Effects of Nerve Growth Factor (NGF) on Human Ocular Surface Epithelium

Abstract

Abstract: : Purpose: The aim of this study is to evaluate the effects of NGF on primary culture of human corneal and conjunctival epithelia and, particularly, its role in proliferation and differentiation events. Methods: NGF concentration was evaluated by a specific ELISA in culture medium of corneal and conjunctival epithelial cells at different time. NGF levels were related to proliferation and differentiation markers of the cells culture. In order to investigate the effect of NGF on proliferative compartment, exogenous NGF at concentrations between 0.01 to 250 ng/ml was added to the culture medium. The total colony forming efficiency and the percentage of aborted colonies were evaluated at different NGF concentrations. The inhibition of endogenous NGF was evaluated in parallel cultures by adding neutralizing anti–NGF antibody in concentration ranging between 100 to 250 ng/ml. Pathways of NGF involved were analysed by culturing conjunctival epithelium in presence of increasing concentration of NGF and antibodies against the TrkA and the p75 receptors. Effects on differentiation was investigated by RT–PCR analysis, immunocitological stainings and western blot analysis on cell pellets and culture medium. Results: Primary culture of human corneal and conjunctival epithelial cells produce and release endogenous NGF in the culture medium in a time–dependent fashion during clonal growth. The concentration of NGF in the medium raise a 3–5 fold increase before confluence of both conjunctival and corneal cultures with a corresponding increase in the total number of daughter colonies. Addition of exogenous NGF did not show an increase in p63 highly positive cells, but showed an increase in 14–3–3 sigma expressing cells and MUC 5AC expression levels, suggesting a effect on differentiation of conjunctival cells. The biological effect was reversed by the antibody neutralising the endogenous NGF. On going study suggests that different NGF receptors may differentially modulate conjunctival cell differentiation. Conclusions: This study confirms the role of NGF in the pathophysiology of the ocular surface and suggests some likely mechanisms of action. Moreover the demonstration of NGF role in the differentiation of conjunctival epithelium highlights potential involvement of this neurotrophin in a wide spectrum of ocular surface diseases.

Keywords: conjunctiva • cornea: epithelium • growth factors/growth factor receptors