β1 integrin activation inhibits in vitro tube formation:: Effects on cell migration, vacuole coalescence and lumen formation

Human endothelial cells (EC), when plated onto gels of extracellular matrix proteins such as Matrigel or collagen form capillary tubes in a process thought to mimic angiogenesis. We have shown previously that the extent of tube formation and the phenotype of the lumen are regulated by integrins (Gamble et nl 1993) and lumen formation occurs through a process of vacuolization, coalescence and ultimate directional fusion of these vacuoles with the plasma membrane (Meyer 1997 et al). We now show here that activation of beta(1) integrins on endothelial cells inhibits tube formation. On collagen gels, endothelial cells treated with beta(1) activating antibody 8A2 failed to migrate into the gel and tube formation was inhibited. Although several integrins mediate EC attachment to collagen alpha(2)beta(1) is the chief determinant of EC behaviour since a blocking antibody to alpha(2)beta(1) reversed the effect of 8A2. On Matrigel tube formation was also inhibited by 8A2 treatment although cell alignment and sprout formation was still evident. Electron microscopy revealed the organisation of normal numbers of cells into solid sprouts and the formation of small intracellular vacuoles suggesting that initial stages of tube formation including cell migration were unaffected. However, beta(1) integrin activation inhibited the coalescence of these small vacuoles into larger vacuoles, the recruitment of more cells into the sprout and the subsequent formation of mature lumen. The inhibition of capillary tube formation by beta(1) activation was time dependent and long lasting. The critical time for activation of the pi integrin was the initial 1-2h after plating in order to inhibit tube formation although once activated, the beta(1) mediated inhibition on Matrigel was still evident 4 days later. Our results suggest that beta(1) integrins are critical in capillary tube formation in at least two phases. beta(1) integrins are essential for migration of EC through collagen gels. Independently, beta(1) integrins, although not involved in initial vacuole formation, are involved in the process of vacuole coalescence and subsequent lumen formation since beta(1) integrin activation inhibits these processes.