High Glucose Induces Biomolecular Dysregulation in Primary Human Corneal Epithelial Cells: Potential Biomarkers of Hyperglycemia-Driven Corneal Impairment.

Corneal hyperglycemia is associated with epithelial dysfunction characterized by impaired inflammation, oxidative stress, neuropathy and innate immune mechanisms, as observed in diabetic corneal keratopathy. Therefore, the aim of the study was to investigate at cellular level the effects of moderate to severe acute and chronic challenges of glucose on human primary cultures of corneal epithelial cells (CECs). Human CECs (Innoprot, Spain) were exposed to single or repeated 15 mM or 30 mM High Glucose (HG)-supplemented media (CEC Medium containing plain 5 mM glucose), every 2 days from first stimulations and harvested at day 1 (single/acute challenge) or at days 3 and 5 (repeated/chronic challenge). Mannitol was used as osmotic control and cell sustainability were monitored by trypan blue exclusion test and MTT. Conditioned media were subjected to protein analysis while monolayers were processed for relative real-time RT-PCR. Few selected mediators belonging to the inflammatory, complement, innate receptor, oxidative, DNA enzymes and neurotrophic pathways were tested, and protein levels were assayed on selected mediators (IL-6, IL-8, CFH and C5b-9; ELISA). HG exposure induced a time- and concentration-dependent decrease in CEC metabolic activity, without a comparable reduction in cell number. Acute HG exposure promoted an inflammatory response characterized by increased