2007, Volume 23, Number 3, Page(s) 132-136
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
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.
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.
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
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
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
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
Click Here to Zoom
|Figure 1: Immunohistochemical staining with monoclonal antibodies
directed to chymase (a), and tryptase (b) performed
on adjacent sequential sections in a recurrent pterygium
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
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
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.
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
. 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
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
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