ENDOTHELIAL CELL-CONDITIONED MEDIUM MODULATES THE SYNTHESIS AND STRUCTURE OF PROTEOGLYCANS IN VASCULAR SMOOTH-MUSCLE CELLS

We studied the effect of bovine endothelial cell-conditioned medium on proteoglycan synthesis by bovine aorta smooth muscle cells. Confluent cultures were incubated with [S-35]sulfate, [H-3]glucosamine or [H-3]serine in medium alone (control), or medium that had been conditioned on confluent endothelial cells. Metabolically labelled proteoglycans secreted into the culture medium and associated with the cell layer were quantified. During a 24 h incubation, endothelial cell-conditioned medium increased [S-35]sulfate and [H-3]glucosamine incorporation into medium and cell-layer proteoglycans by 59% and 95%, respectively, above controls.[H-3]Serine incorporation into proteoglycan core protein was increased by 150%. The effect of endothelial cell-conditioned medium on [S-35]sulfate incorporation was concentration dependent. The stimulatory effects of the conditioned medium were abolished by cycloheximide and actinomycin D, inhibitors of protein synthesis and transcription, respectively. Endothelial cell-conditioned medium caused no significant change in the degradation or secretion of proteoglycans, indicating that the increase in proteoglycans was due to increased de novo synthesis. TGF-beta neutralizing antibody inhibited 22% of the stimulatory effect of the conditioned medium, suggesting that part of the stimulation was mediated by TGF-beta. Ion-exchange chromatography of [S-35]proteoglycans in the culture medium of smooth muscle cells yielded two major peaks at 0.52 and 0.57 M NaCl in both control and experimental cultures. In both cases the second peak, which represented approx. 80% of the total radioactivity, contained isomeric chondroitin sulfate proteoglycan with chondroitin sulfate and dermatan sulfate accounting for 90% and 10% of the isomers, respectively. The isomeric chondroitin sulfate proteoglycan was fractionated by hydrodynamic size on Sepharose CL-4B, resulting in three fractions (A, B and C). Analytical column chromatography of fractions A and B on Sepharose CL-2B demonstrated that proteoglycans from cultures incubated with endothelial cell-conditioned medium were larger in size than those from control cultures (Mr fraction A, 1700000, compared with 1200000 M(r); fraction B, 540000, compared with 390000). The molecular weights of the core proteins were unchanged. The larger size of proteoglycan A in cultures exposed to endothelial cell-conditioned medium was due to an increase in both the glycosaminoglycan chain number (29 compared to 25) and molecular mass (M(r) 52 000, compared to 40 000). The hydrodynamic size of the glycosaminoglycans in proteoglycan B of control and experimental cultures was identical (M(r) 40 000). Therefore, the increase in the molecular mass of this proteoglycan was attributable to an increase in glycosaminoglycan chain number (12 compared to 9). These results indicate that endothelial cell-conditioned medium contains factor(s) which can modulate the synthesis and structure of proteoglycans in cultured vascular smooth muscle cells.