Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow |
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Authors: | Semino Carlos E Kamm Roger D Lauffenburger Douglas A |
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Institution: | Center for Biomedical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, NE47-383, MIT, Cambridge, MA 02139, USA. semino@mit.edu |
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Abstract: | We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures. |
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Keywords: | EGF epidermal growth factor HB-EGF heparin-binding epidermal growth factor EGFR epidermal growth factor receptor pEGFR phosphorylated EGFR ERK1/2 extracellular signal-regulated protein kinase 1 and 2 pERK1/2 phosphorylated ERK1/2 IGF insulin-like growth factor VEGF vascular endothelial growth factor aFGF acidic fibroblast growth factor bFGF basic fibroblast growth factor PDGF platelet derived growth factor TNF tumor necrosis factor HUVEC human umbilical vein endothelial cells BrdU 5′bromo-2′-deoxyuridine PMA phorbol 12-myristate 13-acetate Ara-C cytosine-arabinoside PFA paraformaldehyde PKC protein kinase C |
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