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1.
FASEB J ; 31(9): 3787-3799, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28522596

RESUMO

Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age-associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)-induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross-linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II-induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II-mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age-dependent disturbance of diastolic function and aggravates Ang II-induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance.-Galán, M., Varona, S., Guadall, A., Orriols, M., Navas, M., Aguiló, S., de Diego, A., Navarro, M. A., García-Dorado, D., Rodríguez-Sinovas, A., Martínez-González, J., Rodriguez, C. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy.


Assuntos
Angiotensina II/farmacologia , Cardiomegalia/induzido quimicamente , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteína-Lisina 6-Oxidase/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/enzimologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Inflamação , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Proteína-Lisina 6-Oxidase/genética , Transdução de Sinais
2.
Hum Mol Genet ; 22(10): 1949-59, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23390133

RESUMO

We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.


Assuntos
Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Neointima/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Animais , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Proteínas de Ligação a DNA/genética , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neointima/genética , Neointima/patologia , Proteínas do Tecido Nervoso/genética , Ratos , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética
3.
Thromb Haemost ; 112(4): 812-24, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24990180

RESUMO

Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.


Assuntos
Proteína-Lisina 6-Oxidase/metabolismo , Remodelação Vascular/genética , Animais , Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Quimiocina CCL2/metabolismo , Colágeno/química , Colágeno/metabolismo , DNA Complementar/metabolismo , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas , Transfecção , Transgenes , Cicatrização
4.
Clin Investig Arterioscler ; 26(2): 66-75, 2014.
Artigo em Espanhol | MEDLINE | ID: mdl-24630523

RESUMO

INTRODUCTION: Previous studies have shown that the loss of NOR-1 function modulates the activation of vascular smooth muscle cells (VSMC). In this study we use a mouse that over-expresses human NOR-1 in VSMC to analyze the effect of a gain of NOR-1 function on the activation of VSMC and in the hyperplasia of the intima induced by hemodynamic stress. METHODS: To generate the transgenic animal the human NOR-1 cDNA was placed under the control of the SM22α promoter. The expression of NOR-1 was analyzed by real time PCR, Western blot, immunohistochemistry and immunocitochemistry, and NOR-1 functionality was evaluated by luciferase activity assays. The incorporation of tritiated thymidine was determined as a cell proliferation index. The left carotid artery was ligated, and cross-sections were subjected to morphometric and immunostaining analysis. RESULTS: The transgenic mouse exhibited significant levels of human NOR-1 in aorta and carotid arteries. In aortic VSMC from transgenic mice an increase in the transcriptional activity of ciclin D2 was detected, as well as higher proliferative rates and increased levels of the marker Myh10. In these animals, carotid artery ligation induced a greater neointimal formation and a higher stenotic grade than in wild-type animals, in accordance with the labelling detected for Myh10 and phosphorylated Histone H3. CONCLUSIONS: These results reinforce the role of NOR-1 in VSMC proliferation and in vascular remodelling, and allow us to propose this model as a useful tool to study the involvement of NOR-1 in vascular function and in vascular diseases such as atherosclerosis and restenosis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Remodelação Vascular/fisiologia , Animais , Aorta/metabolismo , Aorta/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Hiperplasia/patologia , Camundongos , Camundongos Transgênicos , Neointima/etiologia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Estresse Fisiológico/fisiologia , Túnica Íntima/metabolismo
5.
Stem Cells Dev ; 22(3): 459-72, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22897771

RESUMO

Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation development. Our studies indicated that REX1 is expressed at all stages during mouse preimplantation development, with a mixed pattern of nuclear, perinuclear, and cytoplasmic localization. Chromatin association seemed to be altered in 8-cell embryos, and in the blastocyst, we found REX1 localized almost exclusively in the nucleus. A functional role for Rex1 in vivo was assessed by gain- and loss-of-function approaches. Embryos with attenuated levels of Rex1 after injection of zygotes with siRNAs did not exhibit defects in preimplantation development in vitro. In contrast, overexpression of Rex1 interfered with cleavage divisions and with proper blastocyst development, although we failed to detect alterations in the expression of lineage and pluripotency markers. Rex1 gain- and loss-of-function did alter the expression levels of Zscan4, an important regulator of preimplantation development and pluripotency. Our results suggest that Rex1 plays a role during preimplantation development. They are compatible with a role for Rex1 during acquisition of pluripotency in the blastocyst.


Assuntos
Desenvolvimento Embrionário , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Técnicas de Cultura Embrionária , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Transporte Proteico , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
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