RESUMO
Maple syrup urine disease (MSUD) is an intoxication-type inherited metabolic disorder in which hyperleucinemia leads to brain swelling and death without treatment. MSUD is caused by branched-chain alpha-ketoacid dehydrogenase deficiency due to biallelic loss of the protein products from the genes BCKDHA, BCKDHB, or DBT, while a distinct but related condition is caused by loss of DLD. In this case series, eleven individuals with MSUD caused by two pathogenic variants in DBT are presented. All eleven individuals have a deletion of exon 2 (delEx2, NM_001918.3:c.48_171del); six individuals are homozygous and five individuals are compound heterozygous with a novel missense variant (NM_001918.5:c.916 T > C [p.Ser306Pro]) confirmed to be in trans. Western Blot indicates decreased amount of protein product in delEx2;c.916 T > C liver cells and absence of protein product in delEx2 homozygous hepatocytes. Ultrahigh performance liquid chromatography-tandem mass spectrometry demonstrates an accumulation of branched-chain amino acids and alpha-ketoacids in explanted hepatocytes. Individuals with these variants have a neonatal-onset, non-thiamine-responsive, classical form of MSUD. Strikingly, the entire cohort is derived from families who immigrated to the Washington, DC, metro area from Honduras or El Salvador suggesting the possibility of a founder effect.
Assuntos
Doença da Urina de Xarope de Bordo , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , América Central , Genômica , Humanos , Recém-Nascido , Doença da Urina de Xarope de Bordo/genética , MutaçãoRESUMO
Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Mutase/genética , Acidemia Propiônica/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Acil Coenzima A/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Carnitina/metabolismo , Linhagem Celular , Citratos/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Metilmalonil-CoA Mutase/deficiência , Acidemia Propiônica/genética , Acidemia Propiônica/patologiaRESUMO
Propionic acidemia (PA) and methylmalonic acidemia (MMA) are autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, which are caused by a deficiency in the enzyme propionyl-CoA carboxylase or the enzyme methylmalonyl-CoA (MM-CoA) mutase, respectively. The functional consequence of PA or MMA is the inability to catabolize P-CoA to MM-CoA or MM-CoA to succinyl-CoA, resulting in the accumulation of P-CoA and other metabolic intermediates, such as propionylcarnitine (C3), 3-hydroxypropionic acid, methylcitric acid (MCA), and methylmalonic acid (only in MMA). P-CoA and its metabolic intermediates, at high concentrations found in PA and MMA, inhibit enzymes in the first steps of the urea cycle as well as enzymes in the tricarboxylic acid (TCA) cycle, causing a reduction in mitochondrial energy production. We previously showed that metabolic defects of PA could be recapitulated using PA patient-derived primary hepatocytes in a novel organotypic system. Here, we sought to investigate whether treatment of normal human primary hepatocytes with propionate would recapitulate some of the biochemical features of PA and MMA in the same platform. We found that high levels of propionate resulted in high levels of intracellular P-CoA in normal hepatocytes. Analysis of TCA cycle intermediates by GC-MS/MS indicated that propionate may inhibit enzymes of the TCA cycle as shown in PA, but is also incorporated in the TCA cycle, which does not occur in PA. To better recapitulate the disease phenotype, we obtained hepatocytes derived from livers of PA and MMA patients. We characterized the PA and MMA donors by measuring key proximal biomarkers, including P-CoA, MM-CoA, as well as clinical biomarkers propionylcarnitine-to-acetylcarnitine ratios (C3/C2), MCA, and methylmalonic acid. Additionally, we used isotopically-labeled amino acids to investigate the contribution of relevant amino acids to production of P-CoA in models of metabolic stability or acute metabolic crisis. As observed clinically, we demonstrated that the isoleucine and valine catabolism pathways are the greatest sources of P-CoA in PA and MMA donor cells and that each donor showed differential sensitivity to isoleucine and valine. We also studied the effects of disodium citrate, an anaplerotic therapy, which resulted in a significant increase in the absolute concentration of TCA cycle intermediates, which is in agreement with the benefit observed clinically. Our human cell-based PA and MMA disease models can inform preclinical drug discovery and development where mouse models of these diseases are inaccurate, particularly in well-described species differences in branched-chain amino acid catabolism.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Aminoácidos/metabolismo , Citratos/metabolismo , Ciclo do Ácido Cítrico , Hepatócitos/patologia , Ácido Metilmalônico/metabolismo , Acidemia Propiônica/patologia , Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Ácido Cítrico/farmacologia , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Metilmalonil-CoA Descarboxilase/metabolismo , Metilmalonil-CoA Mutase/deficiência , Propionatos/farmacologia , Acidemia Propiônica/tratamento farmacológico , Acidemia Propiônica/metabolismoRESUMO
BACKGROUND: Propionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations. METHODS: In this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease. RESULTS: Primary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5mM) and valine (5mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient-derived fibroblasts. CONCLUSIONS: We demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue.
Assuntos
Hepatócitos/citologia , Hepatócitos/metabolismo , Acidemia Propiônica/metabolismo , Actinas/análise , Aminoácidos de Cadeia Ramificada/metabolismo , Amônia/metabolismo , Carbono-Carbono Ligases/genética , Carbono-Carbono Ligases/metabolismo , Células Cultivadas , Criança , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Hemodinâmica , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Isoleucina/farmacologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Mutação , Ureia/metabolismo , Valina/farmacologiaRESUMO
OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lipoproteínas LDL/efeitos dos fármacos , Animais , Doenças Cardiovasculares/sangue , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Técnicas In Vitro , Lipoproteínas LDL/metabolismo , Macaca fascicularis , Modelos Cardiovasculares , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Especificidade da EspécieRESUMO
Some health concerns are often not identified until late into clinical development of drugs, which can place participants and patients at significant risk. For example, the United States Food and Drug Administration (FDA) labeled the xanthine oxidase inhibitor febuxostat with a"boxed" warning regarding an increased risk of cardiovascular death, and this safety risk was only identified during Phase 3b clinical trials after its approval. Thus, better preclinical assessment of drug efficacy and safety are needed to accurately evaluate candidate drug risk earlier in discovery and development. This study explored whether an in vitro vascular model incorporating human vascular cells and hemodynamics could be used to differentiate the potential cardiovascular risk associated with molecules that have similar on-target mechanisms of action. We compared the transcriptomic responses induced by febuxostat and other xanthine oxidase inhibitors to a database of 111 different compounds profiled in the human vascular model. Of the 111 compounds in the database, 107 are clinical-stage and 33 are FDA-labelled for increased cardiovascular risk. Febuxostat induces pathway-level regulation that has high similarity to the set of drugs FDA-labelled for increased cardiovascular risk. These results were replicated with a febuxostat analog, but not another structurally distinct xanthine oxidase inhibitor that does not confer cardiovascular risk. Together, these data suggest that the FDA warning for febuxostat stems from the chemical structure of the medication itself, rather than the target, xanthine oxidase. Importantly, these data indicate that cardiovascular risk can be evaluated in this in vitro human vascular model, which may facilitate understanding the drug candidate safety profile earlier in discovery and development.
Assuntos
Doenças Cardiovasculares , Estados Unidos , Humanos , Doenças Cardiovasculares/induzido quimicamente , Xantina Oxidase , Febuxostat/farmacologia , Fatores de Risco , Inibidores Enzimáticos/efeitos adversos , Fatores de Risco de Doenças CardíacasRESUMO
Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cells 5 [NFAT5]) is a Rel homology transcription factor classically known for its osmosensitive role in regulating cellular homeostasis during states of hypo- and hypertonic stress. A recently growing body of research indicates that TonEBP is not solely regulated by tonicity, but that it can be stimulated by various tonicity-independent mechanisms in both hypertonic and isotonic tissues. Physiological and pathophysiological stimuli such as cytokines, growth factors, receptor and integrin activation, contractile agonists, ions, and reactive oxygen species have been implicated in the positive regulation of TonEBP expression and activity in diverse cell types. These new data demonstrate that tonicity-independent stimulation of TonEBP is critical for tissue-specific functions like enhanced cell survival, migration, proliferation, vascular remodeling, carcinoma invasion, and angiogenesis. Continuing research will provide a better understanding as to how these and other alternative TonEBP stimuli regulate gene expression in both health and disease.
Assuntos
Osmose/fisiologia , Fatores de Transcrição/metabolismo , Animais , Humanos , Camundongos , Fatores de Transcrição/fisiologiaRESUMO
Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.
Assuntos
Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Fatores de Transcrição NFATC/genética , Ligação Proteica , RatosRESUMO
RATIONALE: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. OBJECTIVE: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. METHODS AND RESULTS: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2(-/-) mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR(-/-)) mice. CONCLUSIONS: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.
Assuntos
Aterosclerose/metabolismo , Imunofenotipagem , Macrófagos/fisiologia , Fator 2 Relacionado a NF-E2/fisiologia , Fosfolipídeos/fisiologia , Animais , Aterosclerose/genética , Células Cultivadas , Feminino , Macrófagos/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Fosfolipídeos/metabolismoRESUMO
OBJECTIVE: We hypothesized that (1) neointima formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin microbubbles (RMBs), and (2) the addition of dual-mode ultrasound decreases the total amount of drug needed to reduce neointima formation. METHODS AND RESULTS: Balloon injury was performed in rat carotids to induce neointima formation. High or low doses of RMBs were injected intravenously and ruptured at the site of injury with ultrasound. Compared with nontreated injured arteries, neointima formation was reduced by 0% and 35.9% with 10(8) RMBs and by 28.7% and 34.9% in arteries treated with 10(9) RMBs with and without ultrasound, respectively. CONCLUSIONS: Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 10(8) RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect, demonstrating that this technology may have promise for localized potent drug therapy.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Microbolhas/uso terapêutico , Neointima/tratamento farmacológico , Sirolimo/administração & dosagem , Sirolimo/uso terapêutico , Ultrassom/métodos , Animais , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Cateterismo/efeitos adversos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Modelos Animais , Neointima/etiologia , Neointima/prevenção & controle , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologiaRESUMO
OBJECTIVE: Calcineurin (Cn) and the nuclear factor of activated T cells (NFAT) family of transcription factors are critical in vascular smooth muscle cell (SMC) development and pathology. Here, we used a genomics approach to identify and validate NFAT gene targets activated during platelet-derived growth factor-BB (PDGF-BB)-induced SMC phenotypic modulation. METHODS AND RESULTS: Genome-wide expression arrays were used to identify genes both (1) differentially activated in response to PDGF-BB and (2) whose differential expression was reduced by both the Cn inhibitor cyclosporin A and the NFAT inhibitor A-285222. The 20 most pharmacologically sensitive genes were validated by quantitative reverse transcription-polymerase chain reaction analysis of PDGF-BB-stimulated SMCs in the presence of Cn/NFAT inhibitors, including the VIVIT peptide. In all experiments, protein C receptor (PROCR) gene activation was reduced. We showed that PROCR expression was virtually absent in untreated, quiescent SMCs. PDGF-BB stimulation, however, induced significant PROCR promoter activation and downstream protein expression in a Cn/NFAT-dependent manner. Mutation of a species-conserved, NFAT binding motif significantly attenuated PDGF-BB-induced PROCR promoter activity, thereby distinguishing NFAT as the first PROCR transcriptional activator to date. Moreover, SMC PROCR expression was upregulated in the neointima as early as 7 days following acute vascular injury in rat carotid arteries. CONCLUSION: We hereby report PROCR as a novel, NFAT-dependent gene that may be implicated in vascular restenosis and consequent inward remodeling.
Assuntos
Fatores de Coagulação Sanguínea/genética , Calcineurina/genética , Genoma/genética , Músculo Liso Vascular/patologia , Fatores de Transcrição NFATC/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores de Superfície Celular/genética , Animais , Sequência de Bases , Becaplermina , Fatores de Coagulação Sanguínea/metabolismo , Calcineurina/metabolismo , Inibidores de Calcineurina , Lesões das Artérias Carótidas/metabolismo , Cateterismo/efeitos adversos , Células Cultivadas , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Animais , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Pirazóis/farmacologia , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
OBJECTIVE: The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs. METHODS AND RESULTS: We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle α actin (SMαA). Bioinformatic analysis of SMαA revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SMαA promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SMαA promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SMαA mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration. CONCLUSIONS: We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SMαA expression and PDGF-BB-stimulated SMC migration.
Assuntos
Aterosclerose/metabolismo , Lesões das Artérias Carótidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Túnica Íntima/metabolismo , Actinas/genética , Actinas/metabolismo , Angiotensina II/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Becaplermina , Sítios de Ligação , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Técnicas de Cocultura , Biologia Computacional , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genes Reporter , Humanos , Hiperplasia , Íntrons , Camundongos , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fatores de Transcrição NFATC/genética , Fenótipo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Proto-Oncogênicas c-sis , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Fatores de Transcrição/metabolismo , Transfecção , Túnica Íntima/patologiaRESUMO
OBJECTIVE: The goal of this study was to assess the activity of ß-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. METHODS AND RESULTS: Histological staining identified preferential nuclear localization of ß-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with ß-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of ß-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required ß-catenin, TCF, and the transcriptional coactivator CRBP-binding protein. Finally, we identified platelet endothelial cell adhesion molecule-1 as a critical regulator of constitutive ß-catenin and glycogen synthase kinase-3ß activities. CONCLUSIONS: These data reveal novel constitutive activation of the endothelial ß-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Hemodinâmica , Inflamação/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , beta Catenina/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Núcleo Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Genes Reporter , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Interferência de RNA , Estresse Mecânico , Fatores de Transcrição TCF/genética , Fatores de Tempo , Ativação Transcricional , Transfecção , beta Catenina/genéticaRESUMO
OBJECTIVE: Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. METHODS AND RESULTS: An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice. CONCLUSIONS: These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/etiologia , Hiperglicemia/metabolismo , Músculo Liso Vascular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteopontina/metabolismo , Animais , Apirase/farmacologia , Artérias/metabolismo , Sítios de Ligação , Calcineurina/metabolismo , Inibidores de Calcineurina , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Teste de Tolerância a Glucose , Humanos , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/deficiência , Fatores de Transcrição NFATC/genética , Osteopontina/deficiência , Osteopontina/genética , Regiões Promotoras Genéticas , Pirazóis/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Ativação Transcricional , Transfecção , Uridina Trifosfato/metabolismoRESUMO
Lung cancer rates are rising globally and non-small cell lung cancer (NSCLC) has a five year survival rate of only 24%. Unfortunately, the development of drugs to treat cancer is severely hampered by the inefficiency of translating pre-clinical studies into clinical benefit. Thus, we sought to apply a tumor microenvironment system (TMES) to NSCLC. Using microvascular endothelial cells, lung cancer derived fibroblasts, and NSCLC tumor cells in the presence of in vivo tumor-derived hemodynamic flow and transport, we demonstrate that the TMES generates an in-vivo like biological state and predicts drug response to EGFR inhibitors. Transcriptomic and proteomic profiling indicate that the TMES recapitulates the in vivo and patient molecular biological state providing a mechanistic rationale for the predictive nature of the TMES. This work further validates the TMES for modeling patient tumor biology and drug response indicating utility of the TMES as a predictive tool for drug discovery and development and potential for use as a system for patient avatars.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Endoteliais/metabolismo , Neoplasias Pulmonares/metabolismo , Modelos Biológicos , Microambiente Tumoral , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Células Endoteliais/patologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Camundongos SCIDRESUMO
Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Butiratos/uso terapêutico , Acidemia Propiônica/tratamento farmacológico , Acil Coenzima A/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Área Sob a Curva , Butiratos/química , Butiratos/metabolismo , Células Cultivadas , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Camundongos , Modelos Biológicos , Acidemia Propiônica/patologia , Curva ROC , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND & AIMS: Increasing evidence highlights dietary fructose as a major driver of non-alcoholic fatty liver disease (NAFLD) pathogenesis, the majority of which is cleared on first pass through the hepatic circulation by enzymatic phosphorylation to fructose-1-phosphate via the ketohexokinase (KHK) enzyme. Without a current approved therapy, disease management emphasises lifestyle interventions, but few patients adhere to such strategies. New targeted therapies are urgently required. METHODS: We have used a unique combination of human liver specimens, a murine dietary model of NAFLD and human multicellular co-culture systems to understand the hepatocellular consequences of fructose administration. We have also performed a detailed nuclear magnetic resonance-based metabolic tracing of the fate of isotopically labelled fructose upon administration to the human liver. RESULTS: Expression of KHK isoforms is found in multiple human hepatic cell types, although hepatocyte expression predominates. KHK knockout mice show a reduction in serum transaminase, reduced steatosis and altered fibrogenic response on an Amylin diet. Human co-cultures exposed to fructose exhibit steatosis and activation of lipogenic and fibrogenic gene expression, which were reduced by pharmacological inhibition of KHK activity. Analysis of human livers exposed to 13C-labelled fructose confirmed that steatosis, and associated effects, resulted from the accumulation of lipogenic precursors (such as glycerol) and enhanced glycolytic activity. All of these were dose-dependently reduced by administration of a KHK inhibitor. CONCLUSIONS: We have provided preclinical evidence using human livers to support the use of KHK inhibition to improve steatosis, fibrosis, and inflammation in the context of NAFLD. LAY SUMMARY: We have used a mouse model, human cells, and liver tissue to test how exposure to fructose can cause the liver to store excess fat and become damaged and scarred. We have then inhibited a key enzyme within the liver that is responsible for fructose metabolism. Our findings show that inhibition of fructose metabolism reduces liver injury and fibrosis in mouse and human livers and thus this may represent a potential route for treating patients with fatty liver disease in the future.
RESUMO
Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise alpha and beta subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , Hibridização In Situ , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Ligação Proteica , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Krüppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle alpha-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Aorta/citologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Diferenciação Celular , Células Cultivadas , Ciclosporina/efeitos adversos , Regulação para Baixo , Imunossupressores/efeitos adversos , Fator 4 Semelhante a Kruppel , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Túnica Média/efeitos dos fármacos , Túnica Média/patologia , Regulação para CimaRESUMO
Vascular smooth muscle cell (SMC) phenotypic modulation plays a key role in atherosclerosis and is classically defined as a switch from a 'contractile' phenotype to a 'synthetic' phenotype, whereby genes that define the contractile SMC phenotype are suppressed and proliferation and/or migratory mechanisms are induced. There is also evidence that SMCs may take on a 'proinflammatory' phenotype, whereby SMCs secrete cytokines and express cell adhesion molecules, e.g. IL-8, IL-6, and VCAM-1, respectively, which may functionally regulate monocyte and macrophage adhesion and other processes during atherosclerosis. Factors that drive the inflammatory phenotype are not limited to cytokines but also include hemodynamic forces imposed on the blood vessel wall and intimate interaction of endothelial cells with SMCs, as well as changes in matrix composition in the vessel wall. However, it is critical to recognize that our understanding of the complex interaction of these multiple signal inputs has only recently begun to shed light on mechanisms that regulate the inflammatory SMC phenotype, primarily through models that attempt to recreate this environment ex vivo. The goal of this review is to summarize our current knowledge in this area and identify some of the key unresolved challenges and questions requiring further study.