RESUMO
Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.
Assuntos
Reprogramação Celular , Ácidos Graxos , Coração , Regeneração , Animais , Camundongos , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Hipóxia Celular , Proliferação de Células , Metabolismo Energético , Ativação Enzimática , Epigênese Genética , Ácidos Graxos/metabolismo , Coração/fisiologia , Histona Desmetilases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutação , Miocárdio , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxirredução , Regeneração/fisiologia , Traumatismo por Reperfusão , Transcrição GênicaRESUMO
Long noncoding RNAs (lncRNAs) influence the transcription of gene networks in many cell types, but their role in tumor-associated macrophages (TAMs) is still largely unknown. We found that the lncRNA ADPGK-AS1 was substantially upregulated in artificially induced M2-like human macrophages, macrophages exposed to lung cancer cells in vitro, and TAMs from human lung cancer tissue. ADPGK-AS1 is partly located within mitochondria and binds to the mitochondrial ribosomal protein MRPL35. Overexpression of ADPGK-AS1 in macrophages upregulates the tricarboxylic acid cycle and promotes mitochondrial fission, suggesting a phenotypic switch toward an M2-like, tumor-promoting cytokine release profile. Macrophage-specific knockdown of ADPGK-AS1 induces a metabolic and phenotypic switch (as judged by cytokine profile and production of reactive oxygen species) to a pro-inflammatory tumor-suppressive M1-like state, inhibiting lung tumor growth in vitro in tumor cell-macrophage cocultures, ex vivo in human tumor precision-cut lung slices, and in vivo in mice. Silencing ADPGK-AS1 in TAMs may thus offer a novel therapeutic strategy for lung cancer.
Assuntos
Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.
Assuntos
Sulfeto de Hidrogênio , Telomerase , Animais , Humanos , Camundongos , Senescência Celular , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Células Endoteliais/metabolismo , Sulfeto de Hidrogênio/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We conducted a phase I trial in newly diagnosed acute myeloid leukaemia (AML) to investigate the combination of two novel targeted agents, gemtuzumab ozogamicin (GO) and midostaurin, with intensive chemotherapy in FLT3-mutated AML and CBF leukaemia. Three dose levels of midostaurin and one to three sequential doses of 3 mg/m2 GO in combination with '7 + 3' induction were evaluated. Based on safety findings in 12 patients, our results show that 3 mg/m2 GO on Days 1 + 4 and 100 mg midostaurin on Days 8-21 can be safely combined with IC in newly diagnosed AML.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Gemtuzumab , Leucemia Mieloide Aguda , Estaurosporina , Humanos , Estaurosporina/análogos & derivados , Estaurosporina/administração & dosagem , Estaurosporina/uso terapêutico , Estaurosporina/efeitos adversos , Gemtuzumab/administração & dosagem , Gemtuzumab/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Adulto , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/efeitos adversos , Quimioterapia de Indução , Tirosina Quinase 3 Semelhante a fms/genética , Aminoglicosídeos/administração & dosagem , Aminoglicosídeos/uso terapêuticoRESUMO
BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.
Assuntos
Ciclo do Ácido Cítrico , Diabetes Mellitus Tipo 2 , Hipoglicemiantes , Rim , Fígado , Metformina , Inibidores do Transportador 2 de Sódio-Glicose , Metformina/farmacologia , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Humanos , Hipoglicemiantes/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/sangue , Masculino , Fígado/metabolismo , Fígado/efeitos dos fármacos , Rim/metabolismo , Rim/efeitos dos fármacos , Feminino , Quimioterapia Combinada , Camundongos Endogâmicos C57BL , Metabolômica , Biomarcadores/sangue , Pessoa de Meia-Idade , Glicemia/metabolismo , Glicemia/efeitos dos fármacos , Estudos Longitudinais , Camundongos , Idoso , Resultado do TratamentoRESUMO
In newly diagnosed acute myeloid leukemia (AML), immediate initiation of treatment is standard of care. However, deferral of antileukemic therapy may be indicated to assess comorbidities or pretherapeutic risk factors. We explored the impact of time from diagnosis to treatment on outcomes in newly diagnosed AML undergoing venetoclax-based therapy in two distinct cohorts. By querying the Study Alliance Leukemia database and the global health network TriNetX, we identified 138 and 717 patients respectively with an average age of 76 and 72 years who received venetoclax-based first-line therapy. When comparing patients who started treatment earlier or later than 10 days after initial diagnosis, no significant difference in median overall survival was observed - neither in the SAL cohort (7.7 vs. 9.6 months; P=0.42) nor in the TriNetX cohort (7.5 vs. 7.2 months; P=0.41). Similarly, severe infections, bleeding, and thromboembolic events were equally observed between early and later treatments, both in the overall patient groups and specific subgroups (age ≥75 years or leukocytes ≥20x109/L). This retrospective analysis indicates that delaying the start of venetoclax-based therapy in newly diagnosed AML might be a safe option for selected patients, provided that close clinical monitoring is performed.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Mieloide Aguda , Sulfonamidas , Humanos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Sulfonamidas/uso terapêutico , Sulfonamidas/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/diagnóstico , Idoso , Masculino , Feminino , Idoso de 80 Anos ou mais , Pessoa de Meia-Idade , Tempo para o Tratamento , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Resultado do Tratamento , Adulto , Estudos Retrospectivos , Antineoplásicos/uso terapêuticoRESUMO
We retrospectively studied 97 acute myeloid leukemia patients with trisomy 19 (median age at diagnosis 57 years; range, 17- 83 years) treated between 2001 and 2019 within two multicenter study groups. Trisomy 19 occurred alone in ten (10.5%) patients, with additional abnormalities being present in non-complex karyotypes in eight (8%) patients and in complex karyotypes in 79 (82%) patients. Altogether, karyotypes characterized by trisomies only were present in 27 (28%) patients. Data on response and outcome of intensively treated patients were available for 92 cases. The median follow-up was 6.4 years (95% confidence interval [95% CI]: 2.9-9.0 years). The complete remission (CR) rate after induction therapy was 52% (48 patients); the early death rate was 10% (n=9). Notably, patients with trisomy 19 as the sole abnormality had a CR rate of 89%. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was performed in 34 (35%) patients (CR, n=19; active disease, n=15). Five-year relapse-free and overall survival rates were 26% (95% CI: 16-43%) and 20% (95% CI: 13-31%), respectively. Overall survival rates were significantly higher in patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time-dependent covariable on overall survival revealed that trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only was a favorable factor (hazard ratio [HR]=0.47; P=0.021); higher age at diagnosis had an adverse impact (10 years difference; HR=1.29; P=0.002), whereas allo-HCT did not have a beneficial impact (odds ratio=1.45; P=0.21). In our cohort, patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only had a high CR rate and better clinical outcome.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Humanos , Pessoa de Meia-Idade , Criança , Trissomia/genética , Estudos Retrospectivos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/tratamento farmacológico , Indução de Remissão , Cariótipo AnormalRESUMO
We retrospectively studied 125 patients with acute myeloid leukemia and trisomy 4 (median age at diagnosis, 58 years; range, 16-77 years) treated between 2000 and 2019 within a multicenter study. Trisomy 4 was the sole abnormality in 28 (22%) patients and additional abnormalities were present in 97 (78%) patients. Twenty-two (22%) and 15 (15%) of 101 tested patients harbored NPM1 and FLT3-ITD mutations. Two (3%) of 72 tested patients had double CEBPA mutations. Data on response to intensive anthracycline-based induction therapy were available for 119 patients. Complete remission was achieved in 67% (n=80) and the early death rate was 5% (n=6). Notably, patients with trisomy 4 as sole abnormality had a complete remission rate of 89%. Allogeneic hematopoietic cell transplantation was performed in 40 (34%) patients, of whom 19 were transplanted in first complete remission. The median follow-up of the intensively treated cohort was 5.76 years (95% confidence interval [95% CI]: 2.99-7.61 years). The 5-year overall survival and relapse-free survival rates were 30% (95% CI: 22-41%) and 27% (95% CI: 18-41%), respectively. An Andersen-Gill regression model on overall survival revealed that favorable-risk according to the European LeukemiaNet classification (hazard ratio [HR]=0.34; P=0.006) and trisomy 4 as sole abnormality (HR=0.41; P=0.01) were favorable factors, whereas age with a difference of 10 years (HR=1.15; P=0.11), female gender (HR=0.74; P=0.20) and allogeneic hematopoietic cell transplantation (HR=0.64; P=0.14) did not have an significant impact. In our cohort, patients with trisomy 4 as their sole abnormality had a high complete remission rate and favorable clinical outcome. Allogeneic hematopoietic cell transplantation did not seem to improve overall survival.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Feminino , Humanos , Pessoa de Meia-Idade , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação , Nucleofosmina , Prognóstico , Estudos Retrospectivos , Trissomia/genética , Masculino , Adolescente , Adulto Jovem , Adulto , IdosoRESUMO
Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.
Assuntos
Retinopatia Diabética/enzimologia , Retinopatia Diabética/prevenção & controle , Epóxido Hidrolases/antagonistas & inibidores , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Progressão da Doença , Ácidos Docosa-Hexaenoicos/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Ependimogliais , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/patologia , Presenilina-1/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/metabolismo , Retina/patologia , Solubilidade , Corpo Vítreo/metabolismoRESUMO
Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44-/- mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMTΔ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMTΔ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.
Assuntos
Neoplasias da Mama/metabolismo , Família 2 do Citocromo P450/metabolismo , Linfangiogênese/fisiologia , Prostaglandinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Família 2 do Citocromo P450/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Ácidos Graxos Insaturados/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangiogênese/genética , Macrófagos , Camundongos , Camundongos Knockout , Monócitos , Processos Neoplásicos , Proteômica , Transdução de Sinais , Receptores Toll-Like , Regulação para CimaRESUMO
BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
Assuntos
Cadeias beta de Integrinas/química , Compostos de Sulfidrila/química , Animais , Cromatografia Líquida de Alta Pressão , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Sulfeto de Hidrogênio/farmacologia , Cadeias beta de Integrinas/metabolismo , Mecanotransdução Celular , Camundongos , Resistência ao Cisalhamento , Espectrometria de Massas em Tandem , Vasodilatação/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Polyunsaturated fatty acids (PUFAs) are used as traditional remedies to treat hair loss, but the mechanisms underlying their beneficial effects are not well understood. Here, we explored the role of PUFA metabolites generated by the cytochrome P450/soluble epoxide hydrolase (sEH) pathway in the regulation of the hair follicle cycle. Histological analysis of the skin from wild-type and sEH-/- mice revealed that sEH deletion delayed telogen to anagen transition, and the associated activation of hair follicle stem cells. Interestingly, EdU labeling during the late anagen stage revealed that hair matrix cells from sEH-/- mice proliferated at a greater rate which translated into increased hair growth. Similar effects were observed in in vitro studies using hair follicle explants, where a sEH inhibitor was also able to augment whisker growth in follicles from wild-type mice. sEH activity in the dorsal skin was not constant but altered with the cell cycle, having the most prominent effects on levels of the linoleic acid derivatives 12,13-epoxyoctadecenoic acid (12,13-EpOME), and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Fitting with this, the sEH substrate 12,13-EpOME significantly increased hair shaft growth in isolated anagen stage hair follicles, while its diol; 12,13-DiHOME, had no effect. RNA sequencing of isolated hair matrix cells implicated altered Wnt signaling in the changes associated with sEH deletion. Taken together, our data indicate that the activity of the sEH in hair follicle changes during the hair follicle cycle and impacts on two stem cell populations, i.e., hair follicle stem cells and matrix cells to affect telogen to anagen transition and hair growth.
Assuntos
Epóxido Hidrolases , Folículo Piloso , Células-Tronco , Animais , Epóxido Hidrolases/metabolismo , Cabelo/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
RNA editing by adenosine deaminases acting on dsRNA (ADAR) has become of increasing medical relevance, particularly because aberrant ADAR1 activity has been associated with autoimmunity and malignancies. However, the role of ADAR1 in dendritic cells (DC), representing critical professional APCs, is unknown. We have established conditional murine CD11c Cre-mediated ADAR1 gene ablation, which did not induce general apoptosis in CD11c+ cells but instead manifests in cell type-specific effects in DC subpopulations. Bone marrow-derived DC subset analysis revealed an incapacity to differentiate CD103 DC+ in both bulk bone marrow and purified pre-DC lineage progenitor assays. ADAR1 deficiency further resulted in a preferential systemic loss of CD8+/CD103+ DCs, revealing critical dependency on ADAR1, whereas other DC subpopulations were moderately affected or unaffected. Additionally, alveolar macrophages were depleted and dysfunctional, resembling pulmonary alveolar proteinosis. These results reveal an unrecognized role of ADAR1 in DC subset homeostasis and unveils the cell type-specific effects of RNA editing.
Assuntos
Adenosina Desaminase/metabolismo , Células Dendríticas/imunologia , Homeostase/imunologia , Macrófagos Alveolares/imunologia , Animais , Proliferação de Células , Células Dendríticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Edição de RNA , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
Assuntos
Neoplasias da Mama/metabolismo , Epóxido Hidrolases/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Epóxido Hidrolases/genética , Compostos de Epóxi/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Deleção de Genes , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismoRESUMO
BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.
Assuntos
Aterosclerose/enzimologia , Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/enzimologia , Cistationina gama-Liase/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Células Endoteliais/enzimologia , Sulfeto de Hidrogênio/metabolismo , Placa Aterosclerótica , Idoso , Idoso de 80 Anos ou mais , Animais , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/prevenção & controle , Catepsinas/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cistationina gama-Liase/deficiência , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Progressão da Doença , Proteína Semelhante a ELAV 1/genética , Células Endoteliais/patologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Cetoglutáricos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Oxigenases de Função Mista/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Timina DNA Glicosilase/metabolismo , Animais , Células Cultivadas , Citosina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácidos Cetoglutáricos/antagonistas & inibidores , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução/efeitos dos fármacosRESUMO
RATIONALE: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. OBJECTIVE: To determine the role of the AMPKα2 subunit in vascular repair. METHODS AND RESULTS: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. CONCLUSIONS: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Apoptose/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Isquemia/metabolismo , Neutrófilos/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Membro Posterior/irrigação sanguínea , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Isquemia/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aterosclerose/etiologia , Aterosclerose/metabolismo , Expressão Gênica , Monócitos/metabolismo , Células Mieloides/metabolismo , Animais , Aterosclerose/patologia , Metilação de DNA , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Macrófagos/metabolismo , Metionina/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologiaRESUMO
Macrophages in adipose tissue contribute to inflammation and the development of insulin resistance in obesity. Exposure of macrophages to saturated fatty acids alters cell metabolism and activates pro-inflammatory signaling. How fatty acids influence macrophage mitochondrial dynamics is unclear. We investigated the mechanism of palmitate-induced mitochondrial fragmentation and its impact on inflammatory responses in primary human macrophages. Fatty acids, such as palmitate, caused mitochondrial fragmentation in human macrophages. Increased mitochondrial fragmentation was also observed in peritoneal macrophages from hyperlipidemic apolipoprotein E knockout mice. Fatty acid-induced mitochondrial fragmentation was independent of the fatty acid chain saturation and required dynamin-related protein 1 (DRP1). Mechanistically, mitochondrial fragmentation was regulated by incorporation of palmitate into mitochondrial phospholipids and their precursors. Palmitate-induced endoplasmic reticulum stress and loss of mitochondrial membrane potential did not contribute to mitochondrial fragmentation. Macrophages treated with palmitate maintained intact mitochondrial respiration and ATP levels. Pharmacological or genetic inhibition of DRP1 enhanced palmitate-induced mitochondrial ROS production, c-Jun phosphorylation, and inflammatory cytokine expression. Our results indicate that mitochondrial fragmentation is a protective mechanism attenuating inflammatory responses induced by palmitate in human macrophages.