RESUMO
In the retina, angiogenesis is an important component of normal physiological events such as embryonic vascular development. It is also involved in pathological processes including diabetic retinopathies and age-related macular degeneration, and tumour growth such as choroidal melanoma. Fibroblast growth factor (FGF) 2 and vascular endothelial cell growth factor (VEGF) are the two major angiogenic factors in the retina. We investigated the mechanism of proliferation and the regulation of the mitogenic properties of FGF2 and VEGF in cultures of chorocapillary endothelial cells (CEC). FGF2 is a strong mitogen for CEC and induced a 2.5-fold increase in cell proliferation after 4 days in culture in the absence of serum. In contrast, VEGF is a poor mitogen for CEC. FGF2, but not VEGF induces a large activation of MEK1, ERK1/2 and P90(RSK) during CEC proliferation. Pharmacological inhibition of Ras processing, and of MEK1 and ERK1/2 activation reduced only by 50% FGF2-induced cell proliferation, suggesting that there is another signalling pathway for CEC proliferation. Pharmacological inhibition of the PI 3-Kinase also inhibits by half FGF2-induced CEC proliferation. FGF2 stimulates the activation of the PI 3-K, P70(S6K) and Akt. Inhibition of both ERK1/2 and PI 3-K activities suppressed FGF2-induced CEC proliferation, demonstrating that CEC proliferation requires both ERKs and PI 3-K pathways. These data on the molecular mechanism and signalling may have important implications for providing more selective methods for anti-angiogenic and anti-tumoural therapy.
Assuntos
Capilares/fisiologia , Corioide/irrigação sanguínea , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Linfocinas/farmacologia , Animais , Capilares/citologia , Bovinos , Divisão Celular , Corioide/citologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Separação Imunomagnética , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/farmacologia , Modelos Biológicos , Neovascularização Fisiológica , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Cross-Talk , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
PURPOSE: To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro. METHODS: Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 microg/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates. Cells were treated with various specific pharmacologic inhibitors of the mitogen-activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase (PI 3-K) pathways to analyze signaling involved in CEC proliferation. Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies. RESULTS: FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation. Individually, these three components were not mitogenic. EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90(RSK) cascade. Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46%. The PI 3-K/p70(S6K)/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation. Inhibition of PI 3-K/p70(S6K) activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation. CONCLUSIONS: CEC proliferation involves both ERK and PI 3-K. That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity. These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.
Assuntos
Corioide/irrigação sanguínea , Endotélio Vascular/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Animais , Western Blotting , Bovinos , Divisão Celular/fisiologia , Células Cultivadas , Endotélio Vascular/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-aktRESUMO
PURPOSE: To establish a pure culture of choriocapillary endothelial cells as a model of angiogenesis in vitro. MATERIAL AND METHODS: Bovine choriocapillary endothelial cells (BCEC) were obtained by the method described by Hoffmann et al. (6) using the polystyrene paramagnetic beads coated with Lycopersicon esculentum, which attach specifically to the rest of fucose on the surface of microvascular endothelial cells. The endothelial characteristic of the cultured cells was evaluated by immunocytochemistry using anti von Willebrand factor and anti-CD 31 antibodies. Proliferation and survival of BCEC were tested using haemacytometer of Mallasez. RESULTS: The purity of obtained BCEC culture was confirmed by positive immunocytochemical staining with anti von Willebrand and anti factor CD 31 antibodies in more than 95% of cells. The proliferation of cells in Endothelial Cell Medium resulted in twofold increase of number of cells during 4-day observation period. After reaching the confluence, the cells continued to proliferate with increase of the cell number by 60% during 4-day observation. CONCLUSION: The use of paramagnetic beads coated with specific lectine provide a pure isolation of BCEC, which can be maintained in culture with preservation of their characteristic.