Vascular endothelial growth factor mediates corneal nerve repair.

The cornea, with primarily sensory and some autonomic innervation, is among the most densely innervated tissue of the human body.1 In humans, most corneal nerves originate from the ophthalmic branch of the trigeminal nerve and form a subbasal plexus under the corneal epithelium. This plexus extends thin nerve leashes that contain nociceptors at the surface of the cornea.2 Corneal nerves are of great importance because of their role in protecting the cornea from irritants and their trophic properties, which are necessary to maintain a healthy ocular surface.3 Disruption of the corneal nerves has been shown to impair corneal healing significantly.4 Vascular endothelial growth factor (VEGF) has long been known as a potent growth factor that stimulates the proliferation of blood vessels. Specifically, it is an endothelial cell mitogen and angiogenic factor and a potent mediator of vascular permeability.5 Signaling is mediated by the flt-1 (VEGFR1), flk-1 (VEGFR2), neuropilin (NRP)-1, and NRP2 receptors.6 VEGF is essential for blood vessel development and has been strongly implicated in the pathogenesis of diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and corneal neovascularization.7-12 The healthy cornea has minimal free VEGF.13 However, in injured states, VEGF expression has been shown to be upregulated and can contribute to corneal neovascularization.14 Anti–VEGF therapies have become important tools in slowing the growth of some cancerous tumors and have been used to treat ocular diseases such as macular degeneration. Recently, investigators have shown that the anti–VEGF neutralizing antibody bevacizumab can bind murine VEGF164 and human VEGF165 and that topical or systemic application of bevacizumab inhibits inflammation-induced angiogenesis and lymphangiogenesis in the cornea.15 VEGF has also been implicated as an important player in nerve growth. In vitro experiments have shown that VEGF and its receptor are expressed by neurons and stimulate neurogenic, neuroprotective, and neurotrophic activities.16-18 These activities include proliferation of cortical neurons, protection of central and peripheral neurons from hypoxia-induced death, and promotion of axonal outgrowth in peripheral neurons.19 VEGF has also been shown to have an important role in neurodegenerative diseases such as amyotrophic lateral sclerosis.20-22 Studies have indicated that systemic inhibition of VEGF with bevacizumab may cause apoptosis in retinal neurons.23 However, implications for anti–VEGF therapy on corneal innervation or nerve repair have not been explored. Here we used a transgenic mouse displaying corneal neurofluorescence24 to characterize the pattern of nerve regeneration in the cornea after epithelial debridement. Then, with the use of RT-PCR, immunohistochemistry, and Western blot, we investigated the expression of VEGF (VEGF164) and its receptors in the cornea and trigeminal ganglia. Last, we observed the effect of VEGF on trigeminal neuron outgrowth in vitro and the effects of anti–VEGF neutralizing antibodies on the axonal extension in vitro and on the regeneration of the subbasal nerve plexus after injury.