Project Grant R21EY034650

MOLECULAR MECHANISMS OF COLD STORAGE-INDUCED DAMAGE TO THE CORNEAL ENDOTHELIUM - MOLECULAR MECHANISMS OF COLD STORAGE-INDUCED DAMAGE TO THE CORNEAL ENDOTHELIUM THE ENDOTHELIUM AT THE POSTERIOR SURFACE OF THE CORNEA IS A NON-REGENERATIVE MONOLAYER. IT MAINTAINS CORNEAL TRANSPARENCY THROUGH ITS BARRIER AND FLUID PUMP FUNCTIONS. THUS, A RAPID DECLINE IN ENDOTHELIAL CELL DENSITY (ECD), OBSERVED DURING GENETIC DISORDERS OR IN RESPONSE TO INTRAOCULAR SURGERY, DISTURBS CORNEAL TRANSPARENCY. MOREOVER, SINCE THERE ARE NO DRUGS TO HALT ENDOTHELIAL CELL LOSS (ECL) DURING THESE DISORDERS, CORNEAL TRANSPLANTATION IS THE STANDARD OF CARE WHEN ECD < 600-800 CELLS/MM2. IN 2021, > 49,000 TRANSPLANTS WERE CARRIED OUT IN THE USA AT THE COST OF ~ $40,000/TRANSPLANT. IN ADDITION TO THE GIFT OF SIGHT, THE PROCEDURE SAVED AN ESTIMATED LIFETIME VALUE OF $5.8 BILLION. ALTHOUGH CORNEAL TRANSPLANTS ARE MORE SUCCESSFUL THAN OTHER SOLID ORGAN TRANSPLANTS, DELAYED RECOVERY OF CORNEAL TRANSPARENCY AND A RAPID DECLINE IN ECD AFTER THE SURGERY ARE TWO SIGNIFICANT CHALLENGES YET TO BE FULLY UNDERSTOOD. THE DECREASE IN THE ECD AFTER SURGERY IS IN ADDITION TO ECL THAT OCCURS DURING COLD STORAGE (CS). HOWEVER, THE MOLECULAR MECHANISMS FOR ECL DURING CS OR ACUTE ECL AFTER SURGERY ARE UNKNOWN. OUR RECENT STUDIES HAVE SHOWN THAT CS ADVERSELY IMPACTS THE ENDOTHELIUM. SPECIFICALLY, WHEN FRESHLY ISOLATED PORCINE CORNEAS WERE HELD IN PROLONGED CS (> 3 DAYS AT 4 C), ENDOTHELIUM EXHIBITED MICROTUBULE (MT) DISASSEMBLY, DESTRUCTION OF ITS PERI-JUNCTIONAL ACTOMYOSIN RING (PAMR), LIPID PEROXIDATION, AND CELL DEATH. THESE EFFECTS WERE INHIBITED BY INCLUDING ANTIOXIDANTS OR IRON CHELATORS (DEFEROXAMINE) IN THE CS MEDIUM. THUS, WE HYPOTHESIZE THAT CS INDUCES OXIDATIVE STRESS IN THE ENDOTHELIUM, LEADING TO (A) A BREAKDOWN OF ITS TIGHT JUNCTIONS VIA THE DESTRUCTION OF ITS PAMR AND (B) THE FORMATION OF ISLANDS OF DENUDED DESCEMET'S MEMBRANE (IDDMS) BECAUSE OF CELL DEATH. TO REGAIN CORNEAL TRANSPARENCY, THE EXTREME LOSS IN BARRIER FUNCTION MUST BE CORRECTED BY THE REPOPULATION OF IDDMS AND NORMALIZATION OF THE TIGHT JUNCTIONS. THE MAIN GOAL OF THIS PROJECT IS TO TEST ALL OUR HYPOTHESES BASED ON TWO SPECIFIC AIMS. FIRST, IN AIM 1, WE WILL DETERMINE HOW CS INDUCES MITOCHONDRIAL DYSFUNCTION IN THE ENDOTHELIUM, LEADING TO OXIDATIVE STRESS. WE WILL ALSO ASSESS IF THE MODE OF CELL DEATH ALSO INVOLVES FERROPTOSIS. SUBSEQUENTLY, IN AIM 2, WE WILL DETERMINE HOW OXIDATIVE STRESS DESTROYS THE PAMR, THEREBY CAUSING A BREAKDOWN OF TIGHT JUNCTIONS. IN ADDITION, WE WILL TEST THE ASSUMPTION THAT THE REPOPULATION OF IDDMS IS SLOWED BY OXIDATIVE STRESS PREVAILING IN THE ENDOTHELIUM, LEADING TO A DELAY IN THE RAPID RECOVERY OF CORNEAL TRANSPARENCY AFTER CS. OVERALL, THIS PROJECT WILL DISSECT THE MOLECULAR MECHANISMS THAT ADVERSELY AFFECT THE ENDOTHELIUM DURING CS. WE EXPECT OUR FINDINGS TO ENABLE PHARMACOLOGICAL APPROACHES BY WHICH WE CAN LIMIT ECL DURING CS AND ACUTELY AFTER SURGERY FOR ENHANCED CORNEAL TRANSPLANTATION.