Hyaluronan in Corneal Regeneration and Pathology

PURPOSE: The cornea is a multilayered tissue essential for vision. The extracellular matrix component hyaluronan (HA) serves various functions in tissues, ranging from modulating cell phenotypes and activities, and tissue stiffness, to protecting tissues from reactive oxygen species (ROS). This dissertation aims to understand previously unexplored functions of HA in the cornea, with a focus on the role of HA in supporting transient amplifying cells’ (TACs) phenotype (Aim1), corneal stiffness (Aim2), and physiological properties of oxidized HA (oxHA) generated by ROS (Aim3). METHODS: (1) To test the role of HA on TACs’ phenotype, cells associated with HA (HA clusters) in the cornea were characterized using EdU label retaining model, lineage tracing model, and cell assays. (2) To study whether HA contributes to corneal stiffness, corneas of wild-type (wt) mice and mice lacking enzymes involved in the biosynthesis of HA were analyzed before and after a corneal alkali burn (AB), using quantitative wave-based optical coherence elastography (OCE) and immunofluorescence. (3) To investigate whether oxHA presents altered properties, high molecular weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl) and the oxHA’s structure and physiological properties were compared to naïve size-matched HA fragments. RESULTS: The results showed that (1) HA clusters in the peripheral corneal epithelium contain a significantly higher proportion of label retaining cells compared with the remaining peripheral cornea. HA+ cells are significantly smaller than HA- cells. Only HA+ cells and limbal epithelial cells form holoclones. (2) Mice lacking Has2 present stiffer cornea compared with wt mice. However, these mice presented a significant decrease in corneal stiffness and increased HA deposition at 14 days after AB. (3) HA chloramide (HACl) was found for the oxHA produced by HOCl based on NMR spectra. Native HA of all molecular weight (MWs) promotes corneal wound healing in vivo, while oxHA does not. CONCLUSIONS: HA is associated with a subset of TACs with stem-cell like properties. HA directly affects corneal stiffness. HA fragmentation by ROS alters its physiological activity on corneal epithelial wound healing and such change can be attributed to chemical structural change.