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

Abstract

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 al 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 β1 integrins on endothelial cells inhibits tube formation. On collagen gels, endothelial cells treated with β1 activating antibody 8A2 failed to migrate into the gel and tube formation was inhibited. Although several integrins mediate EC attachment to collagen α2β1 is the chief determinant of EC behaviour since a blocking antibody to α2β1 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, β1 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 β1 activation was time dependent and long lasting. The critical time for activation of the β1 integrin was the initial 1–2h after plating in order to inhibit tube formation although once activated, the β1 mediated inhibition on Matrigel was still evident 4 days later. Our results suggest that β1 integrins are critical in capillary tube formation in at least two phases, β1 integrins are essential for migration of EC through collagen gels. Independently, β1 integrins, although not involved in initial vacuole formation, are involved in the process of vacuole coalescence and subsequent lumen formation since β1 integrin activation inhibits these processes.