Novel Role for CGRP in Aqueous Humor Outflow

Purpose: Calcitonin gene related peptide (CGRP)-positive nerve fibers are the major type of sensory neurons innervating the trabecular meshwork (TM) and Schlemm's canal (SC). Upon activation, these neurons secrete CGRP locally. Here, we determined the role of CGRP in regulating homeostatic TM functions and the relation of CGRP-positive neurite innervation to segmental outflow regions. Methods: CGRP receptor expression in primary human TM (HTM) and primary human SC (HSC) cells in culture was determined by western blot analysis. HTM cells were treated with CGRP (0.1-3 µM) and fluorescent beads for 24 hours or were treated with CGRP (1 µM), TGFβ2 (5 ng/mL), and CGRP+TGFβ2 for 48 hours and processed for western blot analysis of alpha-smooth muscle actin (α-SMA) and collagen type 1 alpha 1 (COL1A1) expression. Segmental flow regions were determined in 9-month-old C57BL/6J mice using fluorescent tracer beads. Anterior segment flatmounts were co-labeled with antibodies against CGRP, CALCRL (CGRP receptor), and PECAM-1 (SC endothelium) and imaged by confocal microscopy. The density of CGRP neurites and CALCRL expression in each flow region were quantified by ImageJ analysis. Results: CGRP receptors are expressed in human TM and SC cells. CGRP treatment significantly increased phagocytosis and blocked TGFβ2-induced COL1A1 and α-SMA expression. Significantly more CGRP-positive neurites were identified in high-flow regions compared to all other flow regions. CALCRL expression was significantly associated with high-flow and moderate-flow regions compared to no-flow regions. Conclusions: These data suggest that CGRP modulates important homeostatic mechanisms of TM function and may influence the development and regulation of segmental regions of aqueous humor outflow.