Proteoglycans synthesized by cultured bovine aortic smooth muscle cells after exposure to lead: lead selectively inhibits the synthesis of versican, a large chondroitin sulfate proteoglycan

Abstract

To investigate the effects of lead on the formation of extracellular matrix in the vascular wall, we characterized proteoglycans synthesized by cultured vascular smooth muscle cells after exposure to the metal by biochemical techniques. Confluent cultures of bovine aortic smooth muscle cells were metabolically labeled with [ 35S]sulfate or [ 35S]methionine/cysteine in the presence of lead nitrate. The amount of glycosaminoglycans (GAGs) was evaluated by the incorporation of [ 35S]sulfate into GAGs by the cetylpyridinium chloride precipitation method. The labeled proteoglycans were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-2B molecular sieve chromatography. The GAG M r and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M r was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain or chondroitin ABC lyase. Lead significantly decreased the [ 35S]sulfate incorporation into GAGs accumulated in the cell layer and the conditioned medium. [ 35S]Sulfate-labeled proteoglycans obtained from the cell layer and the conditioned medium were separated into three peaks on DEAE-Sephacel chromatography and only the peak with the highest charge density was decreased by lead. The highly charged peak was eluted near the void volume on Sepharose CL-2B molecular sieve chromatography and sensitive to chondroitin ABC lyase on Sepharose CL-6B chromatography, indicating that lead selectively inhibits the synthesis of large and highly charged chondroitin/dermatan sulfate proteoglycans (CS/DSPGs). However, the size of chondroitin/dermatan sulfate chains of the CS/DSPGs was M r≈47 000 in both the control and lead-treated cultures. On the other hand, lead decreased the accumulation of a large CS/DSPG with a core protein of ≈450 kDa in the cell layer and the conditioned medium; the core protein was identified as versican core by Western blot analysis. It is therefore suggested that lead inhibits the synthesis of the versican core protein in vascular smooth muscle cells without a change in length of chondroitin/dermatan sulfate side chains. As a result, versican-poor extracellular matrix would be induced by lead in vascular smooth muscle cells.