Semino Carlos E, Kamm Roger D, Lauffenburger Douglas A
Center for Biomedical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, NE47-383, MIT, Cambridge, MA 02139, USA.
Exp Cell Res. 2006 Feb 1;312(3):289-98. doi: 10.1016/j.yexcr.2005.10.029. Epub 2005 Dec 7.
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.
我们在此表明，表皮生长因子（EGF）受体的自分泌配体激活与间质流相结合，在血管内皮细胞对血管内皮生长因子（VEGF）刺激的形态发生反应中起关键作用。在胶原凝胶上培养并在无EGF和VEGF的情况下暴露于低间质流的人脐静脉内皮细胞（HUVEC）单层细胞仍保持存活和有丝分裂能力，但几乎没有血管形态发生的迹象。添加VEGF在48至72小时内产生了一种依赖于流的形态发生反应，其特征为分支的毛细血管样结构。与自分泌EGF受体途径相关的抑制剂，包括加拉迪恩、AG1478、PD98059和一种EGF受体阻断抗体，可基本消除该反应，这表明形态发生过程的调节是通过自分泌EGF受体激活来进行的。此外，我们观察到在我们的系统中，EGF受体总是独立于间质流被激活，而且，上述使用的EGF受体抑制剂降低了受体的磷酸化状态，这与毛细血管形态发生的抑制相关。最后，5'-溴-2'-脱氧尿苷（BrdU）标记在单层中识别出分裂细胞，但在延伸的毛细血管样结构中未识别出。EGF途径抑制剂加拉迪恩和AG1478并未减少单层中BrdU的掺入，这表明EGF受体介导的形态发生行为主要是由于细胞迁移而非增殖。基于这些结果，我们提出了一个体外毛细血管形态发生对VEGF刺激和间质液流反应的两步模型：通过独立于EGF受体的有丝分裂活性维持单层，以及由自分泌EGF受体激活介导的迁移反应，其中细胞形成毛细血管样结构。
