Overexpressed alpha(3)beta(1) and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells

ECV304, a spontaneously transformed cell line derived from the human umbili cal vein endothelial cell (HUVEC) (Takahashi ct nl., 1990), has been develo ped as an ill vitro angiogenesis model. In the present study, we further ch aracterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellu lar adhesion, slower but reproducible progression of angiogenesis on Matrig el, and resistance to apoptosis. Thus, the expression of integrin and activ ation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream e ffector of the integrin pathway, were examined. Flow cytometry revealed tha t alpha(3)beta(1) integrin was markedly upregulated in ECV304 cells, while alpha(v)beta(1) and alpha(5)beta(1) integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial st age of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 ce lls, rendering them resistent to the inhibitory effect of PD98059 on prolif eration. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 mu M of P D98059, no significant changes in cell binding and tubulogenesis on Matrige l was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative I-I-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha(3)beta(1) integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.