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1.
Physiol Rep ; 11(24): e15866, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38114067

RESUMEN

Human bone mesenchymal stem cell-derived extracellular vesicles (HBMSC-EV) have been used successfully in animal models of myocardial ischemia, yet have dampened effects in metabolic syndrome through unknown mechanisms. This study demonstrates the basal differences between non-diabetic human coronary artery endothelial cells (HCAEC) and diabetic HCAEC (DM-HCAEC), and how these cells respond to the treatment of HBMSC-EV. HCAEC and DM-HCAEC were treated with HBMSC-EV for 6 h. Proteomics, western blot analysis, and tube formation assays were performed. Key metabolic, growth, and stress/starvation cellular responses were significantly altered in DM-HCAEC in comparison to that of HCAEC at baseline. Proteomics demonstrated increased phosphorus metabolic process and immune pathways and decreased RNA processing and biosynthetic pathways in DM-HCAEC. Similar to previous in vivo findings, HCAEC responded to the HBMSC-EV with regenerative and anti-inflammatory effects through the upregulation of multiple RNA pathways and downregulation of immune cell activation pathways. In contrast, DM-HCAEC had a significantly diminished response to HBMSC-EV, likely due to the baseline abnormalities in DM-HCAEC. To achieve the full benefits of HBMSC-EV and for a successful transition of this potential therapeutic agent to clinical studies, the abnormalities found in DM-HCAEC will need to be further studied.


Asunto(s)
Diabetes Mellitus , Vesículas Extracelulares , Células Madre Mesenquimatosas , Animales , Humanos , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Células Cultivadas , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Diabetes Mellitus/metabolismo
2.
Int J Mol Sci ; 24(2)2023 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-36675188

RESUMEN

Human bone marrow mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) are known for their regenerative and anti-inflammatory effects in animal models of myocardial ischemia. However, it is not known whether the efficacy of the EVs can be modulated by pre-conditioning of HBMSC by exposing them to either starvation or hypoxia prior to EV collection. HBMSC-EVs were isolated following normoxia starvation (NS), normoxia non-starvation (NNS), hypoxia starvation (HS), or hypoxia non-starvation (HNS) pre-conditioning. The HBMSC-EVs were characterized by nanoparticle tracking analysis, electron microscopy, Western blot, and proteomic analysis. Comparative proteomic profiling revealed that starvation pre-conditioning led to a smaller variety of proteins expressed, with the associated lesser effect of normoxia versus hypoxia pre-conditioning. In the absence of starvation, normoxia and hypoxia pre-conditioning led to disparate HBMSC-EV proteomic profiles. HNS HBMSC-EV was found to have the greatest variety of proteins overall, with 74 unique proteins, the greatest number of redox proteins, and pathway analysis suggestive of improved angiogenic properties. Future HBMSC-EV studies in the treatment of cardiovascular disease may achieve the most therapeutic benefits from hypoxia non-starved pre-conditioned HBMSC. This study was limited by the lack of functional and animal models of cardiovascular disease and transcriptomic studies.


Asunto(s)
Enfermedades Cardiovasculares , Vesículas Extracelulares , Células Madre Mesenquimatosas , Animales , Humanos , Enfermedades Cardiovasculares/metabolismo , Proteómica , Vesículas Extracelulares/metabolismo , Hipoxia/metabolismo , Células Madre Mesenquimatosas/metabolismo
3.
Basic Res Cardiol ; 118(1): 3, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36639609

RESUMEN

Recent studies demonstrated that mitochondrial antioxidant MnSOD that reduces mitochondrial (mito) reactive oxygen species (ROS) helps maintain an optimal balance between sub-cellular ROS levels in coronary vascular endothelial cells (ECs). However, it is not known whether EC-specific mito-ROS modulation provides resilience to coronary ECs after a non-reperfused acute myocardial infarction (MI). This study examined whether a reduction in endothelium-specific mito-ROS improves the survival and proliferation of coronary ECs in vivo. We generated a novel conditional binary transgenic animal model that overexpresses (OE) mitochondrial antioxidant MnSOD in an EC-specific manner (MnSOD-OE). EC-specific MnSOD-OE was validated in heart sections and mouse heart ECs (MHECs). Mitosox and mito-roGFP assays demonstrated that MnSOD-OE resulted in a 50% reduction in mito-ROS in MHEC. Control and MnSOD-OE mice were subject to non-reperfusion MI surgery, echocardiography, and heart harvest. In post-MI hearts, MnSOD-OE promoted EC proliferation (by 2.4 ± 0.9 fold) and coronary angiogenesis (by 3.4 ± 0.9 fold), reduced myocardial infarct size (by 27%), and improved left ventricle ejection fraction (by 16%) and fractional shortening (by 20%). Interestingly, proteomic and Western blot analyses demonstrated upregulation in mitochondrial complex I and oxidative phosphorylation (OXPHOS) proteins in MnSOD-OE MHECs. These MHECs also showed increased mitochondrial oxygen consumption rate (OCR) and membrane potential. These findings suggest that mito-ROS reduction in EC improves coronary angiogenesis and cardiac function in non-reperfused MI, which are associated with increased activation of OXPHOS in EC-mitochondria. Activation of an energy-efficient mechanism in EC may be a novel mechanism to confer resilience to coronary EC during MI.


Asunto(s)
Infarto del Miocardio , Fosforilación Oxidativa , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/metabolismo , Células Endoteliales/metabolismo , Proteómica , Infarto del Miocardio/metabolismo , Mitocondrias/metabolismo , Endotelio/metabolismo
4.
J Thorac Cardiovasc Surg ; 166(1): e5-e14, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-36244819

RESUMEN

OBJECTIVE: Our recent studies using a porcine model of metabolic syndrome (MS) and chronic myocardial ischemia show that extracellular vesicle (EV) therapy improves blood flow and arteriogenesis in ischemic myocardium, although mechanisms of these changes are unclear. We hypothesized that in the setting of MS, EV therapy would decrease antiangiogenic signaling to mediate increased blood flow to chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-fat diet for 4 weeks to induce MS, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs underwent intramyocardial injection of vehicle (control, n = 6) or human bone marrow-derived EVs (n = 8). Five weeks later, left ventricular myocardium in ischemic territory was harvested. Protein expression was measured using immunoblot analysis, and data were analyzed using Wilcoxon rank sum test. Myocardial perfusion was measured with isotope-labeled microspheres, and correlation data were analyzed using Spearman rank correlation coefficient. RESULTS: EV treatment was associated with decreased expression of antiangiogenic proteins, angiostatin (P < .001) and endostatin (P = .043) in ischemic myocardium compared with control. In EV-treated pigs, there was a negative correlation between blood flow to ischemic myocardium and angiostatin (rs = -0.76; P = .037), but not endostatin expression (rs = .02; P = .98). EV treatment was also associated with decreased cathepsin D, which cleaves precursors to produce angiostatin and endostatin, in ischemic myocardium (P = .020). CONCLUSIONS: In the setting of MS and chronic myocardial ischemia, EV therapy is associated with decreased expression of antiangiogenic proteins, which might contribute to increased blood flow to chronically ischemic myocardium.


Asunto(s)
Vesículas Extracelulares , Síndrome Metabólico , Isquemia Miocárdica , Porcinos , Humanos , Animales , Síndrome Metabólico/metabolismo , Angiostatinas/metabolismo , Modelos Animales de Enfermedad , Isquemia Miocárdica/complicaciones , Miocardio/metabolismo , Vesículas Extracelulares/metabolismo , Circulación Coronaria
5.
J Thorac Cardiovasc Surg ; 165(5): e225-e236, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36028364

RESUMEN

OBJECTIVE: Extracellular vesicle (EV) therapy has been shown to mitigate inflammation in animal models of acute myocardial ischemia/reperfusion. This study evaluates the effect of EV therapy on inflammatory signaling in a porcine model of chronic myocardial ischemia and metabolic syndrome. METHODS: Yorkshire swine were fed a high-cholesterol diet for 4 weeks to induce metabolic syndrome, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs received intramyocardial injection of either saline (control) (n = 6) or EVs (n = 8). Five weeks later, pigs were put to death and left ventricular myocardial tissue in ischemic and nonischemic territories were harvested. Protein expression was measured with immunoblotting, and macrophage count was determined by immunofluorescent staining of cluster of differentiation 68. Data were statistically analyzed via Wilcoxon rank-sum test. RESULTS: EV treatment was associated with decreased expression of proinflammatory markers nuclear factor kappa B (P = .002), pro-interleukin (IL) 1ß (P = .020), and cluster of differentiation 11c (P = .001) in ischemic myocardium, and decreased expression of nuclear factor kappa B in nonischemic myocardium (P = .03) compared with control. EV treatment was associated with increased expression of anti-inflammatory markers IL-10 (P = .020) and cluster of differentiation 163 (P = .043) in ischemic myocardium compared with control. There were no significant differences in expression of IL-6, tumor necrosis factor alpha, arginase, HLA class II histocompatibility antigen DR alpha chain, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, or phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in ischemic myocardium or pro-IL1ß, IL-6, tumor necrosis factor alpha, IL-10, or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in nonischemic myocardium of EV-treated pigs compared with control. There were no differences in macrophage count in ischemic myocardium between EV-treated pigs and control. CONCLUSIONS: In the setting of metabolic syndrome and chronic myocardial ischemia, intramyocardial EV therapy attenuates proinflammatory signaling.


Asunto(s)
Vesículas Extracelulares , Síndrome Metabólico , Isquemia Miocárdica , Porcinos , Animales , Interleucina-10 , FN-kappa B/metabolismo , Síndrome Metabólico/terapia , Síndrome Metabólico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/patología , Vesículas Extracelulares/metabolismo , Modelos Animales de Enfermedad
6.
J Thorac Cardiovasc Surg ; 164(6): e371-e384, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-34756431

RESUMEN

OBJECTIVE: The burden of mortality and morbidity of cardiovascular disease is in part due to substantial fibrosis accelerated by coexisting risk factors. This study aims to evaluate the effect of extracellular vesicle therapy on diastolic function and myocardial fibrosis in the setting of chronic myocardial ischemia with and without a high-fat diet. METHODS: Forty male Yorkshire swine were administered a normal or high-fat diet. At 11 weeks of age, they underwent placement of an ameroid constrictor on their left circumflex coronary artery. Both dietary groups then received either intramyocardial injection of vehicle saline as controls or extracellular vesicles as treatment into the ischemic territory (normal diet control, n = 8; high-fat diet controls, n = 11) or extracellular vesicles (normal diet extracellular vesicles, n = 9; high-fat diet extracellular vesicles, n = 12). Five weeks later, hemodynamic parameters, histology, and selected protein expression were evaluated. RESULTS: Extracellular vesicles reduced end-diastolic pressure volume relationship (P = .002), perivascular collagen density (P = .031), calcium mineralization (P = .026), and cardiomyocyte diameter (P < .0001), and upregulated osteopontin (P = .0046) and mechanistic target of rapamycin (P = .021). An interaction between extracellular vesicles and diet was observed in the vimentin area (P = .044) and fraction of myofibroblast markers to total vimentin (P = .049). Significant changes across diet were found with reductions in muscle fiber area (P = .026), tumor necrosis factor α (P = .0002), NADPH oxidase 2 and 4 (P = .0036, P = .008), superoxide dismutase 1 (P = .034), and phosphorylated glycogen synthase kinase 3ß (P = .020). CONCLUSIONS: Extracellular vesicle therapy improved the myocardium's ability to relax and is likely due to structural improvements at the extracellular matrix and cellular levels.


Asunto(s)
Vesículas Extracelulares , Isquemia Miocárdica , Masculino , Porcinos , Animales , Dieta Alta en Grasa/efectos adversos , Vimentina/metabolismo , Vimentina/farmacología , Circulación Coronaria , Modelos Animales de Enfermedad , Isquemia Miocárdica/complicaciones , Vesículas Extracelulares/metabolismo , Fibrosis , Miocardio/patología
7.
Am J Physiol Heart Circ Physiol ; 321(5): H839-H849, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34506225

RESUMEN

Yorkshire swine were fed standard diet (n = 7) or standard diet containing applesauce rich in caffeic acid with Lactobacillus plantarum (n = 7) for 3 wk. An ameroid constrictor was next placed around the left coronary circumflex artery, and the dietary regimens were continued. At 14 wk, cardiac function, myocardial perfusion, vascular density, and molecular signaling in ischemic myocardium were evaluated. The L. plantarum-applesauce augmented NF-E2-related factor 2 (Nrf2) in the ischemic myocardium and induced Nrf2-regulated antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase quinone 1 (NQO-1), and thioredoxin reductase (TRXR-1). Improved left ventricular diastolic function and decreased myocardial collagen expression were seen in animals receiving the L. plantarum-applesauce supplements. The expression of endothelial nitric oxide synthase (eNOS) was increased in ischemic myocardial tissue of the treatment group, whereas levels of asymmetric dimethyl arginine (ADMA), hypoxia inducible factor 1α (HIF-1α), and phosphorylated MAPK (pMAPK) were decreased. Collateral-dependent myocardial perfusion was unaffected, whereas arteriolar and capillary densities were reduced as determined by α-smooth muscle cell actin and CD31 immunofluorescence in ischemic myocardial tissue. Dietary supplementation with L. plantarum-applesauce is a safe and effective method of enhancing Nrf2-mediated antioxidant signaling cascade in ischemic myocardium. Although this experimental diet was associated with a reduction in hypoxic stimuli, decreased vascular density, and without any change in collateral-dependent perfusion, the net effect of an increase in antioxidant activity and eNOS expression resulted in improvement in diastolic function.NEW & NOTEWORTHY Colonization of the gut microbiome with certain strains of L. Plantarum has been shown to convert caffeic acid readily available in applesauce to 4-vinyl-catechol, a potent activator of the Nrf2 antioxidant defense pathway. In this exciting study, we show that simple dietary supplementation with L. Plantarum-applesauce-mediated Nrf2 activation supports vascular function, ameliorates myocardial ischemic diastolic dysfunction, and upregulates expression of eNOS.


Asunto(s)
Lactobacillus plantarum/metabolismo , Isquemia Miocárdica/terapia , Miocardio/enzimología , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Probióticos , Disfunción Ventricular Izquierda/terapia , Función Ventricular Izquierda , Alimentación Animal , Animales , Circulación Coronaria , Diástole , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Femenino , Fibrosis , Hemo-Oxigenasa 1/metabolismo , Masculino , Densidad Microvascular , Isquemia Miocárdica/enzimología , Isquemia Miocárdica/microbiología , Isquemia Miocárdica/fisiopatología , Miocardio/patología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Recuperación de la Función , Transducción de Señal , Sus scrofa , Tiorredoxinas/metabolismo , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/microbiología , Disfunción Ventricular Izquierda/fisiopatología
8.
STAR Protoc ; 2(3): 100753, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34458871

RESUMEN

Reactive oxygen species (ROS) are implicated in endothelial dysfunction and cardiovascular disease. Endothelial cells (ECs) produce most ATP through glycolysis rather than oxidative phosphorylation; thus mitochondrial ROS production is lower than in other cell types. This makes quantification of changes in EC mitochondrial oxidative status challenging. Here, we present an optimized protocol using mitochondrial-targeted adenovirus-based redox sensor for ratiometric quantification of specific changes in mitochondrial ROS in live human coronary artery EC. For complete details on the use and execution of this protocol, please refer to Waypa et al. (2010); Liao et al. (2020); Gao et al. (2021).


Asunto(s)
Vasos Coronarios/citología , Células Endoteliales/citología , Proteínas Fluorescentes Verdes/genética , Mitocondrias/metabolismo , Biología Molecular/métodos , Adenoviridae/genética , Células Cultivadas , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mitocondrias/genética , Biología Molecular/instrumentación , Especies Reactivas de Oxígeno/metabolismo , Transducción Genética
9.
Am J Physiol Heart Circ Physiol ; 320(5): H1999-H2010, 2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33861149

RESUMEN

Cardiovascular disease (CVD) is the leading cause of death globally. Current treatment options include lifestyle changes, medication, and surgical intervention. However, many patients are unsuitable candidates for surgeries due to comorbidities, diffuse coronary artery disease, or advanced stages of heart failure. The search for new treatment options has recently transitioned from cell-based therapies to stem-cell-derived extracellular vesicles (EVs). A number of challenges remain in the EV field, including the effect of comorbidities, characterization, and delivery. However, recent revolutionary developments and insight into the potential of personalizing EV contents by bioengineering methods to alter specific signaling pathways in the ischemic myocardium hold promise. Here, we discuss the past limitations of cell-based therapies and recent EV studies involving in vivo, in vitro, and omics, and future challenges and opportunities in EV-based treatments in CVD.


Asunto(s)
Vesículas Extracelulares/metabolismo , Insuficiencia Cardíaca/metabolismo , Células Madre Mesenquimatosas/metabolismo , Isquemia Miocárdica/metabolismo , Animales , Humanos , Miocitos Cardíacos/metabolismo
10.
Int J Mol Sci ; 22(7)2021 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-33918396

RESUMEN

Cardiovascular diseases continue to be the leading cause of death worldwide, with ischemic heart disease as the most significant contributor. Pharmacological and surgical interventions have improved clinical outcomes, but are unable to ameliorate advanced stages of end-heart failure. Successful preclinical studies of new therapeutic modalities aimed at revascularization have shown short lasting to no effects in the clinical practice. This lack of success may be attributed to current challenges in patient selection, endpoint measurements, comorbidities, and delivery systems. Although challenges remain, the field of therapeutic angiogenesis is evolving, as novel strategies and bioengineering approaches emerge to optimize delivery and efficacy. Here, we describe the structure, vascularization, and regulation of the vascular system with particular attention to the endothelium. We proceed to discuss preclinical and clinical findings and present challenges and future prospects in the field.


Asunto(s)
Isquemia Miocárdica/terapia , Neovascularización Fisiológica , Animales , Ensayos Clínicos como Asunto , Humanos
11.
J Am Heart Assoc ; 10(4): e017437, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33559477

RESUMEN

Background Mesenchymal stem cell-derived extracellular vesicles (EVs) promote angiogenesis in the ischemic myocardium. This study examines the difference in vascular density, myocardial perfusion, molecular signaling, and gene expression between normal diet (ND) and high fat diet (HFD) groups at baseline and following intramyocardial injection of EVs. Methods and Results Intact male Yorkshire swine fed either an ND (n=17) or HFD (n=14) underwent placement of an ameroid constrictor on the left circumflex coronary artery. Subsequently, animals received either intramyocardial injection of vehicle-saline as controls; (ND-controls n=7, HFD-controls, n=6) or EVs; (ND-EVs n=10, HFD-EVs n=8) into the ischemic territory. Five weeks later, myocardial function, perfusion, vascular density, cell signaling, and gene expression were examined. EVs improved indices of myocardial contractile function, myocardial perfusion, and arteriogenesis in both dietary cohorts. Interestingly, quantification of alpha smooth muscle actin demonstrated higher basal arteriolar density in HFD swine compared with their ND counterparts; whereas EVs were associated with increased CD31-labeled endothelial cell density only in the ND tissue, which approached significance. Levels of total endothelial nitric oxide synthase, FOXO1 (forkhead box protein O1) , transforming growth factor-ß, phosphorylated VEGFR2 (vascular endothelial growth factor receptor 2), and phosphorylated MAPK ERK1/ERK2 (mitogen-activated protein kinase) were higher in ischemic myocardial lysates from ND-controls compared with HFD-controls. Conversely, HFD-control tissue showed increased expression of phosphorylated endothelial nitric oxide synthase, phosphorylated FOXO1, VEGFR2, and MAPK ERK1/ERK2 with respect to ND-controls. Preliminary gene expression studies indicate differential modulation of transcriptional activity by EVs between the 2 dietary cohorts. Conclusions HFD produces a profound metabolic disorder that dysregulates the molecular mechanisms of collateral vessel formation in the ischemic myocardium, which may hinder the therapeutic angiogenic effects of EVs.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Circulación Coronaria/fisiología , Vasos Coronarios/diagnóstico por imagen , Dieta Alta en Grasa/efectos adversos , Vesículas Extracelulares/patología , Isquemia Miocárdica/etiología , Miocardio/metabolismo , Animales , Enfermedad Crónica , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Masculino , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/metabolismo , Miocardio/patología , Neovascularización Patológica/diagnóstico , Neovascularización Patológica/etiología , Neovascularización Patológica/metabolismo , Fosforilación , Porcinos
12.
FEBS Open Bio ; 11(1): 35-47, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33179452

RESUMEN

Cardiovascular disease has been associated with increased levels of reactive oxygen species (ROS). Recently, we have shown that a critical balance between cytosolic ROS and mitochondrial ROS is crucial in cardiovascular health and that modulation of mitochondrial ROS helps prevent detrimental effects of cytosolic ROS on endothelial cells (EC) in transgenic animals. Here, we report the development of a controlled delivery system for a mitochondria-targeted antioxidant, JP4-039, from an electrospun scaffold made of FDA-approved biocompatible polymeric nanofibers. We demonstrate that the active antioxidant moiety was preserved in released JP4-039 for over 72 h using electron paramagnetic resonance. We also show that both the initial burst release of the drug within the first 20 min and the ensuing slow and sustained release that occurred over the next 24 h improved tube formation in human coronary artery ECs (HCAEC) in vitro. Taken together, these findings suggest that electrospinning methods can be used to upload mitochondrial antioxidant (JP4-039) onto a biocompatible nanofibrous PLGA scaffold, and the uploaded drug (JP4-039) retains nitroxide antioxidant properties upon release from the scaffold, which in turn can reduce mitochondrial ROS and improve EC function in vitro.


Asunto(s)
Antioxidantes/administración & dosificación , Portadores de Fármacos/química , Nanofibras/química , Óxidos de Nitrógeno/administración & dosificación , Antioxidantes/farmacocinética , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/patología , Línea Celular , Vasos Coronarios/citología , Vasos Coronarios/patología , Liberación de Fármacos , Células Endoteliales , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxidos de Nitrógeno/farmacocinética , Especies Reactivas de Oxígeno/metabolismo
13.
Front Chem ; 8: 592688, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33330380

RESUMEN

Cardiovascular disease (CVD) has been the leading cause of death for many decades, highlighting the importance of new research and treatments in the field. The role of hypoxia and subsequent free radical production [reactive oxygen species (ROS)] have become an area of particular interest in CVD. Interestingly, our laboratory and other laboratories have recently reported positive roles of subcellular ROS in modulating endothelial cell (EC) metabolism, proliferation, and angiogenesis. This bidirectional relationship between ROS and EC metabolism, as well as functional changes, continues to be an area of active research. Interestingly, ECs have been shown to rely on anaerobic processes for ATP generation, despite their direct access to oxygen. This paradox has proven to be beneficial as the major reliance on glycolysis produces ATP faster, preserves oxygen, and results in reduced ROS levels in contrast to oxidative phosphorylation. This review will address the relationship between ROS and carbohydrate, lipid, and nitrogen metabolism in ECs, and their effects on EC phenotype such as sprouting angiogenesis.

14.
mBio ; 10(6)2019 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-31719185

RESUMEN

Macrolide antibiotics bind to 23S rRNA within the peptide exit tunnel of the ribosome, causing the translating ribosome to stall when an appropriately positioned macrolide arrest motif is encountered in the nascent polypeptide. Tylosin is a macrolide antibiotic produced by Streptomyces fradiae Resistance to tylosin in S. fradiae is conferred by methylation of 23S rRNA by TlrD and RlmAII Here, we demonstrate that yxjB encodes RlmAII in Bacillus subtilis and that YxjB-specific methylation of 23S rRNA in the peptide exit tunnel confers tylosin resistance. Growth in the presence of subinhibitory concentrations of tylosin results in increased rRNA methylation and increased resistance. In the absence of tylosin, yxjB expression is repressed by transcription attenuation and translation attenuation mechanisms. Tylosin-dependent induction of yxjB expression relieves these two repression mechanisms. Induction requires tylosin-dependent ribosome stalling at an RYR arrest motif at the C terminus of a leader peptide encoded upstream of yxjB Furthermore, NusG-dependent RNA polymerase pausing between the leader peptide and yxjB coding sequences is essential for tylosin-dependent induction. Pausing synchronizes the position of RNA polymerase with ribosome position such that the stalled ribosome prevents transcription termination and formation of an RNA structure that sequesters the yxjB ribosome binding site. On the basis of our results, we are renaming yxjB as tlrBIMPORTANCE Antibiotic resistance is a growing health concern. Resistance mechanisms have evolved that provide bacteria with a growth advantage in their natural habitat such as the soil. We determined that B. subtilis, a Gram-positive soil organism, has a mechanism of resistance to tylosin, a macrolide antibiotic commonly used in the meat industry. Tylosin induces expression of yxjB, which encodes an enzyme that methylates 23S rRNA. YxjB-dependent methylation of 23S rRNA confers tylosin resistance. NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling induce yxjB expression, and hence tylosin resistance, by preventing transcription termination upstream of the yxjB coding sequence and by preventing repression of yxjB translation.


Asunto(s)
Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Factores de Elongación de Péptidos/metabolismo , ARN Ribosómico 23S/genética , Ribosomas/metabolismo , Tilosina/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Metilación , Regiones Promotoras Genéticas , Unión Proteica , ARN Ribosómico 23S/química , Transcripción Genética/efectos de los fármacos
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