Glycosaminoglycan distribution in substratum adhesion sites of aging human skin fibroblasts, including papillary and reticular subpopulations

Abstract

Glycosaminoglycan (GAG) distribution has been analyzed in the adhesion sites, left substratum-bound after EGTA-mediated detachment, of various human skin fibroblast populations grown in vitro in the presence (asc +) or absence (asc −) of ascorbate. Examination of these skin fibroblasts during the EGTA treatment by scanning electron microscopy reveals that (a) asc + cells detach much more rapidly than asc − cells, but (b)asc − or asc + cells leave the same two types of structures in longterm culture-generated substratum-attached material (L-SAM) — long linear restraction fibers and “footpat-like” structures. Most of the [ 3H]glucosamine-radiolabeled polysaccharides in L-SAM were shown to be GAGs. Fibroblasts from a full-thickness skin sample from a very young patient (AG4449) have similar distributions of the GAGs in both the EGTA-suspended cell and L-SAM fractions; however, asc + cell and L-SAM fractions contain relatively more heparan sulfate than the asc − fractions. In contrast, full-thickness skin fibroblasts from an elderly patient (AG2261) generate GAG distributions in their L-SAMs (with greatly elevated levels of hyaluronate and chondroitin sulfate) that are very different from those of the cell fractions and from those of AG44449: furthermore these distributions in AG2261 fractions do not change when shifted from asc − to asc + medium. These studies led to analyses of teh two major fibroblast subsets — papillary (PAP) or reticular (RET) — that can be isolated from the dermis of a newborn infant (patient 5). The GAG distributions in the RET fractions were different from those in PAP fractions; of special note was the greater length of heparan sulfate chains from all RET fractions examined when compared to PAP fractions. There was a remarkable similarity in the GAG distributions of asc + RET fractions when compared to the full-thickness AG2261 cell fractions. In summary, these studies demonstrate that asc − or asc + “young” cells generate different GAG distributions in their substratum adhesion sites, whereas “old” cells from a full thickness skin sample do not alter their distribution when shifted from asc − to asc + (this distribution is different from that of “young” cells). Furthermore, analyses of GAGs in papillary and reticular cell fractions reveal significant differences between the two, with considerable similarity of asc + reticular fractions of the full-thickness AG2261 fibroblasts, which is consistent with the enrichment of reticular fibroblasts in the skin of aging individuals.