Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[aSS]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCI (dissociative solvent) or 0.5 M guanidine HCI (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population ("Kay = 0.31) contained predominantly chondroitin sulfate chains with Mr = ~40,000. The smaller population (Kay = 0.61) contained dermatan sulfate chains of similar Mr (~40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kay = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.Proteoglycans and their constituent glycosaminoglycans are present in blood vessel walls (1-14) and form important structural links between the fibrous components of the intercellular matrix (collagen and elastic fibers) and the arterial endothelial and smooth muscle cells (15). Recently, considerable attention has focused on these macromolecules since they have been shown to influence the structural integrity of the vascular wall and to affect several arterial properties such as viscoelasticity, permeability, lipid metabolism, hemostasis, and thrombosis (15,16). Furthermore, their participation in such cell processes as proliferation (17), migration (18), and adhesion (19) highlight their importance in many events that are common to both blood vessel development and disease.Although several studies have investigated changes in the glycosaminoglycan component of arterial proteoglycans during development and disease (10, 11,15,20), only recently have attempts been made to define the physical-chemical nature of entire proteoglycan molecules isolated from arteries. This information is important since it is thought that the structure of the whole molecule, and not merely the component parts, dictates the functional properties of the proteoglycans. Employing methods used to isolate proteoglycans from cartila...