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The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-β1(TGF-β1) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-β1on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-β1also 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 [3H]glucosamine, [35S]sulfate, or35S-labeled amino acids in the presence of TGF-β1. The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan Mrand composition were analyzed by Sepharose CL-6B chromatography, and the core protein Mrwas analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-β1on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-β1induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of ∼50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of ∼400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-β1cause 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 perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-β1promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing “S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (Ml. Cl) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) ~38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (Mll, Cll) that was included on Sepharose CL-4B at Kav= 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (Mlla, Clla) and an included peak eluting at Kav= 0.39 (Mllb, Cllb). The void peaks (Mlla, Clla) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroiti-nase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt ~38,000. The included peaks on Sepha-rose CL-6B (Mllb, Cllb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt ~38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chon-drotinase AC (~60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only ~10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H-activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of ~70% of the 36S-sulfate-labeled material from the cell layer. This culture system is offered as a model to investigate the role of proteoglycans in hematopoiesis.