Correlation between NGF/TrkA and microvascular density in human pterygium

Abstract

Pterygium is a surface ocular lesion that is associated with chronic UV exposure. The primary effect is a solar actinic elastosis within the stroma. All the other changes are secondary. Pterygium is characterized by proliferation, inflammatory infiltrates, fibrosis, angiogenesis and extracellular matrix breakdown. The aim of this study was to correlate microvascular density and nerve growth factor (NGF)/NGF-receptor transmembrane tyrosine kinase (TrkA) expression in endothelial cells in human pterygium. Specimens of human pterygium obtained from 30 patients who had undergone surgical excision and of 10 normal bulbar conjunctiva were investigated immunohistochemically by using anti-CD31, anti-NGF and anti-TrkA antibodies. Results showed that endothelial cells in human pterygium are immunoreactive to both NGF and its receptor TrkA, and that this immunoreactivity is correlated to microvascular density. The results of this study suggest that an autocrine loop between NGF and its receptor TrkA is activated in pterygium and that it is involved in the angiogenic response taking place in this pathological condition. These data are in accord with recent evidences, which have clearly established that NGF plays a role as an angiogenic factor in several pathological conditions. Understanding the mechanism of angiogenesis in pterygium provides a basis for a rational approach to the development of anti-angiogenic therapy in patients affected by this disease.

Pterygium is a surface ocular lesion that is associated with chronic UV exposure. The primary effect is a solar actinic elastosis within the stroma. All the other changes are secondary. Pterygium is characterized by proliferation, inflammatory infiltrates, fibrosis, angiogenesis and extracellular matrix breakdown. The ocular lesion begins to grow from limbal epithelium and invades the cornea centripetally followed by conjunctival epithelium, exhibiting degenerative and hyperplastic changes as well as proliferative, inflammatory features and a rich vasculature.

Seifert and Sekundo (1998) identified intraepithelial capillaries in the optical half of 42.3% pteryigia, providing evidence of neovascularization in pterygium. Many angiogenic cytokines, such as fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), tumour necrosis factor alpha (TNF-α) and interleukin-6 and -8 (IL-6 and IL-8), have been implicated and immunoreactivity for these growth factors has been demonstrated in epithelial cells, endothelial cells, fibroblasts and inflammatory cells (Kria et al. 1996; Lee et al. 2001; Di Girolamo et al. 2002, 2006; Marcovich et al. 2002; Aspiotis et al. 2007). We have correlated the extent of angiogenesis, measured as microvessel counts, with the number of tryptase-positive mast cells in tissue fragments from pterygium and normal bulbar conjunctiva and have demonstrated that the degree of angiogenesis is highly correlated with tryptase-positive mast cell counts (Ribatti et al. 2007).

Nerve growth factor (NGF) is a member of neurotrophin family. It was discovered on account of its ability to regulate growth, differentiation and survival of peripheral neurons during embryo development (Levi-Montalcini 1987). Later, the effects of NGF on neuronal cells of the peripheral and central nervous systems, and on several non-neuronal cells were also determined (Cassiman et al. 2001; Tabakman et al. 2004). The biological effect of NGF is mediated by two classes of receptors. p75, a 75-kDa glycoprotein that belongs to a superfamily of cytokine receptors, and a transmembrane tyrosine kinase of 140 kDa (TrkA), phosphorylated on tyrosine after binding to its ligand (Ebendal 1992). A general agreement seems to confirm that a TrkA expression is necessary for NGF signal transduction and NGF biological actions. NGF receptors have been demonstrated on different human tissues, not only of the central and peripheral nervous systems, but also on immune cells (Levi-Montalcini et al. 1990). Recent evidences have established that NGF plays a role as an angiogenic factor (Nico et al. 2008). NGF is able to stimulate migration and proliferation of endothelial cells, remodelling of extracellular matrix and functional maturation of newly formed blood vessels, and plays a crucial role in angiogenesis associated with several pathological conditions, such as cardiovascular diseases and tumours.

Here, we have investigated the immunoreactivity to NGF and its receptor TrkA in bioptic specimens of human pterygium and we have correlated this immunoreactivity to microvascular density.

Materials and methods

Patient population

The study group included 30 cases of surgically excised pterygium (15 males and 15 females), whose ages ranged from 25 to 67 years. All patients underwent excision by the bare sclera technique at the Department of Pathology, Cancer Center of Solca, Cuenca, Ecuador. Most of the lesions were located on the nasal side and only the head of primary pterygium was included as pterygium sample. Ten nasal epibulbar conjunctival segments, excised during cataract surgery near the limbus, were used as control tissues. The study protocol was approved by both the Ecuadorian and Italian Research Ethic Committees, because tissues were obtained from patients in Ecuador and subsequent laboratory studies were performed in Italy, and informed consent was obtained from all patients.

Immunohistochemistry

A murine monoclonal antibody (MAb) against the endothelial cell marker CD31 (MAb 1A10, Dako, Glostrup, Denmark) and two polyclonal antibodies against NGF (AB1526, Chemicon International Inc., Temecula, CA, USA) and TrkA (ab37837, Abcam, Cambridge, UK) were used. Briefly, 5 μm sections were collected on poly-L-lysine coated slides, deparaffinized by the xylene-ethanol sequence, rehydrated in a graded ethanol scale and rinsed for 10 min in 0.1 M phosphate-buffered saline (PBS). Thereafter, the sections were sequentially incubated with: (i) the antibody anti-CD31 (1:25 in PBS) and the antibodies anti-NGF and anti-TrkA (1:100 in PBS) overnight at 4 °C; (ii) biotinylated swine anti-rabbit Ig (Dako Italia, Milan, Italy) diluted 1:300 in PBS for 15 min at room temperature and (iii) streptavidin–peroxidase conjugate (Vector, Burlingame, CA, USA) diluted 1:250 in PBS for 15 min at room temperature. The immunodetection was performed in 0.05 M acetate buffer, pH 5.1, 0.02% 3-amino-9-ethylcarbazole grade II (Sigma Chemicals Co., St. Louis, MO, USA) and 0.05% H₂O₂ for 20 min at room temperature. Afterwards, the sections were washed in the same buffer and counterstained with Gill’s hematoxylin number 2 (Polysciences, Warrington, PA, USA), and mounted in buffered glycerin. Negative controls included an unrelated monoclonal IgG1 produced by the P3X63/Ag8 mouse secretory myeloma, replacing the antibody for the antibody anti-CD31, and pre-incubation with a 10-fold excess of specific blocking peptide (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for the polyclonal antibodies against NGF and TrkA.

Microvessel density, NGF and TrkA expression counting

These were simultaneously assessed by two investigators with a double-headed light microscope (Axioplan II, Zeiss, Oberkochen, Germany). Four to six 200× fields covering almost the whole of each of three sections per sample were examined with a 144-intersection point square reticulum (0.78 mm²) inserted in the eyepiece. Care was taken to select microvessels, i.e. capillaries and small venules, from all the CD31-stained vessels. They were identified as transversally sectioned tubes with a single layer of endothelial cells, with or without a thin basement membrane. Each assessment was agreed upon in turn. Microvessels were counted with a planimetric point-count method with slight modifications to restrict counting to transversally cut microvessels occupying the reticulum intersection points. As the microvessel diameter was smaller than the distance between adjacent points, only one transversally sectioned microvessel could occupy a given point. Microvessels transversally sectioned outside the points and those longitudinally or tangentially sectioned were omitted. Therefore, it was sufficiently certain that a given microvessel was counted only once, even in the presence of several of its section planes. As almost the entire section was analysed per sample, and as transversally sectioned microvessels hit the intersection points randomly, the method allowed objective counts. Endothelial cells stained with anti-NGF and anti-TrkA antibodies were counted on 4–6 fields covering the whole of each of three sections adjacent to those stained for microvessels, and means ± 1SD and medians were determined for each section, sample and group of samples. The relationship between microvessel density, and NGF and TrkA expression was examined by chi-square test or logistic regression analysis. Statistical significance was defined as P < 0.05.

Results

Pterygium tissues were more vascularized than normal conjunctiva (Figure 1a,b). Intense angiogenic response was observed particularly in the subepithelial area of pterygium (Figure 1a). Immunoreactivity to both NGF and NGF receptor TrkA was detectable in endothelial cells of the blood vessels in both pterygium and normal conjunctiva (Figure 1c–f). Immunoreactivity for NGF and TrkA was detectable also in epithelial cells of pterygium, whereas in normal conjunctiva, epithelial cells were negative to NGF and positive to TrkA (data not shown), according to recent data published by Di Girolamo et al. (2008). Table 1 shows the correlation between microvascular density, and NGF and TrkA counts, in both pterygium and normal conjunctiva. There are significant differences between these two groups concerning the microvessel density and the number of endothelial cells positive to NGF and TrkA. In fact, the within-group comparison showed that both counts were always significantly correlated.

Figure 1.

Immunohistochemical staining for CD31, NGF and NGF-receptor TrkA in bioptic specimens of human pterygium (a, c, e) and normal conjunctiva (b, d, f). Note a higher microvessel density in pterygium as compared with normal conjunctiva and that NGF and TrkA are both expressed by endothelial cells. Original magnification: ×250.

Table 1.

Microvascular density (MVD), immunoreactivity to nerve growth factor (NGF) and NGF-receptor TrkA (TrkA) in endothelial cells in human pterygium and normal conjunctiva

	MVD	NGF	TrkA
Pterygium (n = 30)	20 ± 5*	15 ± 3*	14 ± 2*
Normal conjunctiva (n = 10)	10 ± 3	4 ± 1	3 ± 1

^*P < 0.001 vs. normal conjunctiva.

Discussion

Pterygium is characterized by excessive neovascularization. In support of cumulative UV exposure in the pathogenesis of pterygium are the production of angiogenic cytokines induced by UV, including by FGF-2, PDGF, TGF-β, TNF-α, VEGF, IL-6 and IL-8 (Kria et al. 1996; Lee et al. 2001; Di Girolamo et al. 2002, 2006; Marcovich et al. 2002; Aspiotis et al. 2007). These cytokines that are induced by UV radiation may play a key role in the development of pterygium, by stimulating angiogenesis, cellular proliferation and tissue invasion. As concerns the role of UV in the pathogenesis of pterygium, Di Girolamo et al. (2002, 2006) have clearly demonstrated that in vitro expression of IL-6, IL-8 and VEGF mRNA and protein was induced in UV-irradiated pterygium epithelial cells in a time- and dose-dependent manner. Moreover, inhibitors of ERK1/2, JNK and p38 mitogen-activated protein kinases (MAPKs) significantly abolished the UV-mediated increase of IL-6, IL-8 and VEGF (Di Girolamo et al. 2006). Pterygium is often inflamed, with prominent infiltrates of lymphocytes, plasma cells and mast cells. Mast cells, for instance, are a source of angiogenic cytokines and proteases (Ribatti et al. 2004) and their presence contributes to neovascularization occuring in pterygium (Ribatti et al. 2007).

In this study, we have demonstrated that endothelial cells in human pterygium are immunoreactive to both NGF and its receptor TrkA and that this immunoreactivity is correlated to microvascular density. It may be supposed that an autocrine loop between NGF and its receptor TrkA is activated in pterygium and that it is involved in the angiogenic response taking place in this pathological condition. A higher expression of both NGF and TrkA on endothelial cells together with a higher expression of other angiogenic cytokines contribute to the pathogenesis of pterygium through an increase in microvascular density, which in turn, sustains the disease progression.

The number of endothelial cells immunoreactive to CD31 is more higher as compared with the number of endothelial cells positive to NGF and TrkA, because not all endothelial cells stain for these two antigens and may be also positive to other angiogenic cytokines, such as FGF-2, PDGF, TGF-β, TNF-α, VEGF and IL-8, as its has been previously demonstrated by other authors (Kria et al. 1996; Di Girolamo et al. 2002; Aspiotis et al. 2007).

NGF is a pleiotropic factor acting at both neuronal and vascular levels. Recent evidence obtained in vitro and in vivo and in pathological conditions clearly indicates that NGF acts as an angiogenic factor (Nico et al. 2008). NGF has been shown to stimulate in vitro the proliferation of human endothelial cells of different origin (Raychaudhuri et al. 2001; Cantarella et al. 2002; Steinle & Granger 2003) and the expression of both TrkA and p75 in human umbilical vein endothelial cells was demonstrated by RT-PCR analysis and Western blotting (Cantarella et al. 2002). NGF induces a dose-dependent angiogenic response in vivo in the rat cornea (Seo et al. 2001) and in the chick embryo chorioallantoic membrane assay (Cantarella et al. 2002), where NGF-induced neovascularization is significantly inhibited by neutralizing NGF-antibodies (Cantarella et al. 2002). Whether these processes occur directly via NGF-induced angiogenesis or indirectly, via the induction of classical angiogenic factors such as VEGF, remains to be clarified. Increasing evidence suggests that a least VEGF and NGF possess reciprocal angiogenic and neurotrophic effects on blood vessels and neurons (Lazarovici et al. 2006).

Several anti-angiogenic approaches have been proposed for the treatment of pterygium. One case report documented the successful treatment of an early recurrent pterygium using interferon alpha 2 beta (Esquenazi 2005), where the therapeutic effect may be related to its anti-angiogenic activity (Sidky & Borden 1987). TNP-470, a synthetic analogue of Fumagillin and a potent fungus-derived inhibitor of angiogenesis (Minischetti et al. 2000), inhibited the proliferation of fibroblasts isolated from active pterygium in vitro (Kria et al. 1998). More recently, bevacizumab, a monoclonal antibody anti-VEGF, has been proposed in the treatment of pterygium (Hosseini et al. 2007; Bahar et al. 2008). Although surgical techniques are currently the preferred option in the treatment of pterygium, future treatments targeting mediators of angiogenesis may provide more effective strategies for medical management of this pathological condition and the results of this study may have implications for the treatment of pterygium by inhibition of the NGF-TrkA system.