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1.
FASEB J ; 33(5): 6667-6681, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30779601

RESUMO

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-ß. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing.-Andrews, D., Oliviero, G., De Chiara, L., Watson, A., Rochford, E., Wynne, K., Kennedy, C., Clerkin, S., Doyle, B., Godson, C., Connell, P., O'Brien, C., Cagney, G., Crean, J. Unravelling the transcriptional responses of TGF-ß: Smad3 and EZH2 constitute a regulatory switch that controls neuroretinal epithelial cell fate specification.

2.
Eur J Med Chem ; 162: 80-108, 2018 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-30419493

RESUMO

Lipoxins (LXs) are endogenously generated eicosanoids with potent bio-actions consistent with attenuation of inflammation. The costly synthesis and metabolic instability of LXs may limit their therapeutic potential. Here we report the synthesis and characterization of novel imidazole-/oxazole-containing synthetic-LX-mimetics (sLXms). The key steps of asymmetric synthesis of putative sLXms include a Suzuki reaction and an asymmetric ketone reduction. The effect of the novel compounds on inflammatory responses was assessed using a human monocyte cell line stably expressing a Nuclear Factor Kappa B (NFkB) reporter gene, by investigating downstream cytokine secretion. The potential interaction of the imidazoles/oxazoles with the molecular target of LXs, i.e. G-protein coupled receptor (GPCR) Formyl Peptide Receptor 2 (ALX/FPR2) was investigated using a cell system where ALX/FPR2 is coupled to the Gαq subunit and receptor interaction determined by mobilisation of intracellular calcium. In vivo anti-inflammatory effects were assessed using a murine zymosan-induced peritonitis model. Overall, structure-activity relationship (SAR) studies demonstrated that the (R)-epimer of 6C-dimethyl-imidazole (1R)-11 was the most potent and efficient anti-inflammatory agent, among the ten compounds tested. This molecule significantly attenuated LPS-induced NFkB activity, reduced the release of several pro-inflammatory cytokines and inhibited peritonitis-associated neutrophil infiltration in vivo. The underlying mechanism for those actions appeared to be through FPR2 activation. These data support the therapeutic potential of imidazole-containing sLXms in the context of novel inflammatory regulators.

3.
Front Cardiovasc Med ; 5: 148, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30460244

RESUMO

Finding genetic variants that cause functional disruption or regulatory change among the many implicated GWAs variants remains a key challenge to translating the findings from GWAs to therapeutic treatments. Defining the causal mechanisms behind the variants require functional screening experiments that can be complex and costly. Prioritizing variants for functional characterization using techniques that capture important functional and regulatory elements can assist this. The genetic architecture of complex traits such as cardiovascular disease and type II diabetes comprise an enormously large number of variants of small effect contributing to heritability and spread throughout the genome. This makes it difficult to distinguish which variants or core genes are most relevant for prioritization and how they contribute to the regulatory networks that become dysregulated leading to disease. Despite these challenges, recent GWAs for CAD prioritized genes associated with lipid metabolism, coagulation and adhesion along with novel signals related to innate immunity, adipose tissue and, vascular function as important core drivers of risk. We focus on three examples of novel signals associated with CAD which affect risk through missense or UTR mutations indicating their potential for therapeutic modification. These variants play roles in adipose tissue function vascular function and innate immunity which form the cornerstones of immuno-metabolism. In addition we have explored the putative, but potentially important interactions between the environment, specifically food and nutrition, with respect to key processes.

4.
Semin Immunol ; 40: 36-48, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30293857

RESUMO

Inflammation is an essential response to injury and its timely and adequate resolution permits tissue repair and avoidance of chronic inflammation. Ageing is associated with increased inflammation, sub-optimal resolution and these act as drivers for a number of ageing-associated pathologies. We describe the role played by specialised proresolving lipid mediators (SPMs) in the resolution of inflammation and how insufficient levels of these mediators, or compromised responsiveness may play a role in the pathogenesis of many ageing-associated pathologies, e.g. Alzheimer's Disease, atherosclerosis, obesity, diabetes and kidney disease. Detailed examination of the resolution phase of inflammation highlights the potential to harness these lipid mediators and or mimetics of their bioactions, in particular, their synthetic analogues to promote effective resolution of inflammation, without compromising the host immune system.

5.
Diabetes ; 67(12): 2657-2667, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30213823

RESUMO

Increasing evidence points to the fact that defects in the resolution of inflammatory pathways predisposes individuals to the development of chronic inflammatory diseases, including diabetic complications such as accelerated atherosclerosis. The resolution of inflammation is dynamically regulated by the production of endogenous modulators of inflammation, including lipoxin A4 (LXA4). Here, we explored the therapeutic potential of LXA4 and a synthetic LX analog (Benzo-LXA4) to modulate diabetic complications in the streptozotocin-induced diabetic ApoE-/- mouse and in human carotid plaque tissue ex vivo. The development of diabetes-induced aortic plaques and inflammatory responses of aortic tissue, including the expression of vcam-1, mcp-1, il-6, and il-1ß, was significantly attenuated by both LXA4 and Benzo-LXA4 in diabetic ApoE-/- mice. Importantly, in mice with established atherosclerosis, treatment with LXs for a 6-week period, initiated 10 weeks after diabetes onset, led to a significant reduction in aortic arch plaque development (19.22 ± 2.01% [diabetic]; 12.67 ± 1.68% [diabetic + LXA4]; 13.19 ± 1.97% [diabetic + Benzo-LXA4]). Secretome profiling of human carotid plaque explants treated with LXs indicated changes to proinflammatory cytokine release, including tumor necrosis factor-α and interleukin-1ß. LXs also inhibited platelet-derived growth factor-stimulated vascular smooth muscle cell proliferation and transmigration and endothelial cell inflammation. These data suggest that LXs may have therapeutic potential in the context of diabetes-associated vascular complications.

6.
J Am Soc Nephrol ; 29(5): 1437-1448, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29490938

RESUMO

Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation.Methods We investigated the potential of LXA4 and a synthetic LX analog (Benzo-LXA4) as therapeutics in a murine model of diabetic kidney disease, ApoE-/- mice treated with streptozotocin.Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1ß, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-α, NF-κB) and novel (early growth response-1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α-driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-ß1 and TNF-αConclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.

7.
Cardiovasc Diabetol ; 16(1): 143, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-29110715

RESUMO

BACKGROUND: Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response. METHODS: Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE-/-) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE-/- mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 µg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE-/- mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry. RESULTS: Liraglutide decreased atherosclerotic lesion formation in ApoE-/- mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 µg/kg liraglutide treatment in ApoE-/- mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells. CONCLUSIONS: This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Hipoglicemiantes/uso terapêutico , Liraglutida/uso terapêutico , Macrófagos/metabolismo , Fenótipo , Animais , Aterosclerose/tratamento farmacológico , Linhagem Celular , Humanos , Hipoglicemiantes/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Liraglutida/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3095-3104, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28923783

RESUMO

Bone morphogenetic protein-7 (BMP-7) counteracts pro-fibrotic effects of TGFß1 in cultured renal cells and protects from fibrosis in acute and chronic renal injury models. Using the unilateral ureteral obstruction (UUO) model of chronic renal fibrosis, we investigated the effect of exogenous-rhBMP-7 on pro-fibrotic signaling pathways mediated by TGFß1 and hypoxia. Mice undergoing UUO were treated with vehicle or rhBMP-7 (300µg/kg i.p.) every other day for eight days and kidneys analysed for markers of fibrosis and SMAD, MAPK, and PI3K signaling. In the kidney, collecting duct and tubular epithelial cells respond to BMP-7 via activation of SMAD1/5/8. Phosphorylation of SMAD1/5/8 was reduced in UUO kidneys from vehicle-treated animals yet maintained in UUO kidneys from BMP-7-treated animals, confirming renal bioactivity of exogenous rhBMP-7. BMP-7 inhibited Collagen Iα1 and Collagen IIIα1 gene expression and Collagen I protein accumulation, while increasing expression of Collagen IVα1 in UUO kidneys. Activation of SMAD2, SMAD3, ERK, p38 and PI3K/Akt signaling occurred during fibrogenesis and BMP-7 significantly attenuated SMAD3 and Akt signaling in vivo. Analysis of renal collecting duct (mIMCD) and tubular epithelial (HK-2) cells stimulated with TGFß1 or hypoxia (1% oxygen) to activate Akt provided further evidence that BMP-7 specifically inhibited PI3K/Akt signaling. PTEN is a negative regulator of PI3K and BMP-7 increased PTEN expression in vivo and in vitro. These data demonstrate an important mechanism by which BMP-7 orchestrates renal protection through Akt inhibition and highlights Akt inhibitors as anti-fibrotic therapeutics.

9.
Mol Aspects Med ; 58: 102-113, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28479307

RESUMO

Inflammation and its timely resolution play a critical role in effective host defence and wound healing. Unresolved inflammatory responses underlie the pathology of many prevalent diseases resulting in tissue fibrosis and eventual organ failure as typified by kidney, lung and liver fibrosis. The role of autocrine and paracrine mediators including cytokines, prostaglandins and leukotrienes in initiating and sustaining inflammation is well established. More recently a physiological role for specialized pro-resolving lipid mediators [SPMs] in modulating inflammatory responses and promoting the resolution of inflammation has been appreciated. As will be discussed in this review, SPMs not only attenuate the development of fibrosis through promoting the resolution of inflammation but may also directly suppress fibrotic responses. These findings suggest novel therapeutic paradigms to treat intractable life-limiting diseases such as renal fibrosis.


Assuntos
Mediadores da Inflamação/metabolismo , Nefropatias/etiologia , Nefropatias/metabolismo , Animais , Biomarcadores , Progressão da Doença , Fibrose , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/uso terapêutico , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Metabolismo dos Lipídeos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais
10.
Diabetes ; 66(8): 2266-2277, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28487436

RESUMO

The let-7 miRNA family plays a key role in modulating inflammatory responses. Vascular smooth muscle cell (SMC) proliferation and endothelial cell (EC) dysfunction are critical in the pathogenesis of atherosclerosis, including in the setting of diabetes. Here we report that let-7 levels are decreased in diabetic human carotid plaques and in a model of diabetes-associated atherosclerosis, the diabetic ApoE-/- mouse. In vitro platelet-derived growth factor (PDGF)- and tumor necrosis factor-α (TNF-α)-induced vascular SMC and EC activation was associated with reduced let-7 miRNA expression via Lin28b, a negative regulator of let-7 biogenesis. Ectopic overexpression of let-7 in SMCs inhibited inflammatory responses including proliferation, migration, monocyte adhesion, and nuclear factor-κB activation. The therapeutic potential of restoring let-7 levels using a let-7 mimic was tested: in vitro in SMCs using an endogenous anti-inflammatory lipid (lipoxin A4), ex vivo in murine aortas, and in vivo via tail vein injection in a 24-h murine model. Furthermore, we delivered let-7 mimic to human carotid plaque ex vivo and observed significant changes to the secretome in response to let-7 therapy. Restoration of let-7 expression could provide a new target for an anti-inflammatory approach in diabetic vascular disease.


Assuntos
Aterosclerose/genética , Estenose das Carótidas/genética , Complicações do Diabetes/genética , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Apolipoproteínas E/genética , Artérias Carótidas/citologia , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/administração & dosagem , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Diabetologia ; 60(4): 729-739, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28188334

RESUMO

AIMS/HYPOTHESIS: In this study, we aimed to evaluate the therapeutic potential of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase, for ameliorating high-fat diet (HFD)-induced pathophysiology in mice. We also aimed to determine whether the beneficial effects of AICAR were dependent on adiponectin. Furthermore, human adipose tissue was used to examine the effect of AICAR ex vivo. METHODS: Six-week-old male C57BL/6J wild-type and Adipoq -/- mice were fed a standard-fat diet (10% fat) or an HFD (60% fat) for 12 weeks and given vehicle or AICAR (500 µg/g) three times/week from weeks 4-12. Diet-induced pathophysiology was examined in mice after 11 weeks by IPGTT and after 12 weeks by flow cytometry and western blotting. Human adipose tissue biopsies from obese (BMI 35-50 kg/m2) individuals were incubated with vehicle or AICAR (1 mmol/l) for 6 h at 37°C, after which inflammation was characterised by ELISA (TNF-α) and flow cytometry. RESULTS: AICAR attenuated adipose inflammation in mice fed an HFD, promoting an M1-to-M2 macrophage phenotype switch, while reducing infiltration of CD8+ T cells. AICAR treatment of mice fed an HFD partially restored glucose tolerance and attenuated hepatic steatosis and kidney disease, as evidenced by reduced albuminuria (p < 0.05), urinary H2O2 (p < 0.05) and renal superoxide levels (p < 0.01) in both wild-type and Adipoq -/- mice. AICAR-mediated protection occurred independently of adiponectin, as similar protection was observed in wild-type and Adipoq -/- mice. In addition, AICAR promoted an M1-to-M2 macrophage phenotype switch and reduced TNF-α production in tissue explants from obese human patients. CONCLUSIONS/INTERPRETATION: AICAR may promote metabolic health and protect against obesity-induced systemic diseases in an adiponectin-independent manner. Furthermore, AICAR reduced inflammation in human adipose tissue explants, suggesting by proof-of-principle that the drug may reduce obesity-induced complications in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT02322073.


Assuntos
Adiponectina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Adiponectina/genética , Animais , Inflamação/imunologia , Inflamação/metabolismo , Nefropatias/imunologia , Nefropatias/metabolismo , Hepatopatias/imunologia , Hepatopatias/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/imunologia , Obesidade/metabolismo
12.
Front Immunol ; 8: 7, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28167941

RESUMO

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1-3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1-3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.

13.
Sci Rep ; 6: 31355, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27531581

RESUMO

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.


Assuntos
Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência de RNA/métodos , Transcrição Genética , Hipóxia Celular , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Transdução de Sinais
14.
Mol Ther ; 24(7): 1290-301, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27203438

RESUMO

The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-ß1, and TGF-ß type 1 receptor (TGF-ßR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-ß1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-ßR1 confirmed that miR-let7c attenuates TGF-ß1-driven TGF-ßR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.


Assuntos
Exossomos/metabolismo , Nefropatias/genética , Nefropatias/patologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Actinas/metabolismo , Animais , Transporte Biológico , Engenharia Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Fibrose , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Nefropatias/metabolismo , Nefropatias/terapia , Masculino , Camundongos , Ratos , Transdução Genética
15.
Am J Physiol Renal Physiol ; 311(1): F35-45, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27122540

RESUMO

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3ß (Ser9), nuclear accumulation of ß-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-ß (TGF-ß); Wnt6 reversed TGF-ß-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-ß-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/ß(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.


Assuntos
Diferenciação Celular/genética , Nefropatias/genética , Nefropatias/patologia , Rim/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/fisiologia , Animais , Células Epiteliais/patologia , Feminino , Fibrose , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas I-kappa B/genética , Rim/embriologia , Nefropatias/induzido quimicamente , Túbulos Renais/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética , Fator de Transcrição RelA/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Vimentina/biossíntese
16.
Diabetes ; 64(12): 4238-46, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26307587

RESUMO

Obesity has been posited as an independent risk factor for diabetic kidney disease (DKD), but establishing causality from observational data is problematic. We aimed to test whether obesity is causally related to DKD using Mendelian randomization, which exploits the random assortment of genes during meiosis. In 6,049 subjects with type 1 diabetes, we used a weighted genetic risk score (GRS) comprised of 32 validated BMI loci as an instrument to test the relationship of BMI with macroalbuminuria, end-stage renal disease (ESRD), or DKD defined as presence of macroalbuminuria or ESRD. We compared these results with cross-sectional and longitudinal observational associations. Longitudinal analysis demonstrated a U-shaped relationship of BMI with development of macroalbuminuria, ESRD, or DKD over time. Cross-sectional observational analysis showed no association with overall DKD, higher odds of macroalbuminuria (for every 1 kg/m(2) higher BMI, odds ratio [OR] 1.05, 95% CI 1.03-1.07, P < 0.001), and lower odds of ESRD (OR 0.95, 95% CI 0.93-0.97, P < 0.001). Mendelian randomization analysis showed a 1 kg/m(2) higher BMI conferring an increased risk in macroalbuminuria (OR 1.28, 95% CI 1.11-1.45, P = 0.001), ESRD (OR 1.43, 95% CI 1.20-1.72, P < 0.001), and DKD (OR 1.33, 95% CI 1.17-1.51, P < 0.001). Our results provide genetic evidence for a causal link between obesity and DKD in type 1 diabetes. As obesity prevalence rises, this finding predicts an increase in DKD prevalence unless intervention should occur.


Assuntos
Albuminúria/etiologia , Diabetes Mellitus Tipo 1/complicações , Nefropatias Diabéticas/etiologia , Falência Renal Crônica/etiologia , Obesidade/fisiopatologia , Adulto , Albuminúria/epidemiologia , Albuminúria/genética , Índice de Massa Corporal , Estudos de Casos e Controles , Estudos de Coortes , Estudos Transversais , Nefropatias Diabéticas/epidemiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/fisiopatologia , Feminino , Finlândia/epidemiologia , Loci Gênicos , Predisposição Genética para Doença , Humanos , Incidência , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/genética , Estudos Longitudinais , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/genética , Prevalência , Fatores de Risco
17.
Cell Metab ; 22(1): 125-37, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26052006

RESUMO

The role of inflammation in obesity-related pathologies is well established. We investigated the therapeutic potential of LipoxinA4 (LXA4:5(S),6(R),15(S)-trihydroxy-7E,9E,11Z,13E,-eicosatetraenoic acid) and a synthetic 15(R)-Benzo-LXA4-analog as interventions in a 3-month high-fat diet (HFD; 60% fat)-induced obesity model. Obesity caused distinct pathologies, including impaired glucose tolerance, adipose inflammation, fatty liver, and chronic kidney disease (CKD). Lipoxins (LXs) attenuated obesity-induced CKD, reducing glomerular expansion, mesangial matrix, and urinary H2O2. Furthermore, LXA4 reduced liver weight, serum alanine-aminotransferase, and hepatic triglycerides. LXA4 decreased obesity-induced adipose inflammation, attenuating TNF-α and CD11c(+) M1-macrophages (MΦs), while restoring CD206(+) M2-MΦs and increasing Annexin-A1. LXs did not affect renal or hepatic MΦs, suggesting protection occurred via attenuation of adipose inflammation. LXs restored adipose expression of autophagy markers LC3-II and p62. LX-mediated protection was demonstrable in adiponectin(-/-) mice, suggesting that the mechanism was adiponectin independent. In conclusion, LXs protect against obesity-induced systemic disease, and these data support a novel therapeutic paradigm for treating obesity and associated pathologies.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Inflamação/complicações , Inflamação/tratamento farmacológico , Nefropatias/complicações , Lipoxinas/uso terapêutico , Hepatopatias/complicações , Obesidade/complicações , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/patologia , Animais , Autofagia/efeitos dos fármacos , Inflamação/imunologia , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/imunologia , Nefropatias/patologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/imunologia , Hepatopatias/patologia , Camundongos Endogâmicos C57BL , Obesidade/imunologia
18.
Semin Immunol ; 27(3): 169-76, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26048571

RESUMO

Inflammation is a complex process governed by the interaction of multiple cell types of the innate immune system and secreted mediators. Such mediators may act in a paracrine or autocrine fashion on target effector cells. An appropriate inflammatory response is characterised by dynamically regulated initiation, propagation and eventual resolution and restoration of tissue homeostasis. Dysregulation of any of these processes may underlie chronic inflammatory conditions such as atherosclerosis, diabetes and arthritis. Our growing understanding of the active processes underlying the resolution of inflammation suggest novel therapeutic paradigms. Here we review specialised lipid mediators and their targets which regulate such innate processes.


Assuntos
Citocinas/imunologia , Ácidos Docosa-Hexaenoicos/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Lipoxinas/imunologia , Doença Crônica , Homeostase/imunologia , Humanos , Imunidade Inata/imunologia
19.
Trends Cell Biol ; 25(5): 249-64, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25592806

RESUMO

Bone morphogenetic proteins (BMPs) are secreted extracellular matrix (ECM)-associated proteins that regulate a wide range of developmental processes, including limb and kidney formation. A critical element of BMP regulation is the presence of secreted antagonists that bind and inhibit BMP binding to their cognate Ser/Thr kinase receptors at the plasma membrane. Antagonists such as Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) have been shown to inhibit BMP action in a range of different cell types and developmental stage-specific contexts. Here we review new developments in the field of BMP and BMP antagonist biology during mammalian development and suggest strategies for targeting these proteins in human disease.


Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/química , MicroRNAs/química , Transdução de Sinais , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
20.
Exp Cell Res ; 330(2): 371-81, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25107382

RESUMO

Epithelial injury and tubulointerstitial fibrosis (TIF) within a hypoxic microenvironment are associated with progressive loss of renal function in chronic kidney disease [CKD]. Transforming growth factor beta-1 (TGF-ß1) is an important mediator of renal fibrosis. Growing evidence suggests that Vitamin D [1,25-(OH)2D] and its analogues may have a renoprotective effect in CKD. Here we examined the protective effect of the vitamin D analogue paricalcitol [PC; 19-nor-1α,3ß,25-trihydroxy-9,10-secoergosta-5(Z),7(E) 22(E)-triene] on the responses of human renal epithelial cells to TGF-ß1. PC attenuated TGF-ß1-induced Smad 2 phosphorylation and upregulation of the Notch ligand Jagged-1, α-smooth muscle actin and thrombospondin-1 and prevented the TGF-ß1-mediated loss of E-Cadherin. To mimic the hypoxic milieu of CKD we cultured renal epithelial cells in hypoxia [1% O2] and observed similar attenuation by PC of TGF-ß1-induced fibrotic responses. Furthermore, in cells cultured in normoxia [21% O2], PC induced an accumulation of hypoxia-inducible transcription factors (HIF) 1α and HIF-2α in a time and concentration [1 µM-2 µM] dependent manner. Here, PC-induced HIF stabilisation was dependent on activation of the PI-3Kinase pathway. This is the first study to demonstrate regulation of the HIF pathway by PC which may have importance in the mechanism underlying renoprotection by PC.


Assuntos
Células Epiteliais/efeitos dos fármacos , Ergocalciferóis/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Actinas/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Hipóxia Celular , Linhagem Celular Transformada , Células Epiteliais/patologia , Fibrose , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteína Jagged-1 , Proteínas de Membrana/biossíntese , Nefrite Intersticial/patologia , Fosforilação , Estabilidade Proteica , Interferência de RNA , Proteínas Serrate-Jagged , Proteína Smad2/metabolismo , Trombospondina 1/biossíntese , Fator de Crescimento Transformador beta1/metabolismo
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