Rapid Induction of Hyaluronan Synthesis and Hyaluronan Synthase in Corneal Wound Healing

Abstract

Abstract: : Purpose: Corneal wounds deposit non–transparent stromal matrix containing abnormal proteoglycans. Hyaluronan (HA), a high molecular weight glycosaminoglycan not present in the normal stroma, is frequently observed in corneas with various pathological conditions. HA is highly bioactive, involved in cell migration and proliferation as well as in mediation of inflammatory response. The presence of HA in wounds suggests an active participation in the cellular events of scar formation. HA is synthesized in mammalian tissues by three different hyaluronan synthase (HAS) enzymes. This study was initiated to determine the time course during which HA appears in the corneal stroma after wounding, and to identify the cells and HAS isoforms involved in HA stromal biosynthesis. Methods: The temporal appearance of HA in mouse corneal scalpel wounds was examined by staining with biotin labelled hyaluronan binding protein (bHABP). HA in corneal extracts was quantified using fluorophore assisted–carbohydrate electrophoresis (FACE). Cellular HAS enzyme expression was detected by immunostaining with peptide antibodies to HAS1 and 2. HAS gene expression in cultured keratocytes was determined using real time RT–PCR. Results: Unwounded and 1 hr wounded corneas showed no HA by staining with bHABP but by 3 hr a diffuse stain was detected throughout the wounded cornea. Intensity of the staining increased up to 48 hr becoming granular. Staining decreased after 2 weeks but was clearly detected in the healed corneas for up to 3 months. HA in the early wounds was confirmed by FACE analysis. Immunohistochemistry of the wounded region showed strong expression of HAS2 protein on cells near the wound within 24 hr. Many of these cells were also CD45 positive. HAS2 mRNA in cultured keratocytes was markedly upregulated within 1–3 hr of serum stimulation. Conclusions: Accumulation of HA in the cornea represents the most rapid and pervasive change in stomal extracellular matrix yet observed in response to wounding. Staining and RT–PCR suggest that HAS2 enzyme is induced in stromal keratocytes in response to wounding, however inflammatory cells may also participate. The rapid, abundant, and persistent presence of HA in the wounded tissue is likely an important factor in several of the cellular healing processes including cell migration cell division and localization of inflammatory cells in the healing tissue.

Keywords: proteoglycans/glycosaminoglycans • cornea: stroma and keratocytes • gene/expression