Article Text
Abstract
Aims To analyse the relationship between mast cells and vascularisation in pterygia and to determine whether mast cells play an important role in the vascularisation of pterygia through the secretion of vascular endothelial growth factor (VEGF).
Methods Fifty-two pterygia and forty-four normal conjunctiva samples were obtained. Formalin-fixed, paraffin wax-embedded tissues were analysed by immunohistochemistry with CD31 and VEGF antibodies. Dual-immunofluorescence was used to see the location of mast cells and microvessels. To prove that mast cells have the function of secreting VEGF, we used dual-immunofluorescence, toluidine blue stain and immunohistochemisty study.
Results Mast cells are located near the microvessels. The numbers of mast cells in pterygia (10.8±2.7) were significantly higher compared with those in conjunctiva (4.7±2.4, p<0.01). The numbers of microvessels in pterygia (20.7±5.4) were also significantly higher than those in conjunctiva (9.3±2.9, p<0.01). There was an association between mast cell count and microvessel density in pterygia (r=0.77, p<0.001). The cells were positive for toluidine blue staining and could express VEGF through a serial section stain. Dual-immunofluorescence showed that VEGF and mast cell tryptase (MCT) were expressed in the same cell.
Conclusion The results suggest that mast cells have a function in the vascularisation of pterygia through the secretion of VEGF.
- Angiogenesis
- biochemistry
- conjunctiva
- genetics
- glaucoma
- immunology
- retina
Statistics from Altmetric.com
Introduction
Pterygium is one of the most common surface ocular lesions. It is a fibrovascular neoformation characterised by a triangular or wing-shaped overgrowth of abnormal conjunctiva onto the cornea. It shows degenerative, hyperplastic changes, has proliferative, inflammatory features and is highly vascular.1 In severe cases, a pterygium can grow into the central cornea, which can induce irregular corneal astigmatism, resulting in vision loss.
Although the pathogenesis of pterygium has not yet been clarified, several mechanisms have been reported to participate in the pathology of pterygium, including immunological mechanisms, genetic mechanisms, extracellular matrix remodelling, viral infections, apoptotic mechanisms and proliferous mechanisms.2 ,3 In spite of the development of surgical techniques to treat this disease, pterygia will recur and become aggressive after resection.4 Previous research has proved that this disease was a neoplastic-like growth disorder and more than a simple degenerative condition of the conjunctiva.5
Angiogenesis is defined as the formation of new blood vessels from pre-existing vasculature and occurs both in chronic inflammations and tumours.6 ,7 Aspiotis et al 8 showed that the density of microvessels in pterygia was significantly higher than that in normal conjunctiva. Moreover, they demonstrated the overexpression of vascular endothelial growth factor (VEGF) in pterygia, which was considered to be one of the most potent angiogenic factors. There is an increased number of mast cells in pterygia compared with normal conjunctiva.9 ,10 The density of mast cells is highly correlated with the extent of pathological angiogenesis in tumours.11 Ribatti and colleagues12 have proved that mast cells have been implicated in the angiogenesis of pterygia, which may be one reason for the pathogenesis of pterygia. Mast cells release a number of angiogenic factors. Among these, VEGF is considered to be one of the most active factors.13
Given the above background, our purpose was to study whether mast cells promote the angiogenesis of pterygia through the secretion of VEGF. We also correlated the extent of angiogenesis with the number of mast cells in pterygia.
Materials and methods
Patients and study design
The patients were selected prospectively by making a clinical diagnosis of pterygium. The patients participating in this research had to have two features. First, a wing-shaped fold of conjunctiva encroaching upon the cornea from either side within the interpalpebral fissure must be found in these patients through slit lamp microscope examination. Second, these patients must have characteristics of the overgrowth of a wing-shaped fold of conjunctiva onto the centre of the cornea more than 3 mm.14 The study group included 52 cases (28 men and 24 women). Ages ranged between 42 and 83 years (mean age 63.3 years; SD 11.1). All patients underwent excision by the bare sclera technique. Primary pterygia tissues were harvested from these 52 patients. Normal conjunctiva samples were collected from the superior conjunctivas of 44 patients (23 men and 21 women) who had other eye diseases, but exhibited no characteristics of pterygia at the time of their cataract or vitreoretinal surgeries. Ages ranged from 38 to 79 years (mean age 62.2 years; SD 10.5). All patients underwent a complete ophthalmic examination. Patients did not receive any medication before surgery, except for a topical anaesthetic, and no drugs or chemical agents were used during surgical operation. The study protocol was approved by the ethics committee of Anhui Medical University, all procedures were performed under the tenets of the Declaration of Helsinki, and informed consent was obtained from all patients.
Immunohistochemistry, double-immunofluorescence and toluidine blue staining study
Tissue segments were fixed by immersion in cold 10% formalin in 0.2 M phosphate buffer with a pH of 7.3 for 12 h and then processed for paraffin embedding. Microtome sections (4 μm) were treated for immunohistochemical and double-immunofluorescence analysis. Adjacent microtome sections were treated for the immunohistochemical analysis of VEGF and toluidine blue stain.
Immunohistochemistry study
All sections were deparaffinised in xylene, sequentially rehydrated in alcohol and washed in phosphate-buffered saline (PBS). The sections were heated twice in a microwave oven for 5 min in citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was blocked with hydrogen peroxide in methanol. Then the sections were incubated with normal goat serum at room temperature. The sections were incubated with rabbit polyclonal anti-VEGF monoclonal antibody (1:150; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) and rabbit polyclonal anti-CD31 antibody (ready-to-use; Zhongshan Biologic and Technical Co., Beijing, China) in a humidified chamber. The incubation was performed at 4°C overnight, followed by washing with PBS. The tissues were incubated with biotinylated goat anti-rabbit antibody (ready-to-use; Zhongshan Biologic and Technical Co.) for 30 min at room temperature and further incubated with streptavidin-alkaline phosphatase conjugate (Zhongshan Biologic and Technical Co.). The reaction was visualised with a diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Biologic and Technical Co.) and counterstained with haematoxylin. Negative controls were performed with normal mouse or rabbit sera that were diluted to the same concentration as the primary antibody.
Double-immunofluorescence study
Double immunostaining was performed using the method described above.15 Briefly, paraffin sections were treated with xylene and ethanol, washed with PBS and then antigen retrieval. Slices were blocked with 10% goat serum. Monoclonal mouse antiserum against VEGF (Santa Cruz Biotechnology Inc.) was used at a dilution of 1:200; the same dilution was used for polyclonal rabbit antiserum against mast cell tryptasel (Santa Cruz Biotechnology Inc.); monoclonal mouse anti-CD31 antibody (ready-to-use; Zhongshan Biologic and Technical Co.) was also used. Sections were incubated with the two antibodies (anti-MCT/anti-VEGF or anti-MCT/anti-CD31) at 4°C overnight, washed with PBS three times for 5 min each, and then incubated with rhodamine-conjugated secondary antibody (goat anti-mouse IgG, diluted 1:200; Zhongshan Biologic and Technical Co.) and FITC-conjugated secondary antibody (goat anti-rabbit IgG, diluted 1:200; Zhongshan Biologic and Technical Co.) for 60 min at 37°C. Various negative controls were performed. After a final wash, the slices were coverslipped and examined with a confocal microscope (Leica SP5; Leica Microsystems, Mannheim, Germany).
Microvessel counts
Microvessel counts were performed by using the method described by Ribatti et al. 12 Two investigators counted the positive microvessels simultaneously, without knowledge of the final pathological diagnosis. Five fields (×200) per sample were examined with a light microscope (80i; Nikon, Tokyo, Japan). We selected the microvessels strictly. The CD31-positive capillaries and small venules were identified as transversally sectioned tubes with a single layer of endothelial cells, either with or without a thin basement membrane. Each assessment was agreed on in turn. Microvessels were counted with a planimetric point-count method, according to which only microvessels transversally cut and occupying the reticulum points were counted. As almost the entire section was analysed for each sample and transversally sectioned microvessels hit the intersection points randomly, the method allowed objective counts. Means±SD and medians were determined for each section, sample and group of samples.
Mast cell counts
Mast cells were highlighted in every second section adjacent to that stained for microvessels with toluidine blue, counted in six (250×) fields, covering almost the whole section, inside a square reticulum (0.25 mm2), and calculated as means±SD and medians for each group of samples.
Statistics
We used SPSS 13.0 for data analysis, and p<0.05 was considered to be significant. The significance of changes in the counts of microvessels and mast cells were assessed with parametric (t test) and non-parametric (Mann–Whitney U test) analyses of variance to compare the groups. Correlations between counts were assessed with Pearson's (r) coefficient analysis.
Results
Correlation of mast cells and microvessels
From the H&E staining, more microvessels could been seen in pterygium tissues than in normal conjunctival tissues (figure 1A,B). The expression of CD31 was observed as a marker for microvessels in all subjects, but the number of immunopositive microvessels in pterygium specimens (20.7±5.4) was significantly higher than that in normal conjunctiva specimens (9.3±2.9, p<0.01, table 1, figure 1C,D). From the toluidine blue staining, the cytoplasm of mast cells was stained dark blue (figure 1E,F). Through double immunostaining, mast cells were stained green with anti-tryptase (FITC, figure 2A,D) and microvessels were stained red with anti-CD31 (rhodamine, figure 2B,E). Mast cells located near the microvessels in both pterygium (figure 2A,B,C) and conjunctiva tissues (figure 2D,E,F). The number of these mast cells was quantitated in the two groups. Mast cells in pterygium tissues (10.8±2.7) were shown to be significantly higher compared with those in the normal conjunctiva group (4.7±2.4, p<0.01; table 1, figure 1E,F) by toluidine blue staining. Furthermore, the mast cell count was highly correlated with microvessel density (r=0.77, p<0.001) in pterygium tissues.
Mast cells secreting VEGF
The cytoplasm of mast cells was stained dark blue by toluidine blue staining (figure 3A,C). Some cells were shown to be positive for VEGF through an immunohistochemical study (figure 3B,D). The cells that were positive for toluidine blue staining might secrete VEGF in both pterygium (figure 3A,B) and conjunctiva tissues (figure 3C,D), as shown in the serial sections study. Furthermore, double immunostaining showed that mast cells, which were stained green with antitryptase (FITC, figure 4A,D) and VEGF, which was stained red with anti-VEGF (rhodamine, figure 4B,E), were expressed in the same cell (figure 4C,F).
Discussion
In this study, we tested the expression of three biomarkers (CD31, VEGF and MCT) in pterygium and normal conjunctiva samples. The results revealed that the numbers of microvessels were significantly increased in the pterygium samples compared with those in the normal conjunctiva samples. The numbers of mast cells were significantly increased in the pterygium samples, as shown by the toluidine blue staining. Through a dual-immunofluorescence study of CD31 and MCT in pterygia and conjunctiva, mast cells were found to be located near the microvessels. Through adjacent section studies, the results suggested that mast cells might have the function of secreting VEGF. To confirm this result, double-immunostaining analysis was used. The results showed that MCT-positive cells were also positive for VEGF, which demonstrated that mast cells in pterygium tissues have the function of secreting VEGF. These results indicated that mast cells are involved in the development of neovessels in pterygia.
Pterygium, which invades the cornea, forming a wing-like shape, shows degenerative and hyperplasic changes, has proliferative, inflammatory features and is highly vascular.16 Previous studies have shown that VEGF and neovessels play an important role in the pathogenesis of pterygia.17 ,18 Moreover, the important role of mast cells in the development of pterygia has also been reported.12 ,19 However, to our knowledge, the relationship between mast cells and microvessel density in the pathogenesis of pterygia has not been demonstrated. This study was designed to clarify this issue in the Han Chinese population.
The number of mast cells and microvessels increased in the formation of pterygia. Similar results were reported in previous research.10 ,20 Moreover, there was a striking association between mast cell and microvessel counts in this study. These results indicated that mast cells promoted neovascularisation in pterygia. Some previous studies showed that many cytokines were involved in the pathogenesis of neovascularisation in pterygia, including VEGF, tumour necrosis factor alpha, tissue factor and basic fibroblast growth factor.5 ,21 Among these factors, VEGF seems to be one of the most important regulators of the angiogenic mediators. Moreover, previous studies had already proved that bevacizumab, one kind of anti-VEGF monoclonal antibody, could suppress the neovascularisation that occurs in the development of pterygia.22 ,23 As VEGF seems to be one of the most effective angiogenic mediators, we chose it to investigate whether mast cells have the function of secreting this growth factor in pterygia tissues. Through the study of adjacent sections, we could see that mast cells had the function of secreting VEGF. This was similar to the function of mast cells in other diseases, including tumours and inflammation disorders.24 ,25 Moreover, epithelial cells and some stroma cells also had the function of secreting VEGF, as observed by the immunohistochemisty and double-immunofluorescence analysis of both pterygia and controls of this study (figure 3, figure 4). However, the aim of this study was merely to prove that the increased mast cells in pterygia tissues have the function of promoting vasculature through the secretion of VEGF. The other cells that can secret VEGF are an interesting part of the pathogenesis of pterygia. We will clarify the process by which they do so in future study. Ribatti et al 12 reported that tryptase is the major mast cell protease and one of the most powerful angiogenic mediators released by human mast cells. Tryptase was used as a marker for mast cells, and mast cells were involved in the development of neovessels in this study. Whether MCT promoted the neovessels directly or by promoting the secretion of VEGF was not fully understood. In order to ensure that the mast cells had the function of secreting VEGF, we made the following efforts. First, through serial sections staining, we found that the cells secreting VEGF might be mast cells. Second, we directly demonstrated that mast cells could secret VEGF by using the double-immunostaining method.
Like other studies in this area, there were some limitations in our study. Although many kinds of cells have the function of secreting VEGF, we only tested for it in mast cells. The VEGF from other kinds of cells must be tested in the future. In addition, all the patients tested came from a Chinese Han population. Studies on multi-ethnic populations may clarify this function of mast cells in the neovascularisation of pterygia.
In conclusion, we showed that mast cells secreting VEGF are involved in the pathogenesis of neovascularisation in pterygia. The presence of mast cells was also correlated with the capillary density in this disease. Therefore, the new anti-angiogenic strategy by the inhibition of mast cell degranulation or activation might yield new methods for the prevention and treatment of pterygia, which is worthy of further investigation.
Acknowledgments
The authors would like to thank all the donors enrolled in the present study.
References
Footnotes
-
Funding This work was supported in part by the Anhui Provincial Natural Science Foundation (1208085MH178), the Natural Science Foundation of Higher Educational Bureau of Anhui Province (kj2011z189) and the Talent Foundation of the Department of Personnel Anhui Province (2010–45). This work was supported in part by the three foundations.
-
Competing interests None.
-
Patient consent Obtained.
-
Ethics approval Ethics approval was provided by the ethics committee of Anhui Medical University.
-
Provenance and peer review Not commissioned; externally peer reviewed.
Linked Articles
- At a glance