Thesis Abstract - Robciuc Alexandra

Environmental Stress and the Corneal Epithelium

The cornea is the first optical element of the eye and, together with the eyelids, eye socket, tears, and sclera, shares an important part in ocular protection. It is a thin, transparent, avascular tissue with a rigorous layered structure. The continuous contact with the outside environment exposes the ocular surface tissues, such as the corneal epithelium, to pathogens, mechanical traumas, irritants, toxins, allergens, or radiation from the sun. The cellular stress response represents an adaptive reaction to environmental stimuli and defines the health-state of the tissue, the absence/presence of clinical manifestations. We have aimed in this thesis project to study the stress response of the corneal epithelium to environmental stimuli and to determine its contribution to ocular surface diseases such as climatic droplet keratopathy (CDK), infection, or dry eye disease. A cell culture model of the corneal epithelium was exposed to environmental stress – UV radiation, LPS, or hyperosmolarity (HO) – to identify macromolecular alterations: mRNA expression, protein localization, enzyme activation, lipid conversions.

CDK is a degenerative disease of the cornea with increased prevalence in warm, dry climate. Examination of corneal tissue and tears from patients with CDK suggested an involvement of metalloproteinases (MMPs) in the disease-associated tissue degradation. Our cellular model helped reveal the connexion between UV radiation and the unbalanced secretion of gelatinases (MMP-2 and MMP-9) and thus explain in part the pathogenesis of this rare disease. The evaluation of the inflammatory response to UV, initially, and then to LPS, or HO, highlighted IL-8 secretion as an acute stress marker and followed throughout the studies. Human corneal epithelial cells were found also to release lipid-modifying enzymes into the cell culture medium as a response to stress. Of particular importance to us were the enzymes of the sphingolipid metabolism, a lipid signalling pathway of great importance in the stress response. These enzymes were released as part of cell-derived extracellular vesicles, the vesicle-lipids, however, were the mediators of a significant decrease in IL-8 levels. The same sphingolipid enzymes appeared responsible for the intracellular response to HO, controlling the IL-8 production but also the stress-induced neutral lipid loading.

We have therefore succeeded to establish a causative link between UV radiation and tissue degeneration in CDK, to determine the role of the sphingolipid signalling pathway in ocular surface stress and to discover more about HO consequences in the corneal epithelium. The stress response at the ocular surface is a thin balance between tissue protection and maintenance of function. Inflammation represents one of the most relevant clinical signs of distress and we aimed to identify targets for therapies that seek to restore tissue homeostasis.