Role of Extracellular Matrix Component Hyaluronan in Corneal Homeostasis and Pathology

Purpose: Defects in the ability to repair and restore corneal integrity after injury can lead to loss of corneal transparency and visual impairment. Extracellular matrix components play an important role in regulation of normal corneal physiology and response to injuries and diseases. This dissertation evaluated the role of hyaluronan (HA) in the limbus in regulating limbal stem cells (LSCs) and lymphangiogenesis, and, therefore, the maintenance of corneal homeostasis and regeneration after corneal damage. Methods: (1) To determine the role of HA in maintenance of LSCs during ex vivo expansion, limbal stem/progenitor cells isolated from the human and mouse corneas were cultured under different conditions with or without HA. (2) To assess the role of HA in the initial phase of corneal epithelial wound healing, HAS2Δ/ΔCorEpi mice (lacking hyaluronan synthase 2 (Has2) expression in K14 expressing cells), and wild-type mice were subjected to different corneal injury types. (3) To investigate whether HA plays a role in corneal lymphangiogenesis, effect of HA on lymphatic endothelial cell viability, proliferation and tube formation ability were assessed in vitro and mice lacking hyaluronan synthases were subjected to alkali burn for in vivo experiments. Results: (1) LSCs cultured ex vivo in the presence of HA have higher proliferation, colony-forming efficiency, migration and expression of putative LSC markers compared to other coatings. (2) Both LSCs and corneal epithelial cells contribute towards closure of corneal wounds. The removal of limbal epithelium did not delay closure of smaller wounds, indicating that smaller wounds do not rely on LSCs for wound closure as do larger wounds. In HAS2Δ/ΔCorEpi mice, an experimental model of limbal stem cell deficiency (LSCD), removal of the limbal rim did not further affect wound healing, irrespective of the wound size. (3) There is an upregulation of HA throughout the cornea after chemical injury and lymphatic vessels grow into HA rich regions of the cornea. HA also enhances the viability, proliferation and tube formation ability of lymphatic endothelial cells in vitro. Conclusion: HA is a superior substrate for culturing LSCs ex vivo, when compared to those commonly used in research for LSC expansion. The dimensions and location of corneal wounds dictates the involvement of LSCs during wound healing, specifically smaller wounds do not rely on LSCs. HA also plays a role in regulating corneal lymphangiogenesis. HA in the limbus is important for the maintenance of corneal homeostasis and transparency.