In vitro analysis of fetal fibroblast collagenolytic activity

Abstract

Tissue repair in the rabbit fetus is remarkably rapid and occurs without significant inflammation or excessive collagen deposition. The objective of this study was to compare the ability of rabbit fetal and adult fibroblasts to express the matrix metalloproteinases which are thought to be critical to scar tissue remodeling. In vitro, both fetal and adult rabbit fibroblasts express procollagenase messenger RNA in a constitutive manner. Mechanical disruption of fetal fibroblast monolayers caused a twofold increase in procollagenase mRNA. In contrast, the adult rabbit fibroblast procollagenase mRNA remained unchanged. The mRNA data correlated well with enzyme protein levels. Quantitation by immunoprecipitation showed a 2.3-fold increase in fetal fibroblast procollagenase protein after mechanical injury, whereas the level in adult rabbit fibroblasts remained unchanged. However, it was noted that the constitutive levels of procollagenase mRNA and protein were higher in adult fibroblasts. Analysis of enzyme activity, by means of a fluorogenic substrate, showed that adult fibroblasts had 2.2 times more collagenase activity compared with fetal cells. After mechanical injury, the fetal fibroblast collagenase activity increased 1.3-fold compared with 1.7-fold in the adult fibroblasts. In contrast, fetal fibroblast gelatinase activity was 1.25 times greater than in adult cells and increased 1.4-fold after mechanical injury, whereas the adult profile remained unchanged. Immunolocalization studies indicated that 1 hour after mechanical injury, procollagenase was produced primarily by fibroblasts along the mechanical injury ridge. By 4 hours after injury, the ridge cells began to migrate out into the open area and procollagenase was noted in adjacent cells of both adult and fetal origin. By 12 and 18 hours, all cells throughout the monolayer were expressing procollagenase. These findings show that in vitro fetal fibroblasts actually had lower levels of procollagenase, but higher levels of gelatinase compared with adult fibroblasts. The increased gelatinase expression may explain why fetal wounds do not have excessive collagen accumulation and heal without a visible scar.