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BACKGROUND: We recently showed that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1) activity, based on TM5614, increases cell motility and induces the detachment of hematopoietic stem cells from their niches. In this TM5614 phase II clinical trial, we investigated whether the combination of a PAI-1 inhibitor and tyrosine kinase inhibitors (TKIs) would induce a deep molecular response (DMR) in patients affected by chronic myeloid leukemia (CML) by quantifying BCR-ABL1 transcripts. METHODS: Patients with chronic phase CML treated with a stable daily dose of TKIs for at least 1 year and yielding a major molecular response (MMR) but not achieving MR4.5 were eligible for this study. After inclusion, patients began to receive TM5614 as well as a TKI. The primary objective was an evaluation of the cumulative incidence of patient progression from an MMR/MR4 to MR4.5 by 12 months. RESULTS: Thirty-three patients were enrolled in the study. The median age was 59.0 years and 58% were male. No Sokal high-risk patients were enrolled in this trial. The median TKI treatment duration was 4.8 years. At the start of this study, seven patients and 26 patients received imatinib and second-generation TKIs, respectively. The cumulative MR4.5 incidence by 12 months was 33.3% (95% confidence interval, 18.0%-51.8%). The cumulative MR4.5 spontaneous conversion over 12 months was estimated as 8% with TKIs alone based on historical controls. The halving time of BCR-ABL1 at 2 months was significantly shorter for patients who achieved an MR4.5 , by 12 months than for the other patients (cutoff value: 48 days; sensitivity: 0.80; specificity: 0.91; ROC-AUC: 0.83). During this study, bleeding events and abnormal coagulation related to the drug were not reported, and TM5614 was found to be highly safe. CONCLUSION: TM5614 combined with TKI was well tolerated and induced MR4.5 in more patients than stand-alone TKI treatment.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide de Fase Crônica , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Proteínas de Fusão bcr-abl/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidores de Proteínas Quinases/efeitos adversos , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genéticaRESUMO
Metabolic dysfunction is thought to contribute to the severity of psychiatric disorders; however, it has been unclear whether current highsimple sugar diets contribute to pathogenesis of these diseases. Here, we demonstrate that a high-sucrose diet during adolescence induces psychosis-related behavioral endophenotypes, including hyperactivity, poor working memory, impaired sensory gating, and disrupted interneuron function in mice deficient for glyoxalase-1 (GLO1), an enzyme involved in detoxification of sucrose metabolites. Furthermore, the high-sucrose diet induced microcapillary impairments and reduced brain glucose uptake in brains of Glo1-deficient mice. Aspirin protected against this angiopathy, enhancing brain glucose uptake and preventing abnormal behavioral phenotypes. Similar vascular damage to our model mice was found in the brains of randomly collected schizophrenia and bipolar disorder patients, suggesting that psychiatric disorders are associated with angiopathy in the brain caused by various environmental stresses, including metabolic stress.
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Methylglyoxal (MG) is a reactive and cytotoxic α-dicarbonyl byproduct of glycolysis. Our bodies have several bio-defense systems to detoxify MG, including an enzymatic system by glyoxalase (GLO) 1 and GLO2. We identified a subtype of schizophrenia patients with novel mutations in the GLO1 gene that results in reductions of enzymatic activity. Moreover, we found that vitamin B6 (VB6) levels in peripheral blood of the schizophrenia patients with GLO1 dysfunction are significantly lower than that of healthy controls. However, the effects of GLO1 dysfunction and VB6 deficiency on the pathophysiology of schizophrenia remains poorly understood. Here, we generated a novel mouse model for this subgroup of schizophrenia patients by feeding Glo1 knockout mice VB6-deficent diets (KO/VB6(-)) and evaluated the combined effects of GLO1 dysfunction and VB6 deficiency on brain function. KO/VB6(-) mice accumulated homocysteine in plasma and MG in the prefrontal cortex (PFC), hippocampus, and striatum, and displayed behavioral deficits, such as impairments of social interaction and cognitive memory and a sensorimotor deficit in the prepulse inhibition test. Furthermore, we found aberrant gene expression related to mitochondria function in the PFC of the KO/VB6(-) mice by RNA-sequencing and weighted gene co-expression network analysis (WGCNA). Finally, we demonstrated abnormal mitochondrial respiratory function and subsequently enhanced oxidative stress in the PFC of KO/VB6(-) mice in the PFC. These findings suggest that the combination of GLO1 dysfunction and VB6 deficiency may cause the observed behavioral deficits via mitochondrial dysfunction and oxidative stress in the PFC.
Assuntos
Lactoilglutationa Liase , Esquizofrenia , Deficiência de Vitamina B 6 , Animais , Humanos , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Córtex Pré-Frontal/metabolismo , Esquizofrenia/genéticaRESUMO
Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(-) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(-) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(-) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.
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Leptin resistance is an important mechanism underlying the development and maintenance of obesity and is thus regarded as a promising target of obesity treatment. Plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue-type and urokinase-type plasminogen activators, is produced at high levels in adipose tissue, especially in states of obesity, and is considered to primarily be involved in thrombosis. PAI-1 may also have roles in inter-organ tissue communications regulating body weight, because PAI-1 knockout mice reportedly exhibit resistance to high fat diet (HFD)-induced obesity. However, the role of PAI-1 in body weight regulation and the underlying mechanisms have not been fully elucidated. We herein studied how PAI-1 affects systemic energy metabolism. We examined body weight and food intake of PAI-1 knockout mice fed normal chow or HFD. We also examined the effects of pharmacological inhibition of PAI-1 activity by a small molecular weight compound, TM5441, on body weight, leptin sensitivities, and expressions of thermogenesis-related genes in brown adipose tissue (BAT) of HFD-fed wild type (WT) mice. Neither body weight gain nor food intake was reduced in PAI-1 KO mice under chow fed conditions. On the other hand, under HFD feeding conditions, food intake was decreased in PAI-1 KO as compared with WT mice (HFD-WT mice 3.98 ± 0.08 g/day vs HFD-KO mice 3.73 ± 0.07 g/day, P = 0.021), leading to an eventual significant suppression of weight gain (HFD-WT mice 40.3 ± 1.68 g vs HFD-KO mice 34.6 ± 1.84 g, P = 0.039). Additionally, TM5441 treatment of WT mice pre-fed the HFD resulted in a marked suppression of body weight gain in a PAI-1-dependent manner (HFD-WT-Control mice 37.6 ± 1.07 g vs HFD-WT-TM5441 mice 33.8 ± 0.97 g, P = 0.017). TM5441 treatment alleviated HFD-induced systemic and hypothalamic leptin resistance, before suppression of weight gain was evident. Moreover, improved leptin sensitivity in response to TM5441 treatment was accompanied by increased expressions of thermogenesis-related genes such as uncoupling protein 1 in BAT (HFD-WT-Control mice 1.00 ± 0.07 vs HFD-WT-TM5441 mice 1.32 ± 0.05, P = 0.002). These results suggest that PAI-1 plays a causative role in body weight gain under HFD-fed conditions by inducing hypothalamic leptin resistance. Furthermore, they indicate that pharmacological inhibition of PAI-1 activity is a potential strategy for alleviating diet-induced leptin resistance in obese subjects.
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Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α) is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions, many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1) has also been shown to have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were evaluated in SK-N-BE(2)c cells by measuring HIF response element (HRE) promoter activity. Several furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological HIF-α/HRE transcriptional activity.
Assuntos
Furanos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxigenases de Função Mista/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Tiofenos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Furanos/síntese química , Furanos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Oxigenases de Função Mista/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Repressoras/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiofenos/síntese química , Tiofenos/química , Ativação Transcricional/efeitos dos fármacosRESUMO
Pharmacotherapy shows striking individual differences in pharmacokinetics and pharmacodynamics, involving drug efficacy and adverse reactions. Recent genetic research has revealed that genetic polymorphisms are important intrinsic factors for these inter-individual differences. This pharmacogenomic information could help develop safer and more effective precision pharmacotherapies and thus, regulatory guidance/guidelines were developed in this area, especially in the EU and US. The Project for the Promotion of Progressive Medicine, Medical Devices, and Regenerative Medicine by the Ministry of Health, Labour and Welfare, performed by Tohoku University, reported scientific information on the evaluation of genetic polymorphisms, mainly on drug metabolizing enzymes and transporters, during non-clinical studies and phase I clinical trials in Japanese subjects/patients. We anticipate that this paper will be helpful in drug development for the regulatory usage of pharmacogenomic information, most notably pharmacokinetics.
Assuntos
Ensaios Clínicos Fase I como Assunto , Preparações Farmacêuticas/metabolismo , Polimorfismo Genético/genética , Descoberta de Drogas , Humanos , Japão , Preparações Farmacêuticas/química , FarmacogenéticaRESUMO
Novel plasminogen activator inhibitor-1 (PAI-1) inhibitors with highly improved oral bioavailability were discovered by structure-activity relationship studies on N-acyl-5-chloroanthranilic acid derivatives. Because lipophilic N-acyl groups seemed to be important for the anthranilic acid derivatives to strongly inhibit PAI-1, synthesis of compounds in which 5-chloroanthranilic acid was bound to a variety of highly lipophilic moieties with appropriate linkers was investigated. As the result it appeared that some of the derivatives possessing aryl- or heteroaryl-substituted phenyl groups in the acyl chain had potent in vitro PAI-1 inhibitory activity. Oral absorbability of typical compounds was also evaluated in rats, and compounds 40, 55, 60 and 76 which have diverse chemical structure with each other were selected for further pharmacological evaluation.
Assuntos
Anticoagulantes/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , ortoaminobenzoatos/farmacologia , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/síntese química , Anticoagulantes/farmacocinética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Masculino , Estrutura Molecular , Ratos Wistar , Relação Estrutura-Atividade , ortoaminobenzoatos/administração & dosagem , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/farmacocinéticaRESUMO
AIM: The aim of this clinical trial was to obtain proof of concept for high-dose pyridoxamine as a novel treatment for schizophrenia with enhanced carbonyl stress. METHODS: Ten Japanese schizophrenia patients with high plasma pentosidine, which is a representative biomarker of enhanced carbonyl stress, were recruited in a 24-week, open trial in which high-dose pyridoxamine (ranging from 1200 to 2400 mg/day) was administered using a conventional antipsychotic regimen. Main outcomes were the total change in Positive and Negative Syndrome Scale score and the Brief Psychiatric Rating Scale score from baseline to end of treatment at week 24 (or at withdrawal). RESULTS: Decreased plasma pentosidine levels were observed in eight patients. Two patients showed marked improvement in their psychological symptoms. A patient who harbors a frameshift mutation in the Glyoxalase 1 gene also showed considerable reduction in psychosis accompanied with a moderate decrease in plasma pentosidine levels. A reduction of greater than 20% in the assessment scale of drug-induced Parkinsonism occurred in four patients. Although there was no severe suicide-related ideation or behavior, Wernicke's encephalopathy-like adverse drug reactions occurred in two patients and were completely suppressed by thiamine supplementation. CONCLUSION: High-dose pyridoxamine add-on treatment was, in part, effective for a subpopulation of schizophrenia patients with enhanced carbonyl stress. Further randomized, placebo-controlled trials with careful monitoring will be required to validate the efficacy of high-dose pyridoxamine for these patients.
Assuntos
Antipsicóticos/farmacologia , Arginina/análogos & derivados , Lisina/análogos & derivados , Avaliação de Resultados em Cuidados de Saúde , Estresse Oxidativo/efeitos dos fármacos , Piridoxamina/farmacologia , Esquizofrenia/sangue , Esquizofrenia/tratamento farmacológico , Complexo Vitamínico B/farmacologia , Adulto , Arginina/sangue , Arginina/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Humanos , Lactoilglutationa Liase/genética , Lisina/sangue , Lisina/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Piridoxamina/administração & dosagem , Piridoxamina/efeitos adversos , Esquizofrenia/genética , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/efeitos adversosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-ß-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-ß signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-ß-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-ß-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Espaço Intracelular/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Nicho de Células-Tronco , Fator de Crescimento Transformador beta/metabolismo , Animais , Medula Óssea/metabolismo , Movimento Celular , Espaço Extracelular/metabolismo , Furina/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/metabolismo , Transdução de SinaisRESUMO
Adhesive small bowel obstruction remains a common problem for surgeons. After surgery, platelet aggregation contributes to coagulation cascade and fibrin clot formation. With clotting, fibrin degradation is simultaneously enhanced, driven by tissue plasminogen activator-mediated cleavage of plasminogen to form plasmin. The aim of this study was to investigate the cellular events and proteolytic responses that surround plasminogen activator inhibitor (PAI-1; Serpine1) inhibition of postoperative adhesion. Peritoneal adhesion was induced by gauze deposition in the abdominal cavity in C57BL/6 mice and those that were deficient in fibrinolytic factors, such as Plat-/- and Serpine1-/- In addition, C57BL/6 mice were treated with the novel PAI-1 inhibitor, TM5275. Some animals were treated with clodronate to deplete macrophages. Epidermal growth factor (EGF) experiments were performed to understand the role of macrophages and how EGF contributes to adhesion. In the early phase of adhesive small bowel obstruction, increased PAI-1 activity was observed in the peritoneal cavity. Genetic and pharmacologic PAI-1 inhibition prevented progression of adhesion and increased circulating plasmin. Whereas Serpine1-/- mice showed intra-abdominal bleeding, mice that were treated with TM5275 did not. Mechanistically, PAI-1, in combination with tissue plasminogen activator, served as a chemoattractant for macrophages that, in turn, secreted EGF and up-regulated the receptor, HER1, on peritoneal mesothelial cells, which led to PAI-1 secretion, further fueling the vicious cycle of impaired fibrinolysis at the adhesive site. Controlled inhibition of PAI-1 not only enhanced activation of the fibrinolytic system, but also prevented recruitment of EGF-secreting macrophages. Pharmacologic PAI-1 inhibition ameliorated adhesion formation in a macrophage-dependent manner.-Honjo, K., Munakata, S., Tashiro, Y., Salama, Y., Shimazu, H., Eiamboonsert, S., Dhahri, D., Ichimura, A., Dan, T., Miyata, T., Takeda, K., Sakamoto, K., Hattori, K., Heissig, B. Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice.
Assuntos
Receptores ErbB/metabolismo , Macrófagos/fisiologia , Piperazinas/uso terapêutico , Serpina E2/antagonistas & inibidores , Aderências Teciduais/patologia , para-Aminobenzoatos/uso terapêutico , Animais , Antígeno CD11b , Ensaios de Migração Celular , Movimento Celular/efeitos dos fármacos , Cetuximab/farmacologia , Fator de Crescimento Epidérmico , Receptores ErbB/genética , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias/prevenção & controle , Células RAW 264.7 , Serpina E2/genética , Serpina E2/metabolismo , Transdução de Sinais , Aderências Teciduais/metabolismo , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
BACKGROUND: Hypoxia exposure is known to induce an alteration in skeletal muscle fiber-type distribution mediated by hypoxia-inducible factor (HIF)-α. The downstream pathway of HIF-α leading to fiber-type shift, however, has not been elucidated. The calcineurin pathway is one of the pathways responsible for slow muscle fiber transition. Because calcineurin pathway is activated by vascular endothelial growth factor (VEGF), one of the factors induced by HIF-1α, we hypothesized that the stabilization of HIF-1α may lead to slow muscle fiber transition via the activation of calcineurin pathway in skeletal muscles. To induce HIF-1α stabilization, we used a loss of function strategy to abrogate Prolyl hydroxylase domain protein (PHD) 2 responsible for HIF-1α hydroxylation making HIF-1α susceptible to ubiquitin dependent degradation by proteasome. The purpose of this study was therefore to examine the effect of HIF-1α stabilization in PHD2 conditional knockout mouse on skeletal muscle fiber-type transition and to elucidate the involvement of calcineurin pathway on muscle fiber-type transition. RESULTS: PHD2 deficiency resulted in an increased capillary density in skeletal muscles due to the induction of vascular endothelial growth factor. It also elicited an alteration of skeletal muscle phenotype toward the type I fibers in both of the soleus (35.8 % in the control mice vs. 46.7 % in the PHD2-deficient mice, p < 0.01) and the gastrocnemius muscle (0.94 vs. 1.89 %, p < 0.01), and the increased proportion of type I fibers appeared to correspond to the area of increased capillary density. In addition, calcineurin and nuclear factor of activated T cell (NFATc1) protein levels were increased in both the gastrocnemius and soleus muscles, suggesting that the calcineurin/NFATc1 pathway was responsible for the type I fiber transition regardless of PGC-1α, which responded minimally to PHD2 deficiency. Indeed, we found that tacrolimus (FK-506), a calcineurin inhibitor, successfully suppressed slow fiber-type formation in PHD2-deficient mice. CONCLUSIONS: Taken together, stabilized HIF-1α induced by PHD2 conditional knockout resulted in the transition of muscle fibers toward a slow fiber type via a calcineurin/NFATc1 signaling pathway. PHD2 conditional knockout mice may serve as a model for chronic HIF-1α stabilization as in mice exposed to low oxygen concentration.
Assuntos
Calcineurina/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/deficiência , Fibras Musculares de Contração Lenta/enzimologia , Músculo Esquelético/enzimologia , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Animais , Inibidores de Calcineurina/farmacologia , Capilares/metabolismo , Hipóxia Celular , Linhagem Celular , Genótipo , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Camundongos Knockout , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Neovascularização Fisiológica , Fenótipo , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/farmacologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.
Assuntos
Hipóxia Celular/fisiologia , Eritropoetina/biossíntese , Rim/citologia , Miofibroblastos/metabolismo , Animais , Camundongos , Transdução de SinaisRESUMO
Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducible transcription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible therapeutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo production, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1, PHD2, and PHD3) that mediate the inactivation of HIF1α and HIF2α under normal oxygen conditions. The loss of all PHD isoforms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combination of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform induces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2α or the hepatocyte-specific Epo gene enhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure after HIF2α activation. HIF2α also induces the expression of HIF3α, which is involved in the attenuation of Epo production. These results demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for hepatic Epo expression regulated by a PHD-HIF2α-EpoHE cascade in vivo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Eritropoetina/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Anemia/etiologia , Animais , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Policitemia/genética , Proteínas Repressoras , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.
Assuntos
Doenças Desmielinizantes/patologia , Esclerose Múltipla/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Animais , Axônios , Disponibilidade Biológica , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Colágeno Tipo IV/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Camundongos , Esclerose Múltipla/tratamento farmacológico , Paralisia/tratamento farmacológico , Paralisia/etiologia , Permeabilidade , Desempenho Psicomotor/efeitos dos fármacosRESUMO
The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.
Assuntos
Genes Reporter , Ensaios de Triagem em Larga Escala/métodos , Animais , Sequência de Bases , Hipóxia Celular/genética , Linhagem Celular , Humanos , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Ratos , Elementos de Resposta/genética , Estresse Fisiológico/genéticaRESUMO
Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inativadores de Plasminogênio/farmacologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Piperazinas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Regulação para Cima , para-Aminobenzoatos/farmacologiaRESUMO
Podocyte-endothelial cell cross-talk is paramount for maintaining the filtration barrier. The present study investigated the endothelial response to podocyte injury and its subsequent role in glomerulosclerosis using the podocyte-specific injury model of NEP25/LMB2 mice. NEP25/LMB2 mice showed proteinuria and local podocyte loss accompanied by thrombotic microangiopathy on day 8. Mice showed an increase of glomerular plasminogen activator inhibitor type 1 (PAI-1) mRNA and aberrant endothelial PAI-1 protein already on day 1, before thrombosis and proteinuria. A PAI-1-specific inhibitor reduced proteinuria and thrombosis and preserved podocyte numbers in NEP25/LMB2 mice by stabilization of ß1-integrin translocation. Heparin loading significantly reduced thrombotic formation, whereas proteinuria and podocyte numbers were unchanged. Immortalized podocytes treated with PAI-1 and the urokinase plasminogen activator (uPA) complex caused significant cell detachment, whereas podocytes treated with PAI-1 or uPA alone or with the PAI-1/uPA complex pretreated with an anti-uPA receptor (uPAR) antibody failed to cause detachment. Confocal microscopy and cell surface biotinylation experiments showed that internalized ß1-integrin was found together with uPAR in endocytotic vesicles. The administration of PAI-1 inhibitor or uPAR-blocking antibody protected cultured podocytes from cell detachment. In conclusion, PAI-1/uPA complex-mediated uPAR-dependent podocyte ß1-integrin endocytosis represents a novel mechanism of glomerular injury leading to progressive podocytopenia. This aberrant cross-talk between podocytes and endothelial cells represents a feedforward injury response driving podocyte loss and progressive glomerulosclerosis.
Assuntos
Endocitose , Cadeias beta de Integrinas/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Podócitos/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular , Heparina , Humanos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Trombose/metabolismo , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/metabolismoAssuntos
Descoberta de Drogas , Fibrinolíticos/uso terapêutico , Terapia de Alvo Molecular , Piperazinas/uso terapêutico , Inibidor 1 de Ativador de Plasminogênio/fisiologia , para-Aminobenzoatos/uso terapêutico , Envelhecimento/genética , Animais , Coagulação Sanguínea/genética , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Inflamação/genética , Camundongos , Neovascularização Fisiológica/genética , Inibidor 1 de Ativador de Plasminogênio/deficiência , Fibrose Pulmonar/tratamento farmacológico , Ratos , Regeneração , Trombose/tratamento farmacológicoRESUMO
The prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT) depends on the rapid recovery and sustained life-long hematopoiesis. The activation of the fibrinolytic pathway promotes hematopoietic regeneration; however, the role of plasminogen activator inhibitor-1 (PAI-1), a negative regulator of the fibrinolytic pathway, has not yet been elucidated. We herein demonstrate that bone marrow (BM) stromal cells, especially osteoblasts, produce PAI-1 in response to myeloablation, which negatively regulates the hematopoietic regeneration in the BM microenvironment. Total body irradiation in mice dramatically increased the local expression levels of fibrinolytic factors, including tissue-type plasminogen activator (tPA), plasmin, and PAI-1. Genetic disruption of the PAI-1 gene, or pharmacological inhibition of PAI-1 activity, significantly improved the myeloablation-related mortality and promoted rapid hematopoietic recovery after HSCT through the induction of hematopoiesis-promoting factors. The ability of a PAI-1 inhibitor to enhance hematopoietic regeneration was abolished when tPA-deficient mice were used as recipients, thus indicating that PAI-1 represses tPA-dependent hematopoietic regeneration. The PAI-1 inhibitor not only accelerated the expansion of the donor HSCs during the early-stage of regeneration, but also supported long-term hematopoiesis. Our results indicate that the inhibition of PAI-1 activity could be a therapeutic approach to facilitate the rapid recovery and sustained hematopoiesis after HSCT.