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1.
Genomics ; 113(5): 3128-3140, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34245829

RESUMO

The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increases in oxygen (O2) with the first breaths, resulting in functional DA closure, with anatomic closure occurring within the first days of life. Failure of DA closure results in persistent patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA's response to O2, though modulated by the endothelium, is intrinsic to the DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondrial-derived reactive oxygen species, which increase in proportion to arterial partial pressure of oxygen (PaO2). The resulting redox changes inhibit voltage-gated potassium channels (Kv) leading to cell depolarization, calcium influx and DASMC constriction. To date, there has not been an unbiased assessment of the human DA O2-sensors using transcriptomics, nor are there known molecular mechanisms which characterize DA closure. DASMCs were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorting using CD90 and CD31 to eliminate fibroblasts or endothelial cells, respectively. The purity of the DASMC population was confirmed by positive staining for α-smooth muscle actin, smoothelin B and caldesmon. Cells were grown for 96 h in hypoxia (2.5% O2) or normoxia (19% O2) and confocal imaging with Cal-520 was used to determine oxygen responsiveness. An oxygen-induced increase in intracellular calcium of 18.1% ± 4.4% and SMC constriction (-27% ± 1.5% shortening) occurred in all cell lines within five minutes. RNA sequencing of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p < 0.05). We examined these genes using Gene Ontology (GO). This unbiased assessment of altered gene expression indicated significant enrichment of the following GOterms: mitochondria, cellular respiration and transcription. The top regulated biologic process was generation of precursor metabolites and energy. The top regulated cellular component was mitochondrial matrix. The top regulated molecular function was transcription coactivator activity. Multiple members of the NADH-ubiquinone oxidoreductase (NDUF) family are upregulated in human DASMC (hDASMC) following normoxia. Several of our differentially regulated transcripts are encoded by genes that have been associated with genetic syndromes that have an increased incidence of PDA (Crebb binding protein and Histone Acetyltransferase P300). This first examination of the effects of O2 on human DA transcriptomics supports a putative role for mitochondria as oxygen sensors.


Assuntos
Permeabilidade do Canal Arterial , Canal Arterial , Nascimento Prematuro , Canal Arterial/metabolismo , Permeabilidade do Canal Arterial/etiologia , Permeabilidade do Canal Arterial/metabolismo , Células Endoteliais/metabolismo , Humanos , Recém-Nascido , Mitocôndrias/genética , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Nascimento Prematuro/metabolismo , Transcriptoma , Vasoconstrição/fisiologia
2.
Circ Res ; 124(12): 1727-1746, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-30922174

RESUMO

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H2O2) ([H2O2]mito). Decreases in [H2O2]mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca2+]i) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O2 sensor is unknown. OBJECTIVE: To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I's rotenone binding site, in pulmonary vascular oxygen-sensing. METHODS AND RESULTS: Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H2O2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H2O2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca2+]i. Hypoxia had no effects on [Ca2+]i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H2O2 in PASMC while increasing cytosolic H2O2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H2O2, prevented hypoxic increases in [Ca2+]i, and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD+) ratio and decreasing expression of the O2-sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III's Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca2+]i. In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. CONCLUSIONS: Ndufs2 is essential for oxygen-sensing and HPV.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Hipóxia/metabolismo , NADH Desidrogenase/metabolismo , Oxigênio/metabolismo , Resistência Vascular/fisiologia , Vasoconstrição/fisiologia , Animais , Células Cultivadas , Hipóxia/patologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Técnicas de Cultura de Órgãos , Oxigênio/análise , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley
3.
Arterioscler Thromb Vasc Biol ; 40(11): 2605-2618, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32998516

RESUMO

OBJECTIVE: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2-the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2EC-/-). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFß (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2EC+/-) and caused excessive angiogenesis in both vascular beds for Bmpr2EC-/- mice. CONCLUSIONS: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/deficiência , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Adulto , Idoso , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Estudos de Casos e Controles , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Fator 2 de Diferenciação de Crescimento/toxicidade , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Transdução de Sinais , Adulto Jovem
4.
Int J Mol Sci ; 19(9)2018 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-30213070

RESUMO

Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (-0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (-3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (-2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (-2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation.


Assuntos
Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Transcriptoma/genética , Disfunção Ventricular Direita/genética , Disfunção Ventricular Direita/fisiopatologia , Animais , Anexina A1/genética , Anexina A1/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Masculino , Camundongos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Trombospondinas/genética , Trombospondinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Pflugers Arch ; 468(1): 43-58, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26395471

RESUMO

The mammalian homeostatic oxygen sensing system (HOSS) initiates changes in vascular tone, respiration, and neurosecretion that optimize oxygen uptake and tissue oxygen delivery within seconds of detecting altered environmental or arterial PO2. The HOSS includes carotid body type 1 cells, adrenomedullary cells, neuroepithelial bodies, and smooth muscle cells (SMCs) in pulmonary arteries (PAs), ductus arteriosus (DA), and fetoplacental arteries. Hypoxic pulmonary vasoconstriction (HPV) optimizes ventilation-perfusion matching. In utero, HPV diverts placentally oxygenated blood from the non-ventilated lung through the DA. At birth, increased alveolar and arterial oxygen tension dilates the pulmonary vasculature and constricts the DA, respectively, thereby transitioning the newborn to an air-breathing organism. Though modulated by endothelial-derived relaxing and constricting factors, O2 sensing is intrinsic to PASMCs and DASMCs. Within the SMC's dynamic mitochondrial network, changes in PO2 alter the reduction-oxidation state of redox couples (NAD(+)/NADH, NADP(+)/NADPH) and the production of reactive oxygen species, ROS (e.g., H2O2), by complexes I and III of the electron transport chain (ETC). ROS and redox couples regulate ion channels, transporters, and enzymes, changing intracellular calcium [Ca(2+)]i and calcium sensitivity and eliciting homeostatic responses to hypoxia. In PASMCs, hypoxia inhibits ROS production and reduces redox couples, thereby inhibiting O2-sensitive voltage-gated potassium (Kv) channels, depolarizing the plasma membrane, activating voltage-gated calcium channels (CaL), increasing [Ca(2+)]i, and causing vasoconstriction. In DASMCs, elevated PO2 causes mitochondrial fission, increasing ETC complex I activity and ROS production. The DASMC's downstream response to elevated PO2 (Kv channel inhibition, CaL activation, increased [Ca(2+)]i, and rho kinase activation) is similar to the PASMC's hypoxic response. Impaired O2 sensing contributes to human diseases, including pulmonary arterial hypertension and patent DA.


Assuntos
Canal Arterial/metabolismo , Pulmão/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Canais de Cálcio/metabolismo , Humanos , Pulmão/irrigação sanguínea , Oxigênio/sangue , Canais de Potássio/metabolismo
7.
Biochem J ; 455(2): 157-67, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23924350

RESUMO

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.


Assuntos
Volume Cardíaco/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Animais , Dano ao DNA , DNA Mitocondrial/metabolismo , Metabolismo Energético , Predisposição Genética para Doença , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
8.
J Clin Invest ; 134(11)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38573824

RESUMO

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP affects risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2-knockout (Tet2-/-) and floxed control mice (Tet2fl/fl) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that, while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake, and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.


Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Imunidade Inata , Neutrófilos , Streptococcus pneumoniae , Animais , Feminino , Humanos , Masculino , Camundongos , Dioxigenases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Camundongos Knockout , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/microbiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Streptococcus pneumoniae/imunologia
9.
Trends Endocrinol Metab ; 34(9): 554-570, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37414716

RESUMO

Mitochondria operate as hubs of cellular metabolism that execute important regulatory functions. Damaged/dysfunctional mitochondria are recognized as major pathogenic contributors to many common human diseases. Assessment of mitochondrial function relies upon invasive tissue biopsies; peripheral blood cells, specifically platelets, have emerged as an ideal candidate for mitochondrial function assessment. Accessibility and documented pathology-related dysfunction have prompted investigation into the role of platelets in disease, the contribution of platelet mitochondria to pathophysiology, and the capacity of platelets to reflect systemic mitochondrial health. Platelet mitochondrial bioenergetics are being investigated in neurodegenerative and cardiopulmonary diseases, infection, diabetes, and other (patho)physiological states such as aging and pregnancy. Early findings support the use of platelets as a biomarker for mitochondrial functional health.


Assuntos
Metabolismo Energético , Mitocôndrias , Humanos , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Plaquetas/metabolismo , Biomarcadores , Biópsia Líquida
10.
Endocrinol Diabetes Metab ; 6(1): e385, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36300606

RESUMO

INTRODUCTION: Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is the most common inherited metabolic disorder of ß-oxidation. Patients with MCADD present with hypoketotic hypoglycemia, which may quickly progress to lethargy, coma, and death. Prognosis for MCADD patients is highly promising once a diagnosis has been established, though management strategies may vary depending on the severity of illness and the presence of comorbidities. METHODS AND RESULTS: Given the rapid developments in the world of gene therapy and implementation of newborn screening for inherited metabolic disorders, the provision of concise and contemporary knowledge of MCADD is essential for clinicians to effectively manage patients. Thus, this review aims to consolidate current information for physicians on the pathogenesis, diagnostic tools, and treatment options for MCADD patients. CONCLUSION: MCADD is a commonly inherited metabolic disease with serious implications for health outcomes, particularly in children, that may be successfully managed with proper intervention.


Assuntos
Hipoglicemia , Erros Inatos do Metabolismo Lipídico , Recém-Nascido , Criança , Humanos , Acil-CoA Desidrogenase/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/terapia , Erros Inatos do Metabolismo Lipídico/complicações , Triagem Neonatal/efeitos adversos , Hipoglicemia/diagnóstico , Hipoglicemia/etiologia , Hipoglicemia/terapia
11.
J Am Heart Assoc ; 12(13): e029131, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37345832

RESUMO

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells' mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion to oxygen tension, causing vasoconstriction by regulating redox-sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2-induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2-induced constriction (P=0.0034) without reducing phenylephrine-induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC-I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2-induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2-induced ROS generation (P=0.02). In vivo, S1QEL prevented O2-induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2-induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2-dependent changes in electron leak at site IQ in ETC-I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency.


Assuntos
Canal Arterial , Animais , Humanos , Coelhos , Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Transporte de Elétrons , Cálcio/metabolismo , Elétrons , Rotenona/metabolismo , Rotenona/farmacologia , Microtomografia por Raio-X
12.
Data Brief ; 40: 107736, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35005134

RESUMO

The Ductus Arteriosus (DA) is a fetal vessel that connects the aorta to the pulmonary artery ensuring that placental oxygenated blood is diverted from the lungs to the systemic circulation. Following exposure to oxygen (O2), in the first few days of life, the DA responds with a functional closure that is followed by anatomical closure. Here, we study human DA smooth muscle cells (DASMC) taken from 10 term infants during congenital heart surgery. Purification of these cells using flow cytometry ensured a pure population of DASMCs, which we confirmed as responsive to O2. An oxygen-induced increase in intracellular calcium of 18.1%±4.4% and SMC constriction (-27%±1.5% shortening) occurred in all cell lines within five minutes. These cells were maintained in either hypoxia (2.5% O2), mimicking in utero conditions or in normoxia (19% O2) mimicking neonate conditions. We then used 3' RNAsequencing to identify the transcriptome of DASMCs in each condition [1]. In this paper, we present the full differentially regulated gene list from this experiment.

13.
Redox Biol ; 49: 102225, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34959099

RESUMO

BACKGROUND: Neutrophils play a role in innate immunity and are critical for clearance of Staphylococcus aureus. Current understanding of neutrophil bactericidal effects is that NADPH oxidase produces reactive oxygen species (ROS), mediating bacterial killing. Neutrophils also contain numerous mitochondria; since these organelles lack oxidative metabolism, their function is unclear. We hypothesize that mitochondria in human neutrophils contribute to the bactericidal capacity of S. aureus. METHODS: and Findings: Using human neutrophils isolated from healthy volunteers (n = 13; 7 females, 6 males), we show that mitochondria are critical in the immune response to S. aureus. Using live-cell and fixed confocal, and transmission electron microscopy, we show mitochondrial tagging of bacteria prior to ingestion and surrounding of phagocytosed bacteria immediately upon engulfment. Further, we demonstrate that mitochondria are ejected from intact neutrophils and engage bacteria during vital NETosis. Inhibition of the mitochondrial electron transport chain at Complex III, but not Complex I, attenuates S. aureus killing by 50 ± 7%, comparable to the NADPH oxidase inhibitor apocynin. Similarly, mitochondrial ROS scavenging using MitoTEMPO attenuates bacterial killing 112 ± 60% versus vehicle control. Antimycin A treatment also reduces mitochondrial ROS production by 50 ± 12% and NETosis by 53 ± 5%. CONCLUSIONS: We identify a previously unrecognized role for mitochondria in human neutrophils in the killing of S. aureus. Inhibition of electron transport chain Complex III significantly impairs antimicrobial activity. This is the first demonstration that vital NETosis, an early event in the antimicrobial response, occurring within 5 min of bacterial exposure, depends on the function of mitochondrial Complex III. Mitochondria join NADPH oxidase as bactericidal ROS generators that mediate the bactericidal activities of human neutrophils.


Assuntos
Neutrófilos , Staphylococcus aureus , Feminino , Humanos , Masculino , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/metabolismo
14.
Redox Biol ; 58: 102508, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334378

RESUMO

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.


Assuntos
COVID-19 , Infecções por Papillomavirus , Animais , Camundongos , Humanos , SARS-CoV-2 , Hipóxia/complicações , Poro de Transição de Permeabilidade Mitocondrial , Trifosfato de Adenosina
15.
Lab Invest ; 91(8): 1122-35, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21647091

RESUMO

While there is general agreement that cardiovascular disease (CVD) development is influenced by a combination of genetic, environmental, and behavioral contributors, the actual mechanistic basis of how these factors initiate or promote CVD development in some individuals while others with identical risk profiles do not, is not clearly understood. This review considers the potential role for mitochondrial genetics and function in determining CVD susceptibility from the standpoint that the original features that molded cellular function were based upon mitochondrial-nuclear relationships established millions of years ago and were likely refined during prehistoric environmental selection events that today, are largely absent. Consequently, contemporary risk factors that influence our susceptibility to a variety of age-related diseases, including CVD were probably not part of the dynamics that defined the processes of mitochondrial-nuclear interaction, and thus, cell function. In this regard, the selective conditions that contributed to cellular functionality and evolution should be given more consideration when interpreting and designing experimental data and strategies. Finally, future studies that probe beyond epidemiologic associations are required. These studies will serve as the initial steps for addressing the provocative concept that contemporary human disease susceptibility is the result of selection events for mitochondrial function that increased chances for prehistoric human survival and reproductive success.


Assuntos
Doenças Cardiovasculares/etiologia , DNA Mitocondrial/genética , Mitocôndrias/fisiologia , Evolução Biológica , Dieta/efeitos adversos , Suscetibilidade a Doenças , Humanos , Oxirredução , Seleção Genética
16.
Redox Biol ; 47: 102164, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34656823

RESUMO

Iron-sulfur (Fe-S) clusters are essential cofactors most commonly known for their role mediating electron transfer within the mitochondrial respiratory chain. The Fe-S cluster pathways that function within the respiratory complexes are highly conserved between bacteria and the mitochondria of eukaryotic cells. Within the electron transport chain, Fe-S clusters play a critical role in transporting electrons through Complexes I, II and III to cytochrome c, before subsequent transfer to molecular oxygen. Fe-S clusters are also among the binding sites of classical mitochondrial inhibitors, such as rotenone, and play an important role in the production of mitochondrial reactive oxygen species (ROS). Mitochondrial Fe-S clusters also play a critical role in the pathogenesis of disease. High levels of ROS produced at these sites can cause cell injury or death, however, when produced at low levels can serve as signaling molecules. For example, Ndufs2, a Complex I subunit containing an Fe-S center, N2, has recently been identified as a redox-sensitive oxygen sensor, mediating homeostatic oxygen-sensing in the pulmonary vasculature and carotid body. Fe-S clusters are emerging as transcriptionally-regulated mediators in disease and play a crucial role in normal physiology, offering potential new therapeutic targets for diseases including malaria, diabetes, and cancer.


Assuntos
Proteínas Ferro-Enxofre , Ferro , Biologia , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Enxofre/metabolismo
17.
Free Radic Biol Med ; 170: 150-178, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33450375

RESUMO

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies.


Assuntos
Hipertensão Pulmonar , Neoplasias , Biologia , Humanos , Peróxido de Hidrogênio , Hipertensão Pulmonar/genética , Neoplasias/genética , Neoplasias/terapia , Oxigênio
18.
Compr Physiol ; 10(2): 713-765, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32163206

RESUMO

In lung vascular cells, mitochondria serve a canonical metabolic role, governing energy homeostasis. In addition, mitochondria exist in dynamic networks, which serve noncanonical functions, including regulation of redox signaling, cell cycle, apoptosis, and mitochondrial quality control. Mitochondria in pulmonary artery smooth muscle cells (PASMC) are oxygen sensors and initiate hypoxic pulmonary vasoconstriction. Acquired dysfunction of mitochondrial metabolism and dynamics contribute to a cancer-like phenotype in pulmonary arterial hypertension (PAH). Acquired mitochondrial abnormalities, such as increased pyruvate dehydrogenase kinase (PDK) and pyruvate kinase muscle isoform 2 (PKM2) expression, which increase uncoupled glycolysis (the Warburg phenomenon), are implicated in PAH. Warburg metabolism sustains energy homeostasis by the inhibition of oxidative metabolism that reduces mitochondrial apoptosis, allowing unchecked cell accumulation. Warburg metabolism is initiated by the induction of a pseudohypoxic state, in which DNA methyltransferase (DNMT)-mediated changes in redox signaling cause normoxic activation of HIF-1α and increase PDK expression. Furthermore, mitochondrial division is coordinated with nuclear division through a process called mitotic fission. Increased mitotic fission in PAH, driven by increased fission and reduced fusion favors rapid cell cycle progression and apoptosis resistance. Downregulation of the mitochondrial calcium uniporter complex (MCUC) occurs in PAH and is one potential unifying mechanism linking Warburg metabolism and mitochondrial fission. Mitochondrial metabolic and dynamic disorders combine to promote the hyperproliferative, apoptosis-resistant, phenotype in PAH PASMC, endothelial cells, and fibroblasts. Understanding the molecular mechanism regulating mitochondrial metabolism and dynamics has permitted identification of new biomarkers, nuclear and CT imaging modalities, and new therapeutic targets for PAH. © 2020 American Physiological Society. Compr Physiol 10:713-765, 2020.


Assuntos
Endotélio Vascular/metabolismo , Hipertensão Pulmonar/fisiopatologia , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Artéria Pulmonar/metabolismo , Animais , Humanos , Dinâmica Mitocondrial
19.
EBioMedicine ; 36: 316-328, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30232024

RESUMO

We hypothesized that changes in the mitochondrial DNA (mtDNA) would significantly influence whole body metabolism, adiposity and gene expression in response to diet. Because it is not feasible to directly test these predictions in humans we used Mitochondrial-Nuclear eXchange mice, which have reciprocally exchanged nuclear and mitochondrial genomes between different Mus musculus strains. Results demonstrate that nuclear-mitochondrial genetic background combination significantly alters metabolic efficiency and body composition. Comparative RNA sequencing analysis in adipose tissues also showed a clear influence of the mtDNA on regulating nuclear gene expression on the same nuclear background (up to a 10-fold change in the number of differentially expressed genes), revealing that neither Mendelian nor mitochondrial genetics unilaterally control gene expression. Additional analyses indicate that nuclear-mitochondrial genome combination modulates gene expression in a manner heretofore not described. These findings provide a new framework for understanding complex genetic disease susceptibility.


Assuntos
Adiposidade/genética , Metabolismo Energético/genética , Epistasia Genética , Regulação da Expressão Gênica , Genes Mitocondriais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Tecido Adiposo/metabolismo , Animais , Biomarcadores , Composição Corporal , Feminino , Perfilação da Expressão Gênica , Patrimônio Genético , Genoma Mitocondrial , Masculino , Camundongos , Transcriptoma
20.
Front Cardiovasc Med ; 5: 195, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30740395

RESUMO

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51). Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs. Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

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