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1.
Eur Heart J ; 43(45): 4739-4750, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36200607

RESUMO

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.


Assuntos
Insuficiência Cardíaca , Remodelação Ventricular , Camundongos , Animais , Fibrose , Miofibroblastos/metabolismo , Cardiomegalia/metabolismo , Fibroblastos/metabolismo , Insuficiência Cardíaca/patologia , Miocárdio/patologia , Camundongos Endogâmicos C57BL
2.
Development ; 144(9): 1698-1711, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28356311

RESUMO

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.


Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Fatores de Transcrição/metabolismo , Vertebrados/metabolismo , Células Amácrinas/citologia , Células Amácrinas/metabolismo , Animais , Apoptose/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Contagem de Células , Divisão Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Células Cultivadas , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/metabolismo , Eletroporação , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Brn-3B/deficiência , Fatores de Transcrição/deficiência
3.
Can J Physiol Pharmacol ; 97(6): 503-514, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30576226

RESUMO

Cardiovascular disease (CVD) is the number one cause of death in both men and women. Younger women have a lower risk for CVD, but their risk increases considerably after menopause when estrogen levels decrease. The cardiovascular protective properties of estrogen are mediated through decreasing vascular inflammation and progression of atherosclerosis, decreasing endothelial cell damage by preventing apoptosis and anti-hypertrophic mechanisms. Estrogen also regulates glucose and lipid levels, which are 2 important risk factors for CVD. Resveratrol (RES), a cardioprotective polyphenolic compound, is classified as a phytoestrogen due its capacity to bind to and modulate estrogen receptor signalling. Due to its estrogen-like property, we speculate that the cardioprotective effects of RES treatment could be sex-dependent. Based on earlier reports and more recent data from our lab presented here, we found that RES treatment may have more favourable cardiovascular outcomes in females than in males. This review will discuss estrogen- and phytoestrogen-mediated cardioprotection, with a specific focus on sex-dependent effects reported in preclinical and clinical studies.


Assuntos
Cardiotônicos/farmacologia , Fitoestrógenos/farmacologia , Resveratrol/farmacologia , Caracteres Sexuais , Animais , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/prevenção & controle , Humanos
4.
Can J Physiol Pharmacol ; 97(12): 1132-1140, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31374178

RESUMO

Long-chain saturated fatty acids, especially palmitic acid (PA), contribute to cardiomyocyte lipotoxicity. This study tests the effects of PA on adult rat cardiomyocyte contractile function and proteins associated with calcium regulating cardiomyocyte contraction and relaxation. Adult rat cardiomyocytes were pretreated with resveratrol (Resv) and then treated with PA. For the reversal study, cardiomyocytes were incubated with PA prior to treatment with Resv. Cardiomyocyte contractility, ratio of rod- to round-shaped cardiomyocytes, and Hoechst staining were used to measure functional and morphological changes in cardiomyocytes. Protein expression of sarco-endoplasmic reticulum ATPase 2a (SERCA2a), native phospholamban (PLB) and phosphorylated PLB (pPLB ser16 and pPLB thr17), and troponin I (TnI) and phosphorylated TnI (pTnI) were measured. SERCA2a activity was also measured. Our results show that PA (200 µM) decreased the rate of cardiomyocyte relaxation, reduced the number of rod-shaped cardiomyocytes, and increased the number of cells with condensed nuclei; pre-treating cardiomyocytes with Resv significantly prevented these changes. Post-treatment with Resv did not reverse morphological changes induced by PA. Protein expression levels of SERCA2a, PLB, pPLBs, TnI, and pTnI were unchanged by PA or Resv. SERCA2a activity assay showed that Vmax and Iono ratio were increased with PA and pre-treatment with Resv prevented this increase. In conclusion, our results show that Resv protect cardiomyocytes from contractile dysfunction induced by PA.


Assuntos
Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ácido Palmítico/efeitos adversos , Resveratrol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Troponina I/metabolismo
5.
Can J Physiol Pharmacol ; 97(8): 786-795, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237455

RESUMO

Exercise enhances cardiac sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) function through unknown mechanisms. The present study tested the hypothesis that the positive effects of exercise on SERCA2a expression and function in the left ventricle is dependent on adenosine-monophosphate-activated protein kinase (AMPK) α2 function. AMPKα2 kinase-dead (KD) transgenic mice, which overexpress inactivated AMPKα2 subunit, and wild-type C57Bl/6 (WT) mice were randomized into sedentary groups or groups with access to running wheels. After 5 months, exercised KD mice exhibited shortened deceleration time compared with sedentary KD mice. In left ventricular tissue, the ratio of phosphorylated AMPKαThr172:total AMPKα was 65% lower (P < 0.05) in KD mice compared with WT mice. The left ventricle of KD mice had 37% lower levels of SERCA2a compared with WT mice. Although exercise increased SERCA2a protein levels in WT mice by 53%, this response of exercise was abolished in exercised KD mice. Exercise training reduced total phospholamban protein content by 23% in both the WT and KD mice but remained 20% higher overall in KD mice. Collectively, these data suggest that AMPKα influences SERCA2a and phospholamban protein content in the sedentary and exercised heart, and that exercise-induced changes in SERCA2a protein are dependent on AMPKα function.


Assuntos
Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Condicionamento Físico Animal , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Diástole/fisiologia , Masculino , Camundongos , Fosforilação , Comportamento Sedentário
6.
Molecules ; 24(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319579

RESUMO

In this study, we tested the potential cardioprotective effects of the phytoalexin resveratrol (Rsv) on primary adult rat cardiac fibroblasts (CF), myofibroblasts (MF) and cardiomyocytes. Adult rat CF and cardiomyocytes were isolated from male 10-week old Sprague-Dawley rats, cultured for either 24 h (cardiomyocytes) or 48 h (CF) before treatments. To isolate MF, CF were trypsinized after 48 h in culture, seeded in fresh plates and cultured for 24 h prior to treatment. All three cells were then treated for a further 24 h with a range of Rsv doses. In CF and MF, cell proliferation, viability, apoptosis assays were performed with or without Rsv treatment for 24 h. In cardiomyocytes, cell viability and apoptosis assay were performed 24 h after treatment. In separate experiments, CF was pre-incubated with estrogen, tamoxifen and fulvestrant for 30 min prior to Rsv treatment. Rsv treatment decreased proliferation of both fibroblasts and myofibroblasts. Rsv treatment also increased the proportion of dead CF and MF in a dose dependent manner. However, treatment with Rsv did not induce cell death in adult cardiomyocytes. There was an increase in the percentage of cells with condensed nuclei with Rsv treatment in both CF and MF, but not in cardiomyocytes. Treatment with estrogen, tamoxifen and fulvestrant alone or in combination with Rsv did not have any additional effects on CF survival. Our results demonstrate that treatment with Rsv can inhibit cell proliferation and induce cell death in rat CF and MF, while not affecting cardiomyocyte survival. We also demonstrated that the induction of cell death in CF with Rsv treatment was independent of estrogen receptor alpha (ERα) signaling.


Assuntos
Proliferação de Células/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Receptor alfa de Estrogênio/genética , Humanos , Ratos , Sesquiterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Tamoxifeno/farmacologia , Fitoalexinas
7.
J Mol Cell Cardiol ; 120: 64-73, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29750994

RESUMO

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFß occurs via a scleraxis-dependent mechanism: TGFß induces recruitment of scleraxis to the MMP2 promoter, and TGFß was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , Miocárdio/citologia , Miofibroblastos/fisiologia , Análise de Variância , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Elementos E-Box/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Knockout , Células NIH 3T3 , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
8.
Int J Mol Sci ; 19(10)2018 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-30336567

RESUMO

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and α-smooth muscle actin (α-SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.


Assuntos
Fibroblastos/metabolismo , Miocárdio/citologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Biomarcadores/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Miofibroblastos/metabolismo , Fenótipo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley
9.
Can J Physiol Pharmacol ; 95(10): 1067-1077, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28727928

RESUMO

In the vascular system, ageing is accompanied by the accrual of senescent cells and is associated with an increased risk of vascular disease. Endothelial cell (EC) dysfunction is a hallmark of vascular disease and is characterized by decreased angiogenic potential, reduced nitric oxide bioavailability, impaired vasodilation, increased production of ROS, and enhanced inflammation. In ECs, the major producer of nitric oxide is the endothelial nitric oxide synthase (eNOS) enzyme that is encoded by the NOS3 gene. NOS3/eNOS function is tightly regulated at both the transcriptional and post-transcriptional levels to maintain normal vascular function. A key transcriptional regulator of eNOS expression is p53, which has been shown to play a central role in mediating cellular senescence and thereby vascular dysfunction. Herein, we show that, in ECs, the MEOX homeodomain transcription factors decrease the expression of genes involved in angiogenesis, repress eNOS expression at the mRNA and protein levels, and increase the expression of p53. These findings support a role for the MEOX proteins in promoting endothelial dysfunction.


Assuntos
Envelhecimento/metabolismo , Vasos Sanguíneos/metabolismo , Endotélio Vascular/metabolismo , Hemodinâmica , Proteínas de Homeodomínio/metabolismo , Doenças Vasculares/metabolismo , Fatores Etários , Envelhecimento/genética , Animais , Vasos Sanguíneos/fisiopatologia , Senescência Celular , Endotélio Vascular/fisiopatologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Doenças Vasculares/genética , Doenças Vasculares/fisiopatologia
10.
J Mol Cell Cardiol ; 92: 140-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26883788

RESUMO

Cardiac fibroblasts are the major extracellular matrix producing cells in the heart. Our laboratory was the first to demonstrate that the transcription factor scleraxis induces collagen 1α2 expression in both cardiac fibroblasts and myofibroblasts. Here we identify a novel post-translational mechanism by which scleraxis activity is regulated and determine its effect on transcription of genes targeted by scleraxis. Putative serine phosphorylation sites on scleraxis were revealed by in silico analysis using motif prediction software. Mutation of key serine residues to alanine, which cannot be phosphorylated, significantly attenuated the expression of fibrillar type I collagen and myofibroblast marker genes that are normally induced by scleraxis. Down-regulation of collagen 1α2 expression was due to reduced binding of the non-phosphorylated scleraxis mutant to specific E-box DNA-binding sites within the promoter as determined by chromatin immunoprecipitation in human cardiac myofibroblast cells and by electrophoretic mobility shift assay. This is the first evidence suggesting that scleraxis is phosphorylated under basal conditions. The phosphorylation sequence matched that targeted by Casein Kinase 2, and inhibition of this kinase activity disrupted the ability of scleraxis to modulate the expression of its target genes while also attenuating TGFß-induced expression of type I collagen and myofibroblast phenotype conversion marker genes. These results demonstrate a novel mechanism for regulation of scleraxis activity, which may prove to be tractable for pharmacologic manipulation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Colágeno Tipo I/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Substituição de Aminoácidos/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Caseína Quinase II/antagonistas & inibidores , Colágeno Tipo I/biossíntese , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Miofibroblastos/metabolismo , Fosforilação , Serina/genética , Serina/metabolismo , Ativação Transcricional/genética , Fator de Crescimento Transformador beta1/biossíntese
11.
Am J Physiol Cell Physiol ; 311(2): C297-307, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27357547

RESUMO

The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-ß, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Coração/fisiologia , Miofibroblastos/metabolismo , Miofibroblastos/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Miocárdio/metabolismo , Células NIH 3T3 , Fenótipo , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
12.
J Cell Sci ; 127(Pt 1): 40-9, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24155330

RESUMO

Cardiac fibrosis is linked to fibroblast-to-myofibroblast phenoconversion and proliferation but the mechanisms underlying this are poorly understood. Ski is a negative regulator of TGF-ß-Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-ß-mediated cellular processes and is repressed by Zeb2. Here, we investigated whether Ski diminishes the myofibroblast phenotype by de-repressing Meox2 expression and function through repression of Zeb2 expression. We show that expression of Meox1 and Meox2 mRNA and Meox2 protein is reduced during phenoconversion of fibroblasts to myofibroblasts. Overexpression of Meox2 shifts the myofibroblasts into fibroblasts, whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Overexpression of Ski partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. Expression of Zeb2 increased during phenoconversion and Ski overexpression reduces Zeb2 expression in first-passage myofibroblasts. Furthermore, expression of Meox2 is decreased in scar following myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by upregulating the expression of Meox2. This cascade might regulate cardiac myofibroblast phenotype and presents therapeutic options for treatment of cardiac fibrosis.


Assuntos
Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Musculares/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Proteínas de Homeodomínio/agonistas , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas Musculares/agonistas , Proteínas Musculares/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miofibroblastos/patologia , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de Zinco
13.
Am J Physiol Heart Circ Physiol ; 310(2): H239-49, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26566727

RESUMO

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFß1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFß/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFß1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFß1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFß1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFß1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Miócitos Cardíacos/metabolismo , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Células 3T3 , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Butadienos/farmacologia , Células COS , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Chlorocebus aethiops , Fibrose/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
14.
Dev Biol ; 393(2): 195-208, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25035933

RESUMO

Homeobox genes are an evolutionarily conserved class of transcription factors that are critical for development of many organ systems, including the brain and eye. During retinogenesis, homeodomain-containing transcription factors, which are encoded by homeobox genes, play essential roles in the regionalization and patterning of the optic neuroepithelium, specification of retinal progenitors and differentiation of all seven of the retinal cell classes that derive from a common progenitor. Homeodomain transcription factors control retinal cell fate by regulating the expression of target genes required for retinal progenitor cell fate decisions and for terminal differentiation of specific retinal cell types. The essential role of homeobox genes during retinal development is demonstrated by the number of human eye diseases, including colobomas and anophthalmia, which are attributed to homeobox gene mutations. In the following review, we highlight the role of homeodomain transcription factors during retinogenesis and regulation of their gene targets. Understanding the complexities of vertebrate retina development will enhance our ability to drive differentiation of specific retinal cell types towards novel cell-based replacement therapies for retinal degenerative diseases.


Assuntos
Oftalmopatias Hereditárias/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Retina , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mutação , Retina/citologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Células-Tronco/citologia , Fatores de Transcrição , Vertebrados
15.
Biochim Biophys Acta ; 1813(1): 201-12, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21040746

RESUMO

The homeobox transcription factor PROX1 is essential for the development and maintenance of lymphatic vasculature. How PROX1 regulates lymphatic endothelial cell fate remains undefined. PROX1 has been shown to upregulate the expression of Cyclin E, which mediates the G(1) to S transition of the cell cycle. Here we demonstrate that PROX1 activates the mouse Cyclin E1 (Ccne1) promoter via two proximal E2F-binding sites. We have determined that the N-terminal region of PROX1 is sufficient to activate a 1-kb Ccne1 promoter, whereas the homeodomain is dispensable for activation. We have identified that the Prospero domain 1 (PD1) is required for the nuclear localization of PROX1. Our comparison of two DNA-binding-deficient constructs of PROX1 showed a cell-type-specific difference between these two proteins in both their localization and function. We demonstrated that siRNA-mediated knockdown of PROX1 in lymphatic endothelial cells decreases progression from G(1) to S phase of the cell cycle. We conclude that PROX1 activates the Ccne1 promoter independent of DNA binding, and our results illustrate a novel role for PROX1 in the regulation of lymphatic endothelial cell proliferation.


Assuntos
Ciclo Celular , Ciclina E/genética , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Sítios de Ligação , Western Blotting , Proliferação de Células , Células Cultivadas , Ciclina E/metabolismo , Fator de Transcrição E2F1/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Rim/citologia , Rim/metabolismo , Luciferases/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo
16.
Can J Physiol Pharmacol ; 90(8): 1029-59, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22646022

RESUMO

This review focuses on the role of adipokines in the maintenance of a healthy cardiovascular system, and the mechanisms by which these factors mediate the development of cardiovascular disease in obesity. Adipocytes are the major cell type comprising the adipose tissue. These cells secrete numerous factors, termed adipokines, into the blood, including adiponectin, leptin, resistin, chemerin, omentin, vaspin, and visfatin. Adipose tissue is a highly vascularised endocrine organ, and different adipose depots have distinct adipokine secretion profiles, which are altered with obesity. The ability of many adipokines to stimulate angiogenesis is crucial for adipose tissue expansion; however, excessive blood vessel growth is deleterious. As well, some adipokines induce inflammation, which promotes cardiovascular disease progression. We discuss how these 7 aforementioned adipokines act upon the various cardiovascular cell types (endothelial progenitor cells, endothelial cells, vascular smooth muscle cells, pericytes, cardiomyocytes, and cardiac fibroblasts), the direct effects of these actions, and their overall impact on the cardiovascular system. These were chosen, as these adipokines are secreted predominantly from adipocytes and have known effects on cardiovascular cells.


Assuntos
Adipocinas/fisiologia , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/metabolismo , Adipócitos/metabolismo , Adipócitos/fisiologia , Adipocinas/metabolismo , Animais , Doenças Cardiovasculares/complicações , Humanos , Modelos Cardiovasculares , Obesidade/complicações , Obesidade/fisiopatologia
17.
Cells ; 11(16)2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-36010555

RESUMO

Endothelial cells regulate vascular homeostasis through the secretion of various paracrine molecules, including bioactive lipids, but little is known regarding the enzymes responsible for generating these lipids under either physiological or pathophysiological conditions. Arachidonate lipoxygenase (ALOX) expression was therefore investigated in confluent and nonconfluent EA.h926 endothelial cells, which represent the normal quiescent and proliferative states, respectively. mRNAs for ALOX15, ALOX15B, and ALOXE3 were detected in EA.hy926 cells, with the highest levels present in confluent cells compared to nonconfluent cells. In contrast, ALOX5, ALOX12, and ALOX12B mRNAs were not detected. At the protein level, only ALOX15B and ALOXE3 were detected but only in confluent cells. ALOXE3 was also observed in confluent human umbilical artery endothelial cells (HUAEC), indicating that its expression, although previously unreported, may be a general feature of endothelial cells. Exposure to laminar flow further increased ALOXE3 levels in EA.hy926 cells and HUAECs. The evidence obtained in this study indicates that proliferative status and shear stress are both important factors that mediate endothelial ALOX gene expression. The presence of ALOX15B and ALOXE3 exclusively in quiescent human endothelial cells suggests their activity likely contributes to the maintenance of a healthy endothelium.


Assuntos
Araquidonato Lipoxigenases , Células Endoteliais , Araquidonato Lipoxigenases/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Endotélio , Humanos , Lipídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Front Neurosci ; 16: 843794, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35546872

RESUMO

Forebrain development in vertebrates is regulated by transcription factors encoded by homeobox, bHLH and forkhead gene families throughout the progressive and overlapping stages of neural induction and patterning, regional specification and generation of neurons and glia from central nervous system (CNS) progenitor cells. Moreover, cell fate decisions, differentiation and migration of these committed CNS progenitors are controlled by the gene regulatory networks that are regulated by various homeodomain-containing transcription factors, including but not limited to those of the Pax (paired), Nkx, Otx (orthodenticle), Gsx/Gsh (genetic screened), and Dlx (distal-less) homeobox gene families. This comprehensive review outlines the integral role of key homeobox transcription factors and their target genes on forebrain development, focused primarily on the telencephalon. Furthermore, links of these transcription factors to human diseases, such as neurodevelopmental disorders and brain tumors are provided.

19.
Biomolecules ; 13(1)2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36671414

RESUMO

A decrease in the circulating levels of adiponectin in obesity increases the risk of metabolic complications, but the role of globular adiponectin, a truncated form produced by proteolytic cleavage, has not been defined. The objective of this investigation was to determine how globular adiponectin is generated and to determine whether this process impacts obesity. The cleavage of recombinant full-length adiponectin into globular adiponectin by plasma in vitro was used to identify Gly-93 as the N-terminal residue after proteolytic processing. The amino acid sequence of the cleavage site suggested thrombin was the protease responsible for cleavage, and inhibitors confirmed its likely involvement. The proteolytic site was modified, and this thrombin-resistant mutant protein was infused for 4 weeks into obese adiponectin-knockout mice that had been on a high-fat diet for 8 weeks. The mutation of the cleavage site ensured that globular adiponectin was not generated, and thus did not confound the actions of the full-length adiponectin. Mice infused with the mutant adiponectin accumulated less fat and had smaller adipocytes compared to mice treated with globular adiponectin, and concurrently had elevated fasting glucose. The data demonstrate that generation of globular adiponectin through the action of thrombin increases both adipose tissue mass and adipocyte size, but it has no effect on fasting glucose levels in the context of obesity.


Assuntos
Adiponectina , Trombina , Camundongos , Animais , Trombina/metabolismo , Obesidade/metabolismo , Glucose/metabolismo , Tecido Adiposo/metabolismo
20.
Nucleic Acids Res ; 36(3): 872-84, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18086710

RESUMO

Dlx homeobox genes are first expressed in embryonic retina at E11.5. The Dlx1/Dlx2 null retina has a reduced ganglion cell layer (GCL), with loss of late-born differentiated retinal ganglion cells (RGCs) due to increased apoptosis. TrkB signaling is proposed to regulate the dynamics of RGC apoptosis throughout development. DLX2 expression markedly precedes the onset of TrkB expression in the GCL; TrkB co-expression with Dlx2 and RGC markers is well-established by E13.5. In the Dlx1/Dlx2 null retina, TrkB expression is significantly reduced by E16.5. We demonstrated that DLX2 binds to a specific region of the TrkB promoter in retinal neuroepithelium during embryogenesis. In vitro confirmation and the functional consequences of DLX2 binding to this TrkB regulatory region support TrkB as a Dlx2 transcriptional target. Furthermore, ectopic Dlx2 expression in retinal explants activates TrkB expression and Dlx2 knockdown in primary retinal cultures results in reduced TrkB expression. RGC differentiation and survival require the coordinated expression of transcription factors. This study establishes a direct transcriptional relationship between a homeodomain protein involved in RGC differentiation and a neurotrophin receptor implicated in RGC survival. Signaling mediated by TrkB may contribute to survival of late-born RGCs whose terminal differentiation is regulated by Dlx gene function.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição/metabolismo , Células Amácrinas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Retina/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Ativação Transcricional
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