The response of endothelial cells to TGF beta-1 is dependent upon cell shape, proliferative state and the nature of the substratum

Endothelial cells plated on two-dimensional (2-D) substrata proliferate until they form a tightly apposed confluent monolayer of quiescent cells that display a typical 'cobblestone' morphology. When added to proliferating cultures TGF beta-1 (transforming growth factor beta-1) inhibited cell growth and caused marked morphological changes, with the cells becoming enlarged and ragged. These effects were dose-dependent and reversible. TGF beta-1 also reduced the cloning efficiency and colony size of these cells, indicating that TGF beta-1 is cytotoxic and cytostatic for endothelial cells. By contrast, TGF beta-1 added to quiescent cobblestone cultures did not affect cell morphology or cell numbers. In the presence of 20% serum, the level of total protein synthesis per cell was significantly increased by TGF beta-1 in a dose-dependent manner when the cells were cultured on a 2-D substratum, regardless of whether the cells were proliferating or cobblestone quiescent. The level of plasminogen activator inhibitor type 1 was specifically increased in these cultures, as demonstrated by reverse fibrin zymography and immunoprecipitation. Endothelial cells embedded within a 3-D collagen gel display an elongated 'sprouting' morphology. Such cells self-associate to form three-dimensional cellular networks within the gel, but do not proliferate. The addition of TGF beta-1 to these quiescent sprouting cells initially induced rounding-up without altering protein synthesis, and cell death occurred later. The effects of TGF beta-1 on sprouting endothelial cells were also examined using two culture systems where both the cobblestone and the sprouting phenotypes were present. TGF beta-1 reduced the number of cells present and the extent of migration of sprouting cells embedded within a type I collagen gel, but had no effect upon sprouting cells embedded within a complex endothelial-produced extracellular matrix. Large vessel (aortic) and microvessel (retinal) endothelial cells responded in a similar way to TGF beta-1; the only difference being that an increased synthesis of PAI-1 was not observed with sub-confluent BREC cultures. Our results suggest that the effects of TGF beta-1 upon endothelial cells depend on the shape (cobblestone or sprouting), on the proliferative state of the cells, and on the nature of the matrix surrounding the cells. The response of these cells to TGF beta-1 in vivo may be similarly modulated during angiogenesis by changes in the cell phenotype and the composition of the surrounding matrix.