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Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm-1), proteins (1662 and 1608 cm-1) and fatty acids (2854, 2923, 2956 and 3012 cm-1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.
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Retinopatia Diabética/diagnóstico por imagem , Estresse Oxidativo , Espectroscopia de Infravermelho com Transformada de Fourier , Síncrotrons , Animais , Biomarcadores/análise , Diabetes Mellitus Experimental/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Retina/fisiopatologiaRESUMO
Metformin hydrochloride, an antihyperglycemic agent, and sulindac, a nonsteroidal anti-inflammatory drug, are FDA-approved drugs known to exert anticancer effects. Previous studies demonstrated sulindac and metformin's anticancer properties through mitochondrial dysfunction and inhibition of mitochondrial electron transport chain complex I and key signaling pathways. In this study, various drugs were administered to A549 lung cancer cells, and results revealed that a combination of sulindac and metformin enhanced cell death compared to the administration of the drugs separately. To measure superoxide production over time, we employed a time-lapse fluorescence imaging technique using mitochondrial-targeted hydroethidine. Fluorescence microscopy data showed the most significant increases in superoxide production in the combination treatment of metformin and sulindac. Results showed significant differences between the combined drug treatment and control groups and between the positive control and control groups. This approach can be utilized to quantify the anticancer efficacy of drugs, creating possibilities for additional therapeutic options.
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Neoplasias Pulmonares , Metformina , Humanos , Sulindaco/farmacologia , Sulindaco/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Superóxidos , Preparações Farmacêuticas , Imagem com Lapso de Tempo , Linhagem Celular Tumoral , Microscopia de Fluorescência , Metformina/farmacologia , Metformina/uso terapêuticoRESUMO
OBJECTIVE: Metabolic changes have been extensively documented in neurodegenerative brain disorders, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, a disorder that, like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting a broader functional insult arising from reduced MBLAC1 protein expression and one possibly linked to metabolic alterations. METHODS: Our current studies, utilizing Mblac1 knockout (KO) mice, seek to determine whether mitochondrial respiration is affected in the peripheral tissues of these mice. We quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in livers and kidneys of wild-type (WT) mice and their homozygous KO littermates of males and females, using 3D optical cryo-imaging. RESULTS: Compared to WT, the RR of livers from KO mice was significantly reduced, without an apparent sex effect, driven predominantly by significantly lower NADH levels. In contrast, no genotype and sex differences were observed in kidney samples. Serum analyses of WT and KO mice revealed significantly elevated glucose levels in young and aged KO adults and diminished cholesterol levels in the aged KOs, consistent with liver dysfunction. DISCUSSION/CONCLUSION: As seen with C. elegans swip-10 mutants, loss of MBLAC1 protein results in metabolic changes that are not restricted to neural cells and are consistent with the presence of peripheral comorbidities accompanying neurodegenerative disease in cases where MBLAC1 expression changes impact risk.
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Caenorhabditis elegans , Doenças Neurodegenerativas , Animais , Feminino , Humanos , Camundongos , Masculino , Idoso , Camundongos Knockout , Caenorhabditis elegans/genética , Doenças Neurodegenerativas/diagnóstico por imagem , NAD/metabolismo , Neurônios Dopaminérgicos/metabolismo , Imagem ÓpticaRESUMO
Introduction: Obesity is a major risk factor associated with multiple pathological conditions including diabetes and cardiovascular disease. Endothelial dysfunction is an early predictor of obesity. However, little is known regarding how early endothelial changes trigger obesity. In the present work we report a novel endothelial-mediated mechanism essential for regulation of metabolic homeostasis, driven by c-Myc. Methods: We used conditional knockout (EC-Myc KO) and overexpression (EC-Myc OE) mouse models to investigate the endothelial-specific role of c-Myc in metabolic homeostasis during aging and high-fat diet exposure. Body weight and metabolic parameters were collected over time and tissue samples collected at endpoint for biochemical, pathology and RNA-sequencing analysis. Animals exposed to high-fat diet were also evaluated for cardiac dysfunction. Results: In the present study we demonstrate that EC-Myc KO triggers endothelial dysfunction, which precedes progressive increase in body weight during aging, under normal dietary conditions. At endpoint, EC-Myc KO animals showed significant increase in white adipose tissue mass relative to control littermates, which was associated with sex-specific changes in whole body metabolism and increase in systemic leptin. Overexpression of endothelial c-Myc attenuated diet-induced obesity and visceral fat accumulation and prevented the development of glucose intolerance and cardiac dysfunction. Transcriptome analysis of skeletal muscle suggests that the protective effects promoted by endothelial c-Myc overexpression are associated with the expression of genes known to increase weight loss, energy expenditure and glucose tolerance. Conclusion: Our results show a novel important role for endothelial c-Myc in regulating metabolic homeostasis and suggests its potential targeting in preventing obesity and associated complications such as diabetes type-2 and cardiovascular dysfunction.
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Metabolic changes have been extensively documented in brain tissue undergoing neurodegeneration, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, that like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting the possibility of a broader functional insult arising from reduced MBLAC1 protein expression, and one possibly linked to metabolic alterations. Our current studies, utilizing Mblac1 knockout (KO) mice, seeks to determine whether mitochondrial respiration is affected in peripheral tissues of these animals in this model. To initiate these studies, we quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in the livers of wild type (WT) mice and their homozygous KO littermates, using 3D optical cryo-imaging. We found that Mblac1 KO mice exhibited a greater oxidized redox state compared to WT mice. When compared to the WT group, the redox ratio of KO mice was decreased by 46.32%, driven predominantly by significantly lower NADH levels (more oxidized state). We speculate that, as seen with C. elegans swip-10 mutants, that loss of MBLAC1 protein results in deficits in tricarboxylic acid cycle (TCA) production of NADH and FAD TCA that leads to diminished cellular ATP production and oxidative stress. Such observations are consistent with changes that in the central nervous system (CNS) could support neurodegeneration and in the periphery account for comorbidities.
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Doença de Alzheimer , Caenorhabditis elegans , Animais , Humanos , Camundongos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Dopamina/metabolismo , Fígado , Camundongos Knockout , NAD/metabolismo , Imagem ÓpticaRESUMO
Though angiogenesis has been investigated in depth, vascular regression and rarefaction remain poorly understood. Regression of renal vasculature accompanies many pathological states such as diabetes, hypertension, atherosclerosis, and radiotherapy. Radiation decreases microvessel density in multiple organs, though the mechanism is not known. By using a whole animal (rat) model with a single dose of partial body irradiation to the kidney, changes in the volume of renal vasculature were recorded at two time points, 60 and 90 days after exposure. Next, a novel vascular and metabolic imaging (VMI) technique was used to computationally assess 3D vessel diameter, volume, branch depth, and density over multiple levels of branching down to 70 µm. Four groups of rats were studied, of which two groups received a single dose of 12.5 Gy X-rays. The kidneys were harvested after 60 or 90 days from one irradiated and one non-irradiated group at each time point. Measurements of the 3D vasculature showed that by day-90 post-radiation, when renal function is known to deteriorate, total vessel volume, vessel density, maximum branch depth, and the number of terminal points in the kidneys decreased by 55%, 57%, 28%, and 53%, respectively. Decreases in the same parameters were not statistically significant at 60 days post-irradiation. Smaller vessels with internal diameters of 70-450 µm as well as large vessels of diameter 451-850 µm, both decreased by 90 days post-radiation. Vascular regression in the lungs of the same strain of irradiated rats has been reported to occur before 60 days supporting the hypothesis that this process is regulated in an organ-specific manner and occurs by a concurrent decrease in luminal diameters of small as well as large blood vessels.
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Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2's rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.
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Ureter , Endotélio , Epitélio , Morfogênese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ureter/metabolismoRESUMO
Mitochondrial dysfunction contributes to various injuries and diseases. A mechanistic understanding of how dysfunctional mitochondria modulates metabolism is of paramount importance. Three-dimensional (3D) optical cryo-imager is a custom-designed device that can quantify the volumetric bioenergetics of organs in small animal models. The instrument captures the autofluorescence of bioenergetics indices (NADH and FAD) from tissues at cryogenic temperature. The quantified redox ratio (NADH/FAD) is used as an optical indicator of mitochondrial redox state.
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Flavina-Adenina Dinucleotídeo/análise , Imageamento Tridimensional/métodos , Rim/química , Mitocôndrias/química , NAD/análise , Imagem Óptica/métodos , Animais , Criopreservação , Metabolismo Energético , Flavina-Adenina Dinucleotídeo/metabolismo , Secções Congeladas , Rim/metabolismo , Rim/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NAD/metabolismo , OxirreduçãoRESUMO
BACKGROUND: Mitochondrial [Formula: see text]-oxidation of fatty acids is the primary energy source for the heart and carried out by Hydroxy Acyl-CoA Dehydrogenase (HADH) encoded trifunctional protein. Mutations in the genes encoding mitochondrial proteins result in functionally defective protein complexes that contribute to energy deficiencies, excessive reactive oxygen species (ROS) production, and accumulation of damaged mitochondria. We hypothesize that a dramatic alternation in redox state and associated mitochondrial dysfunction is the underlying cause of Fatty Acid Oxidation (FAO) deficiency mutant, resulting in heart failure. Mitochondrial co-enzymes, NADH and FAD, are autofluorescent metabolic indices of cells when imaged, yield a quantitative assessment of the cells' redox status and, in turn, that of the tissue and organ. METHOD: We utilized an optical cryo-imager to quantitively evaluate the three-dimensional distribution of mitochondrial redox state in newborn rats' hearts and kidneys. Redox ratio (RR) assessment shows that mitochondrial dysfunction is extreme and could contribute to severe heart problems and eventual heart failure in the mutants. RESULTS: Three-dimensional redox ratio (NADH/FAD) rendering, and the volumetric mean value calculations confirmed significantly decreased cardiac RR in mutants by 31.90% and 12.32%, in renal mitochondrial RR compared to wild-type control. Further, histological assessment of newborn heart myocardial tissue indicated no significant difference in myocardial tissue architecture in both control and severe (HADHAe4-/-) conditions. CONCLUSION: These results demonstrate that optical imaging can accurately estimate the redox state changes in newborn rat organs. It is also apparent that the FAO mutant's heart tissue with a low redox ratio is probably more vulnerable to cumulative damages than kidneys and fails prematurely, contributing to sudden death.
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Mitocôndrias , Miocárdio , Acil-CoA Desidrogenase/metabolismo , Animais , Animais Recém-Nascidos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Oxirredução , RatosRESUMO
SIGNIFICANCE: Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network. AIM: Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution. APPROACH: By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways. RESULTS: The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the "minimum work" hypothesis of the vascular network fitting to Murray's law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation. CONCLUSIONS: Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.
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Imageamento Tridimensional , NAD , Animais , Flavina-Adenina Dinucleotídeo , Mitocôndrias , Imagem Óptica , RatosRESUMO
BACKGROUND: Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to accelerate diabetic wound healing in preclinical and clinical studies. Mitochondrial dysfunction and oxidative stress play key roles in impaired diabetic wound healing, and the effect of PBM on the metabolic state of diabetic wounds remains to be elucidated. METHODS: In this study, a custom-designed in vivo fluorescence imaging technique was used to quantitatively assess the effect of FR-PBM on the mitochondrial bioenergetics of diabetic wounds. The intrinsic fluorescence of two mitochondrial co-enzymes, nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), was monitored to quantify the redox ratio (RR) (NADH/FAD) of wounds over time. RESULTS: Using an excisional model of wound healing, we demonstrated that 670 nm (FR) PBM improved mitochondrial bioenergetics and stimulated the rate of wound healing in diabetic db/db mice. Wound closure and the RR of diabetic wounds in response to 670 nm PBM (4.5 J/cm2, 60 mW/cm2 for 90 s per day, 5 days/week) were compared to the sham-treated group. At day 9 of post-wounding, we observed a 43% decrease in the wound area and a 75% increase in RR in FR-treated diabetic mice compared to sham-treated diabetic mice. CONCLUSIONS: We conclude that the increase in mitochondrial RR and the related decrease in oxidative stress may be an important factor in FR-PBM mediated acceleration of wound healing in diabetic mice.
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Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom-designed fluorescence cryo-imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap-frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH-oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt-sensitive (SS) rats and SS rats in which NADPH-oxidase isoform 4 (Nox4) was knocked out (SSNox4-/- ). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SSNox4-/- rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SSNox4-/- rats indicates that mechanisms independent of H2 O2 produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.
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Rim , Imagem Óptica , Animais , Rim/diagnóstico por imagem , Rim/cirurgia , Oxirredução , Ratos , Ratos Endogâmicos Dahl , Ratos Sprague-DawleyRESUMO
Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to restore the function of damaged mitochondria, increase the production of cytoprotective factors and prevent cell death. Our laboratory has shown that FR PBM improves functional and structural outcomes in animal models of retinal injury and retinal degenerative disease. The current study tested the hypothesis that a brief course of NIR (830 nm) PBM would preserve mitochondrial metabolic state and attenuate photoreceptor loss in a model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (Quantum Devices, Barneveld, WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained, but not treated with 830 nm light. Retinal metabolic state, function and morphology were assessed at p30 by measurement of mitochondrial redox (NADH/FAD) state by 3D optical cryo-imaging, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and histomorphometry. PBM preserved retinal metabolic state, retinal function, and retinal morphology in PBM-treated animals compared to the sham-treated group. PBM protected against the disruption of the oxidation state of the mitochondrial respiratory chain observed in sham-treated animals. Scotopic ERG responses over a range of flash intensities were significantly greater in PBM-treated rats compared to sham controls. SD-OCT studies and histological assessment showed that PBM preserved the structural integrity of the retina. These findings demonstrate for the first time a direct effect of NIR PBM on retinal mitochondrial redox status in a well-established model of retinal disease. They show that chronic proteotoxic stress disrupts retinal bioenergetics resulting in mitochondrial dysfunction, and retinal degeneration and that therapies normalizing mitochondrial metabolism have considerable potential for the treatment of retinal degenerative disease.
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Metabolismo Energético/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , Mitocôndrias/efeitos da radiação , Degeneração Retiniana/radioterapia , Retinose Pigmentar/radioterapia , Animais , Modelos Animais de Doenças , Eletrorretinografia , Flavina-Adenina Dinucleotídeo/metabolismo , Raios Infravermelhos , Mitocôndrias/metabolismo , NAD/metabolismo , Oxirredução , Ratos , Ratos Transgênicos , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Retinose Pigmentar/diagnóstico por imagem , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Tomografia de Coerência Óptica , Resultado do TratamentoRESUMO
The kidney is one of the most radiosensitive organs; it is the primary dose-limiting organ in radiotherapies for upper abdominal cancers. The role of mitochondrial redox state in the development and treatment of renal radiation injury, however, remains ill-defined. This study utilizes 3D optical cryo-imaging to quantify renal mitochondrial bioenergetics dysfunction after 13 Gy leg-out partial body irradiation (PBI). Furthermore, the mitigating effects of lisinopril (lisino), an anti-hypertensive angiotensin converting enzyme inhibitor, is assessed in renal radiation-induced injuries. Around day 150 post-irradiation, kidneys are harvested for cryo-imaging. The 3D images of the metabolic indices (NADH, nicotinamide adenine dinucleotide, and FAD, flavin adenine dinucleotide) are acquired, and the mitochondrial redox states of the irradiated and irradiated + lisino kidneys are quantified by calculating the volumetric mean redox ratio (NADH/FAD). PBI oxidized renal mitochondrial redox state by 78%. The kidneys from the irradiated + lisino rats showed mitigation of mitochondrial redox state by 93% compared to the PBI group. The study provides evidence for an altered bioenergetics and energy metabolism in the rat model of irradiation-induced kidney damage. In addition, the results suggest that lisinopril mitigates irradiation damage by attenuating the oxidation of mitochondria leading to increase redox ratio.
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Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Nefropatias/tratamento farmacológico , Rim/efeitos da radiação , Lisinopril/uso terapêutico , Mitocôndrias/efeitos da radiação , Lesões por Radiação/tratamento farmacológico , Animais , Feminino , Flavina-Adenina Dinucleotídeo/metabolismo , Raios gama , Imageamento Tridimensional , Rim/metabolismo , Nefropatias/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Lesões por Radiação/metabolismo , RatosRESUMO
Background: Diabetes is known to cause delayed wound healing, and chronic non-healing lower extremity ulcers may end with lower limb amputations and mortalities. Given the increasing prevalence of diabetes mellitus worldwide, it is critical to focus on underlying mechanisms of these debilitating wounds to find novel therapeutic strategies and thereby improve patient outcome. Methods: This study aims to design a label-free optical fluorescence imager that captures metabolic indices (NADH and FAD autofluorescence) and monitors the in vivo wound healing progress noninvasively. Furthermore, 3D optical cryo-imaging of the mitochondrial redox state was utilized to assess the volumetric redox state of the wound tissue. Results: The results from our in vivo fluorescence imager and the 3D cryo-imager quantify the differences between the redox state of wounds on diabetic mice in comparison with the control mice. These metabolic changes are associated with mitochondrial dysfunction and higher oxidative stress in diabetic wounds. A significant correlation was observed between the redox state and the area of the wounds. Conclusion: The results suggest that our developed novel optical imaging system can successfully be used as an optical indicator of the complex wound healing process noninvasively.
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Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time-lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.
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Glucose/farmacologia , Microscopia , Estresse Oxidativo/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Retina/citologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion ( O2â¢- ) production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O2â¢- production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O2â¢- production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O2â¢- . After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O2â¢- fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O2â¢- production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O2â¢- production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O2â¢- production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O2â¢- production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.
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Postural orthostatic tachycardia syndrome (POTS) is a disabling condition characterized by orthostatic intolerance with tachycardia in the absence of drop-in blood pressure. A custom-built near-infrared spectroscopy device (NIRS) is applied to monitor the muscle oxygenation, noninvasively in patients undergoing incremental head-up tilt table (HUT). Subjects (6 POTS patients and 6 healthy controls) underwent 30 mins of 70°on a HUT. The results showed a significant difference in deoxyhemoglobin (Hb), change-in-oxygenation (ΔOxy) and blood volume (ΔBV) between patients and healthy controls. However, oxyhemoglobin (HbO2) showed a significantly faster rate of change in the healthy controls during the first 10 mins of the tilt and during the recovery. This NIRS muscle oximetry tool provides quantitative measurements of blood oxygenation monitoring in diseases such as POTS.
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Tissues with high-energy demand including the heart are rich in the energy-producing organelles, mitochondria, and sensitive to mitochondrial dysfunction. While alterations in mitochondrial function are increasingly recognized in cardiovascular diseases, the molecular mechanisms through which changes in mitochondria lead to heart abnormalities have not been fully elucidated. Here, we report that transgenic mice overexpressing a novel regulator of mitochondrial dynamics, transmembrane protein 135 (Tmem135), exhibit increased fragmentation of mitochondria and disease phenotypes in the heart including collagen accumulation and hypertrophy. The gene expression analysis showed that genes associated with ER stress and unfolded protein response, and especially the pathway involving activating transcription factor 4, are upregulated in the heart of Tmem135 transgenic mice. It also showed that gene expression changes in the heart of Tmem135 transgenic mice significantly overlap with those of aged mice in addition to the similarity in cardiac phenotypes, suggesting that changes in mitochondrial dynamics may be involved in the development of heart abnormalities associated with aging. Our study revealed the pathological consequence of overexpression of Tmem135, and suggested downstream molecular changes that may underlie those disease pathologies.
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Expressão Gênica , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Miocárdio/metabolismo , Animais , Biomarcadores , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/mortalidade , Cardiopatias/patologia , Imuno-Histoquímica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Análise de Sequência de DNARESUMO
Whole thoracic irradiation (WTI) is known to cause deterioration in cardiac function. Whether irradiation predisposes the heart to further ischemia and reperfusion (IR) injury is not well known. The aim of this study is to examine the susceptibility of rat hearts to IR injury following a single fraction of 15 Gy WTI and to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. After day 35 of irradiation, ex vivo hearts from irradiated and nonirradiated rats (controls) were exposed to 25-min global ischemia followed by 60-min IR, or hearts were perfused without IR for the same protocol duration [time controls (TC)]. Online fluorometry of metabolic indices [redox state: reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and NADH/FAD redox ratio] and functional variables [systolic left ventricular pressure (LVP), diastolic LVP (diaLVP), coronary flow (CF), and heart rate were recorded in the beating heart; developed LVP (dLVP) and rate pressure product (RPP)] were derived. At the end of each experimental protocol, hearts were immediately snap frozen in liquid N2 for later three-dimensional imaging of the mitochondrial redox state using optical cryoimaging. Irradiation caused a delay in recovery of dLVP and RPP after IR when compared to nonirradiated hearts but recovered to the same level at the end of reperfusion. CF in the irradiated hearts recovered better than the control hearts after IR injury. Both fluorometry and 3-D cryoimaging showed that in WTI and control hearts, the redox ratio increased during ischemia (reduced) and decreased on reperfusion (oxidized) when compared to their respective TCs; however, there was no significant difference in the redox state between WTI and controls. In conclusion, our results show that although irradiation of rat hearts compromised baseline cardiovascular function, it did not alter cardiac mitochondrial redox state and induce greater susceptibility of these hearts to IR injury.