Growth regulatory activities of endothelial extracellular matrix: Mediation by transforming growth factor-β

The potential of transforming growth factor-β (TGF-β) to modulate the growth of endothelial cells via alterations in the cell's extracellular matrix (ECM) was examined. Rat brain endothelial cells were cultured in the presence or absence of TGF-β, and subsequently ECM was prepared from the cell cultures by hypotonic lysis of the cells. Untreated endothelial cells were then cultured on the various matrices. Cells grown on TGF-β-treated ECM showed a significant decrease in cell number (41 ± 6% mean growth inhibition at 6 days, P < 0.005 by paired T-test) compared with cells grown on untreated ECM. The growth inhibitory activity of the ECM was depleted by 9 days of culture, and resumption of exponential cell growth was observed. A similar phenomenon was observed if anti-TGF-β neutralizing antibodies were incubated with the ECM. When the TGF-β-treated matrix was exposed to a brief dithiothreitol treatment in order to inactivate residual TGF-β, an approximately equal degree of growth inhibition was observed initially, but the reversal of inhibition occurred at an earlier time point than that with unreduced TGF-β-treated matrix. Analyses of the composition of matrices synthesized in the presence or absence of TGF-β revealed about a twofold increase in the accumulation of various radioactive metabolic precursors in the TGF-β-treated matrices. However, no qualitative alterations in the matrix or cellular-associated proteins or glycoproteins were observed, as analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. An increase in cell-associated heparan sulfate, however, was observed in TGF-β-treated cells. The results suggest that certain growth regulatory effects of TGF-β may be mediated, at least in part, by alterations in the ECM.