Abstract
Purpose :
Hyaluronan (HA) is a key component of the corneal limbal extracellular matrix and is upregulated after injury, playing an important role in corneal wound healing. Excess reactive oxygen species (ROS) are toxic to cells and HA is susceptible to oxidation by ROS, yet the precise chemical structures of oxidized HA (oxHA) and their physiological properties remain unknown. The aim of this study was to characterize the molecular weight and structure of oxHA generated using different ROS, and to compare the effects of oxHA and unoxidized HA of different molecular weights on cellular responses of corneal epithelial cells that are related to wound healing.
Methods :
High molecular weight HA (HMWHA - 2670kDa) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). Thereafter, the oxHA was purified using chromatography by Sephadex PD 10 columns. The size of oxHA was characterized by agarose gel electrophoresis and gel filtration high-pressure liquid chromatography. The structure of the oxHA was characterized by 1H and 13C NMR at 500 MHz. Human corneal epithelial cells (HTCEs) were treated with oxHA, or size-matched unoxidized HA, and cell viability, proliferation, and migration assayed.
Results :
Smaller HA fragments were generated with increasing molar ratios of both H2O2 and HOCl. Chlorinated HA was detected after treatment with HOCl, according to 1H NMR spectra. HMWHA significantly promoted corneal epithelial cell viability when compared to LMWHA, ULMWHA and oxHA. LMWHA and oxHA promoted cell migration, while HMWHA and ULMWHA did not.
Conclusions :
HA fragmentation by ROS alters its physiological activity on corneal epithelial cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.