Glycosaminoglycan production in cultures of early and late passage human endothelial cells: The influence of an anionic endothelial cell growth factor and the extracellular matrix

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Abstract
An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL ⩽ 20) and late (CPDL > 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on matrix from late passage cells. We conclude that (1) EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; (2) early passage cells contain more GAG but less heparan sulfate than late passage cells; (3) extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix; (4) cells grown in the presence of EC growth factor are more adhesive to the substratum and more resistant to detachment by proteolytic enzymes than cells grown in medium without EC growth factor.