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1.
Physiol Rep ; 12(12): e16012, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38959068

RESUMO

Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.


Assuntos
Bleomicina , Pulmão , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Fibrose Pulmonar , Transglutaminases , Animais , Bleomicina/toxicidade , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Transglutaminases/metabolismo , Transglutaminases/genética , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Camundongos , Pulmão/patologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Camundongos Endogâmicos C57BL , Glicólise , Masculino
2.
Am J Physiol Lung Cell Mol Physiol ; 324(6): L863-L869, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37039378

RESUMO

Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatography/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyruvate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle deficiency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the development of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.


Assuntos
Fibrose Pulmonar Idiopática , Lesão Pulmonar , Neoplasias Pulmonares , Humanos , Projetos Piloto , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Ácido Láctico/análise , Neoplasias Pulmonares/radioterapia , Testes Respiratórios/métodos , Pulmão/metabolismo , Biomarcadores/análise
3.
Pathog Dis ; 79(1)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33220685

RESUMO

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources.


Assuntos
Anaplasma phagocytophilum/efeitos dos fármacos , Anaplasma phagocytophilum/fisiologia , Anaplasmose/genética , Anaplasmose/microbiologia , Desipramina/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Anaplasmose/tratamento farmacológico , Anaplasmose/imunologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Células HL-60 , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/microbiologia
4.
Am J Pathol ; 190(6): 1211-1223, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32194052

RESUMO

Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1fl/fl/PodoCre mice compared with their littermates. By crossbreeding Asah1fl/fl/PodoCre mice with Smpd1-/- mice, we also produced a double knockout strain, Smpd1-/-/Asah1fl/fl/PodoCre, that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1fl/fl/PodoCre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes.


Assuntos
Ceramidase Ácida/genética , Ceramidas/metabolismo , Glomérulos Renais/metabolismo , Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Ceramidase Ácida/metabolismo , Animais , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Podócitos/patologia , Podócitos/ultraestrutura
5.
Int J Mol Sci ; 21(5)2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32138242

RESUMO

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1-/- mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1-/- mice as compared to their wild-type littermates. Besides, Mcoln1-/- mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1-/- mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1-/- mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.


Assuntos
Vesículas Extracelulares/metabolismo , Lisossomos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Calcificação Vascular/metabolismo , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Vesículas Extracelulares/patologia , Imuno-Histoquímica , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Canais de Potencial de Receptor Transitório/genética
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