In this study, we investigated the effects of chronic hyperglycemia on the corneal epithelial wound response and healing in a rat model of type 2 diabetes. Consistent with previous findings in other laboratories, we observed that the closure of a corneal epithelial debridement wound in DM rats maintained in a hyperglycemia state for 6 months was significantly delayed compared with that in weight-matched normoglycemic controls. The epithelial cells derived from both nonwounded and healing corneas of hyperglycemic rats had much reduced EGFR phosphorylation as well as activation of its downstream effectors, PI3K-AKT, and ERK, compared with controls. Of note, we showed for the first time, that the phosphorylation of the proapoptotic protein BAD, indicative of its inactivation, was also impaired in uninjured and to a lesser extent, in the healing corneal epithelia of diabetic rats. Consistent with decreases in AKT activation and BAD phosphorylation, there were decreases in PCNA-positive cells, especially in the wound's leading edge, and increases in TUNEL-positive cells in the diabetic rat corneas. To our knowledge, this is the first report directly linking the decrease in cell proliferation and increase in apoptosis to delayed epithelial wound healing in diabetic cornea. Taken together, we conclude that hyperglycemia perturbs the EGFR-PI3K-AKT and ERK signaling pathways in normal and healing corneas and that increased cellular apoptosis and decreased cell proliferation may be the contributing factors in the impairment of corneal epithelial wound healing in diabetic corneas.
In the literature, age-matched animals were used in most studies of diabetic wound healing.6,36–38
In this study, we maintained the STZ rats in a hyperglycemic state (~500 mg/dL) for ~6 months (age, 7.5 months) and chose weight-matched normoglycemic SD rats (age, 4.5 months) as the controls for wound-healing experiments. Because of delayed growth of STZ rats (), choosing a proper control for a corneal wound-healing study was problematic. On the one hand, aging is known to affect wound healing. On the other hand, eye size may be related to body mass; hence, the size of sublimbal corneal epithelial debridement is likely to be smaller in diabetic rats than in age-matched control rats. Given that rats age approximately 35 times faster than humans, neither age group is considered to be of advanced age. Hence, we chose weight-matched normal and DM rats for this study with diabetic rats at 7.5 months of age. We created limbus-to-limbus epithelial debridement wounds in the cornea. Similar to other studies using age-matched STZ rats,6,36
we observed delayed wound closure within the first 24 hours; however, the difference at this time point did not reach statistical significance (P
> 0.5). At 48 hours pw, the average remaining wound area in the diabetic rats was significantly larger than those in the normoglycemic rats. Forty-eight hours was chosen for the biochemical studies, because at that time point most whole corneal wounds were nearly healed, or delayed healing was observed. We conclude that the healing of limbus-to-limbus corneal epithelial debridement wounds was impaired in SZT-induced diabetic rats. Our results, similar to those in other studies,6,37–39
indicated that chronic hyperglycemia leads to diabetic complications, including poor wound healing, in animal models of human type I DM.
Diabetic keratopathy has been linked to defective basement membrane assembly,40–42
alterations of the corneal extracellular matrix, elevated matrix metalloproteinase,43
and, most important, defects in innervation due to diabetic neuropathy (neurotrophic keratopathy).2,44
Although animal models, primarily rat and mice, of human DM have been the subject of many studies,3,6,36–38,45–48
few studies have gone beyond histochemical analysis, because of the limitation of sample size (the amount of cells collected from the cornea). In our laboratory, we have established an effective Western blot analysis system that allows detection of the activation of multiple signaling molecules simultaneously. In this study, we used an animal model of type I diabetes and epithelial debridement wounding. We observed that although the levels of phospho-EGFR-PI3K-AKT and -ERK in DM were lower than that observed in normal rats, the healing epithelial cells, unlike those isolated from the unwounded corneas, had clearly detectable phospho-AKT in DM rats. This suggests that the epithelial response to wounding was impaired but still somewhat functional and that defects in EGFR signaling may be a cause for the delayed epithelial wound closure. This result is consistent with a pig organ culture study that showed impairment of EGFR signaling in corneas cultured in high glucose. The mechanisms underlying the impairment of EGFR signaling in diabetic corneas, including the defects in the expression and/or the release of EGFR ligands which are tightly regulated in normal corneas,49–52
are currently under investigation in the laboratory.
Consistent with the Western blot analysis results, immunohistochemistry also revealed a decrease in the staining intensity of phospho-AKT in diabetes, compared with normoglycemic corneas. The staining patterns observed in uninjured corneas were very similar to those in human corneas.28
Recently, Saghizadeh et al.53
reported that the overexpression of matrix metalloproteinase-10 and cathepsin F, two proteinases with elevated expression in human diabetic corneas,54
resulted in a decrease in phosphorylated AKT and delayed epithelial wound closure in cultured, normal human corneas, suggesting that defects in cell–matrix interaction may contribute to impaired EGFR-PI3K-AKT signaling in diabetic corneas. It is of interest to note that two factors, insulin and HGF, which were shown to accelerate delayed diabetic corneal healing, are also known to stimulate EGFR transactivation in vitro.55,56
Although no increases in AKT and ERK phosphorylation have been observed in human diabetic corneas overexpressing c-Met, our preliminary study showed topical insulin accelerated epithelial wound healing without altering EGFR-AKT signaling (Yu F-SX, unpublished results, 2009). Further studies to assess cross-talk of these trophic factors in mediating cell signaling and wound healing are warranted.
In addition to changes in activities, we showed alterations in phospho-AKT distribution in healing diabetic cornea. In the nondiabetic corneas, phospho-AKT staining was strong in epithelial cells at the leading edge. The active protein was mostly in the front half of the cells and/or near the sites of cell–matrix adhesion. In the diabetic rat corneas, the staining of phospho-AKT is much weaker, with no clear concentration of phospho-AKT within the cells at the leading edge. Another intriguing alteration in phospho-AKT staining is the nonbasal distribution of phospho-AKT in the stratified epithelium of the diabetic corneas. In the normoglycemic rats, phospho-AKT was primarily found, either in the basal cells near the leading edge, or in basal and wing cells of the stratified epithelia. In diabetic healing corneas, the phospho-AKT staining was weak in intensity and found in nonbasal cells of stratified epithelia. Hence, although abnormalities in the basement membrane are likely to influence EGFR-PI3K-AKT activation, the lack of active AKT in the basal cells in healing diabetic corneas may in turn retard the disassembly and reassembly of the basement membrane, which probably plays a role in the healing of large-diameter debridement wounds.57
Taken together, these results shed new light on the role of PI3K-AKT in the cornea: regulating and assembling the cell migration apparatus that drives cell–matrix interaction and forward movement. Defects in both AKT activation and/or cellular localization may contribute to the delayed epithelial wound healing in the diabetic cornea.
PI3K/AKT has been shown to control diverse cellular activities, including cell survival, growth, proliferation, metabolism, and migration.58
Similarly, the Ras/Raf/MEK/ERK pathway is involved in the regulation of cell cycle progression and apoptosis.59
Downstream effectors of EGFR, AKT, and ERK promote cell survival by phosphorylating proapoptotic molecules such as Bad and FoxO3a.60
BAD phosphorylation is a major mechanism by which trophic factors inactivate the apoptotic machinery.61,62
Consistent with phospho-AKT levels in rat CECs, AKT-specific site phosphorylation of BAD was observed in normoglycemic CECs. However, the site-specific phosphorylation was not detectable in uninjured epithelia and was reduced in the healing epithelial cells of diabetic rats. Parallel to the AKT pathway, the Ras/Raf/MEK/ERK signal-transduction pathway regulates cell cycle progression and cell survival in diverse types of cells. Decreases in pERK activities were found in DM corneal epithelia with low levels of pBAD (Ser112
). These novel findings reveal that BAD functions as a sentinel for select apoptotic signals in the cornea. Hyperglycemia may tip the balance of BAD phosphorylation, resulting in CEC apoptosis and delayed wound healing.
To determine whether epithelial abnormalities, including basal cell degeneration3
and altered cell proliferation6,38
can be found in a rat model of diabetes, we identified TUNEL-positive cells in diabetic corneas at the apical layer, even in healing corneas, where cell proliferation, required for cell repopulation, is expected. Similarly, TUNEL-positive cells were found in human diabetic, but not age-matched control corneas (Xu K and Yu F-SX, unpublished data, 2010). Detected increases in TUNEL-positive cells suggest that hyperglycemia increases epithelial apoptosis in the cornea, in line with decreases in BAD phosphorylation. We also used PCNA staining as a marker for proliferating cells. We found that wounding greatly increased the number of PCNA-positive cells, and that hyperglycemia significantly decreased the number of PCNA-positive cells in healing epithelia of rat corneas. Decreases in PCNA-positive cells suggest an impaired proliferating capacity in the diabetic corneas in response to wounding. Hence, hyperglycemia appears to impair cell proliferation and to induce epithelial apoptosis in the cornea in an EGFR-PI3K-AKT– and ERK-related manner. To date, the direct link between dysfunctional EGFR signaling and the diabetic pathophysiological condition remains to be established. Using STZ diabetic rats, we recently observed that combination of two EGFR ligands, HB-EGF and TGF-α, applied topically accelerates delayed corneal epithelial wound healing, suggesting that restoring aberrant EGFR-signaling pathways may represent an effective therapeutic strategy for treating diabetic keratopathy and delayed corneal wound healing in the cornea.