RESUMO
BACKGROUND: L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. METHODS AND RESULTS: L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase]-/-) or heterozygous (L2HGDH+/-) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH+/+) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [13C]- and [31P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH-/- and L2HGDH+/- hearts compared with L2HGDH+/+ hearts under ischemic stress conditions. CONCLUSION: L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.
Assuntos
Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Animais , Glucose/farmacologia , Glutaratos , Mamíferos , Camundongos , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Estresse Oxidativo , Oxigênio , Fosfatos/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. METHODS: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. RESULTS: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1ß sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. CONCLUSIONS: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.
Assuntos
Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Glucose/metabolismo , Interferon gama/metabolismo , Triptofano/metabolismo , Biomarcadores , Movimento Celular , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cinurenina/metabolismo , Oxirredução , Ligação Proteica , Transdução de SinaisRESUMO
Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Butiratos/farmacologia , Doenças Cardiovasculares/metabolismo , Doenças Metabólicas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica/efeitos dos fármacos , Animais , Doenças Cardiovasculares/diagnóstico por imagem , Doenças Cardiovasculares/fisiopatologia , Metabolismo Energético/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hidrólise , Espectroscopia de Ressonância Magnética , Masculino , Doenças Metabólicas/diagnóstico por imagem , Doenças Metabólicas/fisiopatologia , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , TermodinâmicaRESUMO
T cells can kill tumor cells by cell surface immunological recognition, but low affinity for tumor-associated antigens could lead to T cell off-target effects. Herein, a universal T cell targeting strategy based on bio-orthogonal chemistry and glycol-metabolic engineering is introduced to enhance recognition and cytotoxicity of T cells in tumor immunotherapy. Three kinds of bicycle [6.1.0] nonyne (BCN)-modified sugars are designed and synthesized, in which Ac4 ManN-BCN shows efficient incorporation into wide tumor cells with a BCN motif on surface glycans. Meanwhile, activated T cells are treated with Ac4 GalNAz to introduce azide (N3 ) on the cell surface, initiating specific tumor targeting through a bio-orthogonal click reaction between N3 and BCN. This artificial targeting strategy remarkably enhances recognition and migration of T cells to tumor cells, and increases the cytotoxicity 2 to 4 times for T cells against different kinds of tumor cells. Surprisingly, based on this strategy, the T cells even exhibit similar cytotoxicity with the chimeric antigen receptor T-cell against Raji cells in vitro at the effector: target cell ratios (E:T) of 1:1. Such a universal bio-orthogonal T cell-targeting strategy might further broaden applications of T cell therapy against tumors and provide a new strategy for T cell modification.
Assuntos
Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Azidas , Linhagem Celular Tumoral , Química Click/métodos , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Microscopia Confocal , Linfócitos T/metabolismoRESUMO
One approach to cancer immunotherapy is the repolarization of immunosuppressive tumor-associated macrophages (TAMs) to antitumor M1 macrophages. The present study developed galactose-functionalized zinc protoporphyrin IX (ZnPP) grafted poly(l-lysine)- b-poly(ethylene glycol) polypeptide micelles (ZnPP PM) for TAM-targeted immunopotentiator delivery, which aimed at in vivo repolarization of TAMs to antitumor M1 macrophages. The outcomes revealed that ROS-inducing ZnPP PM demonstrated specificity for the in vitro and in vivo targeting of macrophages, elevated the level of ROS, and lowered STAT3 expression in BM-TAMs. Poly I:C (PIC, a TLR3 agonist)-loaded ZnPP PM (ZnPP PM/PIC) efficiently repolarized TAMs to M1 macrophages, which were reliant on ROS generation. Further, ZnPP PM/PIC substantially elevated the activated NK cells and T lymphocytes in B16-F10 melanoma tumors, which caused vigorous tumor regression. Therefore, the TAM-targeted transport of an immunologic adjuvant with ZnPP-grafted nanovectors may be a potential strategy to repolarize TAMs to M1 macrophages in situ for effective cancer immunotherapy.
Assuntos
Imunoterapia , Macrófagos/imunologia , Melanoma , Micelas , Poli I-C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptor 3 Toll-Like/imunologia , Animais , Linhagem Celular Tumoral , Imunidade Celular/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Melanoma/imunologia , Melanoma/patologia , Melanoma/terapia , Camundongos , Células RAW 264.7 , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Immunosuppressive tumor-associated dendritic cells (TADCs) are potential targets for cancer therapy. However, their poor responsiveness to TLR stimulation is a major obstacle for achieving successful cancer immunotherapy. In the current study, we reported a dysregulated miR-148a/DNA methyltransferase (DNMT)1/suppressor of cytokine signaling (SOCS)1 axis as a unique mechanism for dampened TLR stimulation in TADCs. The results showed that aberrantly elevated miR-148a in bone marrow-derived TADC (BM-TADC) abolished polyinosinic-polycytidylic acid (poly I:C) or LPS-induced dendritic cell maturation through directly suppressing DNMT1 gene, which consequently led to the hypomethylation and upregulation of SOCS1, the suppressor of TLR signaling. In contrast, miR-148a inhibitor (miR-148ai) effectively rescued the expression of DNMT1 and decreased SOCS1 in BM-TADCs, thereby recovering their sensitivity to TLR3 or TLR4 stimulation. To further reprogram TADCs in vivo, miR-148ai was coencapsulated with poly I:C and OVA by cationic polypeptide micelles to generate integrated polypeptide micelle/poly I:C (PMP)/OVA/148ai nanovaccine, which was designed to simultaneously inhibit miR-148a and activate TLR3 signaling in TADCs. The immunization of PMP/OVA/148ai nanovaccine not only effectively modulated the miR-148a/DNMT1/SOCS1 axis in the spleen, but also significantly increased mature dendritic cells both in the spleen and in tumor microenvironment. Moreover, PMP/OVA/148ai ameliorated tumor immunosuppression through reducing regulatory T cells and myeloid-derived suppressor cells, thereby leading to potent anticancer immune responses and robust tumor regression with prolonged survival. This study proposes a nanovaccine-based immunogene therapy with the integration of miR-148a inhibition and TLR3 stimulation as a novel therapeutic approach to boost anticancer immunity by reprogramming TADCs in vivo.
Assuntos
Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Terapia Genética/métodos , Melanoma Experimental , MicroRNAs/antagonistas & inibidores , Animais , Western Blotting , Técnicas de Reprogramação Celular/métodos , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Camundongos , Camundongos Endogâmicos C57BL , Nanotecnologia/métodos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Supressora da Sinalização de Citocina/metabolismoRESUMO
Optical fluorescence imaging is an important strategy to explore the mechanism of virus-host interaction. However, current fluorescent tag labeling strategies often dampen viral infectivity. The present study explores an in situ fluorescent labeling strategy in order to preserve viral infectivity and precisely monitor viral infection in vivo. In contrast to pre-labeling strategy, mice are first intranasally infected with azide-modified H5N1 pseudotype virus (N3 -H5N1p), followed by injection of dibenzocyclooctyl (DBCO)-functionalized fluorescence 6 h later. The results show that DBCO dye directly conjugated to N3 -H5N1p in lung tissues through in vivo bioorthogonal chemistry with high specificity and efficacy. More remarkably, in situ labeling rather than conventional prelabeling strategy effectively preserves viral infectivity and immunogenicity both in vitro and in vivo. Hence, in situ bioorthogonal viral labeling is a promising and reliable strategy for imaging and tracking viral infection in vivo.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Imagem Óptica/métodos , Química ClickRESUMO
Objective: To establish and assess the feasibility and sensitivity of left ventricular elasticity, compliance and stiffness for study of ischemia/reperfusion injury in an isolated mouse heart model utilizing the pressure-volume curve framework. Methods: An isolated, balloon-in-ventricle, isovolumically contracting, crystalloid-perfused Langendorff heart preparation was set up from 15 male C57/6BL mice aged 12-14 weeks. End-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR) were obtained by measuring left ventricular (LV) systolic pressure (LVSP) and diastolic pressure under different balloon volumes. End-systolic elasticity (E(es)), end-diastolic stiffness (S(ed)) and compliance (C(ed)) were calculated from the slope of ESPVR and EDPVR. Measurements of LVSP, developed pressure (LVDP), end of diastolic pressure (EDP), the rate of pressure development dp/dt(max) and dp/dt(min) were monitored during 30 min stabilization, 20 min global ischemia and 40 min reperfusion. The reliability and coefficient of variation (CV) of these parameters were compared with E(es) and C(ed). Results: During stabilization, the heart rate was (395±40) bpm, LVDP was (126±25) mmHg, + dp/dt(max) was (5 590±625) mmHg/s, -dp/dt(min) was (-4 128±625) mmHg/s, E(es) was 5.7±0.3, C(ed) was 0.7±0.2, S(ed) was 1.4±0.1. Ischemia/reperfusion injury resulted in significant decrease in contractile function parameters. The recovery level of LVSP, LVDP, dp/dt(max) and dp/dt(min) were (57±19)%, (23±6)%, (23±7)% and (21±5)% (all P<0.001), respectively. The EDP increased to (5.4±2.0) times than the baseline after ischemia/reperfusion injury. The E(es) and C(ed) was deceased to (42±2)% and (33±2)%, compared with baseline. The stiffness was increased up to (3.1±0.2) times higher than the baseline. The CV of E(es,)S(ed) and C(ed) were lower than LVSP, LVDP, EDP, dp/dt(max,)dp/dt(min,)while the reliability of E(es), S(ed) and C(ed) were higher than the classic contractile function parameters. Conclusion: Pressure-volume curves are feasible during ischemia/reperfusion injury in the isolated mouse heart model with acceptable reliability and sensitivity.
Assuntos
Traumatismo por Reperfusão Miocárdica , Traumatismo por Reperfusão , Animais , Pressão Sanguínea , Diástole , Frequência Cardíaca , Masculino , Camundongos , Reprodutibilidade dos TestesRESUMO
Today the cross-species transmission of avian influenza viruses (AIV) are a great concern. A number of AIV strains are now enzootic among poultry, with H9N2 and highly pathogenic H5N1 AIV strains prevalent in China. H5N1 strains have been recognized to infect zoo and domestic feline species. In this serological study we sought to examine evidence that H5N1 strains have infected domestic cats in northeastern China. In 2013, we conducted a cross-sectional serological study of 916 healthy cats in Heilongjian, Jilin, and Liaonin Provinces. Sera were screened with a hemagglutinin inhibition (HI) assay and seropositive specimens (HI ≥ 1:20) were further evaluated with a microneutralization (MN) assay against a clade 2.3.2 H5N1 AIV, a H9N2 AIV, A (H1N1)pdm09, and a canine H3N2 virus. While â¼2% of cats had elevated HI assays against H5N1, no elevations were confirmed (MN ≥ 1:80). These data serve as baseline for future surveillance for AIV infections among domestic cats. Conducting such surveillance seems important for geographical areas recognized as endemic for AIVs. This is especially true for countries such as China where domestic cats and poultry are often in close contact.
Assuntos
Anticorpos Antivirais/sangue , Doenças do Gato/epidemiologia , Doenças do Gato/virologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/veterinária , Animais , Anticorpos Neutralizantes/sangue , Gatos , China/epidemiologia , Estudos Transversais , Testes de Inibição da Hemaglutinação , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Testes de Neutralização , Infecções por Orthomyxoviridae/epidemiologia , Estudos SoroepidemiológicosRESUMO
The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial ß-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD(-/-)) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD(-/-) mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Cardiomiopatia Dilatada/enzimologia , Hipotermia/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/metabolismo , Temperatura Baixa , Síndrome Congênita de Insuficiência da Medula Óssea , Modelos Animais de Doenças , Hipotermia/etiologia , Hipotermia/metabolismo , Erros Inatos do Metabolismo Lipídico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Mitocondriais , Doenças Musculares , Oxirredução , Estresse FisiológicoRESUMO
BACKGROUND: In 2013, a novel H7N9 avian influenza virus (AIV) was isolated from ill humans in Shanghai and Anhui Province, China. Since then, the virus has spread quickly throughout China. Previous isolation of H7N2 virus from swine suggests that additional H7 subtype AIVs may be transmitted through pigs. However, prior to the recent zoonosis of H7N9, there were very few studies on the seroprevalence of the H7 subtypes in this species. Thus, there is a need to perform serological surveys for novel H7N9 as well as other H7 subtype AIVs in swine. This surveillance may help us understand risk factors for outbreaks of influenza A (H7N9) virus. RESULTS: Only 2.0% (26/1310) of the pig sera had antibodies with an HI titer ≥1:20, and none had an MN titer ≥1:80, against the H7 antigen. Thus, no samples were found to be positive against H7N9. However, 13.6% (178/1310) of the pig sera had antibodies with HI titer ≥1:20 and 8.5% (112/1310) by MN titer ≥1:80 against H9 antigen. Thirty-seven percent (484/1310) of the pig sera had antibodies with HI titer ≥1:20 and 18.2% (238/1310) had MN titer ≥1:80 against pandemic 2009. CONCLUSIONS: Pigs in southern China have been shown to be infected with multiple avian influenza viruses. As the prevalence of novel influenza A viruses (e.g., H7N9 avian influenza virus) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be conducted in the future.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/sangue , China/epidemiologia , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Estudos Soroepidemiológicos , Testes Sorológicos , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/epidemiologiaRESUMO
Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology.
RESUMO
Glioblastoma (GBM) is the most devastating brain tumor and highly resistant to conventional chemotherapy. Herein, we introduce biomimetic nanosonosensitizer systems (MDNPs) combined with noninvasive ultrasound (US) actuation for orthotopic GBM-targeted delivery and sonodynamic-enhanced chemotherapy. MDNPs were fabricated with biodegradable and pH-sensitive polyglutamic acid (PGA) and the chemotherapeutic agent and sonosensitizer doxorubicin (DOX), camouflaged with human GBM U87 cell membranes. MDNPs presented homologous targeting accumulation and in vivo long-term circulation ability. They effectively passed through the blood-brain barrier (BBB) under US assistance and reached the orthotopic GBM site. MDNPs exhibited controllable US-elicited sonodynamic effect by generation of reactive oxygen species (ROS). ROS not only induced cancer cell apoptosis but also downregulated drug-resistance-related factors to disrupt chemoresistance and increase sensitivity to chemotherapy. The in vivo study of orthotopic GBM treatments further proved that MDNPs exhibited US-augmented synergistic antitumor efficacy and strongly prolonged the survival rate of mice. The use of low-dose DOX and the safety of US enabled repeated treatment (4 times) without obvious cardiotoxicity. This effective and safe US-enhanced chemotherapy strategy with the advantages of noninvasive brain delivery and high drug sensitivity holds great promise for deep-seated and drug-resistant tumors.
Assuntos
Glioblastoma , Nanopartículas , Humanos , Camundongos , Animais , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Biomimética , Ultrassonografia , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Medicamentos , Linhagem Celular TumoralRESUMO
BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.
Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Inibidores do Transportador 2 de Sódio-Glicose , Camundongos , Masculino , Animais , Cardiomiopatias Diabéticas/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Sódio , Cálcio , Desoxicitidina Monofosfato , Contração Miocárdica , Camundongos Endogâmicos C57BL , Miocárdio , Trifosfato de AdenosinaRESUMO
The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ßß-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.
Assuntos
Coração/fisiologia , Contração Miocárdica/fisiologia , Miosinas/fisiologia , Troponina T/fisiologia , Animais , Cardiomiopatia Hipertrófica Familiar , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido IncorretoRESUMO
AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In ß(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or ß-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in ß(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo Marrom/metabolismo , Receptores Adrenérgicos/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Tecido Adiposo Marrom/inervação , Antagonistas Adrenérgicos/farmacologia , Animais , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Adrenérgicos/genética , Proteína Desacopladora 1RESUMO
The inflammatory response that accompanies central nervous system (CNS) injury can affect neurological outcome in both positive and negative ways. In the optic nerve, a CNS pathway that normally fails to regenerate when damaged, intraocular inflammation causes retinal ganglion cells (RGCs) to switch into an active growth state and extend lengthy axons down the nerve. The molecular basis of this phenomenon is uncertain. A prior study showed that oncomodulin (Ocm), a Ca(2+)-binding protein secreted by a macrophage cell line, is a potent axon-promoting factor for RGCs. However, it is not known whether Ocm contributes to the physiological effects of intraocular inflammation in vivo, and there are conflicting reports in the literature regarding its expression and significance. We show here that intraocular inflammation causes infiltrative cells of the innate immune system to secrete high levels of Ocm, and that agents that prevent Ocm from binding to its receptor suppress axon regeneration. These results were verified in different strains, species, and experimental models, and establish Ocm as a potent growth-promoting signal between the innate immune system and neurons in vivo.
Assuntos
Axônios/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Inflamação/imunologia , Regeneração Nervosa , Nervo Óptico/fisiologia , Animais , Axônios/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
BACKGROUND: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents-myotropes-activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. METHODS: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by 31P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. RESULTS: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ATP) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG~ATP. CONCLUSIONS: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.
Assuntos
Dobutamina , Insuficiência Cardíaca , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Dobutamina/farmacologia , Metabolismo Energético , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Contração Miocárdica , Miocárdio/metabolismo , Fosfocreatina/metabolismo , Ratos , Troponina/metabolismoRESUMO
Transplantation of adult bone marrow-derived mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of 5 x 10(6) cells overexpressing Akt into the ischemic rat myocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition and cardiac myocyte hypertrophy, regenerated 80-90% of lost myocardial volume, and completely normalized systolic and diastolic cardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1 restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacZ. Thus, mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance.
Assuntos
Mesoderma/citologia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/terapia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transplante de Células-Tronco/métodos , Animais , Apoptose/fisiologia , Biomarcadores/análise , Células Cultivadas , Colágeno/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Técnicas In Vitro , Injeções , Masculino , Mesoderma/fisiologia , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Ratos , Retroviridae/genética , Transdução Genética , beta-Galactosidase/genéticaRESUMO
Pulmonary obstruction occurs in many common forms of congenital heart disease. In this study, pulmonary artery (PA) banding is used as a model for pulmonary stenosis. Significant remodeling of the vascular bed occurs as a result of a prolonged narrowing of the PAs, and here we quantify the biophysical and molecular changes proximal and distal to the obstruction. Main and branch PAs are harvested from banded and sham rabbits and their mechanical properties are assessed using a biaxial tensile tester. Measurements defined as initial and stiff slopes are taken, assuming a linear region at the start and end of the J-shaped stress-strain curves, along with a transitional knee point. Collagen, elastin assays, Movat's pentachrome staining, and Doppler protocols are used to quantify biochemical, structural, and physiological differences. The banded main PAs have significantly greater initial slopes while banded branch PAs have lower initial slopes; however, this change in mechanical behavior cannot be explained by the assay results as the elastin content in both main and branch PAs is not significantly different. The stiff slopes of the banded main PAs are higher, which is attributed to the significantly greater amounts of insoluble collagen. Shifting of the knee points reveals a decreased toe region in the main PAs but an opposite trend in the branch PAs. The histology results show a loss of integrity of the media, increase in ground substance, and dispersion of collagen in the banded tissue samples. This indicates other structural changes could have led to the mechanical differences in banded and normal tissue.