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1.
Antioxidants (Basel) ; 10(10)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34679682

RESUMO

Mechanisms involved in the individual susceptibility to atherosclerotic coronary artery disease (CAD) beyond traditional risk factors are poorly understood. Here, we describe the utility of cultured patient-derived endothelial colony-forming cells (ECFCs) in examining novel mechanisms of CAD susceptibility, particularly the role of dysregulated redox signalling. ECFCs were selectively cultured from peripheral blood mononuclear cells from 828 patients from the BioHEART-CT cohort, each with corresponding demographic, clinical and CT coronary angiographic imaging data. Spontaneous growth occurred in 178 (21.5%) patients and was more common in patients with hypertension (OR 1.45 (95% CI 1.03-2.02), p = 0.031), and less likely in patients with obesity (OR 0.62 [95% CI 0.40-0.95], p = 0.027) or obstructive CAD (stenosis > 50%) (OR 0.60 [95% CI 0.38-0.95], p = 0.027). ECFCs from patients with CAD had higher mitochondrial production of superoxide (O2--MitoSOX assay). The latter was strongly correlated with the severity of CAD as measured by either coronary artery calcium score (R2 = 0.46; p = 0.0051) or Gensini Score (R2 = 0.67; p = 0.0002). Patient-derived ECFCs were successfully cultured in 3D culture pulsatile mini-vessels. Patient-derived ECFCs can provide a novel resource for discovering mechanisms of CAD disease susceptibility, particularly in relation to mitochondrial redox signalling.

2.
Nat Commun ; 12(1): 4992, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404777

RESUMO

Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a workflow that uses these replicates to remove unwanted variation in a hierarchical manner (hRUV). We use this design to produce a dataset of more than 1000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our tools not only provide a strategy for large scale data normalisation, but also provides guidance on the design strategy for large omics studies.


Assuntos
Metabolômica/métodos , Cromatografia Líquida , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Fluxo de Trabalho
4.
Front Pharmacol ; 12: 666334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33967810

RESUMO

Aims/Hypothesis: Peripheral arterial disease (PAD) is a major burden, resulting in limb claudication, repeated surgical interventions and amputation. There is an unmet need for improved medical management of PAD that improves quality of life, maintains activities of daily life and reduces complications. Nitric oxide (NO)/redox balance is a key regulator of angiogenesis. We have previously shown beneficial effects of a ß 3 adrenergic receptor (ß 3AR) agonist on NO/redox balance. We hypothesized that ß 3AR stimulation would have therapeutic potential in PAD by promoting limb angiogenesis. Methods: The effect of the ß 3AR agonist CL 316,243 (1-1,000 nmol/L in vitro, 1 mg/kg/day s. c) was tested in established angiogenesis assays with human endothelial cells and patient-derived endothelial colony forming cells. Post-ischemia reperfusion was determined in streptozotocin and/or high fat diet-induced diabetic and non-diabetic mice in vivo using the hind limb ischemia model. Results: CL 316,243 caused accelerated recovery from hind limb ischemia in non-diabetic and type 1 and 2 diabetic mice. Increased eNOS activity and decreased superoxide generation were detected in hind limb ischemia calf muscle from CL 316, 243 treated mice vs. controls. The protective effect of CL 316,243 in diabetic mice was associated with >50% decreases in eNOS glutathionylation and nitrotyrosine levels. The ß 3AR agonist directly promoted angiogenesis in endothelial cells in vitro. These pro-angiogenic effects were ß 3AR and NOS-dependent. Conclusion/Interpretation: ß 3AR stimulation increased angiogenesis in diabetic ischemic limbs, with demonstrable improvements in NO/redox balance and angiogenesis elicited by a selective agonist. The orally available ß 3AR agonist, Mirabegron, used for overactive bladder syndrome, makes translation to a clinical trial by repurposing of a ß 3AR agonist to target PAD immediately feasible.

5.
Cells ; 10(5)2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922315

RESUMO

Despite effective prevention programs targeting cardiovascular risk factors, coronary artery disease (CAD) remains the leading cause of death. Novel biomarkers are needed for improved risk stratification and primary prevention. To assess for independent associations between plasma metabolites and specific CAD plaque phenotypes we performed liquid chromatography mass-spectrometry on plasma from 1002 patients in the BioHEART-CT study. Four metabolites were examined as candidate biomarkers. Dimethylguanidino valerate (DMGV) was associated with presence and amount of CAD (OR) 1.41 (95% Confidence Interval [CI] 1.12-1.79, p = 0.004), calcified plaque, and obstructive CAD (p < 0.05 for both). The association with amount of plaque remained after adjustment for traditional risk factors, ß-coefficient 0.17 (95% CI 0.02-0.32, p = 0.026). Glutamate was associated with the presence of non-calcified plaque, OR 1.48 (95% CI 1.09-2.01, p = 0.011). Phenylalanine was associated with amount of CAD, ß-coefficient 0.33 (95% CI 0.04-0.62, p = 0.025), amount of calcified plaque, (ß-coefficient 0.88, 95% CI 0.23-1.53, p = 0.008), and obstructive CAD, OR 1.84 (95% CI 1.01-3.31, p = 0.046). Trimethylamine N-oxide was negatively associated non-calcified plaque OR 0.72 (95% CI 0.53-0.97, p = 0.029) and the association remained when adjusted for traditional risk factors. In targeted metabolomic analyses including 53 known metabolites and controlling for a 5% false discovery rate, DMGV was strongly associated with the presence of calcified plaque, OR 1.59 (95% CI 1.26-2.01, p = 0.006), obstructive CAD, OR 2.33 (95% CI 1.59-3.43, p = 0.0009), and amount of CAD, ß-coefficient 0.3 (95% CI 0.14-0.45, p = 0.014). In multivariate analyses the lipid and nucleotide metabolic pathways were both associated with the presence of CAD, after adjustment for traditional risk factors. We report novel associations between CAD plaque phenotypes and four metabolites previously associated with CAD. We also identified two metabolic pathways strongly associated with CAD, independent of traditional risk factors. These pathways warrant further investigation at both a biomarker and mechanistic level.

6.
Circulation ; 139(13): 1612-1628, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30586761

RESUMO

BACKGROUND: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia. METHODS: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP-/-, global natriuretic peptide receptor (NPR)-B-/- and NPR-C-/- animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease. RESULTS: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP-/- and NPR-C-/-, but not NPR-B-/-, mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of Gi, ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level. CONCLUSIONS: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Animais , Hipóxia Celular , Humanos , Camundongos , Camundongos Knockout , Peptídeo Natriurético Tipo C/genética , Remodelação Vascular
7.
ACS Appl Mater Interfaces ; 9(50): 44124-44133, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29172417

RESUMO

Functionalized poly(ethylene dioxythiophene) (f-PEDOT) was copolymerized with two vinyl monomers of different hydrophilicity, acrylic acid and hydroxyethyl methacrylate, to produce electroconductive hydrogels with a range of physical and electronic properties. These hydrogels not only possessed tailored physical properties, such as swelling ratios and mechanical properties, but also displayed electroactivity dependent on the chemical composition of the network. Raman spectroscopy indicated that the functional PEDOT in the hydrogels is in an oxidized form, most likely accounting for the good electrochemical response of the hydrogels observed in physiological buffer. In vitro cell studies showed that cardiac cells respond differently when seeded on hydrogel substrates with different compositions. This study presents a facile approach for the fabrication of electroconductive hydrogels with a range of properties, paving the way for scaffolds that can meet the requirements of different electroresponsive tissues.

8.
Cells Tissues Organs ; 204(3-4): 191-198, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28772272

RESUMO

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFß1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFß1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFß1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFß1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.


Assuntos
Células Endoteliais/metabolismo , Fibrose/etiologia , Imageamento Tridimensional/métodos , Miócitos Cardíacos/metabolismo , Animais , Apoptose , Matriz Extracelular , Humanos , Camundongos , Miócitos Cardíacos/citologia
9.
Free Radic Biol Med ; 108: 585-594, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28438659

RESUMO

BACKGROUND: The novel synthetic triterpenoid, bardoxolone methyl, has the ability to upregulate cytoprotective proteins via induction of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway. This makes it a promising therapeutic agent in disease states characterized by dysregulated oxidative signalling. We have examined the effect of a Nrf2 activator, dihydro-CDDO-trifluoroethyl amide (DH404), a derivative of bardoxolone methyl, on post-infarct cardiac remodeling in rats. METHODS/RESULTS: DH404, administered from day 2 post myocardial infarction (MI: 30min transient ischemia followed by reperfusion) resulted in almost complete protection against adverse ventricular remodeling as assessed at day 28 (left ventricular end-systolic area: sham 0.14±0.01cm2, MI vehicle 0.29±0.04cm2 vs. MI DH404 0.18±0.02cm2, P<0.05); infarct size (21.3±3.4% MI vehicle vs. 10.9±2.3% MI DH404, P<0.05) with associated benefits on systolic function (fractional shortening: sham 71.9±2.6%, MI vehicle 36.2±1.9% vs. MI DH404 58.6±4.0%, P<0.05). These structural and functional benefits were associated with lower myocardial expression of atrial natriuretic peptide (ANP, P<0.01 vs. MI vehicle), and decreased fibronectin (P<0.01 vs. MI vehicle) in DH404-treated MI rats at 28 days. MI increased glutathionylation of endothelial nitric oxide synthase (eNOS) in vitro - a molecular switch that uncouples the enzyme, increasing superoxide production and decreasing nitric oxide (NO) bioavailability. MI-induced eNOS glutathionylation was substantially ameliorated by DH404. An associated increase in glutaredoxin 1 (Grx1) co-immunoprecipitation with eNOS without a change in expression was mechanistically intriguing. Indeed, in parallel in vitro experiments, silencing of Grx1 abolished the protective effect of DH404 against Angiotensin II-induced eNOS uncoupling. CONCLUSION: The bardoxolone derivative DH404 significantly attenuated cardiac remodeling post MI, at least in part, by re-coupling of eNOS and increasing the functional interaction of Grx1 with eNOS. This agent may have clinical benefits protecting against post MI cardiomyopathy.


Assuntos
Glutarredoxinas/metabolismo , Coração/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Angiotensina II/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Fibronectinas/metabolismo , Glutarredoxinas/genética , Coração/fisiologia , Humanos , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Oleanólico/química , Ácido Oleanólico/uso terapêutico , Oxirredução , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Remodelação Ventricular/efeitos dos fármacos
10.
Free Radic Biol Med ; 109: 61-74, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28188926

RESUMO

Rapid and coordinated release of a variety of reactive oxygen species (ROS) such as superoxide (O2.-), hydrogen peroxide (H2O2) and peroxynitrite, in specific microdomains, play a crucial role in cell signalling in the cardiovascular system. These reactions are mediated by reversible and functional modifications of a wide variety of key proteins. Dysregulation of this oxidative signalling occurs in almost all forms of cardiovascular disease (CVD), including at the very early phases. Despite the heavily publicized failure of "antioxidants" to improve CVD progression, pharmacotherapies such as those targeting the renin-angiotensin system, or statins, exert at least part of their large clinical benefit via modulating cellular redox signalling. Over 250 proteins, including receptors, ion channels and pumps, and signalling proteins are found in the caveolae. An increasing proportion of these are being recognized as redox regulated-proteins, that reside in the immediate vicinity of the two major cellular sources of ROS, nicotinamide adenine dinucleotide phosphate oxidase (Nox) and uncoupled endothelial nitric oxide synthase (eNOS). This review focuses on what is known about redox signalling within the caveolae, as well as endogenous protective mechanisms utilized by the cell, and new approaches to targeting dysregulated redox signalling in the caveolae as a therapeutic strategy in CVD.


Assuntos
Cardiomegalia/metabolismo , Cardiomiopatias/metabolismo , Cavéolas/metabolismo , Caveolinas/metabolismo , Insuficiência Cardíaca/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomiopatias/genética , Cardiomiopatias/patologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Cavéolas/patologia , Caveolinas/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Camundongos , Camundongos Knockout , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Transdução de Sinais
11.
Macromol Biosci ; 17(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-27973756

RESUMO

The authors report the preparation of a novel range of functional polyacrylamide stabilized polystyrene nanoparticles, obtained by surfactant-free reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization, their fluorescent tagging, cellular uptake, and biodistribution. The authors show the versatility of the RAFT emulsion process for the design of functional nanoparticles of well-defined size that can be used as drug delivery vectors. Functionalization with a fluorescent tag offers a useful visualization tool for tracing, localization, and clearance studies of these carriers in biological models. The studies are carried out by labeling the sterically stabilized latex particles chemically with rhodamine B. The fluorescent particles are incubated in a healthy human renal proximal tubular cell line model, and intravenously injected into a mouse model. Cellular localization and biodistribution of these particles on the biological models are explored.


Assuntos
Portadores de Fármacos , Corantes Fluorescentes/química , Nanopartículas/química , Imagem Óptica/métodos , Rodaminas/química , Coloração e Rotulagem/métodos , Resinas Acrílicas/química , Animais , Transporte Biológico , Linhagem Celular , Emulsões , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Nanopartículas/administração & dosagem , Tamanho da Partícula , Polimerização , Poliestirenos/química , Distribuição Tecidual
12.
Pharmacol Ther ; 165: 50-62, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27216365

RESUMO

Pulmonary arterial hypertension (PAH) is a syndrome characterised by an increase in pulmonary vascular resistance. This results in elevated resting pulmonary artery pressure and leads to progressive right ventricular (RV) failure, secondary to increased afterload. Although initially thought to be a disease driven primarily by endothelial dysfunction with a resultant vasoconstrictor versus vasodilator imbalance, it has become increasingly apparent that the rise in pulmonary vascular resistance that causes RV failure is also attributable to pulmonary vascular remodelling. This inflammatory, hyper-proliferative and anti-apoptotic phenotype is accompanied by a metabolic switch from physiological mitochondrial oxidative phosphorylation to aerobic glycolysis. The molecular pathways triggering this cellular metabolic shift have been the subject of extensive investigation, as their discovery will inevitably lead to new therapeutic targets. Reactive oxygen/nitrogen species (ROS/RNS) including hydrogen peroxide, superoxide and peroxynitrite are second messenger molecules that are involved in functional oxidative and nitrosative modification of proteins. Dysregulation of oxidative signalling caused by an excess of ROS and RNS relative to antioxidants has been heavily implicated in the underlying pathophysiology of PAH and likely participates in this metabolic reprogramming. This review will focus on the role of oxidative signalling and redox reactions to the molecular pathology of PAH. In addition, promising novel therapeutic agents targeting these pathways will be discussed.


Assuntos
Anti-Hipertensivos/uso terapêutico , Antioxidantes/uso terapêutico , Pressão Arterial/efeitos dos fármacos , Desenho de Fármacos , Hipertensão Pulmonar/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Animais , Anti-Hipertensivos/efeitos adversos , Antioxidantes/efeitos adversos , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Terapia de Alvo Molecular , Nitrosação , Oxirredução , Artéria Pulmonar/fisiopatologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Biomacromolecules ; 17(3): 965-73, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26807678

RESUMO

We present the preparation of 11 nm polyacrylamide-stabilized polystyrene latex particles for conjugation to a microRNA model by surfactant-free RAFT emulsion polymerization. Our synthetic strategy involved the preparation of amphiphilic polyacrylamide-block-polystyrene copolymers, which were able to self-assemble into polymeric micelles and "grow" into polystyrene latex particles. The surface of these sterically stabilized particles was postmodified with a disulfide-bearing linker for the attachment of the microRNA model, which can be released from the latex particles under reducing conditions. These nanoparticles offer the advantage of ease of preparation via a scaleable process, and the versatility of their synthesis makes them adaptable to a range of applications.


Assuntos
Portadores de Fármacos/síntese química , Látex/química , MicroRNAs/administração & dosagem , Nanopartículas/química , Poliestirenos/química , Resinas Acrílicas/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Emulsões/química , Oxirredução , Polimerização , Tensoativos/química
14.
Am J Physiol Renal Physiol ; 304(10): F1266-73, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23408168

RESUMO

MicroRNAs (miRNAs) comprise of a novel class of endogenous small noncoding RNAs that frequently downregulate the expression of target genes. Recent reports suggest that miRNA-200b prevents epithelial-to-mesenchymal transition (EMT) in cancer cells by targeting the E-box binding transcription factors Zinc finger E-box-binding homeobox 1 (ZEB1) and Zinc finger E-box-binding homeobox 2 (ZEB2). About 35% of active fibroblasts are derived from EMT which is central to the development of progressive renal fibrosis. Hence, this study was designed to assess the effect of miRNA-200b on transforming growth factor-ß (TGF-ß1)-induced fibrotic responses in renal tubular cells. Morphologically, human kidney-2 cells transfected with miRNA-200b retained their epithelial cell characteristics when exposed to TGF-ß1. miRNA-200b significantly increased E-cadherin (P < 0.001) and reduced fibronectin mRNA and protein expression (both P < 0.01) independent of phospho-Smad2/3 and phospho-p38 and p42/44 signaling. Increased E-cadherin expression was associated with decreased expression of ZEB1 and ZEB2 and repression of fibronectin was mediated through direct targeting of the fibronectin mRNA, demonstrated using pMIR luciferase reporter assay and site-directed mutagenesis. These results suggest that miRNA-200b suppresses TGF-ß1-induced EMT via inhibition of ZEB1 and ZEB2 and the extracellular matrix protein fibronectin by directing targeting of its 3'UTR mRNA, independent of pathways directly involved in TGF-ß1 signaling.


Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fibronectinas/metabolismo , Túbulos Renais Proximais/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , MicroRNAs/genética , Fosforilação/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Transfecção
15.
Int J Biochem Cell Biol ; 42(10): 1689-97, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20620220

RESUMO

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFbeta1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFbeta1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1alpha, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1alpha expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1alpha, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling.


Assuntos
Nefropatias Diabéticas/metabolismo , Hiperglicemia/metabolismo , Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Fatores de Transcrição/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Nefropatias Diabéticas/genética , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hiperglicemia/genética , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/patologia , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Regulação para Cima
16.
Blood ; 114(1): 85-94, 2009 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-19436054

RESUMO

Dendritic cells (DCs) play a key role in the pathogenesis of HIV infection. HIV interacts with these cells through 2 pathways in 2 temporal phases, initially via endocytosis and then via de novo replication. Here the transcriptional response of human DCs to HIV-1 was studied in these phases and at different stages of the virus replication cycle using purified HIV-1 envelope proteins, and inactivated and viable HIV-1. No differential gene expression was detected in response to envelope. However, more than 100 genes were differentially expressed in response to entry of viable and inactivated HIV-1 in the first phase. A completely different set of genes was differentially expressed in the second phase, predominantly in response to viable HIV-1, including up-regulation of immune regulation genes, whereas genes encoding lysosomal enzymes were down-regulated. Cathepsins B, C, S, and Z RNA and protein decreased, whereas cathepsin L was increased, probably reflecting a concomitant decrease in cystatin C. The net effect was markedly diminished cathepsin activity likely to result in enhanced HIV-1 survival and transfer to contacting T lymphocytes but decreased HIV-1 antigen processing and presentation to these T cells.


Assuntos
Células Dendríticas/virologia , HIV-1/genética , HIV-1/patogenicidade , Apresentação do Antígeno , Sequência de Bases , Catepsinas/genética , Catepsinas/metabolismo , Cistatinas/genética , Cistatinas/metabolismo , Primers do DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Expressão Gênica , Genes Virais , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Cinética , Lisossomos/enzimologia , Lisossomos/genética , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T/virologia , Internalização do Vírus , Replicação Viral
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