CHARACTERIZATION OF THE ACTIVATION OF LATENT TGF-BETA BY COCULTURES OF ENDOTHELIAL-CELLS AND PERICYTES OR SMOOTH-MUSCLE CELLS - A SELF-REGULATING SYSTEM

The conversion of latent transforming growth factor beta (LTGF-β) to the active species, transforming growth factor beta (TGF-β), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-β in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-β as determined by this assay. The concentration of TGF-β in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-β1 and TGF-β2, while SMCs produced primarily TGF-β1. No change in the expression of these two forms of TGF-β was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-β indicated that most of the active TGF-β was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-β in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-β in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-β formation blocked the activation of the protease required for conversion of LTGF-β to TGF-β as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-β. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-β appears to be a self-regulating system.