NGF and NGF-receptor expression of cultured immortalized human corneal endothelial cells

Abstract

Purpose: Several growth factors, including nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), play an important role in the homeostasis of the ocular surface. The involvement of both these growth factors in the pathophysiology of intraocular tissues has been extensively investigated. Despite the expression of NGF receptors by corneal endothelium, to date the role of NGF on the endothelial cell remains to be determined. Using a clonal cell line of human corneal endothelial cells, the aim of this study was to investigate the expression of the NGF-receptor and the potential partnership of NGF and VEGF in maintaining cell viability in vitro.

Methods: A human endothelial cell line (B4G12), was cultured under serum-free conditions as previously described with and without addition of different concentrations of NGF, anti-NGF-antibody (ANA), or VEGF for 4 days and these cells were used for immuno-istochemical, biochemical, and molecular analyses.

Results: NGF induces overexpression of NGF-receptors and synthesis and release of VEGF by endothelial cells and these cells are able to produce and secrete NGF.

Conclusions: These observations indicate that human corneal endothelial cells are receptive to the action of NGF and that these cells may regulate NGF activity through autocrine/paracrine mechanisms.

Figure 1

Representative immnocytochemical preparations showing the expression of NGF-receptors in HCECs cultured in vitro for 4 days under different conditions. TrkA expression in control condition (A), TrkA expression in cells exposed to NGF (B) and TrkA expression in cells exposed to ANA (C). Note that NGF enhances and ANA down-regulates the expression of TrkA. Panel D illustrate the expression of the NGF-receptor p75 in control conditions, in cells exposed to NGF (E), and in cells exposed to ANA (F). This NGF-receptor is nearly non-expressed in control HCECs (D), it is unaffected after treatment with NGF (E) or ANA (F). Scale bars: A-F 20 μm. Panels G and H report the results of western blot and real-time PCR of HCECs treated with NGF or ANA. As indicated in G and H, compared to control, HCECs exposed to NGF express more, while these exposed to ANA express less TrkA protein. The PCR analysis also revealed that NGF enhances and ANA reduces the expression of TrkA gene expression (I). Panel L reports the amount of NGF and VEGF expressed by HCECs under cultured for 4 days without NGF. This result indicates that these cells constitutively express, though differently, both NGF and VEGF. As illustrated in M, confocal immunohistochemical analysis revealed that HCECs express VEGF (red) and TrkA (green). Scale bar: M=8 μm.

Figure 2

This figure reports the effect of NGF on HCEC survival. A: The result of a time-course analysis of HCECs exposed for 4 days to culture medium alone or with 1 ng, 10 ng, or 100 ng of NGF/ml, revealed that at 1 and 10 ng, NGF enhances cell survival, while 100 ng/ml of NGF down-regulates cell survival, compared to control, (*p<0.05). B-D illustrate the structural aspect of these cells exposed to medium alone (B), 10 ng/ml (C), and 100 ng/ml of NGF (D). Note the decreased in cell number in D. Panels E and F illustrate the effect of endogenous NGF inhibition by ANA on HCEC survival. The supplementation of ANA in the culture medium at concentration of 100 ng/ml of medium, significantly (*p<0.05), reduces the number of cells after 4 days in vitro (F), compared to control (E). This effect was statistically significant (G). Scale bars: B-F=25μm.

Figure 3

Dose–response effect of NGF and ANA on VEGF expression in culture medium and cell pellet of HCECs after supplementation of 1, 10, and 100 ng/ml of NGF or ANA at 1, 10 and 100 μg/ml of medium. NGF enhances the presence of VEGF both in the medium and in pellet at concentration of 1 or 10 ng/ml of NGF. At 100 ng/ml NGF has an inhibitor action on VEGF expression. ANA exposure has no effect on the concentration of VEGF in the medium (C), but significantly reduces (*p<0.05) VEGF presence in the pellet at concentration of 100 ug/ml (D). Confocal microscopic analysis of cells exposed to 10 ng/ml of NGF and 100 µg/ml of ANA are illustrated, respectively in F and G. Note the enhanced expression of VEGF after exposure of NGF (F) and the down-regulation after exposure of ANA (G), compared to control (E). Arrows point to VEGF immunopositivity. Scale bars: E-G=15μm.

Figure 4

Dose–response analysis on the effect of VEGF on HCEC survival. The results indicated that VEGF supplementation into culture medium for 4 days enhances HCEC survival at 1, 10, and 100 ng/ml medium, compared to control (A; *p<0.05). The morphological aspect of HCECs exposed to control medium without and with 100 ng/ml of VEGF is shown in B and C, respectively. Scale bars: B, C=25μm.