RESUMO
Kidneys are intricate three-dimensional structures in the body, yet the spatial and molecular principles of kidney health and disease remain inadequately understood. We generated high-quality datasets for 81 samples, including single-cell, single-nuclear, spot-level (Visium) and single-cell resolution (CosMx) spatial-RNA expression and single-nuclear open chromatin, capturing cells from healthy, diabetic and hypertensive diseased human kidneys. Combining these data, we identify cell types and map them to their locations within the tissue. Unbiased deconvolution of the spatial data identifies the following four distinct microenvironments: glomerular, immune, tubule and fibrotic. We describe the complex organization of microenvironments in health and disease and find that the fibrotic microenvironment is able to molecularly classify human kidneys and offers an improved prognosis compared to traditional histopathology. We provide a comprehensive spatially resolved molecular roadmap of the human kidney and the fibrotic process, demonstrating the clinical utility of spatial transcriptomics.
Assuntos
Microambiente Celular , Progressão da Doença , Fibrose , Nefropatias , Rim , Análise de Célula Única , Humanos , Rim/patologia , Microambiente Celular/genética , Nefropatias/genética , Nefropatias/patologia , Transcriptoma , Perfilação da Expressão Gênica , MultiômicaRESUMO
Hypoxia-inducible factor-1α (HIF1α) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize HIF1α for clinical applications. However, whether HIF1α provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies. We hypothesize that systemic and brain-targeted HIF1α stabilization may have divergent effects. To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all HIF1α target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized HIF1α in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the HIF1α target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted HIF1α-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Hipóxia-Isquemia Encefálica/metabolismo , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Quinolonas/farmacologia , Administração Intranasal , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Eritropoetina/genética , Transportador de Glucose Tipo 1/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Glicina/farmacologia , Heme Oxigenase (Desciclizante)/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções Intraperitoneais , Injeções Intraventriculares , Neurônios/metabolismo , Oligodendroglia/metabolismo , RatosRESUMO
Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat's biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat's 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration-time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.
Assuntos
Barbitúricos/toxicidade , Carcinogênese/induzido quimicamente , Testes de Carcinogenicidade , Avaliação Pré-Clínica de Medicamentos , Glicina/análogos & derivados , Animais , Glicina/toxicidade , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a leading monogenetic cause of end-stage renal disease with limited therapeutic repertoire. A targeted drug delivery strategy that directs a small molecule to renal niches around cysts could increase the safety margins of agents that slow the progression of ADPKD but are poorly tolerated due to extrarenal toxicity. Herein, we determined whether previously characterized lysine-based and glutamic acid-based megalin-binding peptides can achieve renal-specific localization in the juvenile cystic kidney (JCK) mouse model of polycystic kidney disease and whether the distribution is altered compared with control mice. We performed in vivo optical and magnetic resonance imaging studies using peptides conjugated to the VivoTag 680 dye and demonstrated that megalin-interacting peptides distributed almost exclusively to the kidney cortex in both normal and JCK mice. Confocal analysis demonstrated that the peptide-dye conjugate distribution overlapped with megalin-positive renal proximal tubules. However, in the JCK mouse, the epithelium of renal cysts did not retain expression of the proximal tubule markers aquaporin 1 and megalin, and therefore these cysts did not retain peptide-dye conjugates. Furthermore, human kidney tumor tissues were evaluated by immunohistochemistry and revealed significant megalin expression in tissues from patients with renal cell carcinoma, raising the possibility that these tumors could be treated using this drug delivery strategy. Taken together, our data suggest that linking a small-molecule drug to these carrier peptides could represent a promising opportunity to develop a new platform for renal enrichment and targeting in the treatment of ADPKD and certain renal carcinomas.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Rim/efeitos dos fármacos , Peptídeos/administração & dosagem , Doenças Renais Policísticas/tratamento farmacológico , Animais , Aquaporina 1/metabolismo , Corantes , Desenho de Fármacos , Epitélio/metabolismo , Ácido Glutâmico/química , Humanos , Córtex Renal/diagnóstico por imagem , Córtex Renal/metabolismo , Neoplasias Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Lisina/química , Imageamento por Ressonância Magnética , Camundongos , Peptídeos/química , Peptídeos/farmacocinética , Doenças Renais Policísticas/diagnóstico por imagem , Distribuição TecidualRESUMO
Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels. Here, we demonstrate that a biomimetic topography, consisting of microhemispheres arrayed over the cell culture substrate, promotes podocyte differentiation in vitro. We define new methods for fabricating microscale curvature on various substrates, including a thin porous membrane. By growing podocytes on our topographic substrates, we found that these biophysical cues augmented nephrin gene expression, supported full-size nephrin protein expression, encouraged structural arrangement of F-actin and nephrin within the cell, and promoted process formation and even interdigitation compared to the flat substrates. Furthermore, the topography facilitated nephrin localization on curved structures while nuclei lay in the valleys between them. The improved differentiation was also evidenced by tracking barrier function to albumin over time using our custom topomembranes. Overall, our work presents accessible methods for incorporating microcurvature on various common substrates, and demonstrates the importance of biophysical stimulation in supporting higher-fidelity podocyte cultivation in vitro.
Assuntos
Biomimética/instrumentação , Técnicas de Cultura de Células/instrumentação , Podócitos/citologia , Animais , Proteínas de Membrana/metabolismo , Camundongos , Podócitos/metabolismo , Transporte ProteicoRESUMO
There is interest in pharmacologic preconditioning for end-organ protection by targeting the HIF system. This can be accomplished by inhibition of prolyl 4-hydroxylase (PHD). GSK360A is an orally active PHD inhibitor that has been previously shown to protect the failing heart. We hypothesized that PHD inhibition can also protect the brain from injuries and resulting behavioral deficits that can occur as a result of surgery. Thus, our goal was to investigate the effect of pre-stroke surgery brain protection using a verified GSK360A PHD inhibition paradigm on post-stroke surgery outcomes. Vehicle or an established protective dose (30 mg/kg, p.o.) of GSK360A was administered to male Sprague-Dawley rats. Initially, GSK360A pharmacokinetics and organ distribution were determined, and then PHD-HIF pharmacodynamic markers were measured (i.e., to validate the pharmacological effects of the GSK360A administration regimen). Results obtained using this validated PHD dose-regimen indicated significant improvement by GSK360A (30mg/kg); administered at 18 and 5 hours prior to transient middle cerebral artery occlusion (stroke). GSK360A exposure and plasma, kidney and brain HIF-PHD pharmacodynamics endpoints (e.g., erythropoietin; EPO and Vascular Endothelial Growth Factor; VEGF) were measured. GSK360A provided rapid exposure in plasma (7734 ng/ml), kidney (45-52% of plasma level) and brain (1-4% of plasma level), and increased kidney EPO mRNA (80-fold) and brain VEGF mRNA (2-fold). We also observed that GSK360A increased plasma EPO (300-fold) and VEGF (2-fold). Further assessments indicated that GSK360A reduced post-stroke surgery neurological deficits (47-64%), cognitive dysfunction (60-75%) and brain infarction (30%) 4 weeks later. Thus, PHD inhibition using GSK360A pretreatment produced long-term post-stroke brain protection and improved behavioral functioning. These data support PHD inhibition, specifically by GSK360A, as a potential strategy for pre-surgical use to reduce brain injury and functional decline due to surgery-related cerebral injury.
Assuntos
Comportamento Animal , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/etiologia , Transtornos Cognitivos/tratamento farmacológico , Glicina/análogos & derivados , Atividade Motora , Inibidores de Prolil-Hidrolase/uso terapêutico , Quinolonas/uso terapêutico , Acidente Vascular Cerebral/complicações , Administração Oral , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/sangue , Lesões Encefálicas/fisiopatologia , Transtornos Cognitivos/etiologia , Eritropoetina/sangue , Eritropoetina/genética , Glicina/administração & dosagem , Glicina/farmacocinética , Glicina/farmacologia , Glicina/uso terapêutico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Atividade Motora/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Prolil Hidroxilases/metabolismo , Inibidores de Prolil-Hidrolase/administração & dosagem , Inibidores de Prolil-Hidrolase/farmacologia , Quinolonas/administração & dosagem , Quinolonas/farmacocinética , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Sensação/efeitos dos fármacos , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.
Assuntos
Aminoácidos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Insuficiência Cardíaca/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Piperidinas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Quinazolinonas/farmacologia , Estresse Fisiológico , Aminoácidos/deficiência , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the ß-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.
Assuntos
Ventrículos do Coração/citologia , Procedimentos Analíticos em Microchip/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos , Função Ventricular/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Dispositivos Lab-On-A-Chip , Contração Miocárdica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.
Assuntos
Lisina/metabolismo , Sirtuína 1/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli , Vetores Genéticos , Humanos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Sirtuína 1/genética , Sirtuína 1/metabolismo , TransfecçãoRESUMO
Hypoxia inducible factor (HIF) stabilization by HIF-prolyl hydroxylase (PHD) inhibitors may improve ischemic conditions such as peripheral artery disease (PAD). This multicenter, randomized, placebo-controlled study evaluated the safety and efficacy of GSK1278863 (an oral PHD inhibitor) in subjects with PAD. The study assessed two active treatment paradigms: single dosing and subchronic daily dosing (300 mg single dose and 15 mg daily for 14 days, respectively). Neither regimen improved exercise performance compared with placebo (change from baseline in the 6-minute walk test (6MWT; feet), (GSK1278863, placebo): single dose (-46, -44), p=0.96; repeat dose (9, 8), p=0.99; change in number of contractions to onset of claudication (goniometry): single dose (4, -1), p=0.053; repeat dose (-2, 1), p=0.08). A calf-muscle biopsy substudy showed no increases in mRNA or protein levels of HIF target genes. More subjects receiving GSK1278863 than placebo experienced adverse events, particularly following the 300 mg single dose. Thus, assessing the safety of GSK1278863 in this setting would require a larger population exposed to the agent for a longer duration. These data do not support a benefit of GSK1278863 in PAD using the regimens tested. CLINICALTRIALSGOV IDENTIFIER NCT01673555:
Assuntos
Claudicação Intermitente/tratamento farmacológico , Doença Arterial Periférica/tratamento farmacológico , Inibidores de Prolil-Hidrolase/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Teste de Esforço , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Qualidade de Vida , Fatores de Tempo , Resultado do Tratamento , Caminhada/fisiologiaRESUMO
A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Mioblastos/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Mioblastos/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Células-Tronco/fisiologiaRESUMO
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Canais Iônicos/fisiologia , Miócitos Cardíacos/classificação , Fenótipo , Células-Tronco Pluripotentes/classificaçãoRESUMO
Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Alelos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Metilação de DNA , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Corpos Embrioides/fisiologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Desenvolvimento Muscular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologiaRESUMO
BACKGROUND: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI). METHODS AND RESULTS: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). CONCLUSIONS: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.
Assuntos
Vasos Coronários/efeitos dos fármacos , Glicina/análogos & derivados , Fator 1 Induzível por Hipóxia/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Quinolonas/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Linhagem Celular , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Glicina/farmacologia , Hemodinâmica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Quantitative measures are needed to identify diabetic patients at higher risk for CV events. Cell-derived microparticles (MPs) are submicron membrane vesicles released from activated cells that are indicative of cell damage. Progenitor cells (PCs) including proangiogenic cells (PACs), often termed endothelial progenitor cells (EPCs), are mediators of reparative capacity. We examined whether the relationship of MPs to PCs/PACs could be used as an improved and clinically feasible index of vascular pathology. METHODS AND RESULTS: Plasma samples were collected from patients with early-stage (ES, Diagnosis < 1 year) and long-term (LT, Diagnosis > 5 years,) Type 2 diabetes and compared with age related healthy subjects (H). PC and MP subtypes were measured by a combination of flow cytometry and ELISA-based methods. The ratio of procoagulant MPs/CD34(+) PCs proved a valuable index to distinguish between subject groups (P = 0.01). This index of compromised vascular function was highest in the LT group despite intensive statin therapy and was more informative than a range of soluble protein biomarkers. CONCLUSIONS: This is the first report of a relationship between MPs and PCs in Type 2 diabetes. This ratio may provide a quantitative and clinically feasible measurement of vascular dysfunction and cardiovascular risk in patients with diabetes. © 2010 International Clinical Cytometry Society.
Assuntos
Micropartículas Derivadas de Células/patologia , Diabetes Mellitus Tipo 2/patologia , Angiopatias Diabéticas/patologia , Endotélio Vascular/patologia , Células-Tronco/patologia , Adulto , Idoso , Antígenos CD34/análise , Antígenos CD34/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Colesterol/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Células-Tronco/efeitos dos fármacosRESUMO
Heme oxygenase-1 (HO) metabolizes heme to form the vasodilator carbon monoxide and antioxidant biliverdin. Upregulation of HO-1 by hemin, which is also a substrate attenuates thrombosis in rodent models, however, whether protection is due to HO-1 upregulation or to increased substrate availability is unknown. This study tested the hypothesis that treatment of mice with cobalt protoporphyrin (CoPP), a non-substrate HO-1 inducer, would protect the endothelium from laser injury. C57Bl/J6 mice were treated with vehicle, CoPP (20 mg/kg), CoPP plus the HO-1 inhibitor tin protoporphyrin (SnPP; 20 mg/kg) or SnPP alone for 18 h. Intravital microscopy was used to quantitate thrombus formation in cremaster arterioles in response to laser ablation of the endothelium. CoPP treatment inhibited thrombosis by 43% compared to vehicle (P<0.05). SnPP co-treatment negated the inhibitory effect of CoPP while SnPP alone potentiated thrombosis compared to vehicle. In CoPP-treated animals, cremaster HO-1 mRNA expression was increased 59+/-17-fold over vehicle (P<0.001). Co-treatment with CoPP+SnPP attenuated this effect by 36%, however the increase in HO-1 protein induced by CoPP was unaffected by SnPP. Induction of HO-1 by the non-substrate inducer CoPP protects against laser induced endothelial injury without the need for increased substrate. Small molecule, substrate-independent upregulation of HO-1 expression represents a feasible approach to ameliorate endothelial dysfunction in cardiovascular disease.
Assuntos
Arteríolas/efeitos dos fármacos , Arteríolas/patologia , Heme Oxigenase-1/biossíntese , Protoporfirinas/farmacologia , Trombose/enzimologia , Animais , Arteríolas/metabolismo , Indução Enzimática/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hemodinâmica/efeitos dos fármacos , Lasers/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Trombose/etiologia , Trombose/genética , Trombose/fisiopatologia , Regulação para Cima/efeitos dos fármacosRESUMO
Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Administração Oral , Aldeídos/química , Animais , Cristalografia por Raios X , Indazóis/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Ratos , Relação Estrutura-Atividade , Quinases Associadas a rho/metabolismoRESUMO
The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.
Assuntos
Anti-Hipertensivos/síntese química , Benzimidazóis/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxidiazóis/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oxidiazóis/farmacocinética , Oxidiazóis/farmacologia , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.
Assuntos
Amidas/síntese química , Anti-Hipertensivos/síntese química , Indazóis/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridonas/síntese química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Indazóis/farmacocinética , Indazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Proteínas Serina-Treonina Quinases/química , Piridonas/farmacocinética , Piridonas/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.