Background: Understanding wound healing and ways to accelerate the healing process includes understanding the factors that influence the synthesis of granulation tissue, which fills the wound before epithelialization. An important phase of early wound healing involves secretion of glycosaminoglycans (GAGs) by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing. The complexity of GAG structure and function in the extracellular matrix (ECM) remains poorly studied in wound healing. There is no established model for cutaneous wound healing due to variations in donor age, anatomic site, or stage of organ development. Rat embryo fibroblasts (REF) developed as a model to study malignant changes in fibroblasts were used as a model for fibroblasts in early wound healing because they lack the confounding variations based on age, site, and stage present in other fibroblasts used to study early wound healing. The purpose of this study was to identify and characterize the sulfated GAGs synthesized by REF-D.
Materials and methods: Rat embryo fibroblasts (REF-D) were cultured in serum-based medium and radiolabeled during their growth phase with (35)S to identify the GAG chains usually associated with proteoglycans (PGs). The sites of attachment (ECM-rich) were collected with detergent in sodium acetate buffer, pH 5.8, in the presence of protease inhibitors. Sulfated molecules were collected by ion-exchange chromatography and then assayed for GAGs. Nitrous acid deamination was used to determine heparan sulfate GAGs, and chondroitinase was used for chondroitin/dermatan sulfate GAGs. The proportion of individual GAGs was expressed with respect to sulfated molecules isolated. In addition, RNA was isolated from subconfluent REF-D, and core proteins for proteoglycans (decorin, biglycan, syndecan-2, and perlecan) were assayed by reverse transcription polymerase chain reaction.
Results: There were two major configurations of GAGs: free GAG chains (79.7% of sulfated molecules) and GAGs attached to the core protein of a proteoglycan (15.6%). The free GAG chains were composed of chondroitin sulfate (79.1% +/- 3.5) and heparan sulfate (28.7% +/- 2.1). In the smaller group of PGs, both heparan sulfate (94.8% +/- 7.3) and chondroitin sulfate (88.9% +/- 3.2) chains were attached to a core protein. REF-D expressed mRNA for biglycan and decorin, which are chondroitin sulfate-containing PGs. In addition, REF-D expressed mRNA for syndecan-2 and perlecan, which are PGs that contain primarily heparan sulfate chains.
Conclusions: A majority of GAG chains synthesized by subconfluent REF-D are chondroitin sulfate. A smaller proportion of chondroitin sulfate chains associate with a core protein as part of a PG (e.g., biglycan, decorin, syndecan-2). Heparan sulfate chains are also present, with a small proportion associated with a core protein (e.g., the PGs syndecan-2, perlecan). The greater presence of free GAG chains forming weak interactions with surrounding molecules may assist fibroblasts that are moving and replicating during this phase. Therefore, REF-D are particularly well suited to study early wound healing by their expression of chondroitin sulfate chains and associated PGs without the influence of donor age, stage, or anatomic site.
Copyright 2000 Academic Press.