Abstract
Purpose :
Type 2 diabetes is known to cause changes in corneal stiffness, wound repair and a number of other factors. Previous studies showed that glycosylation of P2X7 and pannexin-1 affects the targeting of these proteins to the plasma membrane. Our lab has demonstrated not only the attenuation in localization of pannexin-1 and P2X7 at the leading edge of a wound in pre-type 2 diabetic mice but also increased P2X7, Bcl2, and Ki67 expression in epithelium from human diabetic corneas compared to age-matched controls. We hypothesize that dysregulation of epithelia may depend on proliferation, glycosylation and localization of P2X7 and pannexin-1 and our goal is to evaluate it in a number of diabetic models.
Methods :
To analyze proliferation, intact eyes from male control and NONcNZO10/LtJ (NON) diabetic mice (12 weeks) were assessed, along with human primary, human primary diabetic, and human corneal limbal epithelial (HCLE) cultures. H and E stains are performed on islet cells. Subconfluent control cultures were also stimulated with high glucose or mannitol for 24 to 48 hours. Edu Assays (Abcam) were performed on cells and tissue, counter stained with DAPI and imaged using confocal microscopy and analyzed using the freeware Cell Prolifer. Cells were analyzed for changes in glycosylation using tunicamycin and PNGase F, and analyzed via silver stain, glycosylation assay, and western blot analysis. Protein localization was observed using immunohistochemistry.
Results :
There was an increase in proliferation in the limbal-corneal region of the NON diabetic mice, but none in the central cornea. The mice frequently exhibited corneal abrasions. In the cultures there was no change in proliferation in both primary diabetic and control cells or in cells incubated with high glucose (30 mM) compared to normal glucose levels or mannitol controls. Cells treated with PNGase F revealed an increase in pannexin-1 glycosylation in human diabetic cells compared to HCLEs.
Conclusions :
Our results indicate that the change in proliferative cells in the limbal-corneal region may alter regulation. Stimulation with elevated levels of glucose did not induce a similar change. Differential glycosylation profiles of P2X7 and pannexin-1 are associated with diabetes and may contribute to impaired wound healing. Future studies will examine corneal epithelium from various ages.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.