria-specific spin traps for use in biologically relevant systems.


Assuntos
Óxidos N-Cíclicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Organofosfonatos/metabolismo , Pirróis/metabolismo , Superóxidos/metabolismo , Animais , Óxidos N-Cíclicos/química , Organofosfonatos/química , Pirróis/química , Ratos , Detecção de Spin , Superóxidos/química
10.
J Neurosci ; 32(16): 5500-9, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22514312

RESUMO

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4-72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.


Assuntos
Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Transtornos dos Movimentos/diagnóstico , Transtornos dos Movimentos/etiologia , Traumatismo por Reperfusão/complicações , Fatores Etários , Animais , Animais Recém-Nascidos , Antioxidantes/uso terapêutico , Ácido Ascórbico/metabolismo , Benzimidazóis , Velocidade do Fluxo Sanguíneo , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Mapeamento Encefálico , Carbocianinas , Cromanos/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Citometria de Fluxo , Hipóxia-Isquemia Encefálica , Ionóforos/farmacologia , Fluxometria por Laser-Doppler , Potencial da Membrana Mitocondrial/fisiologia , Metaloporfirinas/uso terapêutico , Microvasos/fisiopatologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Transtornos dos Movimentos/tratamento farmacológico , Hipertonia Muscular/etiologia , Antígenos O/metabolismo , Gravidez , Coelhos , Traumatismo por Reperfusão/prevenção & controle , Superóxidos/metabolismo , Fatores de Tempo , Valinomicina/farmacologia
11.
J Biol Chem ; 287(26): 22174-82, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22544750

RESUMO

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.


Assuntos
DNA Mitocondrial/genética , Complicações do Diabetes/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Cardiopatias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Ecocardiografia/métodos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Variação Genética , Teste de Tolerância a Glucose , Cardiopatias/complicações , Masculino , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Mutação , Miocárdio/patologia , Ratos , Ratos Wistar
13.
Antioxid Redox Signal ; 37(1-3): 171-183, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34806400

RESUMO

Significance: Tetrahydrobiopterin (BH4) is most well known as a required cofactor for enzymes regulating cellular redox homeostasis, aromatic amino acid metabolism, and neurotransmitter synthesis. Less well known are the effects dependent on the cofactor's availability, factors governing its synthesis and recycling, redox implications of the cofactor itself, and protein-protein interactions that underlie cell death. This review provides an understanding of the recent advances implicating BH4 in the mechanisms of cell death and suggestions of possible therapeutic interventions. Recent Advances: The levels of BH4 often reflect the sum of synthetic and recycling enzyme activities. Enhanced expression of GTP cyclohydrolase, the rate-limiting enzyme in biosynthesis, increases BH4, leading to improved cell function and survival. Pharmacologically increasing BH4 levels has similar beneficial effects, leading to enhanced production of neurotransmitters and nitric oxide or reducing oxidant levels. The GTP cyclohydrolase-BH4 pairing has been implicated in a type of cell death, ferroptosis. At the cellular level, BH4 counteracts anticancer therapies directed to enhance ferroptosis via glutathione peroxidase 4 (GPX4) activity inhibition. Critical Issues: Because of the multitude of intertwined mechanisms, a clear relationship between BH4 and cell death is not well understood yet. The possibility that the cofactor directly influences cell viability has not been excluded in previous studies when modulating BH4-producing enzymes. Future Directions: The importance of cellular BH4 variations and BH4 biosynthetic enzymes to cell function and viability makes it essential to better characterize temporal changes, cofactor activity, and the influence on redox status, which in turn would help develop novel therapies. Antioxid. Redox Signal. 37, 171-183.


Assuntos
Biopterinas , GTP Cicloidrolase , Biopterinas/análogos & derivados , Biopterinas/metabolismo , GTP Cicloidrolase/genética , Óxido Nítrico/metabolismo , Oxirredução
14.
Dev Neurosci ; 33(3-4): 312-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21659718

RESUMO

Cerebral palsy and death are serious consequences of perinatal hypoxia-ischemia (HI). Important concepts can now be tested using an animal model of cerebral palsy. We have previously shown that reactive oxygen and nitrogen species are produced in antenatal HI. A novel class of neuronal nitric oxide synthase (nNOS) inhibitors have been designed, and they ameliorate postnatal motor deficits when administered prior to the hypoxic-ischemic insult. This study asks how the new class of inhibitors, using JI-8 (K(i) for nNOS: 0.014 µM) as a representative, compare with the frequently used nNOS inhibitor 7-nitroindazole (7-NI; K(i): 0.09 ± 0.024 µM). A theoretical dose equivalent to 75 K(i) of JI-8 or equimolar 7-NI was administered to pregnant rabbit dams 30 min prior to and immediately after 40 min of uterine ischemia at 22 days gestation (70% term). JI-8 treatment resulted in a significant decrease in NOS activity (39%) in fetal brain homogenates acutely after HI, without affecting maternal blood pressure and heart rate. JI-8 treatment resulted in 33 normal kits, 2 moderately and 13 severely affected kits and 5 stillbirths, compared with 8 normal, 3 moderately affected and 5 severely affected kits and 10 stillbirths in the 7-NI group. In terms of neurobehavioral outcome, 7-NI was not different from saline treatment, while JI-8 was superior to saline and 7-NI in its protective effect (p < 0.05). In the surviving kits, JI-8 significantly improved the locomotion score over both saline and 7-NI scores. JI-8 was also significantly superior to saline in preserving smell, muscle tone and righting reflex function, but 7-NI did not show significant improvement. Furthermore, a 100-fold increase in the dose (15.75 µmol/kg) of 7-NI significantly decreased systolic blood pressure in the dam, while JI-8 did not. The new class of inhibitors such as JI-8 shows promise in the prevention of cerebral palsy and is superior to the previously more commonly used nNOS inhibitor.


Assuntos
Paralisia Cerebral/etiologia , Paralisia Cerebral/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Feto/fisiopatologia , Hipóxia-Isquemia Encefálica/complicações , Indazóis/uso terapêutico , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Paralisia Cerebral/fisiopatologia , Inibidores Enzimáticos/farmacologia , Feminino , Feto/efeitos dos fármacos , Humanos , Hipóxia-Isquemia Encefálica/fisiopatologia , Indazóis/farmacologia , Testes Neuropsicológicos , Gravidez , Coelhos
15.
Front Cell Dev Biol ; 9: 687598, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222255

RESUMO

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis.

17.
Am J Physiol Heart Circ Physiol ; 299(1): H88-96, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20418482

RESUMO

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for tetrahydrobiopterin (BH(4)) synthesis. Decreases in GTPCH activity and expression have been shown in late stages of acute cardiac rejection, suggesting a deficit in BH(4). We hypothesized that increasing intracellular levels of BH(4) by cardiac myocyte-targeted overexpression of GTPCH would diminish acute cardiac allograft rejection. Transgenic mice overexpressing GTPCH in the heart were generated and crossed on C57BL6 background. Wild-type and transgenic mouse donor hearts were transplanted into BALB/c recipient mice. Left ventricular (LV) function, histological rejection, BH(4) levels, and inflammatory cytokine gene expression (mRNA) were examined. Expression of human GTPCH was documented by PCR, Western analysis, and function by a significant (P < 0.001) increase in cardiac BH(4) levels. GTPCH transgene decreased histological rejection (46%; P < 0.003) and cardiac myocyte injury (eosin autofluorescence; 56%; P < 0.0001) independent of changes in inflammatory cytokine expression or nitric oxide content. GTPCH transgene decreased IL-2 (88%; P < 0.002), IL-1R2 (42%; P < 0.0001), and programmed cell death-1 (67%; P < 0.0001) expression, whereas it increased fms-like tyrosine kinase 3 (156%; P < 0.0001) and stromal-derived factor-1 (2; 190%; P < 0.0001) expression. There was no difference in ejection fraction or fractional shortening; however, LV mass was significantly increased (P < 0.05) only in wild-type grafts. The decreases in LV mass, cardiac injury, and histological rejection support a protective role of cardiac GTPCH overexpression and increased BH(4) synthesis in cardiac allografts. The mechanism of the decreased rejection appears related to decreased T cell proliferation and modulation of immune function by higher expression of genes involved in hematopoietic/stromal cell development and recruitment.


Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/metabolismo , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Miócitos Cardíacos/enzimologia , Doença Aguda , Animais , Biopterinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , GTP Cicloidrolase/genética , Genótipo , Rejeição de Enxerto/diagnóstico por imagem , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/fisiopatologia , Transplante de Coração/efeitos adversos , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Óxido Nítrico/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Transplante Homólogo , Ultrassonografia , Função Ventricular Esquerda
18.
Ann Neurol ; 66(3): 323-31, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19798726

RESUMO

OBJECTIVE: Tetrahydrobiopterin (BH(4)) deficiency is a cause of dystonia at birth. We hypothesized that BH(4) is a developmental factor determining vulnerability of the immature fetal brain to hypoxic-ischemic injury and subsequent motor deficits in newborns. METHODS: Pregnant rabbits were subjected to 40-minute uterine ischemia, and fetal brains were investigated for global and focal changes in BH(4). Newborn kits were assessed by neurobehavioral tests following vehicle and sepiapterin (BH(4) analog) treatment of dams. RESULTS: Naive fetal brains at 70% gestation (E22) were severely deficient for BH(4) compared with maternal and other fetal tissues. BH(4) concentration rapidly increased normally in the perinatal period, with the highest concentrations found in the thalamus compared with basal ganglia, frontal, occipital, hippocampus, and parietal cortex. Global sustained 40-minute hypoxia-ischemia depleted BH(4) in E22 thalamus and to a lesser extent in basal ganglia, but not in the frontal, occipital, and parietal regions. Maternal supplementation prior to hypoxia-ischemia with sepiapterin increased BH(4) in all brain regions and especially in the thalamus, but did not increase the intermediary metabolite, 7,8-BH(2). Sepiapterin treatment also reduced incidence of severe motor deficits and perinatal death following E22 hypoxia-ischemia. INTERPRETATION: We conclude that early developmental BH(4) deficiency plays a critical role in hypoxic-ischemic brain injury. Increasing brain BH(4) via maternal supplementation may be an effective strategy in preventing motor deficits from antenatal hypoxia-ischemia.


Assuntos
Biopterinas/análogos & derivados , Encéfalo/embriologia , Encéfalo/metabolismo , Distonia/prevenção & controle , Desenvolvimento Fetal/efeitos dos fármacos , Hipóxia Fetal/metabolismo , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/metabolismo , Hipertonia Muscular/prevenção & controle , Pterinas/uso terapêutico , Animais , Animais Recém-Nascidos , Biopterinas/análise , Biopterinas/deficiência , Biopterinas/uso terapêutico , Química Encefálica/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal/fisiologia , Hipóxia Fetal/tratamento farmacológico , Idade Gestacional , Humanos , Troca Materno-Fetal/efeitos dos fármacos , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/uso terapêutico , Gravidez , Pterinas/farmacologia , Coelhos
19.
Ann Neurol ; 65(2): 209-17, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19235180

RESUMO

OBJECTIVE: To design a new class of selective neuronal nitric oxide synthase (NOS) inhibitors, and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxia-ischemia-induced deaths and reduces the number of newborn kits exhibiting signs of CP. METHODS: We used a novel computer-based drug design method called fragment hopping to identify new chemical entities, synthesized them, and conducted in vitro enzyme inhibition studies with the three isozymes of NOS and in vivo experiments to monitor cardiovascular effects on pregnant rabbit dams, NOS activity, and NO(x) (NO and NO(2)) concentration in fetal brain, and assess neurobehavioral effects on kits born to saline- and compound treated dams. RESULTS: The computer-based design led to the development of powerful and highly selective compounds for inhibition of neuronal NOS over the other isozymes. After maternal administration in a rabbit model of CP, these compounds were found to distribute to fetal brain, to be nontoxic, without cardiovascular effects, inhibit fetal brain NOS activity in vivo, reduce NO concentration in fetal brain, and dramatically ameliorate deaths and number of newborn kits exhibiting signs of CP. INTERPRETATION: This approach may lead to new preventive strategies for CP.


Assuntos
Paralisia Cerebral/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Arginina/metabolismo , Comportamento Animal/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Paralisia Cerebral/metabolismo , Paralisia Cerebral/patologia , Paralisia Cerebral/fisiopatologia , Citrulina/metabolismo , Cristalografia por Raios X/métodos , Modelos Animais de Doenças , Desenho de Fármacos , Inibidores Enzimáticos/química , Feminino , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Gravidez , Coelhos , Relação Estrutura-Atividade
20.
Arterioscler Thromb Vasc Biol ; 29(12): 2161-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19762783

RESUMO

OBJECTIVE: The posttranslational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites. METHODS AND RESULTS: Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167, and threonine (T) 231 in HEK293 cells, whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51, S72, T85, T91, T103, S130, S167 and T231). GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity. CONCLUSIONS: Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103, and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.


Assuntos
GTP Cicloidrolase/química , GTP Cicloidrolase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Linhagem Celular , Núcleo Celular/enzimologia , GTP Cicloidrolase/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
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