Nerve Growth Factor (NGF) modulates in vitro cultured human derived keratocyte activities: a potential collaborative effect with TGF–ß1.

Abstract

Abstract: : Purpose: NGF is a pleiotropic factor contributing to the control of wound–healing and inflammatory responses in different tissues and in the cornea, where NGF and its receptors are expressed by epithelial, endothelial and stromal cells (keratocytes) under normal conditions. While NGF effects on epithelial cells are reported, no data are available on the effect of NGF on keratocytes, which play a central role in wound–healing and tissue remodeling as target/effector cells. Therefore, our aim was to investigate in vitro the pro–fibrogenic effects of NGF on human healthy keratocytes. Methods: The functional effects of NGF were investigated on human monolayers and 3D collagen embedded keratocytes isolated from healthy cornea biopsies (n= 7, Banca degli occhi del Veneto, Venice, Italy). The expression of trkA^NGFR and p75^NTR by in vitro cultured keratocytes was confirmed by confocal analysis. The pro–fibrogenic effects of NGF (1–500ng/mL), were analyzed by studying NGF ability to modulate keratocyte migration (in vitro wound model), proliferation/survival ([³H]–thymidine incorporation/Bcl–2), differentiation (α–SMA expression; confocal analysis and cell surface ELISA), metabolic activity (collagen production) and contraction of a 3D collagen matrix. These effects were compared to those of TGF–ß1, a well–known pro–fibrogenic factor. All data were analyzed using the ANOVA program followed by pos–hocs investigation. Results: Our data demonstrate that NGF is able to stimulate the migration of injured keratocytes across a wound line in a dose dependent manner, the differentiation into α–SMA expressing cells, known as myofibroblasts, and the contraction of keratocyte into a 3D collagen gel lattice, without affecting proliferation or collagen production. These cells were found to express both trkA^NGFR and p75^NTR (confocal analysis). These fibrogenic effects seem to be partially mediated by the release of TGF–ß1 upon NGF stimulation, as supported by neutralization studies and TGF–ß1 ELISA in the conditioned media of NGF stimulated keratocytes. Conclusions: Taken together, these findings indicate that NGF can modulate some fibrogenic activities of human healthy keratocytes. This finding would imply a possible active role of NGF on keratocytes during the healing of corneal ulcers.

Keywords: growth factors/growth factor receptors • cornea: stroma and keratocytes • proliferation