Regulatory Role of CTL-O2/Bcl2 in Epithelial Homeostasis

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DESCRIPTION (provided by applicant): Our past research has shown that contact lens (CTL) wear significantly impacts the normal homeostatic control of the corneal epithelium (EH), leading to reduced surface cell shedding, prolonged basal cell retention and decreased proliferation. The net sum of these effects is to produce a "slowed down" (EH) or "stagnant" epithelial layer characterized by decreased central epithelial corneal thickness. At the same time, CTL-associated hypoxia also produces upregulation of P. aeruginosa (PA) receptors on the corneal surface, thus increasing the risk for microbial keratitis (MK). Based on these findings, two important distinct and related questions remain: (1) what CTL wearing schedule produces the LOWEST risk for MK and least disturbance of EH?; (2) what mechanism(s) regulate normal corneal EH and how does CTL wear perturb them? Recent results suggest that Bcl-2 protein(s) expression and location (cytoplasm, nucleus) may constitute a critical nexus regulatory site for these events. To test this hypothesis, we will use an in vivo CTL model in rabbits and a simulated CTL-wear model in rabbits/mice with eyelid closure that will allow experimental modulation of EH and PA binding without inflammation, injury or use of antimitotic agents. We will also use 3 strains of genetically altered mice (WT controls), which exhibit Bcl-2 overexpression (transgenic gain of function), Bcl-2 KO loss of Bcl-2 function) and a Bax KO mouse to establish a regulatory role for Bcl-2 protein(s) in EH and CTL-induced PA binding. Using our newly developed technologies (in vivo confocal; laser confocal triple fluorescence imaging) to assess EH in vivo and ex vivo, we will pursue 5 specific aims: (1) determine whether daily or de novo continuous CTL-wear produces least risk for MK or disturbance of EH in man; (2) establish a significant correlation between Bcl-2 expression and location, EH and risk for MK (PA binding) in normal rabbit and mouse (open, prolonged closed eye); (3) establish the same correlates as in (2) for prolonged periods (1,2 weeks) in a contact lens exposed rabbit cornea using test CTLs of differing types [rigid, soft, silicone hydrogel (Sih)] and lens-O2 transmission; (4) determine the status of EH in mice with stable TG gain of Bcl-2 function, Bcl-2 KO (loss of function) or the pro-apoptotic Bax KO (WT controls) and correlate changes in EH and MK risk (PA binding) to Bcl-2 expression location; (5) modulate corneal EH and PA binding levels in the TG and KO mice corneas by eyelid closure with/without prolonged CTL exposure with variable-O2, mouse-specific, test CTLs (1, 3, 7, 14 days), determine effects on Bcl-2 expression, and correlate changes in PA binding to EH and Bcl-2 expression/location.