Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells

The regulation of vascular endothelial cell behavior during angiogenesis an d in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex , but it clearly involves growth factor-induced changes in extracellular ma trix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contri bute to the formation of the vascular extracellular matrix and also influen ce cellular proliferation and migration. Since the effects of TGF-beta(1) o n vascular smooth muscle cell growth are dependent on cell density, it is p ossible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [H-3]glucosamine, [S-35]sulfate, or S-35-labeled amino acids i n the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-S ephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chr omatography, The glycosaminoglycan M-r and composition were analyzed by Sep harose CL-6B chromatography, and the core protein M-r was analyzed by SDS-p olyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is depende nt on cell density. Specifically, TGF-beta(1) induced an accumulation of sm all chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of simil ar to 50 kDa in the medium of both dense and sparse cultures, but a cell la yer-associated heparan sulfate PG with a core protein size of similar to 40 0 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlec an and biglycan, respectively, by Western blot analysis. The present data s uggest that TGF-beta(1) promotes the synthesis of both perlecan and biglyca n when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan ch ains are elongated only in biglycan synthesized by the cells at a high cell density.