beta(1) 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 th ought to mimic angiogenesis. We have shown previously that the extent of tu be formation and the phenotype of the lumen are regulated by integrins (Gam ble et nl 1993) and lumen formation occurs through a process of vacuolizati on, coalescence and ultimate directional fusion of these vacuoles with the plasma membrane (Meyer 1997 et al). We now show here that activation of bet a(1) integrins on endothelial cells inhibits tube formation. On collagen ge ls, endothelial cells treated with beta(1) activating antibody 8A2 failed t o migrate into the gel and tube formation was inhibited. Although several i ntegrins mediate EC attachment to collagen alpha(2)beta(1) is the chief det erminant of EC behaviour since a blocking antibody to alpha(2)beta(1) rever sed 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 in to solid sprouts and the formation of small intracellular vacuoles suggesti ng that initial stages of tube formation including cell migration were unaf fected. However, beta(1) integrin activation inhibited the coalescence of t hese small vacuoles into larger vacuoles, the recruitment of more cells int o the sprout and the subsequent formation of mature lumen. The inhibition o f capillary tube formation by beta(1) activation was time dependent and lon g lasting. The critical time for activation of the pi integrin was the init ial 1-2h after plating in order to inhibit tube formation although once act ivated, the beta(1) mediated inhibition on Matrigel was still evident 4 day s later. Our results suggest that beta(1) integrins are critical in capillary tube f ormation in at least two phases. beta(1) integrins are essential for migrat ion 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 a ctivation inhibits these processes.