The PEDF Neuroprotective Domain Plus DHA Induces Corneal Nerve Regeneration After Experimental Surgery

The cornea is densely innervated, and several studies have shown that the health of corneal nerves is vital to maintain homeostasis of the ocular surface and tissue clarity.1–4 Many diseases that affect the cornea can compromise corneal innervation, leading to a decrease in tear production and blink reflex as well as impaired epithelial wound healing.4–8 As a result, neurotrophic keratitis and corneal opacification may ensue. It is estimated that 1.57 million people suffer from corneal blindness worldwide.9 In turn, some of surgical interventions such as refractive surgery and corneal transplantation sever corneal nerves, and studies have shown that corneal sensation can take years to recover, and in some cases it is permanently reduced.10,11 Unfortunately, there are few therapeutic interventions available today that can successfully promote the recovery of corneal sensation. Acknowledging our current limitations, the National Eye Institute (NEI) has identified the need for development of therapeutic agents that can stimulate corneal nerve regeneration as one of the highest priorities in vision research.12 Several molecules have been studied that may have a positive effect on corneal nerve regeneration, including nerve growth factor (NGF), VEGF, semaphorins, neurotrophins 3 and 4 (NT-3; NT-4), growth associated protein-43 (GAP-43), and others, all with variable success.13–17 Neuropeptides, such as substance P (SP) and insulin-like growth factor-1 (IGF-1), have been used to treat delayed wound healing due to corneal nerve damage when traditional treatments have failed.18 Pigment epithelial-derived factor (PEDF) is a glucoprotein discovered in the culture media of retinal pigment epithelial (RPE) cells and is widely expressed in different tissues, including the cornea.19,20 Pigment epithelial-derived factor has broad neurotrophic, neuroprotective, and antiangiogenic activity.21 Work in our laboratory has previously shown that treatment with PEDF in association with the ω-3 fatty acid, docosahexaenoic acid (DHA), after lamellar keratectomy increases regeneration of rabbit corneal nerves.22 We have also shown that corneal sensation returns to normal levels in treated animals only 8 weeks after surgical injury.23 The mechanisms of the neuroregenerative action of PEDF and DHA is not completely understood, but our studies suggest that it involves the synthesis of neuroprotectin D1 (NPD1), a docosanoid synthetized from DHA with strong anti-inflammatory and neuroprotective activity.24–26 Pigment epithelial-derived factor is a 50-kDa protein, and synthetic peptides derived from the sequence of human PEDF, which maintain part of its bioactivity, have been tried in models of choroid and corneal neovascularization, and in diabetic retinopathy.27–29 A 44-amino-acid fragment of PEDF, corresponding to positions Val78-Thr121 of the 418 amino acids of PEDF, has neuroprotective activity and has been shown to stimulate survival and differentiation of spinal motor neurons.30 However, the adjacent 34-amino-acid peptide, corresponding to positions Asp44–Asn77, has been shown to be antiangiogenic.31 Some therapeutic advantages of using small peptides include better tissue penetration, a narrower spectrum of action with reduced side effects, and ease of synthesis in reproducibly large-scale quantities, while a possible disadvantage could be faster biodegradation and lower efficacy of the drug. These are all important considerations in the development of therapeutic agents. In this study we compare the effect of the synthetic 44-mer PEDF and 34-mer PEDF peptides and the whole PEDF molecule in association with DHA on the regeneration of corneal nerves.