An encroachment of a fibrovascular tissue onto the cornea is a neoformation
which is called pterygium. The base of this unilateral or bilateral triangle
is on the nasal conjunctiva and points toward the cornea (Lin
et al., 2013; Bazzazi et al., 2010).
Although, benign in many cases, an aggressive pterygium may cause problems
such as blurred vision, ocular irritation and in rare cases dysplasia to even
carcinoma in situ. Furthermore, high prevalence and surgery recurrence (30-69%)
have been always major concerns to physicians (Liang et
al., 2010). Therefore, effective treatment of the disease is a monumental
goal, which in turn, needs accurate knowledge of its underlying physiopathology
(Mandour et al., 2011).
In spite of large amount of data available in the literature as to pterygium,
its exact etiology is still an intriguing question. Various hypotheses have
being put forward in this regard during the two last decades, such as limbal
stem cell aberrations, apoptosis, metalloproteinases, infections and the immune
system (Chui et al., 2008; Detorakis
and Spandidos, 2009). According to the modern science, however, two wings
have been hypothesized as the most important factors in the pathogenesis of
pterygium including chronic inflammation and angiogenesis (Ribatti
et al., 2007; Aspiotis et al., 2007;
Marcovich et al., 2002). Mast Cells (MCs) play
a key role in allergic inflammation and it has been proposed that they may be
considered as a major contributor in pathogenesis of the disease through chronic
inflammation (Nakagami et al., 1999).
On the other hand, angiogenesis, which is defined as growing new blood vessels
from preceding ones, is modulated by a complex interaction between various regulating
factors. One of these important factors is Vascular Endothelial Growth Factor
(VEGF), a signal protein produced by cells that stimulates vasculogenesis and
angiogenesis (Livezeanu et al., 2011). Platelet
Endothelial Cell Adhesion Molecule (PECAM-1) or the Cluster of Differentiation
31 (CD31) is a structural protein in endothelial cell intercellular junctions.
This protein involves in angiogenesis, integrin activation and leukocyte locomotion
(Jackson, 2003). This study, for the first time in
the literature, aimed to examine both wings of pathogenesis of pterygium in
a simultaneous fashion. For this purpose the count of infiltrated MCs (the wing
of inflammation) as well as the status of CD31/VEGF expression (the wing of
angiogenesis) was considered as target goals. In addition, this is the first
study of its type that tackled the confounding effect of the severity of pterygium
by limiting the cases to stage II disease.
MATERIALS AND METHODS
Patients: In this prospective, case-control study, 24 tissue specimens
were acquired from the patients with primary pterygia (the case group). For
the control set 15 normal conjunctival tissues were examined, which were obtained
from the nasal bulbar region next to the limbus during cataract surgery.
In the case group the including criteria were as follow: primary moderate (grade
II according to Awdeh et al. (2008) eye nasal
pterygia with encroachment onto the cornea and an apex passing the limbus ≥1
mm, indication of surgery, no systemic immune diseases and/or previous use of
immunosuppressants and no history of previous ocular surgery, injury or disease.
The controls were age-and sex-matched counterparts without ocular diseases and/or
All the patients with pterygium and cataract were operated in Tabriz Nikookari
Teaching Hospital from April 2012 through February 2013. The histopathological
assessments were carried out in Tabriz Teaching Sina Hospital. This study is
approved by the ethics committee of Tabriz University of medical Sciences and
informed consents are obtained from the participants.
Toluidine blue staining and MC counting: After sectioning the specimens
along the longitudinal axis, both case and control tissues were fixed in a mixed
solution (2.5% formalin plus 1% glutaraldehyde) for 24 h. Then the embedded
specimens in glycol methacrylate were cut into 1 μm thick section and stained
with diluted 1% toluidine blue for 10 min. Metachromatic cells were considered
as MCs (Fig. 1a).
For MC counting, sections were photographed at 200X and the MCs were marked
on the photographs while the entire tissue was skimming through at 400X. Finally,
all the marked spots were counted by two observers and the mean count was reported
Immunohistochemical staining: Paraffin-embedded specimens were fixed
in 10% buffered formalin for a day.
|| (a) Infiltration of mast cells in a specimen of pterygium
(Toluidine blue staining, original magnification 200X), (b) CD31-positivity
in a specimen of pterygium (Hematoxylin eosin staining, original magnification
200X) and (c) Vascular endothelial growth factor (VEGF)-positivity in a
specimen of pterygium (Hematoxylin eosin staining, original magnification
After dehydration, these specimens were prepared and examined for expression
of CD31 and VEGF by a skilled dermatopathologist.
Then 4 μm sections were prepared and dewaxed in an oven for 30 min at
56°C. After being washed in Phosphate Buffered Saline (PBS), the specimens
were pretreated with 0.5% H2O2 in 70% methanol for 30
min in order to ensure that the endogenous peroxidase was blocked. An avidin-biotin
block provided by the manufacturer (DAKO) was used to block endogenous biotin.
Following the manufacturers
instructions the antigens were retrieved and the samples were incubated with
monoclonal mouse anti-human CD31 antibody (DAKO A/S, Glostrup, Denmark; Clone
JC70A; dilution 1:30) and monoclonal mouse anti-human VEGF antibody (DAKO A/S,
Glostrup, Denmark; clone VG1; dilution 1:25) separately for 30 min at room temperature.
Tissues were then incubated with avidin-biotin-peroxidase complex (ABC, Vector
Laboratories Inc, Burlington, USA). Accordingly prepared slides were treated
with diaminobenzidene (DAB) chromagen substrate according to the manufacturer's
instructions and counterstained with hematoxylin.
The slides were examined by a light microscope (Siemens, Munich, Germany) at
different magnifications (Fig. 1b, c).
According to previously established criteria (Jasani and
Schmidt, 1993), the staining was considered positive when it was at least
of focal or of moderate intensity, clearly visible only with medium magnification.
This means that only moderate to severe staining was considered positive in
Statistical analysis: Data were shown as Mean±standard deviation
or number (%). The SPSS software for Windows (ver. 16) was used. Independent
samples t test (for age and MC count) and the Chi-square test (for sex and the
status of CD31/VEGF staining) were employed for analyzing. p≤0.05 was considered
In the case group there were 21 males (84%) and 4 females (16%) with a mean
age of 58.08±10.03 (range: 41-72) years at the time of surgery. In the
control group there were 12 males (80%) and 3 females (20%) with the mean age
of 62.33±9.19 (range: 46-76) years. Both the case and control groups
were matched for there subjects
sex (p = 0.75) and age (p = 0.19).
The mean MC count was 27.72±15.19 (range: 5-51) cells/mm2
in the case group, which was significantly higher than the mean MC count in
the controls (12.00±7.09, range: 4-30 cells mm-2; p = 0.001).
||Rate of CD31-positivity in the specimens of pterygium and
normal bulbar conjunctiva
||Rate of VEGF-positivity in the specimens of pterygium and
normal bulbar conjunctiva
The study of immunoreactivity at CD31 proved that this marker was significantly
more frequent in the cases (22 patients) than in the controls (only 4 subjects)
(p<0.001; Odds ratio = 20, 95% confidence interval 3.85-100). The percentage
of the specimens with positive immunoreaction to CD31 in the two groups is shown
in Fig. 2.
For the VEGF, there was again a significantly higher rate of positivity in
the cases (22 patients) than in the controls (only 3 subjects) (p<0.001;
Odds ratio = 33.3, 95% confidence interval 5.00-100). The percentage of the
specimens with positive immunoreaction to VEGF in the two groups is shown in
Although, there are many hypotheses available in the literature regarding the
pathogenesis of pterygium, its exact underlying physiopathology is still unclear.
However, modern approaches have proposed that two main processes may construct
the basis of this condition; inflammation and angiogenesis (Chui
et al., 2007; Livezeanu et al., 2011).
In the present study, to the best of our knowledge, for the first time both
these wings of pathophysiology have been examined at the same time.
For the wing of inflammation, the count of MCs was compared between the specimens
of pterygium and those of normal bulbar conjunctiva. Accordingly, the mean count
of MC was significantly higher in the first group (27.72±15.19 vs. 12.00±7.09
cells mm-2; p = 0.001). This finding is in line with the outcome
of a previous report by Butrus et al. (1995)
who also showed that the count of MCs is higher in pterygium than normal tissue.
In another series by Nakagami et al. (1999),
the mean count of MC was again higher in the specimens of pterygium comparing
with that in normal conjunctival tissue (34.56±18.0 vs.15.56±9.0
cells mm-2). As seen, these figures are very similar to the mean
count of the MC in the present study.
Zhong et al. (2001) compared the count of MCs
in 17 primary pterygia, 6 recurrent pterygia and 6 normal conjunctival specimens.
The mean numbers of infiltrating MCs were 45.47±5.50 and 48.83±3.19
cells in primary and recurrent pterygium, respectively. These figures were significantly
higher than that in normal connective tissue (4.24±2.36 cells mm-2).
Although the overall outcome is in concord with findings of the present study,
the reported figures seem far different. This may be due to low number of samples
(particularly in the control group), as well as the heterogeneity of the patients
in term of primary and recurred pterygium. As mentioned before, only primary
cases of pterygium were included in the present survey.
This heterogeneity also remains in the findings of another study by Beden
et al. (2003). Based on this study, the difference in mast cell numbers
between the pterygium and control groups was not statistically significant.
However, they posited that MCs may participate in some stages of pterygium development.
To obliterate this conflicting factor, only cases with grade II disease were
recruited in the present study.
For the wing of angiogenesis, on the other hand, the expression of two its
major contributors, i.e., CD31 and VEGF, were compared between the case and
controls. Based on the findings, both the factors were expressed significantly
higher in the specimens with pterygium than in the specimens with normal tissue.
Lee et al. (2001) compared the expression of
VEGF in pterygium and in normal bulbar conjunctiva. They found a significantly
higher rate if VEGF expression in the patients group and asserted the
major contribution of angiogenesis in the development of pterygium. This confirms
the results of our study in this regard.
The role of VEGF in the pathogenesis of pterygium has been proposed in other
studies, as well (Marcovich et al., 2002; Jin
et al., 2003; Van Setten et al., 2003).
Aspiotis et al. (2007) compared the expression
of both CD31 and VEGF in patients with pterygium and in normal bulbar conjunctive.
They also found a higher rate of CD31/VEGF expression in the patients, confirming
the findings of the present study.
Livezeanu et al. (2011) emphasized the presence
of a much richer vascularization in pterygium, compared with normal conjunctiva.
They also used the expression rate of CD31 and VEGF for this propose. Accordingly,
the moderate-to-severe expression of VEGF was seen in 81% of the patients; a
figure which is very close to that in our series (i.e., 88%).
Other studies also confirm that there might be a causative association between
the expression of CD31 and emergence of pterygium (Ling
et al., 2012; Lin et al., 2013; Fukuhara
et al., 2013).
Summing up the results of the present study and available data in the literature,
it is apparent that the angiogenesis is a key factor in pathogenesis of pterygium.
This is in conformity with a newly developed notion which surmises a causative
role of ultraviolet exposure in pterygium, because this exposure has been connected
with induction of angiogenetic factors such as VEGF (Ribatti
et al., 2009).
Likewise, it is suggested that both MCs and angiogenesis may be interconnected
themselves (Ribatti et al., 2007).
Although, this conjecture merits further well-controlled studies, the present
work is the first one which examines both MCs and angiogenesis, simultaneously
in patients with pterygium. In addition, the present survey carries two other
preponderances comparing to available ones in the literature.
First, the patients in the present study were confined to those with primary,
grade II disease. This is pivotal, as it is suggested that the severity of the
disease may play an extra confounding role in pathogenesis of pterygium (Lin
et al., 2013).
Second, the positivity of CD31/VEGF expression was set to moderate-to-severe
definition. It should be born in mind that as far as these two markers are associated
with angiogenesis even in normal tissues, there expressions are expected even
to mild degree. By omitting these normal
expressions, the accuracy of the results seems to be higher.