# Involvement of Vasoactive Intestinal Peptide Family Members in Diabetic Keratopathy
## 4 Discussion  
VIP family members have shown great promise as agents in the treatment of different ocular diseases including DK. In particular, VIP/VPAC receptor signaling exerted multiple positive active roles [83]. VIP, in combination with thymosin beta-4 (T\(\beta\)4), was shown to protect corneal epithelial cells against hyperglycemia-induced damage by promoting tissue wound healing and barrier integrity [171]. Furthermore, the exogenous treatment with the peptide restored corneal epithelial wound closure in locally denervated corneas by inhibiting the overexpressed proinflammatory factors. This aspect is perfectly coherent with the large benefits exerted by VIP in inflammatory diseases [172]. Moreover, several animal and human studies showed that VIP regulates the balance of pro- and anti-inflammatory cytokines by blocking the release of pro-inflammatory cytokines and chemokines and promoting the expression of anti-inflammatory cytokines [173]. Second, VIP/VPAC receptor signaling impairment contributed to the alteration in corneal epithelial wound closure and delayed sensory nerve regeneration in diabetic corneas. Accordingly, VIP increased the release of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) during DK through the activation of Sonic Hedgehog (Shh), which is required for proper wound healing in the corneas [83]. In fact, the mouse eye organ culture demonstrated that adding Shh into the culture medium promotes the proliferation of corneal epithelial cells by inducing the nuclear accumulation of cyclin D1 [174]. Furthermore, it was described that sympathetic overactivation, induced by hyperglycemia, activates the norepinephrine-\(\beta\)2-adrenoceptor pathway, which, in turn, inhibits Shh activity, leading to a reduction of corneal epithelial wound healing in diabetic mice [175]. PACAP has also been shown to have protective effects in DK. A recent study showed that the expression of PACAP and PAC1R is drastically reduced in the whole cornea of diabetic rats, suggesting that PACAP/PAC1R signaling alteration can concur with the corneal epithelium impairment induced by the hyperglycemic state. The exogenous administration of PACAP, in an in vitro model of DK, characterized by human corneal epithelial cells exposed to sustained levels of high glucose, promoted cell viability, and corneal epithelial wound healing through EGFR/ERK1/2 signaling pathway activation [176]. This mechanism, played by PACAP, confirms the key role of EGFR in corneal epithelial homeostasis [177]. In accord, the delay in corneal epithelial healing seems to be also associated with alterations in cell signaling of EGFR due to the hyperglycemic environment [178]. In fact, diabetes compromises EGFR signaling, acting at the level of the receptor, and the addition of ligand (EGF) is not sufficient to boost downstream signaling pathways [25]. Of note, hyperglycemia perturbs not only EGFR phosphorylation but also its downstream effectors, such as ERK1/2. The reduction of ERK1/2 signaling is correlated to the increase in apoptosis and decrease in cell proliferation concurring with the impairment of corneal epithelial wound healing in diabetic corneas [179]. Therefore, PACAP by stimulating EGFR/ERK1/2 signaling activity could promote the physiological state of corneal epithelium damaged by the hyperglycemic environment. In the cornea, NAP treatment was shown to reduce the generation of reactive oxygen species (ROS) by decreasing UV-B-irradiation-induced apoptotic cell death and c-Jun NH2-terminal kinase (JNK) signaling pathway activation [51]. It is well known that the pathological mechanism in diabetes is sustained by hyperglycemia, which in turn is responsible for excess oxidative stress, due to the increase in ROS and altered antioxidant capabilities [180]. Otherwise, the reduction of ROS has been shown to reduce diabetes complications, including complications in the cornea [181]. Therefore, NAP's ability to counteract the generation of ROS sets the stage for investigating its effect against hyperglycemia-induced ROS.