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2007, Volume 23, Number 3, Page(s) 132-136
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Mast cells and angiogenesis in primary and recurrent pterygia
Fatma Hüsniye DİLEK1, Faruk ÖZTÜRK2, Fatma AKTEPE1, Samet ERMİŞ2, Fatih Mehmet MUTLU3
1Departments of Pathology, Kocatepe University, School of Medicine, AFYON
2Departments of Ophtalmology, Kocatepe University, School of Medicine, AFYON
3Department of Ophtalmology, GATA, ANKARA
Keywords: Angiogenesis, chymase, tryptase, mast cell, pterygium
Abstract
Pterygium is a common benign lesion of limbus but the pathogenesis are not completely understood. Pterygia have a chronic inflammatory cellular infiltrate and a rich vasculature. Mast cells are a heterogeneous group of multifunctional tissue-resident cells. It has been suggested that mast cells and their products may be responsible for the formation of new blood vessels. We investigated the number and phenotype of mast cells and neovascularization in pterygia specimens and compared with those in normal conjunctival specimens

Pterygia tissues were obtained during excisional surgery from 32 eyes of 32 consecutive patients. Seventeen of all cases were recurrent pterygia. Superior bulbar conjunctival tissue from the same eye was also sampled as control tissues. The tissue sections were stained with routine hematoxyline-eosin and toluidine blue stain for mast cells. For immunohistochemical studies anti-factor VIII-related antigen, monoclonal anti human mast cell tryptase and chymase were used as an endothelial and mast cell marker.

The mean number of mast cells in pterygia was significantly higher than that in the normal conjunctival tissue and microvessel counts was significantly higher than the counts of the controls in both primary and recurrent pterygia. There was no correlation between microvessel numbers and mast cell numbers. There was no phenotypic difference between the mast cells in the ptergyia and those in the normal conjunctival tissues.

This study confirms that mast cells are prominent in pterygia and our results suggest that mast cells and angiogenesis are independent factors in the genesis and progress of ptergyium.

Introduction
Pterygium is a common lesion of limbus encountered in Turkey. Environmental factors, such as ultraviolet irradiation have been suggested as the main causative factor in the development of the disease. However, the aetiopathogenesis of pterygium remains obscure1.

Pterygium is an active, invasive, inflammatory process. In a two-stage process, ‘conjunctivalization’ of the cornea occurs with tissue characterized by extensive chronic inflammation, cellular proliferation, connective tissue remodeling and angiogenesis. Mast cells (MCs) are known to have important roles not only in allergic-type reactions, but also in chronic inflammatory and collagen-vascular diseases1,2. It has also been suggested that secreting mast cells are able to induce and enhance angiogenesis via multiple, and partly interacting pathways3,4.

Mast cells are composed of groups of cells that are heterogeneous with respect to structure and function. On the basis of their content of neutral proteases, human mast cells have been divided into two phenotypes. One is the tryptase positive containing tryptase but not chymase: This is the predominant type observed in alveoli of the lung and in the small intestinal mucosa. The other is the tryptase positive, chymase positive mast cell, which is the predominant type observed in the skin and in the small intestinal submucosa. The phenotypic characteristics of a mast cell population can be changed by alterations of pathological conditions. Distribution of mast cell subset may have important pathogenetic and therapeutic implications5-7.

In the present study, the number and phenotype of mast cells and their possible role in blood vessel formation in primary and recurrent pterygia specimens were examined and compared with those in normal conjunctival specimens.

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  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Methods
    Pterygia tissues were obtained during excisional surgery from 32 eyes of 32 consecutive patients with primary pterygium and recurrent pterygia tissues were obtained from 17 patients. Duration of the pterygia was not considered. Superior bulbar conjunctival tissue from the same eye was also sampled as control in 49 eyes. Pterygia which invaded less than 1mm into the cornea were excluded. Patients who had ocular pathology other than pterygium or a history of any systemic immune disease and atrophic pterygia were excluded.

    The tissues were fixed in buffered formalin and processed for paraffin embedding. Sections were prepared with HE and stained for 10 minutes with 1% toluidine blue (pH 4.1) for mast cells. Metachromatic cells were counted as mast cells.

    The number of mast cells was counted at three high power (x400) consecutive or nonoverlapping fields by using a light microscope (Olympus BX-50). The mean mast cell number was recorded. Neovascularization was determined in 24 cases of 32 primary pterygium and 17 cases of recurrent pyterygium. Sections of eight samples were inadequate for determination of neovascularization and they were excluded. An immunohistochemical study was carried out using a labeled streptavidin-biotin peroxidase technique. The primary antibodies used in this study were monoclonal mouse anti human mast cell tryptase (Neomarkers, LabVision Corp, CA, USA), monoclonal mouse anti human mast cell chymase (Neomarkers, LabVision Coop, CA, USA), and polyclonal FVIII related protein (Neomarkers, LabVision Corp, CA, USA). Incubation with each of the primary antibodies was performed for 30 minutes at room temperature. Sections were deparaffinized and then enzymatically treated with pepsin for 10 minutes at room temperature, and before incubating with anti FVIII antibody. The slides were immersed in antigen retrieval solution and were heated in a microwave oven for 10 minutes at 650 W for anti-mast cell tryptase or chymase antibodies. The antigen-antibody immunoreaction was visualized using aminoethylcarbazol (AEC) as chromogen.

    Microvessel density was counted using Olympus BX-50 microscope. Areas of the tissue containing the highest density of capillaries and small venules were identified. Large caliber vessels were omitted and even single cells with positive staining were counted as a microvessel. Three different fields were counted with x400 magnification in the most intensely stained area. Mean numbers were based on these three counts for each pterygia and control cases.

    Statistical analysis was performed with SPSS version 9.05 for windows using Wilcoxon signed ranks test. A p value of less than 0.05 was considered significant. Correlation between parameters was studied using the Pearson correlation test.

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  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Results
    The sex distribution of these 49 patients was 28 males, 21 females and the mean age was 55.0 years (range 38 to 73). The mean duration of the lesion was 2.5±1.0 mm and 3.6±1.0 mm for primary and recurrent pterygia respectively. All the pterygia were in nasal localization.

    Histopathologically, all mast cells were identified as mononuclear cells with metachromatic granules in substantia propria. Many of mast cells in pterygia and normal conjunctival tissues were observed beneath the epithelium and around the blood vessels (Figure 1a,b). The mean mast cell counts in primary pterygia was 17.39±8.74 and 8.36±3.0 in control specimens (Table 1).


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    Figure 1: Immunohistochemical staining with monoclonal antibodies directed to chymase (a), and tryptase (b) performed on adjacent sequential sections in a recurrent pterygium (x400).


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    Tablo 1: Mast cell counts in pterygia and normal bulbar conjunctiva.

    In the pterygia and the normal conjunctival tissues, all tryptase-positive cells and all chymase-positive cells were observed beneath the epithelium and around the blood vessels. The mean number of tryptase-positive and chymase positive cells in primary and recurrent pterygia was significantly higher than that in the normal conjunctival tissues (p<0.001). Although mast cell count was different in primary and recurrent pterygia, the difference was not statistically significant (p>0.5).

    The numbers of mast cells which stained with toluidine blue in pterygia or control conjunctival tissues correlated with the immunohistochemical staining. There was no statistically significant correlation between mast cell count and pterygia size (Pearson correlation coefficient r=0.086, p=0.66).

    The average number of blood vessels both of primary and recurrent pterygia was significantly higher than that in control tissues (p<0.05). There was no correlation between microvessel density and mast cell counts.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
  • Discussion
    The exact pathogenesis of pterygium is not yet clearly understood. The most common theory concerning the origin and pathogenesis of pterygium describes the association of the disease with environmental factors such as dust, smoke and ultraviolet radiation1,5. Recent evidence suggests that human papilloma virus may be involved in the pathogenesis of pterygia8. Histologically, actively growing pterygia exhibit degenerative changes. Granular-appearing material beneath the epithelium resembles degenerated collagen and elastic fibers. Pterygia have a chronic inflammatory cellular infiltrate (lymphocytes, mast cells) and a rich vasculature9. Lymphocytes are predominantly T-cells. In addition, deposition of IgE and IgG in pterygium hasbeen reported9,10. Mast cells play an important role in inflammation releasing stored and newly synthesized inflammatory mediators, including heparin, histamin, metallo- and serine proteases, and various growth factors, following activation. Thus, an increase in mast cells has been observed not only in allergy, but also in nonallergic chronic inflammation, angiogenesis, fibrosis, and tissue remodeling2,3,7-10.

    The number of mast cells in pterygia has been reported to be higher than in normal conjunctiva in previous studies in which mast cells were detected by metachromatic dye staining or by morphological characteristics11-14. It has been reported that the number of the mast cells increased in vernal and allergic conjunctivitis, as compared to normal conjunctiva. Phenotype of the increased mast cells, were predominantly tryptase- positive in vernal and allergic conjunctivitis, whereas chymase-positive mast cells were the predominant type in normal conjunctiva7,14. In the present study, although the number of mast cells was confirmed to be increased in the primary and recurrent pterygia, no significant difference was found in the phenotype of mast cells between pterygia and normal conjunctival tissues. Similar results were reported by Nakagami et al.12.

    Beden et al.16 showed that the difference in mast cell numbers between pterygia and control groups was not significant and they suggested that cellular immunity plays an important role in pterygia formation.

    There is much evidence to suggest a link between mast cells and angiogenesis. Mast cells stimulate the proliferation of microvascular endothelial cells in tissue culture and accumulate markedly in tumor angiogenesis3,4,17-20. Many components of mast cells are angiogenic or can modulate the angiogenesis process. These include basic fibroblast growth factor, vascular endothelial growth factor, heparin, heparinase, histamine, tumor necrosis factor-a and various proteases. However, Ghosh et al.21 demonstrated that histamine derived from other inflammatory cells plays a significant role in angiogenesis of the inflammatory granulation tissue. Also, Egozi et al.22 suggested that mast cells modulate the recruitment of neutrophils into sites of injury, yet indicate that mast cells are unlikely to exert a major influence on the proliferative response within healing wounds, including reepithelization, collagen synthesis, and angiogenesis. No correlation was found between the vascular density and the number of mast cells in pathological and surgical scars in another study23.

    Our results confirmed the increase in the number of mast cells in pterygia compared with normal conjunctival tissues. Mast cells may be a factor in pathogenesis of pterygia or may participate during its development. Mast cell and angiogenesis are independent factors in the genesis of ptergyium. Other inflammatory cells may play a significant role in the angiogenesis of the pterygia.

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  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
  • References

    1) Taylor HR, West SK, Rosental FS, Munoz B, Newland HS, Emmett EA. Corneal changes associated with choronic UV irradiation. Arch Ophthalmol 1989; 107:1481-1484.

    2) Coroneo MT, Di Girolamo N, Wakefield D. The pathogenesis of pterygia. Curr Opin Ophthalmol 1999;10:282-288.

    3) Norrby K. Mast cells and angiogenesis. APMIS 2002;110:355-371.

    4) Hiromatsu Y, Toda S. Mast cells and angiogenesis. Microsc Res Tech 2003;60:64-69.

    5) Gali SJ. Biology of disease, new insights into ‘The riddle of the mast cells’: microenviromental regulation of mast cell development and phenotypic heterogeneity. Lab Invest 1990;62:5-33.

    6) Irani AA, Schwartz LB. Mast cell heterogeneity. Clin Exp Allergy 1989;19;143-155.

    7) Church MK, McGill JI. Human ocular mast cells. Curr Opin Allergy Clin Immunol 2002;2:419-422.

    8) Varinli S, Varinli I, Erkisi MK, Doran F. Human papillomavirus in pterygium. Cent Afr J Med 1994;40:24-26

    9) Hill JC, Maske R. Pathogenesis of pterygium. Eye 1989;3:218-226.

    10) Pinkerton OD, Hokoma Y, Shigemura LA. Immunologic basis for pathogenesis of pterygium. Am J Ophtalmol 1984;98:225-228.

    11) Butrus SI, Asraf MF, Laby DM, Rabinowitz AI, Tabbara SO, Hidayat AA. Increased numbers of mast cells in pterygia. Am J Ophtalmol 1995;19:236-237.

    12) NakagamiT, Murakami A, Okisaka S, Ebihara N. Mast cell in pterygium:number and phenotype. Jpn J Ophtalmol 1999;43:75-79.

    13) Powers MR, Qu Z, O’Brien B, Wilson DJ, Thompson JE, Rosenbaum JT. Immunolocalization of bFGF in pterygia: association with mast cells. Cornea 1997;16:545-549.

    14) Bal N, Kayaselcuk F, Pelit A, Bolat F, Demirhan B. Mast cell density in pterygium, and its association with ultraviolet exposure in different climatic conditions: A series of 140 cases. Turkish Journal of Pathology 2006;22:11-16.

    15) Irani AA, Butrus SI, Tabbara KF, Schwartz LB. Human conjunctival mast cells: Distribution of MCT and MCTC in vernal conjunctivitis and giant papillary conjunctivitis. J Allergy Clin Immunol 1990;86:34-39.

    16) Beden U, Irkec M, Orhan D, Orhan M. The roles of Tlymphocyte subpopulations (CD4 and CD8), intercellular adhesion molecule-1 (ICAM-1), HLA-DR receptor, and mast cells in etiopathogenesis of pterygium. Ocul Immunol Inflamm 2003;11:115-122.

    17) Marks RM, Roche WR, Czernieck› M, Penny R, Nelson DS. Mast cell granules cause proliferation of human microvascular endothelial cells. Lab Invest 1999;55:289-294.

    18) Blair RJ, Meng H, Marchese MJ, Ren S, Schwartz LB, Tonnesen MG, Gruber BL. Human mast cells stimulate vascular tube formation. J Clin Invest 1997;99:2691- 2700.

    19) Tataroglu C, Kargi A, Ozkal S, Esrefoglu N, Akkoclu A. Association of macrophages, mast cells and eosinophil leukocytes with angiogenesis and tumor stage in non-small cell lung carcinomas (NSCLC). Lung Cancer 2004;43:47-54.

    20) Yano H, Kinuta M, Tateishi H, Nakano Y, Matsui S, Monden T, Okamura J, Sakai M, Okamoto S. Mast cell infiltration around gastric cancer cells correlates with tumor angiogenesis and metastasis. Gastric Cancer 1999;2:26-33.

    21) Ghosh AK, Hirasawa N, Ohtsu H, Watanabe T, Ohuchi K. Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice. J Exp Med 2002;195:973-982.

    22) Egozi EI, Ferreira AM, Burns AL, Gamelli RL, Dipietro LA. Mast cells modulate the inflammatory but not the proliferative response in healing wounds. Wound Repair Regen 2003;11:46-54.

    23) Beer TW, Baldwin HC, Goddard JR, Gallagher PJ, Wright DH. Angiogenesis in pathological and surgical scars. Hum Pathol 1998;29:1273-1278.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
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