Density-dependent expression of hyaluronic acid binding to vascular cells in vitro

Alterations in glycosaminoglycans and their receptors have been associated with changes in cell proliferation during development, wound healing, regeneration, and remodeling. We have previously found a differential effect of hyaluronic acid on the attachment of vascular cells in vitro; endothelial cell (EC) attachment was improved on hyaluronic acid-coated substrates, whereas that of smooth muscle cells (SMC) was reduced (Orlidge and D'Amore, 1986). To determine if hyaluronic acid binding sites are involved in these different substrate preferences, we have studied specific hyaluronic acid binding to cultured bovine aortic EC and SMC. Since very large numbers of cells are required for these binding assays (3 x 10(6)/data point), and since the level of hyaluronate binding to fixed and native SMC and EC was similar, fixed cells were used throughout this study. The effect of cell density on hyaluronic acid binding was investigated. No significant difference was observed between hyaluronic acid binding to sparse and high density SMC. On the other hand, a more than threefold elevation in specific hyaluronic acid binding was observed on low density EC when compared to binding on high density EC. Hyaluronic acid binding was found to be specific; excesses of heparan sulfate and chondroitin sulfate had no effect on the levels of specific binding. Finally, the effect of cell passage on SMC binding of hyaluronic acid was measured. Specific binding was measured from 1st to 12th passage cells and was found to increase with passage number so that by passage 12, hyaluronic acid binding was fourfold that of 1st passage cells. These data support the concept that SMC may become less differentiated upon continuous culture. Our results indicate quantitative changes in the level of hyaluronic acid binding to vascular cells as a function of their growth state. Further, these data correlate well with in vivo observations which suggest a role for hyaluronic acid in vascular development.