Cell density dependent effects of TGF-? demonstrated by a plasminogen activator-based assay for TGF-?

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Abstract
Transforming growth factor-β1 (TGF-β1) induces a decrease in plasminogen activator (PA) expression in confluent cultures of bovine aortic endothelial (BAE) cells. We describe an assay using the suppression of PA expression in confluent BAE cells by TGF-β1 which detects concentrations of the growth factor ranging from 5 to 200 pg/ml and has an ED50 of 15–20 pg/ml. The assay can be performed in 96-well plates and requires a minimum of 35 ul of solution per sample, thereby limiting the amount of reagents required and allowing many samples to be tested in a single assay. Here we demonstrate that the effect of TGF-β1 on PA expression in BAE cells depends on the length of time the cells are exposed to the growth factor and the density at which the cells are plated. In cells plated at a high density (3.5 × 105 cells/cm2), both 4 h and 24 h exposures to TGF-β1 suppress PA expression. However, with cells plated sparsely (3.5 × 104 cells/cm2), a 4 h exposure to TGF-β1 increases PA expression 2-fold, whereas a 24 h exposure results in an 85% inhibition of basal PA expression. The paradoxical stimulation of PA expression in cells at a sparse density upon 4 h exposure to TGF-β1 occurs in a dose-dependent manner with an ED50 of 15–20 pg/ml. This bifunctional response of PA production in cells exposed to TGF-β1 may have implications with regard to the role of TGF-β1 in angiogenesis.

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