Abstract
Purpose :
Angiogenesis in the normally avascular cornea underlies common corneal diseases. Corneal nerves, derived from trigeminal ganglion (TG), are master regulators of ocular surface homeostasis. Here we seek to determine whether corneal nerves directly modulate angiogenesis and investigate the role of nerve-derived neuropeptides.
Methods :
TG neurons were isolated from BALB/C mice and cultured in Neurobasal A medium. Conditioned media (CM) of TG neurons was collected between Day 3 and 4 of culture. A co-culture system was set up by placing TG neurons in in the top chamber of a transwell and culturing vascular endothelial cells (VEC, MILE SVEN1 cell line) in the bottom chamber. VEC proliferation, migration, and tube formation were determined with and without TG neurons or their CM. in vivo corneal angiogenesis was induced by placing intrastromal sutures in BALB/C mouse corneas. Expression of neuropeptides were determined using immunofluorescence.
Results :
Presence of TG neurons in co-culture decreased VEC proliferation by 35% (P=0.008) and migration by 20% (P=0.046). Similarly, conditioned media (CM) of TG neurons reduced VEC proliferation (P<0.0001), migration (p=0.017), and the number of junctions (P=0.004) and length of tubes (P=0.001) formed by VEC. Topical application of neuron CM led to a 76% reduction in suture induced-corneal angiogenesis in vivo (P=0.025). TG neurons and corneal nerves express neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP). In addition, more than 80% of TG neurons and 90% of subbasal corneal nerves express alpha-melanocyte-stimulating hormone (α-MSH). Exogenous SP (1μM) and CGRP (100nM) promoted VEC proliferation and tube formation in vitro, and local application of their antagonists Spantide I and CGRP837, respectively, led to decreases in corneal angiogenesis in vivo. In contrast, α-MSH (100nM) reduced VEC proliferation, migration, and tube formation in vitro and subconjunctival injection of α-MSH reduced corneal angiogenesis by 56% (P=0.008) in vivo. Antagonizing α-MSH signaling with Agouti-signaling protein or siRNA knock-down in TG neurons reversed the inhibitory effects of neuron CM on VEC proliferation, migration, and tube formation.
Conclusions :
TG neurons and corneal nerves express pro-angiogenic neuropeptides SP and CGRP, and anti-angiogenic peptide α-MSH, which plays a critical role in the direction modulation of corneal angiogenesis by corneal nerves.
This is a 2021 ARVO Annual Meeting abstract.