Sphingolipid Signaling in Vesicating Ocular Injury

About This Grant

Vesicating (blister-forming) chemical-threat agents such as sulfur mustard (SM) or mustard gas, nitrogen mustard (NM), lewisite, and phosgene oxime can cause moderate to severe injuries and pain to the skin, eyes, and lungs. SM and NM are highly reactive bifunctional alkylating agents that can covalently modify all major cellular biomolecules, such as DNA, proteins, and lipids; thus, they are highly toxic. The eyes are particularly vulnerable to vesicant injuries, which cause a biphasic pathology of an acute response of photophobia, corneal erosions and inflammation, and chronic or late effects with significant deterioration of corneal structure and function from neovascularization, epithelial defects, fibrosis, and opacity. No therapeutic drugs are available as Medical Countermeasures (MCMs) for vesicant damage to the eye, eyelid or other organs. The major obstacle in developing potential MCMs is our limited understanding of the complex pathophysiological response of the eye after vesicant exposure. In this application, we propose to test the hypothesis that vesicating ocular injury pathology involves bioactive sphingolipid (SPL) pathways for acute and chronic inflammation and subsequent cornea, conjunctiva, and eyelid damage, causing significant vision impairment and dry-eye symptoms. In preliminary studies, we developed and characterized an NM-induced ocular surface injury (NMOSI) in mice, exposing the entire ocular surface to NM instead of only the cornea. We observed a severe acute inflammatory response that resolves in a month and cause damage to the cornea, atrophied eyelid glands, almost complete loss of vision, and apparent dry-eye symptoms. We found increased activity of acid sphingomyelinase, concurrent reduction in the sphingomyelin, and increased ceramides, suggesting sphingomyelinase activation in ocular surface tissue at three days post-exposure. Here, we propose to characterize NMOSI models in mice and rabbits, focusing on conjunctival goblet cells and epithelial stem cells and how NM affects the eyelids and their glands and causes dry-eye symptoms (SA #1). We will determine the temporal and spatial relationship of NM to SPL pathway for acute toxicity in ocular surface tissue of mice and rabbits separately from the cornea, conjunctiva-sclera, and eyelids at different time points (SA #2). It is unknown whether NM or SM-induced SPL pathways are overlapping. Hence, we propose to study if the NM- induced SPL pathway activation is similar to SM exposure (SA #3). Lastly, we plan to map out the pathway of SPL activation and lipid signaling using in vitro assays with meibomian gland epithelial and corneal cell lines (SA #4). We expect to identify novel associations of bioactive lipids in the inflammatory and wound-healing pathways of vesicating ocular injury, which will aid in improving our understanding of pathophysiological mechanisms of the injury and aid in developing potential MCMs in the future.