SULFATE MOIETIES IN THE SUBENDOTHELIAL EXTRACELLULAR-MATRIX ARE INVOLVED IN BASIC FIBROBLAST GROWTH-FACTOR SEQUESTRATION, DIMERIZATION, ANDSTIMULATION OF CELL-PROLIFERATION

The growth promoting activity of the subendothelial extracellular matr ix (ECM) is attributed to sequestration of basic fibroblast growth fac tor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The prese nt study focuses on the growth promoting activity and bFGF binding cap acity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintain ed in the presence of chlorate, an inhibitor of phosphoadenosine phosp hosulfate synthesis, produced ECM containing 10-15% of the sulfate nor mally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of I-125-bFGF sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to r elease by heparin. Incubation of I-125-bFGF on top of native ECM resul ted in dimerization of the ECM-bound bFGF, but there was a markedly re duced binding and dimerization of bFGF on sulfate-depleted ECM. ECM pr oduced in the presence of chlorate contained a nearly 10-fold less end ogenous bFGF as compared to native ECM and exerted Little or no mitoge nic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulate d the proliferation of chlorate-treated EC seeded on native ECM, sugge sting its interaction with ECM-bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin ha d no effect on chlorate-treated cells seeded in contact with sulfate-d epleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated wi th the cell surface and ECM act in concert to regulate the bioavailabi lity and growth promoting activity of bFGF. While HS in the subendothe lial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to h igh affinity signal transducing receptors.