Keratinocyte-derived factors that stimulate fibroblast hyaluronan synthesis - the role of a low molecular weight factor

Abstract

Hyaluronan-rich matrices are associated with cellular proliferation and migration, with high levels being expressed transiently during adult wound healing, and persistent high levels expressed during scar-free foetal wound healing. Cultured allogeneic keratinocytes and lysed keratinocyte extracts promote chronic wound healing, as does addition of exogenous hyaluronan. It is possible that the keratinocyte-stimulated effects on wound healing may be, at least in part mediated by the release of hyaluronan synthesis-stimulating factors. We have demonstrated that serum-free keratinocyte-conditioned medium (KCM), when added to confluent fibroblast cultures for 24 h stimulated up to an 8-fold increase in hyaluronan synthesis, as measured by 3H-glucosamine incorporation into cetylpyridinium chloride-precipitable glycosaminoglycans. This KCM can be fractionated by ultrafiltration into fractions > 30 kD and < 1 kD which are poorly active individually, but synergise strongly to stimulate fibroblast hyaluronan synthesis. We have examined the activity of both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) to stimulate fibroblast hyaluronan synthesis in the presence and absence of the low molecular weight KCM fraction, and the effect of blocking antibodies to PDGF and bFGF on complete KCM. KCM stimulated a 7-fold increase in 3H-glucosamine incorporation into hyaluronan and a 8.3-fold increase into sulphated glycosaminoglycans. 35SO4 incorporation was increased only 1.5-fold, resulting in a significant decrease in the degree of polymer sulphation of the sulphated glycosaminoglycans. Blocking antibodies to PDGF (all isotypes) reduced the activity of the KCM by 27%, while blocking antibFGF reduced the activity by 58%. Addition of PDGF (20 ng/ml) to the < 1 kD KCM fraction restored full activity, while addition of bFGF had no significant effect. However, addition of heparin with the bFGF resulted in activity being restored. These results demonstrate that cultured human keratinocytes produce potent fibroblast glycosaminoglycan synthesis-stimulating factors. Both PDGF and bFGF appear to be involved, but require the presence of an as yet unidentified KCM low molecular weight factor. Identification of this factor may provide an important therapeutic aid in wound healing