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Pterygia Pathogenesis
Corneal Invasion by Matrix Metalloproteinase Expressing Altered Limbal Epithelial Basal Cells
Nicholas Dushku, MD;
Molykutty K. John, PhD;
Gregory S. Schultz, PhD;
Ted W. Reid, PhD
Arch Ophthalmol. 2001;119:695-706.
ABSTRACT
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Objective To assess the potential role of matrix metalloproteinases (MMPs) in
the pathogenesis of pterygia by comparing the immunolocalization patterns
of MMPs in altered limbal basal stem cells, activated fibroblasts, and areas
of elastotic degeneration adjacent to the pterygia.
Methods Nine primary and 1 recurrent pterygia along with normal superior limbal-conjunctival
tissue and cornea were immunostained with mouse monoclonal antibodies specific
for MMP-1, MMP-2, MMP-3, MMP-9, membrane type 1 (MT1)–MMP (MMP-14),
and membrane type 2–MMP (MMP-15).
Results Normal conjunctival, limbal, and corneal cells lacked significant immunostaining
except for cell surface MT1-MMP. In contrast, altered limbal basal epithelial
cells of the 9 primary and 1 recurrent pterygia immunostained for all 6 MMPs.
Activated and altered fibroblasts associated with the pterygia immunostained
primarily for MMP-1. In contrast, stromal areas of elastotic degeneration
(pingueculae) showed variable immunostaining of MMPs.
Conclusions Altered limbal basal epithelial cells (pterygium cells) immunostained
for multiple types of MMPs in contrast to normal conjunctival, limbal, and
corneal cells. The pterygium cells invading over Bowman's layer produce elevated
MMP-1, MMP-2, and MMP-9 expression, which probably are the main MMPs responsible
for the dissolution of Bowman's layer. Pterygium cells may also cause activation
of fibroblasts at the head of the pterygium, leading to the initial cleavage
of fibrillar collagen in Bowman's layer by the production of MMP-1. Altered
fibroblasts in areas of elastotic degeneration (pingueculae) trailing behind
the pterygium constitute a second type of tumor, which is noninvasive.
Clinical Relevance These data of altered MMP expression support the concept that altered
basal limbal epithelial cells play a key role in the formation and migration
of a pterygium.
INTRODUCTION
IN OUR SEARCH for the pathogenesis of pterygia, several important clinical
and pathologic characteristics of primary and recurrent pterygia have emerged:
- Epidemiological studies have firmly established
that UV-B radiation correlates as the etiologic agent for pterygia1-2 and limbal tumors.3-5
- Pterygia begin growing from limbal epithelium and
not from conjunctival epithelium.6-7
- A segment of limbal epithelium, the migrating limbus,
invades the cornea centripetally followed by conjunctival epithelium.8-10
- A distinct type of corneal cells develops at the
leading edge of the pterygia tissue.9, 11-12
- Vascularization occurs in the conjunctiva adjacent
to pterygia.13
- Bowman's layer is dissolved under the leading edge
of the pterygia.14
- Pterygia have a high recurrence rate.11-12
Since UV-B is known to be mutagenic for the TP53
tumor suppressor gene,15-18
we previously searched for abnormal TP53 expression
in pterygia, limbal tumors, and pingueculae from which these 2 growths seem
to originate.19 We found nuclear p53 expression
without apoptosis in the limbal epithelia of pterygia, limbal tumors, and
most pingueculae. This suggested to us that mutation in TP53 or mutations
in the p53 pathway for apoptosis may occur as an early event in the tumorlike
development in these cells, which is consistent with their causation by UV
radiation. As a consequence of mutational damage to the p53-dependent programmed
cell death mechanism,20 mutations in other
genes could progressively be acquired by the altered limbal basal epithelial
cells. This is consistent with the concept of a multistep21
development of tumorlike pterygium cells that arise from altered limbal epithelial
cells overlying a pinguecula. We also discovered that primary and recurrent
pterygia were characterized by invasion of the cornea by vimentin-expressing
altered limbal epithelial basal cells.8-9
In addition, we discovered there is local infiltration of the adjacent conjunctival
and circumferential limbal epithelia by pterygium cells, which could lead
to a high recurrence rate if not controlled surgically or chemotherapeutically.9 Moreover, using polymerase chain reaction, we found
no human papillomavirus DNA in any of these growths that arise in the UV-exposed
interpalpebral region. We concluded that human papillomavirus DNA is not required
as a cofactor for the etiology of these lesions, either through control of
the action of p53 or through any other mechanism.22-23
These data have led us to propose an integrated model for the formation
and pathophysiology of pterygia and pinguecula.9, 19, 24
A key component of this hypothesis is that the true pterygium cells are tumorlike
altered limbal epithelial basal cells that have altered TP53 tumor suppressor gene expression. With accumulation of sufficient
mutations, the pterygium cells invade onto normal corneal basement membrane
and draw conjunctival epithelial cells along with them.
If this hypothesis is correct, we would predict that the expression
of proteases that degrade basement membrane components, such as type IV collagen
and the fibrillar collagen of corneal stroma, should be elevated in the leading
edges of pterygia, where the degradation of Bowman's layer occurs. Also, the
normal limbal and conjunctival epithelia and stroma should lack these proteases
(or have low levels of the proteases). The primary class of proteases that
degrade matrix are the matrix metalloproteinases (MMPs). The MMPs are a family
of more than 21 genetically distinct proteases, which are normally produced
in small amounts for physiological processes by cells, such as fibroblasts
and epithelial cells.25 Recently, it was reported
that fibroblasts from pterygia when grown in culture exhibited elevated MMP
expression.26-27 In general, invasive
tumor cells are known to overexpress MMPs28-29
of various types depending on the tumor.30-34
These proteases released by tumor cells facilitate invasion by degrading components
of their basement membranes and adjacent stromal matrix. Previously, we proposed
a model for pterygia migration and dissolution of Bowman's layer involving
proteases.19, 24 Recently, elevated
expression of MMPs was demonstrated in pterygia.35-36
Specific localization of MMPs in the altered limbal epithelial basal cells
of pterygia had not been reported previously. In this article, we investigated
MMP expression in the altered limbal epithelial cells. The data are consistent
with these cells contributing to the pathogenesis of pterygia by secreting
MMPs, which promote the invasion by the pterygia and the dissolution of Bowman's
layer.37
MATERIALS AND METHODS
PTERYGIA AND NORMAL TISSUE
In compliance with the World Medical Association Declaration of Helsinki,
9 primary and 1 recurrent pterygia were surgically removed in the ambulatory
surgery center at the Kaiser Permanente Medical Center, Rancho Cordova, Calif,
and processed as described previously.9, 19, 23
Briefly, to identify the invading limbus epithelium with altered limbal basal
cells and the zone of dissolution of Bowman's layer, incisions were made in
the cornea 1 to 2 mm central to the leading edge of the pterygium and deep
enough to include Bowman's layer. The incisions were extended into the adjacent
conjunctiva for 5 to 6 mm posterior to the surgical limbus and 1 to 2 mm beyond
the superior and inferior conjunctival folds. For proper orientation, specimens
were sutured onto sterile cardboard and immediately fixed in 10% neutral buffered
formalin for 6 to 10 hours, then embedded in paraffin. Serial cross sections
of pterygia specimens were made along the longitudinal axis to include the
leading edge of cornea-invading altered limbal basal cells over Bowman's layer,
the migrating limbus, and adjacent conjunctiva. Every tenth section was stained
with hematoxylin-eosin to locate these 3 areas. For immunostaining, sections
were selected that contained cornea-type cells between the dissolved edge
of Bowman's layer and conjunctiva (as indicated by the presence of goblet
cells). A specimen of fresh normal human superior limbal-conjunctival tissue
served as a normal tissue control. In addition, sections of normal cornea
(obtained along with the pterygia) also served as internal controls. Three
cadaver eyes placed in 10% neutral buffered formalin 4 to 5 hours after death
were used for comparison with the fresh surgical specimens (Table 1, only 5-hour specimen results are shown). Human placenta,
which is known to produce MMPs, was used as a positive tissue control.38 Additional negative controls used pterygia tissues
incubated without the primary antibodies to MMPs.
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Table 1. MMP Expression in Fresh Normal Conjunctiva and Limbus (Patient
CF); Cadaver Conjunctiva, Limbus, and Cornea (Patients C34 and C35); and the
Area of Elastotic Degeneration (Pinguecula in Patient C35)*
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IMMUNOHISTOCHEMISTRY
Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded
tissue sections using the avidin-biotin-peroxidase complex method as described
previously.39 Briefly, sections 5 µm
thick were cut and deparaffinized in xylene and descending ethanol series.
Endogenous peroxidase activity was destroyed by a 20-minute treatment at room
temperature with 3% hydrogen peroxide in distilled water. Sections were then
incubated for 1 hour at room temperature in a humidified chamber with primary
mouse monoclonal antibodies directed against the MMPs. The following mouse
monoclonal antibodies were used: MMP-1, MMP-2, MMP-3, and MMP-9, which were
all diluted 50-fold (Oncogene Research Products, Boston, Mass), and membrane
type 1 (MT1)–MMP and membrane type 2 (MT2)–MMP, which were diluted
100-fold (Chemicon International Inc, Temecula, Calif). Sections were washed
and then incubated with a biotinylated secondary antibody directed against
the mouse monoclonal antibodies for 1 hour at room temperature in a humidified
chamber using the Dako LSAB Kit (Dako Corporation, Carpinteria, Calif). Sections
were washed and then incubated with 0.05% 3,3'-diamino-benzidine tetrahydrochloride
in 50-mmol/L Tris at pH 7.6 and 0.01% hydrogen peroxide. Sections were counterstained
with hematoxylin and photographed with a Zeiss Ultraphot photoscope. To evaluate
the specificity of the antibodies, sections were incubated with nonimmune
mouse serum substituted for the primary antibodies. Immunostaining for MMP-1,
MMP-2, MMP-3, and MMP-9 was considered positive when cytoplasmic and stromal
staining was observed. Immunostaining for MT1-MMP and MT2-MMP was considered
positive when membrane staining was observed.
RESULTS
As shown in Figure 1 and Table 1, the specimen of normal conjunctival
and limbal tissue (patient CF) did not display immunostaining for any of the
MMPs in the epithelial basal cells. However, significant cell surface immunostaining
was present with MT1-MMP, and slight staining for MMP-1 and MMP-9 was seen
in the stroma (Figure 1A, D). In
contrast to the fresh surgical specimens, the 2 cadaver specimens (Table 1) immunostained with most MMPs in
the epithelial basal cells and stroma. For example, in the 2 specimens of
normal cadaver conjunctiva, limbus, and cornea (patients C34 and C35), immunostaining
by the 2 membrane-type MMPs (MT1-MMP and MT2-MMP) was primarily restricted
to the membranes of the basal epithelial cells in the cornea, limbus, and
conjunctiva and was not present in the stromal compartments of the tissues.
Immunostaining of MMP-9 also was restricted to the membranes of basal epithelial
cells in the cornea, limbus, and conjunctiva, but in addition, MMP-9 was detected
in the stroma of the conjunctiva, limbus, and cornea. Immunostaining for MMP-2
was present in the epithelium and stroma of the limbus and cornea but was
not detected in the epithelium of the conjunctiva. The stroma of the conjunctiva
was variably positive for MMP-2. Staining for MMP-3 was highly restricted
to the epithelium and stroma of the cornea and was not detected in the conjunctiva
or limbal tissues. Staining for MMP-1 was present in the epithelium of the
cornea and variably present in the corneal stroma and limbal epithelium.
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Figure 1. Matrix metalloproteinase (MMP)
immunostaining in normal limbus epithelium. No MMP staining of basal or suprabasal
cells (brown pigment in some basal cells is melanin). Membrane type 1 (MT1)–MMP
staining of some squamous cells. Mild staining of desquamating surface cells
(A, C, D, F) and stroma (A, D). Staining of all cut stromal edges: (A) MMP-1,
(B) MMP-3, (C) MMP-2, (D) MMP-9, (E) MT1-MMP, and (F) membrane type 2–MMP.
Palisades of Vogt are visible in A through D, indicating that the specimens
came from the limbal region. Limbal basal cells migrate from left to right
(original magnification x325).
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All 10 pterygia specimens (9 primary and 1 recurrent) immunostained
with most of the 6 MMPs studied (Table 2). Immunostaining by the MMPs was consistently high in the invading
limbus epithelium in the altered limbal basal cells and in the adjacent corneal
and conjunctival epithelia, which were infiltrated by invading altered limbal
basal cells (Table 2 and
Figure 2,
Figure 3, and
Figure 4). Matrix metalloproteinase 1 was particularly prominent in the epithelial cells
of the invading limbal epithelium (10/10), the adjacent corneal (9/10), and
conjunctival epithelial cells (10/10) (Table 2 and
Figure 2,
Figure 3, and
Figure 4). The other MMPs were present in the epithelium of about
8 of 10 pterygia specimens. In the recurrent pterygium, we found a single
layer of cuboidal cells, which immunostained with all 6 MMPs, spreading on
top of the surface of terminally differentiated squamous cells (data not shown).
In addition, MMP expression occurred in some corneal stromal sections at cut,
broken, or crushed areas
(Figure 2
and
Figure 4).
Figure 2G is interesting (a higher magnification of
Figure 2A) in that it shows staining of MMP-1 in both the basal
epithelial cells and the epithelial side of Bowman's layer.
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Table 2. Staining of Pterygia With Monoclonal Antibodies to MMPs*
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Figure 2. Corneal invasion by altered limbal
basal cells. A group of matrix metalloproteinase (MMP) immunostaining altered
limbal basal cells (pterygium cells, arrowheads) invading the corneal epithelium
over Bowman's layer: (A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) membrane
type 1–MMP, and (F) membrane type 2–MMP. Altered limbal basal
cells invade from left to right (original magnification x170). Panel
G is a higher magnification (x500) of panel A and shows the basal cells
staining not only for MMP-1 but also on the epithelial side of Bowman's layer.
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Figure 3. The 2 tumors of pterygia. (1)
The pterygium tumor: matrix metalloproteinase (MMP) immunostaining in altered
limbal basal cells (to left of arrowheads that point to the dissolved edges
of Bowman's layer), which are invading corneal epithelium over Bowman's layer
(to right of arrowheads). This tumor is located above the space marked with
an X (see panel A). (2) The pinguecula tumor: stationary noninvading MMP immunostaining
areas of elastotic degeneration (containing altered fibroblasts) that are
dragged onto the cornea by the invading pterygium tumor. Staining of the area
of elastotic degeneration is seen in panel C; however, no fibroblast staining
is observed. The pinguecula is located below the space marked with an X (see
panel A). Before tissue processing these 2 tumors were contiguous. For both
tumors, (A) MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) membrane type 1–MMP,
and (F) membrane type 2–MMP. Altered limbal basal cells invade from
left to right (original magnification x100).
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Figure 4. Dissolution of Bowman's layer.
Matrix metalloproteinase (MMP) immunostaining in altered limbal basal cells
invading corneal epithelium over Bowman's layer, in activated fibroblasts
located at the dissolved edges of Bowman's layer (small arrowheads), and in
fibroblast islands (large arrowhead) within dissolved Bowman's layer: (A)
MMP-1, (B) MMP-3, (C) MMP-2, (D) MMP-9, (E) membrane type 1–MMP, and
(F) membrane type 2–MMP. Altered limbal basal cells invade from left
to right (original magnification x250 for A-F). G, Schematic drawing
of panel A.
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Matrix metalloproteinase 1 was found to be the most frequently expressed
MMP by fibroblasts in pterygia (Table 2 and Figure 4). Matrix
metalloproteinase 1 was often present in fibroblasts at the dissolved edge
of Bowman's layer (7/10) and in lobules of pingueculae (6/10) and less frequently
present in fibroblasts under the migrating limbus (3/10). Fibroblasts found
at the dissolved edges of Bowman's layer (7/10) and in areas of fibroblast
islands frequently immunostained with MMP-1 (Figure 4). In addition, MMP-1 was expressed by 9 of 10 altered limbal
basal cells in cornea over Bowman's layer (Figure 2, Figure 3, and Figure 4) and frequently stained Bowman's
layer beneath these basal cells (Table 2).
Areas of elastotic degeneration (pingueculae) usually immunostained
for all 6 MMPs (Table 3 and Figure 3), although the fibroblasts in these
areas mainly immunostained for MMP-1 and MMP-3.
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Table 3. Staining of Acellular Areas of Pingueculae, Found Within Pterygia,
With Monoclonal Antibodies to MMPs*
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COMMENT
COLLECTING AND ORIENTING THE SPECIMEN
The identification in pterygia of the migrating limbus with its altered
limbal basal cells and their associated activated fibroblasts depends on the
correct surgical collection of the specimen. In addition, proper orientation
of the specimen is required to demonstrate, with serial cross sections, the
key anatomy at the junction where the altered limbal basal cells start to
invade onto corneal basement membrane over dissolving Bowman's layer.
NORMAL TISSUE
As in most normal, resting tissues, conjunctival-limbal-corneal epithelial
tissues express such small amounts of MMPs that they are nearly undetectable
by techniques such as immunohistochemistry40-43
(Figure 1). However, MT1-MMP immunostaining
was detectable in the surface epithelial cells of the fresh normal control
specimens (Figure 1E). Also, MT1-MMP
immunostaining has been found in other normal human tissue.44
In addition, MMP immunostaining occurred in some sections at cut, broken,
or crushed areas (Figure 1, Figure 2, and Figure 4), which may be due to an artifactual translocation of the
MMPs after surgical wounding.45-46
From these data, we conclude that careful collection of specimens is needed
to avoid artifactual MMP staining due to trauma. In addition, fresh surgical
specimens are needed, because cadaver eyes, which were 4 to 5 hours old, tended
to express abnormal levels of MMPs (Table
1).
PTERYGIA
We discovered previously that pterygia consisted of limbal epithelial
tumor cells that expressed p53 and vimentin and displayed a peculiar development
and migration pattern.8-9 We also
previously demonstrated that the pterygium cells had characteristics of limbal
basal epithelial cells.8-9 In
the present study, we found that the altered limbal basal epithelial cells
of pterygia expressed 6 MMPs of various types similar to other invasive tumors,30-34
and we speculate that these MMPs are likely to promote corneal invasion of
this tumor and contribute to the dissolution of Bowman's layer (Figure 2, Figure 3, and Figure 4). In addition to migration of a
segment of altered limbal epithelium and local infiltration of pterygium cells
within adjacent epithelial tissues, we found in our specimen of recurrent
pterygium a pattern of surface spread of MMP-expressing cuboidal cells over
terminally differentiated squamous cells, which is similar to those in one
of our previous studies.19 The spreading of
surface MMP-expressing altered cells has a potential for wider spread than
infiltration in the basal layers and could possibly explain some of the recurrences
with autografts or wide excisions and the need for supplemental topical chemotherapeutic
eyedrops such as mitomycin.19
MIGRATING LIMBUS EPITHELIUM
The invasion of the cornea by an entire segment of limbal epithelium
with altered limbal basal cells can be explained by MMP-2 and MMP-9 expression
by these cells. Elevated expressions of both MMP-2 and MMP-9 are known to
dissolve basement membrane components, such as hemidesmosomes, leading to
migration and invasion of tumor cells.28-29
Consistent with the expression of MMP-2 and MMP-9 by altered limbal cells
is elevated MT1-MMP and MT2-MMP expression, since MT1-MMP and MT2-MMP can
activate latent pro–MMP-2 and pro–MMP-9.
DISSOLUTION OF BOWMAN'S LAYER
We previously described 4 different groups of fibroblasts in pterygia9, 19, 24: (1) a group of collagen-synthesizing
fibroblasts under the migrating limbus near the dissolved edge of Bowman's
layer; (2) a group of collagenase-synthesizing fibroblasts surrounding the
dissolved edges of Bowman's layer (Figure
2, Figure 3, and Figure 4); (3) groups of collagenase-synthesizing
fibroblasts located in islands (Ilots de Fuchs)12
(Figure 4) anterior to the leading
edges of the pterygium and between corneal basement membrane and Bowman's
layer; and (4) groups of elastotic material–synthesizing fibroblasts
in basophilic areas where abnormal elastic-type material was present.
None of these fibroblast groups expressed p53 in pterygia, whereas all
altered limbal basal cells did synthesize p53,19, 23
which suggests that the pterygium cells (ie, altered limbal basal epithelial
cells) are the main tumor cells. We found that the p53 overexpression colocalized
with the MMP expression (data not shown). Most of the fibroblasts in groups
2, 3, and 4 and a few fibroblasts in group 1 expressed mainly MMP-1 and some
MMP-3 but almost none of the other MMPs (Table 2). These findings suggest that in areas of Bowman's layer
dissolution, fibroblasts are making MMPs and most likely play an important
role in helping to dissolve Bowman's layer. These fibroblasts are aided in
the dissolution of Bowman's layer by the MMP-1– and MMP-3–expressing
limbal basal cells (Figure 4 and Figure 5) as indicated by MMP-1 and MMP-3
immunostaining of Bowman's layer in some of the sections. Because the altered
limbal basal epithelial cells (the pterygium cells) express transforming growth
factor  (TGF-),24, 47-49
the adjacent MMP-expressing fibroblasts are most likely TGF-–basic
fibroblast growth factor (bFGF) activated cells24
and are not mutationally altered ones19, 23-24
(Figure 5).
ALTERED FIBROBLASTS IN ELASTOTIC AREA: A SECOND TUMOR
Because fibroblasts in elastotic areas are known to make abnormal elastic
material, they have been considered to be altered cells.50-51
We found these fibroblasts making MMP-1 and MMP-3 but none of the other MMPs
(Table 2). However, since all
areas of elastotic degeneration outside the fibroblasts immunostained for
all 6 MMPs (Table 3), we assume
that the MMPs came from altered fibroblasts. The altered fibroblast lobules
constitute a second stationary tumor (pingueculae) within the main invading
pterygium tumor similar to what is present in other ocular and skin tumors
with associated areas in the stroma of elastotic degeneration.52
In all of these UV-induced growths, the main tumor cell type is the epithelial
cell and not the fibroblast. The fact that tumors consisting of both altered
epithelial cells and altered fibroblasts can exist at the same time has been
demonstrated in animal experiments where ocular tissue was treated with long-term,
low-dose UV radiation.53-54
Recurrent pterygia that return within a few months after surgery do
not usually have sufficient UV exposure to develop areas of elastotic degeneration.
For this reason, they were assumed to be different from primary pterygia and
to produce an exuberant fibroplasia as a result of an abnormal healing reaction.55 In pterygia recurring after several years, we have
found elastotic degeneration in all specimens, including the one reported
herein.
THEORY OF PATHOGENESIS OF PTERYGIA
Based on the data presented in this study and our previous reports,
we propose a theory for the pathogenesis of pterygia. Albedo UV light56 (Figure 5)
causes mutations in both the UV-sensitive TP53 tumor
suppressor genes in the parental limbal basal cells and the elastin gene of
the fibroblasts in the limbal epithelium.19
Because of a damaged p53-dependent programmed cell death mechanism,20 mutations in other genes are progressively acquired.
This allows the multistep21 development of
pterygia and limbal tumor cells from p53-expressing limbal epithelial cells.
These cells overlie a pinguecula of altered fibroblasts that make abnormal
elastotic material and express various MMPs.
Mutations in the TP53 gene or TP53 family in the parental limbal basal cells also result in the overproduction
by the pterygium cells of TGF- via the p53-Rb-TGF- pathway.24, 47 Thus, pterygia are TGF-–secreting
tumors. Excess TGF- secretion by the pterygium cells can explain many
of the tissue changes and MMP expressions seen in pterygia.24, 47-49,57-66
First, pterygium cells (altered limbal basal epithelial cells) produce elevated
MMP-2, MMP-9, MT1-MMP, and MT2-MMP, causing dissolution of hemidesmosome attachments.
Initially, the pterygium cells migrate centrifugally in all directions onto
the adjacent and joined corneal, limbal, and conjunctival basement membranes.
Because of the TGF- production of these cells, they have a reduced number
of cell layers24, 47-49,57
and no tumor mass is seen, resulting in an invisible tumor.19
Later, after an entire group of altered limbal basal cells develop and all
hemidesmosomes are dissolved under these cells, they migrate as a suppressed
growth onto the cornea followed by conjunctival epithelium, expressing all
6 MMPs and contributing to the dissolution of Bowman's layer. In addition,
TGF- synthesized by the pterygium cells causes increased monocytes and
capillaries within the epithelial and stromal layers19, 24, 37, 47-49,57-61
(Figure 5). Second, a group of normal
fibroblasts gather under the invading limbus epithelium next to the dissolved
edges of Bowman's layer and are activated by a TGF-–bFGF pathway24 to produce excess MMP-1 and MMP-362
as they help to dissolve Bowman's layer. Some of these cytokine-activated
fibroblasts migrate anterior to the leading edges of pterygia between Bowman's
layer and the basement membrane of the corneal basal cells to form little
islands of fibroblasts that make MMP-1 and locally help to dissolve Bowman's
layer24, 62 (Figure 4).
The above steps in the formation of a pterygium are seen diagrammatically
in Figure 6. Figure 6 shows the migration of the altered limbal basal epithelial
cells (MMP expressing) within the body of the migrating limbus and their infiltration
into the corneal and conjunctival epithelia. Figure 6 also shows the dissolution of Bowman's layer under the
body of the migrating limbus and the migration of the adjacent conjunctival
epithelial cells and stromal structures, such as pingueculae, within the pterygium.
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Figure 6. Pterygia pathogenesis. Corneal
invasion by matrix metalloproteinase (MMP) expressing altered limbal epithelial
cells and activation of fibroblasts. CJ indicates conjunctiva with goblet
cells infiltrated by pterygium cells; DBL, dissolved Bowman's layer; F I,
fibroblasts making abnormal elastotic material (the pinguecula tumor); F II,
fibroblasts making collagen and possibly elastic materials; F III, fibroblasts
making MMP-1 at dissolved edge of Bowman's layer; F IV, fibroblasts (fibroblast
islands) making MMP-1 at dissolved edges of Bowman's layer; G, goblet cells;
ML, migrating limbus; MMP B, MMP expressing altered limbal basal epithelial
cells invading cornea and conjunctival epithelium; and V, blood vessels (angiogenesis).
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CONCLUSIONS
The main body of the tumor that is located in pterygia is found in the
leading edges and is a migrating transparent microscopic piece of altered
limbal epithelium. The migrating limbal epithelium is the occult tumor. If
sufficient fibroblasts accumulate under the migrating limbus at the leading
edges, the area can be seen clinically with the slitlamp biomicroscope as
a gray, glassy cap.9, 24 Microscopically,
the migrating limbal epithelial tumor is always located between the dissolved
edges of Bowman's layer and conjunctival epithelium (as indicated by the presence
of goblet cells). From this migrating limbus, altered limbal epithelial basal
cells invade centrifugally in all directions into adjacent conjunctival, circumferential
limbal, and corneal epithelia. As the migrating piece of limbal epithelium
moves onto corneal basement membrane over Bowman's layer, the adjacent conjunctival
epithelium infiltrated with the altered limbal basal cells follows, which
creates the gross clinical appearance of the pterygium.
Pterygia are tumors of altered limbal basal cells that secrete TGF-
and produce various types of MMPs similar to other invasive tumors. The tumor
cell proteases degrade components of their basement membranes, which facilitates
invasion. The pterygium cells invading over Bowman's layer produce elevated
MMP-1, MMP-2, and MMP-9 expressions, which contribute to the complete dissolution
of Bowman's layer, which consists primarily of collagen fibril types I and
III.67 Local fibroblasts are activated by the
TGF- and bFGF cytokine pathways to help complete the dissolution of
Bowman's layer by MMP-1. However, MMP-1 makes only a single cut in intact
fibrillar collagen (eg, fibrillar collagen types I, II, III, VII, VIII, and
X), and then the gelatinases MMP-2 and MMP-9 make successive cuts in the altered
type I collagen that eventually produces complete destruction of collagen
strands. As these 2 groups of cells invade into cornea, they drag along the
adjacent conjunctiva and stromal structures, such as pingueculae, which consist
of focal areas of noninvasive stationary fibroblast tumors synthesizing abnormal
elastic material and MMPs.
AUTHOR INFORMATION
Accepted for publication December 28, 2000.
This study was supported in part by grant 61-9783 from the Kaiser Foundation
Research Institute, Oakland, Calif (Dr Dushku), and grant EY05587 from the
National Institutes of Health, Bethesda, Md (Dr Schultz).
Drs Dushku and John contributed equally to this article.
We thank Samuel Woo, Illustration Services, University of California,
Davis, for photography assistance.
Reprints and corresponding author: Nicholas Dushku, MD, Department of
Ophthalmology, Kaiser Permanente Medical Center, 1650 Response Rd, Sacramento,
CA 95815 (e-mail: Nicholas.Dushku{at}kp.org).
From the Department of Ophthalmology, Kaiser Permanente Medical Center,
Sacramento, Calif (Dr Dushku); Department of Obstetrics and Gynecology, Institute
for Wound Healing, University of Florida, Gainesville (Drs John and Schultz);
and Departments of Ophthalmology and Visual Sciences, Texas Tech University,
Lubbock (Dr Reid).
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