RESUMO
Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.
Assuntos
Eritroblastos/metabolismo , Eritropoese , Heme/biossíntese , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Animais , Modelos Animais de Doenças , Eritroblastos/citologia , Ferroquelatase/metabolismo , Teste de Complementação Genética , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Oxirredução , Proteínas/genética , Peixe-Zebra/metabolismo , Proteína Inibidora de ATPaseRESUMO
Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Hemangioblastos/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula/fisiologia , Eletroporação , Embrião de Mamíferos , Embrião não Mamífero , Citometria de Fluxo , Imuno-Histoquímica , Mesonefro/embriologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Peixe-ZebraRESUMO
We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia.
Assuntos
Anemia Macrocítica/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Adolescente , Animais , Criança , Eritropoese/genética , Exoma , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mitocondriais/genética , Mutação , Peixe-Zebra/genéticaRESUMO
Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Epistasia Genética , Eritropoese/genética , Evolução Molecular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.
Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Hematopoese/fisiologia , Hibridização In Situ , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Elementos Reguladores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Directed collective cell migration governs cell orientation during tissue morphogenesis, wound healing, and tumor metastasis. Unfortunately, current methods for initiating collective cell migration, such as scratching, laser ablation, and stencils, either introduce uncontrolled cell-injury, involve multiple fabrication processes, or have utility limited to cells with strong cell-cell junctions. Using three-dimensional (3D) bioprinted gelatin methacryloyl (GelMA) micropatterns on temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) coated interfaces, we demonstrate that directed injury-free collective cell migration could occur in parallel and perpendicular directions. After seeding cells, we created cell-free spaces between two 3D bioprinted GelMA micropatterns by lowering the temperature of PNIPAm interfaces to promote the cell detachment. Unlike conventional collective cell migration methods initiated by stencils, we observed well-organized cell migration in parallel and perpendicular to 3D bioprinted micropatterns as a function of the distance between 3D bioprinted micropatterns. We further established the utility of controlled collective cell migration for directed functional myotube formation using 3D bioprinted fingerprintlike micropatterns as well as iris musclelike concentric circular patterns. Our platform is unique for myoblast alignment and myotube formation because it does not require anisotropic guidance cues. Together, our findings establish how to achieve controlled collective cell migration, even at the macroscale, for tissue engineering and regeneration.
RESUMO
Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Animais , Diferenciação Celular , Respiração Celular , Glicólise , Células-Tronco Hematopoéticas/citologia , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
A series of 5-alkylsulfamoyl benzimidazole derivatives have been designed and synthesized as novel angiotensin II (Ang II) receptor antagonists. The compounds have been evaluated for in vitro Ang II antagonism and for in vivo antihypertensive activity on isolated rat aortic ring and desoxycortisone acetate induced hypertensive rats, respectively. The activity is found related to size of alkyl group. The maximum activity is observed with a compact and bulky alkyl group like tert-butyl and cyclohexyl. The compounds 4g and 4h have shown promising both in vitro and in vivo activities. A receptor binding model is also proposed on the basis on the basis of structure-activity relationship in this study.
Assuntos
Bloqueadores do Receptor Tipo 2 de Angiotensina II , Anti-Hipertensivos/síntese química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Benzimidazóis/química , Pressão Sanguínea/fisiologia , Desenho de Fármacos , Ratos , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
A series of 5-(alkyl and aryl)carboxamido benzimidazole derivatives had been designed, synthesized and evaluated for in vitro angiotensin II--AT1 receptor antagonism and in vivo antihypertensive activities. The pharmacological activities were inversely related to the size of alkyl and aryl substituents. It can be suggested that compounds with lower alkyl groups at 5-position of benzimidazole nucleus demonstrated potent antihypertensive activity.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/síntese química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Desenho de Fármacos , Receptor Tipo 1 de Angiotensina , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Benzimidazóis/química , Benzimidazóis/uso terapêutico , Hipertensão/tratamento farmacológico , Ratos , Padrões de ReferênciaRESUMO
PURPOSE OF REVIEW: The hierarchical nature of the hematopoietic system provides an ideal model system to illustrate the features of lineage tracing. We have outlined the utility of lineage tracing methods in establishing the origin and development of hematopoietic cells. RECENT FINDINGS: Methods such as CRISPR/Cas9, Polylox barcoding, and single-cell RNA-sequencing have improved our understanding of hematopoiesis. SUMMARY: This review chronicles the fate of the hematopoietic cells emerging from the mesoderm that subsequently develops into the adult blood lineages. Specifically, we explain classic techniques utilized in lineage tracing for the hematopoietic system, as well as novel state-of-the-art methods to elucidate clonal hematopoiesis and cell fate mapping at a single-cell level.
Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , HumanosRESUMO
The study has been designed to investigate the effect of 8-Br-cAMP, an activator of protein kinase A (PKA), in diabetes mellitus- and hyperhomocysteinemia-induced vascular endothelial dysfunction. Streptozotocin (55 mg kg-1, i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce diabetes mellitus (serum glucose >200 mg dL-1) and hyperhomocysteinemia (serum homocysteine >10 microM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta, and serum concentration of nitrite/nitrate. The expression of mRNA for p22phox and endothelial nitric oxide synthase (eNOS) was assessed by using reverse transcriptase-polymerase chain reaction (TBARS) (RT-PCR). Serum thiobarbituric acid-reactive substances (TBARS) concentration and aortic superoxide anion concentration were estimated to assess oxidative stress. 8-Br-cAMP (5 mg kg-1, i.p.) or atorvastatin (30 mg kg-1, p.o.) prevented diabetes mellitus- and hyperhomocysteinemia-induced attenuation of acetylcholine-induced endothelium-dependent relaxation, impairment of vascular endothelial lining, decrease in expression of mRNA for eNOS, serum nitrite/nitrate concentration, and increase in expression of mRNA for p22phox, superoxide anion, and serum TBARS. The ameliorative effect of 8-Br-cAMP was prevented by N omega-nitro-L-arginine methyl ester (L-NAME) (25 mg kg-1, i.p.) and glibenclamide (5 mg kg-1, i.p.). Therefore, it may be concluded that 8-Br-cAMP-induced activation of PKA may improve vascular endothelial dysfunction.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Homocisteína/sangue , Homocisteína/metabolismo , Hiper-Homocisteinemia/enzimologia , Hiper-Homocisteinemia/fisiopatologia , Masculino , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/sangue , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The study has been designed to investigate the effect of bis(maltolato) oxovanadium (BMOV), a protein tyrosine phosphatase inhibitor, on hypercholesterolemia and hypertension-induced vascular endothelial dysfunction. High fat diet (8 weeks) and deoxycorticosterone acetate (DOCA; 40 mg kg(-1), s.c.) were administered to rats to produce hypercholesterolemia and hypertension (mean arterial blood pressure >120 mmHg) respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta, and serum concentration of nitrite/nitrate. Serum thiobarbituric acid reactive substances (TBARS) were estimated to assess oxidative stress. BMOV (0.2 mg/ml in drinking water) or atorvastatin (30 mg kg(-1), p.o.) markedly improved acetylcholine-evoked endothelium-dependent relaxation, lining of vascular endothelium, serum nitrite/nitrate concentration, and serum TBARS in hypercholesterolemic and hypertensive rats. However, this ameliorative effect of BMOV has been prevented by L-NAME (25 mg kg(-1), i.p.), an inhibitor of NOS, or by glibenclamide (5 mg kg(-1), i.p.), a blocker of ATP-sensitive K(+) channels. It may be concluded that BMOV-induced inhibition of PTPase may improve vascular endothelial dysfunction.
Assuntos
Pironas/uso terapêutico , Vanadatos/uso terapêutico , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/fisiopatologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Endotelinas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Varredura , Nitratos/sangue , Nitritos/sangue , Pironas/farmacologia , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vanadatos/farmacologia , Doenças Vasculares/patologia , Vasodilatação/efeitos dos fármacosRESUMO
INTRODUCTION: A new use of methylene blue as an ulcerogenic agent and the mechanisms involved were identified with an objective to exploit methylene blue as a pharmacological tool to study investigational antiulcer agents. METHODS: Ulcerogenic potential was assessed using electron microscopy and measurement of an ulcer index after administering methylene blue (5-125 mg kg(-1), p.o.) or absolute ethanol (99%v/v, 2 ml, p.o.) to fasted rats. Estimation of thiobarbituric acid reactive substances, superoxide dismutase, reduced glutathione and catalase was used to assess oxidative stress. H(+)/K(+) ATPase activity, gastric mucosal blood flow and gastric acid secretion were measured to study the mechanism of methylene induced gastric ulcer. RESULTS: Methylene blue (100 mg kg(-1), p.o.) produced marked ulceration of the gastric mucosa due to increased levels of thiobarbituric acid reactive substances, activities of the H(+)/K(+) ATPase and superoxide dismutase, and decreased blood flow to the gastric mucosa, activity of catalase combined with reduced glutathione levels. DISCUSSION: It may be concluded that methylene blue activates the H(+)/K(+) ATPase to increase gastric acid secretion and reduces blood supply to gastric mucosa to produce oxidative stress that subsequently causes ulceration of gastric mucosa. Methylene blue can be used as an ulcerogenic agent to study mechanisms of investigational antiulcer agents.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Mucosa Gástrica/efeitos dos fármacos , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Azul de Metileno/farmacologia , Úlcera Gástrica/induzido quimicamente , Animais , Inibidores Enzimáticos/farmacologia , Etanol/farmacologia , Suco Gástrico/metabolismo , Mucosa Gástrica/irrigação sanguínea , Mucosa Gástrica/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Úlcera Gástrica/patologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The study has been designed to investigate the effect of 8-Br-cAMP, an activator of protein kinase A, in hypertension-induced vascular endothelial dysfunction. Rats were uninephroctomized and desoxycortisone acetate (DOCA) (40 mg/kg, s.c.) was administered to rats to produce hypertension (mean arterial blood pressure > 140 mmHg). Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. The expression of mRNA for p22phox and eNOS was assessed by using reverse transcriptase-polymerase chain reaction. Serum thiobarbituric acid reactive substances concentration and aortic superoxide anion concentration were estimated to assess oxidative stress. 8-Br-cAMP (5 mg/kg, i.p.) or atorvastatin (30 mg/kg, p.o.) prevented hypertension-induced attenuation of acetylcholine-induced endothelium-dependent relaxation, impairment of vascular endothelial lining, decrease in expression of mRNA for endothelial nitric oxide synthase (eNOS), serum nitrite/nitrate concentration and increase in expression of mRNA for p22phox, superoxide anion and serum TBARS. The ameliorative effect of 8-Br-cAMP was prevented by N-nitro-L-arginine methyl ester (25 mg/kg, i.p.) and glibenclamide (30 mg/kg, i.p.). It may be concluded that 8-Br-cAMP may stimulate expression and activity of eNOS and suppress expression of p22phox subunit of NADPH oxidase to reduce oxidative stress and subsequently improve vascular endothelial dysfunction.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Endotélio Vascular/efeitos dos fármacos , Hipertensão/tratamento farmacológico , 8-Bromo Monofosfato de Adenosina Cíclica/administração & dosagem , 8-Bromo Monofosfato de Adenosina Cíclica/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Atorvastatina , Pressão Sanguínea/efeitos dos fármacos , AMP Cíclico/sangue , Esquema de Medicação , Ácidos Heptanoicos/administração & dosagem , Hipertensão/complicações , Masculino , NADPH Oxidases/metabolismo , Nitratos/sangue , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/sangue , Pirróis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Vasodilatação/efeitos dos fármacosRESUMO
The study has been designed to investigate the effect of Bis-(maltolato) oxovanadium (BMOV), an inhibitor of protein tyrosin phosphatase (PTPase), in diabetes mellitus and hyperhomocysteinemia induced vascular endothelial dysfunction. Streptozotocin (55 mg kg(-1), i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce diabetes mellitus (serum glucose >140 mg dl(-1)) and hyperhomocysteinemia (serum homocysteine>10 microM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. Serum thiobarbituric acid reactive substances (TBARS) were estimated to assess oxidative stress. Atorvastatin has been employed in the present study as standard drug to improve vascular endothelial dysfunction. BMOV (0.2 mg/ml in drinking water) or atorvastatin (30 mg kg(-1), p.o.) in diabetic and hyperhomocysteinemic rats significantly reduced serum glucose and homocysteine concentration. BMOV or atorvastatin markedly improved acetylcholine induced endothelium dependent relaxation, vascular endothelial lining, serum nitrite/nitrate concentration and serum TBARS in diabetic and hyperhomocysteinemic rats. However, this ameliorative effect of BMOV has been prevented by l-NAME (25 mg kg(-1), i.p.), an inhibitor of NOS or by glibenclamide (5 mg kg(-1), i.p.), a blocker of ATP sensitive K(+) channels. Therefore, it may be concluded that BMOV induced inhibition of PTPase may activate eNOS due to opening of ATP sensitive K(+) channels and consequently reduce oxidative stress to improve vascular endothelial dysfunction.
Assuntos
Aorta Torácica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Proteínas Tirosina Fosfatases/farmacologia , Pironas/farmacologia , Vanadatos/farmacologia , Vasodilatação , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Glibureto/farmacologia , Hiper-Homocisteinemia/sangue , Hiper-Homocisteinemia/tratamento farmacológico , Hiper-Homocisteinemia/fisiopatologia , Masculino , Metionina , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitritos/sangue , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Proteínas Tirosina Fosfatases/uso terapêutico , Pironas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Estreptozocina , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vanadatos/uso terapêuticoRESUMO
Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.
Assuntos
Blástula , Animais , Movimento Celular , Hematopoese , Camundongos , Parabiose , Peixe-ZebraRESUMO
Transparency of the cornea is indispensable for optimal vision. Ocular trauma is a leading cause of corneal opacity, leading to 25 million cases of blindness annually. Recently, mesenchymal stem cells (MSCs) have gained prominence due to their inflammation-suppressing and tissue repair functions. Here, we investigate the potential of MSCs to restore corneal transparency following ocular injury. Using an in vivo mouse model of ocular injury, we report that MSCs have the capacity to restore corneal transparency by secreting high levels of hepatocyte growth factor (HGF). Interestingly, our data also show that HGF alone can restore corneal transparency, an observation that has translational implications for the development of HGF-based therapy.
Assuntos
Córnea/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Regeneração , Animais , Linhagem Celular , Córnea/citologia , Córnea/efeitos dos fármacos , Lesões da Córnea/genética , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Lesões da Córnea/terapia , Fibroblastos , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Interleucina-1beta/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Modelos Animais , RNA Interferente Pequeno/genética , CicatrizaçãoRESUMO
The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.
Assuntos
Eritropoese/genética , Heme/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Anemia/metabolismo , Animais , Linhagem Celular , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Hemoglobinas/metabolismo , Fígado/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismo , Porfirinas/metabolismo , Protoporfirinas/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.
Assuntos
Anemia/genética , Ferro/metabolismo , Receptores da Transferrina/metabolismo , Nexinas de Classificação/metabolismo , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Fluoresceína-5-Isotiocianato , Imunofluorescência , Inativação Gênica , Camundongos , Nexinas de Classificação/genética , Peixe-ZebraRESUMO
The study has been designed to investigate the effect of demethylasterroquinone B1 (DAQ B1), an activator of Akt, in diabetes mellitus (DM) and hyperhomocysteinemia (HHcy)-induced vascular endothelial dysfunction. Streptozotocin (55 mg kg(-1), i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce DM (serum glucose >140 mg dl(-1)) and HHcy (serum homocysteine >10 microM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. The expression of messenger RNA for p22phox and eNOS was assessed by reverse transcription-polymerase chain reaction. Serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide anion were estimated to assess oxidative stress. DAQ B1 (5 mg kg(-1), p.o.) or atorvastatin (30 mg kg(-1), p.o.) in diabetic and hyperhomocysteinemic rats significantly reduced serum glucose and homocysteine concentration. DAQ B1 or atorvastatin markedly improved acetylcholine-induced endothelium-dependent relaxation, vascular endothelial lining, serum nitrite/nitrate concentration and serum TBARS in diabetic and hyperhomocysteinemic rats. However, this ameliorative effect of DAQ B1 has been prevented by L-NAME (25 mg kg(-1), i.p.), an inhibitor of eNOS. Therefore, it may be concluded that DAQ B1-induced activation of Akt may activate eNOS and consequently reduce oxidative stress to improve vascular endothelial dysfunction.