Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells.

Abstract

Abstract. When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a halflife of ,x,10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-ArgGly-Asp-Ser-coated microbeads (~10 microbeads/ cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.T HE formation and regression of new vascular structures is a regulated process that governs organ development during embryogenesis (for review see reference 11). This suggests that not only capillary proliferation, but also capillary involution, depends on physiological control mechanisms. The composition and organization of the extraceUular matrix are known to markedly influence remodeling of blood vessels. Capillary basement membrane dissolution correlates with microvessel retraction, endothelial cell (EC) 1 rounding, and associated capillary regression (21,24). This series of events are likely caused by the fact that EC must be adherent to matrix proteins to survive and proliferate. When EC are cultured under conditions that prevent adhesion and spreading, they stop growing and lose viability (20,24,31).

Signaling by integrin receptors

“…Addition of microbeads coated with RGD peptides that were too small to permit cell spreading did not rescue these cells. However, plastic surfaces coated with RGD peptides rescued cells from anoikis e ciently (Re et al, 1994). These results suggest that mere attachment of cells through integrins is insu cient for rescue and that cell shape changes are required.…”

“…Cell-matrix interactions result in a cascade of cellular responses which ultimately promote not only cell binding but also lead to cell spreading. Both cell attachment and spreading are critical for cell survival because cells that adhere, but can not spread, may undergo programmed cell death (Bates et al, 1994;Re et al, 1994) Integrin receptors are composed of noncovalently associated a and b chains which form heterodimeric receptor complexes (Hynes, 1992;Ruoslahti, 1991Ruoslahti, , 1996. The a and b subunits contain a large extracellular domain, a short transmembrane domain and a cytoplasmic carboxy terminal domain of variable length.…”

Apoptosis in the vasculature: mechanisms and functional importance

“…Cell ± cell and cell ± matrix interactions Cell-to-cell and cellto-matrix contacts are necessary for the maintenance of survival of anchorage-dependent cells such as endothelial cells (Brooks et al, 1994;Levkau et al, 1998;Meredith et al, 1993;Re et al, 1994;Scatena et al, 1998). Loss of cell-to-cell contact leads to the activation of a default death programme in a variety of cell types.…”

Disruption of integrin-dependent adhesion and survival of endothelial cells by recombinant adenovirus expressing isolated β integrin cytoplasmic domains

“…37 Upon detachment, HUVEC rapidly died by apoptosis. Endothelial cell anoikis has been reported in the presence of integrin antagonists 19 or on non-adhesive substrates 31 but not when pre-existing cell adhesion was disrupted by expressing isolated integrin ␤ subunit cytoplasmic domains. Although isolated ␤ cytoplasmic domains can by themselves induce sustained tyrosine phosphorylation, 28 our data show that these signaling events are not sufficient to maintain non-adherent cells alive.…”