OBJECTIVE: Patients with diabetes are at an increased risk for developing corneal complications and delayed wound healing. This study investigated the effects of high glucose on epidermal growth factor receptor (EGFR) signaling and on epithelial wound healing in the cornea.RESEARCH DESIGN AND METHODS: Effects of high glucose on wound healing and on EGFR signaling were investigated in cultured porcine corneas, human corneal epithelial cells, and human corneas using Western blotting and immunofluorescence. Effects of high glucose on reactive oxygen species (ROS) and glutathione levels and on EGFR pathways were assessed in porcine and primary human corneal epithelial cells, respectively. The effects of EGFR ligands and antioxidants on high glucose–delayed epithelial wound healing were assessed in cultured porcine corneas.RESULTS: High glucose impaired ex vivo epithelial wound healing and disturbed cell responses and EGFR signaling to wounding. High glucose suppressed Akt phosphorylation in an ROS-sensitive manner and decreased intracellular glutathione in cultured porcine corneas. Exposure to high glucose for 24 h resulted in an increase in ROS-positive cells in primary human corneal epithelial cells. Whereas heparin-binding EGF-like growth factor and antioxidant N-acetylcysteine had beneficial effects on epithelial wound closure, their combination significantly accelerated high glucose–delayed wound healing to a level similar to that seen in control subjects. Finally, Akt signaling pathway was perturbed in the epithelia of human diabetic corneas, but not in the corneas of nondiabetic, age-matched donors.CONCLUSIONS: High glucose, likely through ROS, impairs the EGFR–phosphatidylinositol 3-kinase/Akt pathway, resulting in delayed corneal epithelial wound healing. Antioxidants in combination with EGFR ligands may be promising potential therapeutics for diabetic keratopathy.

Summary: The objective of this study was to examine the expression of Toll‐like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic‐polycytidylic acid [poly(I:C)]induces an antiviral response in these cells. Fluorescence‐activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)‐κB and production of the proinflammatory cytokine interleukin (IL)‐6 and the chemokine IL‐8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)‐β mRNA expression (7‐fold). Poly(I:C) stimulation also up‐regulated mRNA expression of the antiviral chemokine IFN‐γ inducible protein 10 (IP10), myxovirus resistance gene A and 2′,5′‐oligoadenylate synthetase (5‐, 10‐ and 9‐fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti‐TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)‐mediated IFN‐β expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.

We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-γ-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-γ-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-γ-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing.

Purpose.: The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). Methods.: Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. Results.: A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser¹³⁶ and Ser¹¹²) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. Conclusions.: In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.