RESUMO
The NFkappaB family of transcription factors, particularly the activated p50/p65 heterodimer, is expressed in vascular cells during intimal thickening formation when hemodynamic conditions are altered. Here, we report that p50, p65, IkappaBalpha and IKKalpha display different spatial and temporal patterns of expression and distribution during both chicken embryo aortic wall remodeling and intimal thickening development. Additionally, we show that both p50 and p65 were located in the nucleus of some mesenchymal cells expressing alpha-smooth muscle actin which are present in the spontaneous intimal thickening observed at embryonic days 12-14 of development. We also demonstrated that both NFkappaB subunits are present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin and stimulated with complete medium. This study demonstrates for the first time the presence of activated NFkappaB during the remodeling of the embryonic aortic wall and the formation of intimal thickening, providing evidence that suggest a possible role for this transcription factor in the EndoMT process.
Assuntos
Aorta/embriologia , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Mesoderma/citologia , NF-kappa B/metabolismo , Neovascularização Fisiológica , Animais , Aorta/citologia , Aspirina/farmacologia , Células Cultivadas , Embrião de Galinha , Células Endoteliais/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microscopia Eletrônica de VarreduraRESUMO
Members of the family of large chondroitin sulfate proteoglycans (CSPGs), such as versican and aggrecan, are involved in early heart development, and in the development and progression of atherosclerosis and restenosis. Given the important roles played by versican and aggrecan in such processes, we sought to determine whether these molecules are present in the aortic wall during the advanced stages of chicken embryo development and the endothelial-mesenchymal transformation (EMT). Immunolabeling of serial cryosections revealed versican immunoreactivity around the cells within the intimal thickening, and the cells organized in lamellar and interlamellar cell layers. In contrast, a weak aggrecan immunoreactivity was limited to the cells arranged into lamellar and interlamellar cell layers. Immunolabeling also demonstrated that V2 is the main versican isoform present at the intimal thickening. According to immunoblotting analysis, the aggrecan content was very low in all stages examined, and two versican isoforms (V0 and V2) were present at day 14 of development. We also investigated whether versican isoforms were present during EMT in vitro. Versican immunoreactivity was detected in patches of endothelial cells; in the detaching and migrating cells, and the extracellular matrix (ECM) deposited by them; and in cells that had acquired mesenchymal characteristics. These data indicate that versican and aggrecan have different spatial and temporal patterns of expression, and they have different functions during remodeling of the aortic wall. Also, the different immunoreactivity and immunolocalization patterns observed for versican both in vivo and in vitro, in addition to being associated with the presence of different versican isoforms, may be related to the predominance of the V2 isoform during intimal thickening formation and EMT.
Assuntos
Aorta/embriologia , Embrião de Galinha/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteoglicanas/metabolismo , Agrecanas , Animais , Western Blotting , Células Cultivadas , Embrião de Galinha/citologia , Embrião de Galinha/fisiologia , Endotélio Vascular/embriologia , Técnicas Imunoenzimáticas , Lectinas Tipo C , Mesoderma/fisiologia , Isoformas de Proteínas/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3 , VersicanasRESUMO
The serine protease thrombin, independently of its participation in hemostasis and thrombosis, has been involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development and progression of atherosclerosis. Many of these functions appear to be mediated by specific thrombin receptors, particularly the protease-activated receptor-1 (PAR1). In this study, we investigated whether both thrombin and PAR1 were present in the aortic wall of chicken embryos at days 11 and 12 of development. We found that PAR1 was limited to some cells of the intimal thickening and the inner media, whereas thrombin appeared distributed across the aortic wall. We also investigated whether PAR1 was present during endothelial-mesenchymal transdifferentiation (EMT) in vitro. A moderate immunoreactivity was detected in the monolayer of endothelial cells. In contrast, a strong cytoplasmic immunoreactivity was observed in the detaching and migrating cells and those that had acquired mesenchymal characteristics. This PAR1 expression was confirmed by flow cytometry. In this study, the addition of thrombin to arrested endothelial cell cultures was assessed. We found that thrombin stimulated endothelial cell spreading and migration, as no migrating cells were observed in serum-free medium (SFM) condition. Immunolocalization of PAR1 in the thrombin-treated cultures showed strong cytoplasmic immunoreactivity in the monolayers and in spreading and migrating cells, whereas in the SFM condition undetectable PAR1 immunoreactivity was observed. Flow cytometry of these cultures revealed an elevated expression of PAR1 in the presence of thrombin, in contrast to that detected in SFM and complete medium. These data indicate that both thrombin and PAR1 are involved in the remodeling of the aortic wall and intimal thickening formation, and in the endothelial-mesenchymal transdifferentiation process.