Abstract
Abstract: :
Purpose: Fibroblasts, which play a central role in wound healing and tissue remodeling as target/effector cells, have been shown to express constitutive Nerve Growth Factor (NGF) and trkA receptor. We have recently shown the ability of NGF to induce the expression of α-smooth muscle actin (α-SMA) in conjunctiva, lung and skin fibroblasts, indicating their phenotype conversion into myofibroblasts. During wound healing, myofibroblasts, usually arising from quiescent fibroblasts/keratocytes, cause wound contraction and subsequently disappear from the repaired tissue. The aim of this study was to examine the effect of NGF on the behavior of in vitro induced conjunctival myofibroblasts in a 3D collagen system. Methods: Confluent cultures of human conjunctiva derived fibroblasts were serum-starved for 3 days (optimized conditions to induce the myofibroblast phenotype in vitro) and treated with TGF-ß1(positive control to induce α-SMA expression) in serum-free conditions. After this pre-treatment, the cells were replated at high density in serum-free conditions (quiescent cells) or at low density in the presence of serum (proliferating cells). The presence of induced myofibroblasts was assessed by evaluating the quantitative expression of α-SMA protein (FACS/ RT-PCR ELISA). The induced myofibroblasts were replated (monolayer or 3D) in either proliferating or quiescent conditions in the presence of TGF-ß1 and/or NGF (6 days). Results: The expression of NGF, trkA and p75 mRNAs by quantitative RT-PCR, and the rate of expression of trkA and p75 proteins by immunocytochemistry and FACS, were evaluated. The p75 expression and the percentage of apoptotic cells (TUNEL positive) present in both culture systems were found to be increased after NGF stimulation, compared to those found in TGF-ß1-induced myofibroblasts before or after additional incubation with TGF-ß1. Conclusion: Our findings demonstrate that NGF is able to stimulate in vitro the expression of trkA+/p75+ in human conjunctival myofibroblasts and that this expression seems to be associated to apoptosis. These data, together with the capability of NGF to cause the myofibroblast phenotype, indicate important roles for NGF both at the beginning and the end of the healing process in human conjunctiva.
Keywords: 365 conjunctiva • 380 cytokines/chemokines • 631 wound healing