April 2007
Volume 48, Issue 4
Free
Cornea  |   April 2007
β-Catenin Activation and Epithelial-Mesenchymal Transition in the Pathogenesis of Pterygium
Author Affiliations
  • Naoko Kato
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
  • Shigeto Shimmura
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
  • Tetsuya Kawakita
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
  • Hideyuki Miyashita
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
  • Yoko Ogawa
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
  • Satoru Yoshida
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
  • Kazunari Higa
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
  • Hideyuki Okano
    Department of Physiology; School of Medicine, Keio University, Tokyo, Japan.
  • Kazuo Tsubota
    From the Department of Ophthalmology; Keio University School of Medicine, Tokyo, Japan; the
    Department of Ophthalmology; Tokyo Dental College, Chiba, Japan; and the
Investigative Ophthalmology & Visual Science April 2007, Vol.48, 1511-1517. doi:10.1167/iovs.06-1060
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      Naoko Kato, Shigeto Shimmura, Tetsuya Kawakita, Hideyuki Miyashita, Yoko Ogawa, Satoru Yoshida, Kazunari Higa, Hideyuki Okano, Kazuo Tsubota; β-Catenin Activation and Epithelial-Mesenchymal Transition in the Pathogenesis of Pterygium. Invest. Ophthalmol. Vis. Sci. 2007;48(4):1511-1517. doi: 10.1167/iovs.06-1060.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate whether β-catenin activation and epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of pterygium.

methods. β-Catenin and E-cadherin expression were examined in surgically excised tissue and eye bank corneas with intact pterygium. Snail and Slug, the transcriptional repressors of E-cadherin, and matrix metalloproteinase (MMP)-7, a down-stream gene regulated by β-catenin were also investigated. Epithelial cells undergoing EMT-like changes were identified by double immunostaining for α-smooth muscle actin (SMA)/vimentin and cytokeratin 14. Transmission electron microscopy was used to examine the ultrastructure of the pterygial head.

results. Histopathology showed aberrant fibrotic proliferation beneath the pterygium epithelium, with epithelial processes extending into the stroma. Transmission electron microscopy revealed the dissociation of epithelial cells, which were surrounded by activated fibroblast-like cells. Characteristic downregulation of E-cadherin and intranuclear accumulation of β-catenin and lymphoid-enhancer-factor-1 in pterygial epithelium were also observed by immunohistochemistry. Of note, epithelial cells extending into the stroma were positive for both α-SMA/vimentin and cytokeratin 14. Snail and Slug were immunopositive in the nuclei of pterygial epithelial cells, but not in normal corneal epithelial cells.

conclusions. EMT of basal epithelial cells may play a key role in the pathogenesis of pterygium.

A pterygium is a wing-shaped, fibrovascular conjunctival outgrowth that centripetally invades the clear cornea. The incidence of pterygium is epidemiologically known to be associated with sun exposure (ultraviolet radiation), and therefore the disease is sometimes regarded as a benign tumor. 1 2 3 A large pterygium approaching the central cornea can cause visual loss, requiring surgical excision, after which it may recur. Although the pathogenesis of pterygium remains unknown, pterygial epithelial cells (PECs) are believed to have acquired an altered balance between proliferation and apoptosis. Many investigators have reported abnormal expression or mutation of p53, a DNA-binding protein with tumor-suppressor properties, or increased expression of the proliferation marker Ki67. 4 5 6 7 8 Shimmura et al. 9 showed abnormally increased telomerase activity in pterygia tissue, indicating hyperproliferative activity of PECs. 
The stromal portion of a pterygium shows aberrant accumulation of extracellular matrix molecules, such as collagen fibers, with pathologic features characteristic of elastotic degeneration. The main component of the stroma has been shown to be accumulated elastin fibers, and pterygial fibroblasts within the lesion secrete significantly higher levels of tropoelastin in vivo. 10 Other investigators have shown that pterygium-derived fibroblasts have characteristics of myofibroblasts (α-smooth muscle actin (SMA)+, vimentin+, and microfilament+), 11 with increased epidermal growth factor (EGF) receptors, 12 and accelerated secretion of matrix metalloproteinases (MMPs), and fibroblast growth factor (FGF). 13 These facts indicate that pterygial fibroblasts and PECs are phenotypically altered from resident cells of the ocular surface, contributing to the fibrotic changes observed in pterygia. 
Fibrosis is a common pathologic event observed in various organs of the body. Recent studies have demonstrated that the cellular origin of fibroblasts during disease are not only remnants of embryonic development, but may also arise from tissue-specific epithelial cells and circulating pools. 14 In particular, the phenomenon of epithelial cells changing their phenotype to fibroblastic cells after morphogenic pressure from injured tissue is called epithelial-mesenchymal transition (EMT). 15 16 EMT is a well-recognized mechanism involved in the dispersion of cells during vertebrate embryogenesis and is also observed in the adult during repair of injured tissue, as well as in the initial stages of cancer metastases. 17 18 19  
Kawakita et al. 20 have reported that corneal limbal epithelial cells (LECs) residing in the peripheral cornea can undergo EMT after exposure to air in vitro. As a result, LECs has been shown to invade the underlying stroma, leading to histologic findings similar to fibrosis. Because the basal limbal epithelium includes putative corneal epithelial/progenitor cells, it is reasonable to hypothesize that altered LECs undergo EMT as one of the mechanisms involved in the pathogenesis of pterygium. To support this hypothesis, we focused on the β-catenin signaling pathway, which is involved in the pathogenesis of several fibrosing diseases. 21 22 23 The β-catenin pathway plays a key role in the induction of EMT, and several pterygium-related genes such as MMPs and cyclin D1 are known to be under the control of β-catenin. 
Materials and Methods
Antibodies
Anti-β-catenin antibody was obtained from Promega (Madison, WI); anti-E-cadherin and anti-lymphoid enhancer factor (LEF)-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-cytokeratin-14 antibody from Abcam (Cambridge, UK); anti-vimentin antibody from NeoMarkers (Fremont, CA); anti-human α-SMA antibody from Dako (Glostrup, Denmark); and anti-Snail and Slug antibodies were obtained from Abgent (San Diego, CA). 
Human Cornea Samples
Four human corneas that were not suitable for transplantation because of pterygium were obtained from Northwest Lions Eye Bank (Seattle, WA) and used for immunohistochemistry (n = 3) and transmission electron microscopy (n = 1). Normal donor human corneas (n = 4) were used as a control. Clinical samples of pterygium were obtained during surgery from nine patients with pterygium after written informed consents were obtained from each patient before surgery. Excised tissue was immediately embedded in OCT compound (Tissue-Tek; Sakura Finetek, Co. Ltd., Tokyo, Japan) and prepared for immunohistochemistry. All research protocols were approved by appropriate ethics committees of the Keio University School of Medicine and Tokyo Dental College, and were performed in accordance with the tenets of the Declaration of Helsinki. 
Immunohistochemistry
Fresh-frozen sections were fixed (Mildform; Wako, Osaka, Japan) and washed in PBS, incubated with primary antibodies or preimmune IgGs for 1 hour at room temperature. Samples were then processed (Vectastain ABC kit; Vector, Burlingame, CA) according to the protocol provided by the manufacturer. Hematoxylin-eosin (HE) staining was performed according to standard procedures. For immunofluorescence, fresh-frozen sections (5–10 μm thick) were air dried and fixed (Mildform 10 N; Wako) for 10 minutes at room temperature. Blocking was performed with 10% donkey or goat serum in phosphate-buffered saline (PBS) for 30 minutes. Sections were then incubated with primary antibodies overnight at 4°C. Immunoreactivity of primary antibodies was visualized by using secondary antibodies conjugated with FITC (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen Corp., Carlsbad, CA). After the sections were washed with PBS, they were mounted (Permafluor; Beckman Coulter Inc., Miami, FL), and images were observed with a microscope (Axiovert 135; Carl Zeiss Inc., Thornwood, NY) equipped with a digital camera (LSM510; Carl Zeiss Inc.). 
Transmission Electron Microscopy
An eye bank donor cornea with pterygium and unaffected limbus at the opposite side of the same corneal button were processed for transmission electron microscopy. Each sample was fixed in 4% paraformaldehyde and 1% glutaraldehyde solution for 4 hours and washed with PBS. The samples were dehydrated in a series of ethanol and embedded in resin (LRWhite; Oken, Tokyo, Japan). Semithin sections (1 μm) were stained with methylene blue. Ultrathin specimens were then sectioned with a microtome (LKB, Gaithersburg, MD) with a diamond knife. Sections in the range of gray to silver were collected on 150-mesh nickel grids, stained with uranyl acetate and lead citrate, and examined under an electron microscope (model 1200 EXII; JEOL, Tokyo, Japan). 
RT-PCR
Surgically excised pterygial tissue (n = 4) and corneal limbal tissue from eye bank corneas (n = 4) were treated with Dispase II (Roche Diagnostics GmbH, Mannheim, Germany) at 37°C for 1 hour, and epithelial sheets were dissociated using surgical forceps. Total RNA was extracted using an SV total RNA isolation system (Promega, Madison, WI), and reverse transcribed (AMV Reverse Transcriptase First-strand cDNA Synthesis Kit; Life Science, Inc., Petersburg, FL). PCR was performed for MMP-7 and GAPDH with the primers listed in Table 1 . Briefly, 20 ng of cDNA was added to a 24-μL reaction volume containing 1 μL of random primers and 23 μL of buffer (Platinum PCR Super Mix; Invitrogen). The amplification conditions were 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute for 40 cycles. Electrophoresis was conducted on 2% (wt/vol) analytical grade agarose gels that were subsequently stained with ethidium bromide. 
Results
Histopathology of Eye Bank Corneas with Pterygium
Unlike surgically excised tissue, an intact pterygium attached to an eye bank cornea offers the unique opportunity to observe the ultrastructure of pterygia in situ. Figure 1Ais a representative HE staining of an eye bank cornea shown along the longitudinal axis of an intact pterygium. Pterygial tissue has invaded centripetally from the conjunctiva toward the central cornea. Toward the head of the pterygium, epithelium thickness was irregular and fibrous proliferation was observed between the basal epithelium and Bowman’s layer. The high-magnification view in Figure 1Breveals the dissolution of the Bowman’s layer behind the leading head of the lesion, which was replaced by massive fibrovascular proliferation. In the pterygial body, massive basophilic amorphous material was observed in the stroma, which is consistent with previous reports. 24  
Further ultrastructural analysis was made by transmission electron microscopy. Figure 2Ashows normal corneal epithelium with epithelial cells forming stratified layers above the Bowman’s layer. The basal cells have a relatively thin basal cytoplasmic membrane, which is attached to the Bowman’s layer through an intact basement membrane and numerous hemidesmosomes. Figure 2Bshows the normal limbus where the Bowman’s layer disappears and epithelial cells are attached directly to the underlying stroma, which contains collagen fibers and fibroblasts. The head of a pterygium (Figs. 2C 2D)consists of irregular collagen fibers with fibroblasts observed between the epithelium and the Bowman’s layer. Basal PECs have irregular cytoplasmic membranes and are attached to the underlying fibrotic tissue containing fibroblast-like cells, sparse melanocytes, and vessels. Breaks were also observed in the basement membrane of the basal PECs. 
Slightly behind the pterygium head, the Bowman’s layer disappeared, and basal PECs attached directly to the fibrous tissue (Fig. 3A) . Basal PECs had a cuboid, relatively small cell contour and high cytoplasmic electron density. Higher magnification (Fig. 3B)showed that these small basal PECs contained numerous cytoplasmic fibrils. The basement membrane is multilaminated and intermingled with irregular extracellular collagen fibers. Examination of epithelial indentations extending into stromal tissue revealed PECs with higher cytoplasmic electron density, enlarged intercellular spaces, and irregular basal cytoplasmic membranes (Figs. 3C 3D) . Subepithelial fibrotic tissue contained numerous enlarged fibroblast-like cells. 
EMT in Pterygium Head PECs
We examined EMT in pterygial epithelium by immunohistochemistry against the mesenchymal markers vimentin, α-SMA, and the basal epithelial marker cytokeratin 14 (K14). Surgically excised tissue and eye bank corneas with pterygium were diffusely immunoreactive to K14 throughout the epithelium (Fig. 4) . K14+ epithelial cells were also found to express α-SMA (Figs. 4A 4B)and vimentin (Fig. 4D)in focal areas of surgical samples, suggesting that these cells were undergoing EMT. Examination of eye bank corneas revealed that these α-SMA+, vimentin+, and K14+ cells were localized at the leading edge of epithelial invaginations into the stroma. Normal corneal epithelium did not express α-SMA (Fig. 4C)or vimentin (Fig. 4E)
Since the loss of membranous localization of E-cadherin protein and transcriptional repression of its mRNA are hallmarks of EMT, 19 we also examined the staining pattern of E-cadherin in pterygium and normal corneal epithelium. E-cadherin was observed lining the cytoplasmic membrane in the central corneal epithelium as well as in pterygium tissue. However, pterygium samples demonstrated the loss of cytoplasmic staining of E-cadherin, as shown in Figure 5A
Nuclear Expression of β-Catenin and LEF-1 and Upregulation of MMP-7 in Pterygium
β-Catenin links E-cadherin and α-catenin to the cytoskeleton to form a complex that maintains normal epithelial polarity and intercellular adhesion. When E-cadherin is downregulated during EMT, β-catenin accumulates in the cytoplasm where it binds to cytosolic T cell-factor/lymphoid enhancer factor (LEF) transcription factors. The resulting complex is shuttled into the nucleus and activates the expression of target genes such as cyclin D1, c-Myc, MMP-7, and membrane type (MT)1-MMP. 25 26 27 28 We therefore examined the localization of β-catenin in pterygium samples. Both surgically excised pterygium (Fig. 5B)and eye bank corneas with pterygium (Fig. 5C)were immunoreactive to β-catenin along the cytosolic membrane of PECs. However, intranuclear staining of β-catenin was observed in sporadic areas of the epithelium in surgically excised pterygium (Fig. 5D) . In eye bank corneas, nuclear transition of β-catenin was observed in epithelial indentations into the underlying stroma (Fig. 5C) . Intranuclear β-catenin was found predominantly in α-SMA/vimentin+ cells. 
Because LEF-1 is reported to be associated with EMT among other transcription factors such as TCF-1, -3, or -4, which work in cooperation with β-catenin, 29 we also examined the expression of LEF-1 in pterygium. Although LEF-1 staining was quite limited, immunoreactivity was observed in the nuclei of epithelial cells in areas similar to where nuclear β-catenin and cytosolic α-SMA were positive (Fig. 5E) . Normal corneal epithelium did not express LEF-1 (Fig. 5F)
We investigated mRNA expression of the β-catenin-driven gene MMP-7 in pterygial and corneal limbal epithelium. MMP-7 was uniquely found in all four pterygium samples examined (Fig. 5G) . MMP-7 is a protease that was suggested to be involved in the pathogenesis of pterygium. 30 Other genes associated with β-catenin such as MT1-MMP, cyclin D1 and c-Myc were investigated; however, no difference in expression was observed by RT-PCR (data not shown). 
Expression of the E-Cadherin Repressor Proteins Snail and Slug
Snail-related zinc finger transcription factors Snail and Slug are repressors of E-cadherin transcription essential for initiating mesodermal development during gastrulation. 19 Of note, strong expression of Snail and Slug was observed in the nucleus of PECs (Figs. 6A 6B 6C) , but not in normal corneal (Fig. 6D)or normal limbal (Fig. 6E)epithelia. 
Discussion
In the present study, we were able to examine the pathologic findings in pterygium tissue found intact on donor corneas unsuitable for transplantation. Previous studies have relied on surgically excised tissue, which offer limited information as to the microscopic architecture of the interface between the pterygium and underlying corneal tissue. We hypothesized that the firm adhesion at the head of an extending pterygium involves EMT-like changes in transformed PECs, and indeed we were able to confirm several histologic findings suggestive of EMT. Both E-cadherin and β-catenin were found to relocate from the cell membrane into the cytoplasm, and nuclear translocation of β-catenin was colocalized with the transcription factor, LEF-1. These cellular events lead to the coexpression of vimentin, α-SMA, and K14 by epithelial cells at the head of the pterygium. Furthermore, Slug and Snail, the upstream regulators of E-cadherin, were also expressed in the nucleus of pterygium epithelium. 
During EMT, epithelial cells show less intercellular adherence junctions, tight junctions, and desmosomes leading to the loss of cellular polarity. Cytokeratin intermediate filaments are also disassembled to rearrange their F-actin stress fibers to express filopodia and lamellipodia. 14 Gross histologic examination of an intact pterygium showed irregular thinning of the epithelium with the appearance of fibrous tissue between the epithelium and Bowman’s layer (Figs. 1A 1B) . Transmission electron microscopy showed newly synthesized collagen fibrils accumulating between the epithelium and Bowman’s layer at pterygial head (Figs. 2C 2D) . Basal PECs had higher cytoplasmic electron density with cytoplasmic fibrils, and PECs found invading the underlying stroma no longer showed adhesion complexes and had enlarged intercellular spaces (Fig. 3D) . Quite impressively, PECs near the leading tip of the indentation into the stroma expressed the basal epithelial marker K14 and the mesenchymal markers vimentin and α-SMA, as shown by double immunofluorescence stains (Fig. 4) . Coexpression of epithelial keratins with vimentin and α-SMA is a classic sign of EMT, supporting our hypothesis that EMT plays a key role in the progression of pterygia. Typical double-positive cells were only observed in limited areas of both surgically excised and intact pterygium. 
We further examined several molecular events associated with EMT. We demonstrated that PECs focally lost membrane-bound E-cadherin and β-catenin (Figs. 5A 5B 5C 5D) . Examination of surgically excised tissue revealed both the membrane-associated expression and cytoplasmic-nuclear localization of both E-cadherin and β-catenin within the same field of view (Figs. 5A 5B) , suggesting that a discontinuity exists between pterygium and normal epithelial tissue. Furthermore, PECs toward the pterygial head simultaneously had nuclear staining for LEF-1 (Fig. 5E) . These findings indicate that the β-catenin signaling pathway is activated in this area. The canonical β-catenin signaling pathway is a necessary component to drive EMT in development and is frequently activated in cancer. 31 β-catenin/LEF-1 complexes play a key role in EMT during the pathogenesis of colon cancer. 29 32 We found that MMP-7, one of the downstream genes of the β-catenin/LEF-1 complex, was uniquely expressed in PECs, and not by corneal limbal epithelial cells taken from the limbus of donor corneas (Fig. 5G) . MMP-7 has been shown to be present in the leading edge of pterygium tissue 30 and may be involved in the destruction of basement membrane and stromal tissue surrounding the pterygium head. 
EMT is triggered by several extracellular signals that include both ligand-dependent signaling by soluble growth factors such as transforming growth factor (TGF)-β, FGF families, and hepatocyte growth factors (HGF), 33 as well as cellular interaction with extracellular matrix proteins such as collagen and hyaluronic acid. 34 Although the signaling pathways leading to EMT after ligand activation are complex, the downregulation of E-cadherin is a major final common pathway. Repression of E-cadherin is driven by the transcriptional regulators Snail and Slug, which translocate into the cell nucleus after phosphorylation of Ser residues. 35 We also found that Snail and Slug were localized in the nucleus of PECs extending from the head toward the body of pterygium samples (Fig. 6) . A clear delineation is observed between the pterygium and corneal epithelial cells suggesting that relevant signaling mechanisms are activated during the progression of pterygium. 
Figure 7shows the proposed mechanisms involved in the pathogenesis of pterygium based on the results of our study. Although our results strongly suggest that EMT is a major factor in the progression of pterygium, specific upstream events triggering Snail activation in PECs are yet to be elucidated. Thecellular changes observed may be the result of an aberrant wound-healing response, since corneal epithelial cells are known to upregulate vimentin during wound healing. 36 Mutations of the genome may be involved, and there is also the possibility that persistent inflammation causes the upregulation of MMP-7, as well as other effectors. Further investigations are needed to link ultraviolet exposure and/or inflammation to the induction of EMT in the pathogenesis of pterygium. 
 
Table 1.
 
Sequences of Primers for RT-PCR
Table 1.
 
Sequences of Primers for RT-PCR
GAPDH Forward primer GAA GGT GAA GGT CGG AGT C
Reverse primer GAA GAT GGT GAT GGG ATT TC
MMP-7 Forward primer GTG GTC ACC TAC AGG ATC GT
Reverse primer ACC ATC CGT CCA GCG TTC AT
Figure 1.
 
Histopathology of pterygium. (A) Representative pathologic appearance of an intact pterygium found in an eye bank cornea. A longitudinal section shows pterygial tissue invading centripetally from the conjunctiva toward the central cornea. The pterygial head (box) shows an epithelium of irregular thickness accompanied by fibrous tissue between basal epithelial cells and Bowman’s layer. Arrowheads: epithelial cells extending into the superficial stroma. In the pterygial body, massive basophilic amorphous material was observed in the stroma ( Image not available ). Hematoxylin-eosin staining. (B) High magnification of the area in the box in (A). Fibrotic tissue with occasional cells is observed between the epithelium and Bowman’s layer, which becomes discontinuous (arrow) and eventually continuous with the loose fibrous tissue of the pterygium body. HE staining. Original magnification: (A) ×40; (B) ×200. Scale bar: (A) 1 mm; (B) 100 μm.
Figure 1.
 
Histopathology of pterygium. (A) Representative pathologic appearance of an intact pterygium found in an eye bank cornea. A longitudinal section shows pterygial tissue invading centripetally from the conjunctiva toward the central cornea. The pterygial head (box) shows an epithelium of irregular thickness accompanied by fibrous tissue between basal epithelial cells and Bowman’s layer. Arrowheads: epithelial cells extending into the superficial stroma. In the pterygial body, massive basophilic amorphous material was observed in the stroma ( Image not available ). Hematoxylin-eosin staining. (B) High magnification of the area in the box in (A). Fibrotic tissue with occasional cells is observed between the epithelium and Bowman’s layer, which becomes discontinuous (arrow) and eventually continuous with the loose fibrous tissue of the pterygium body. HE staining. Original magnification: (A) ×40; (B) ×200. Scale bar: (A) 1 mm; (B) 100 μm.
Figure 2.
 
Transmission electron micrographs of normal limbal epithelium and pterygial epithelium. (A) Normal corneal epithelium. Epithelial cells form stratified layers above the Bowman’s layer. Basal corneal epithelial cells (CEC) are cuboid and have a relatively thin basal cytoplasmic membrane with numerous cytoplasmic organellae. Beneath the basal cells, Bowman’s lamellae (BL) are observed. (B) Normal corneal limbus. Basal limbal epithelial cells (LECs) are smaller in cell size and have an electron-dense cytoplasm. Basal cytoplasmic membrane shows prominent indentations compared with central corneal epithelial cells, which are attached to the underlying stroma (St). (C, D) Pterygial head. Massive fibrous tissue (FT) is observed between basal PECs (PEC) and Bowman’s layer (BL). Fibrous tissue contain irregularly arranged collagen fibers, enlarged fibroblasts (Fb), a few melanocytes (Me), and vessels (V). Bowman’s layer becomes thin and interrupted at the pterygial head. BL, Bowman’s layer; CEC, corneal epithelial cells; LEC, limbal epithelial cells; PECs, pterygial epithelial cells; Fb, fibroblast; FT, fibrous tissue; Me, melanocyte; St, stroma; V, vessel. Original magnification: (A) ×3000; (B) ×4000 (C, D) ×2000.
Figure 2.
 
Transmission electron micrographs of normal limbal epithelium and pterygial epithelium. (A) Normal corneal epithelium. Epithelial cells form stratified layers above the Bowman’s layer. Basal corneal epithelial cells (CEC) are cuboid and have a relatively thin basal cytoplasmic membrane with numerous cytoplasmic organellae. Beneath the basal cells, Bowman’s lamellae (BL) are observed. (B) Normal corneal limbus. Basal limbal epithelial cells (LECs) are smaller in cell size and have an electron-dense cytoplasm. Basal cytoplasmic membrane shows prominent indentations compared with central corneal epithelial cells, which are attached to the underlying stroma (St). (C, D) Pterygial head. Massive fibrous tissue (FT) is observed between basal PECs (PEC) and Bowman’s layer (BL). Fibrous tissue contain irregularly arranged collagen fibers, enlarged fibroblasts (Fb), a few melanocytes (Me), and vessels (V). Bowman’s layer becomes thin and interrupted at the pterygial head. BL, Bowman’s layer; CEC, corneal epithelial cells; LEC, limbal epithelial cells; PECs, pterygial epithelial cells; Fb, fibroblast; FT, fibrous tissue; Me, melanocyte; St, stroma; V, vessel. Original magnification: (A) ×3000; (B) ×4000 (C, D) ×2000.
Figure 3.
 
Transmission electron micrographs of pterygial epithelium. (A) PECs slightly posterior to the pterygial head are small, cuboid basal cells with high cytoplasmic electron density resembling normal basal limbal epithelial cells. The basal cytoplasmic membrane shows indentations associated with the basement membrane (arrowheads). (B) High magnification of basal PECs. Numerous fibrils are observed in the cytoplasm. A multilayered basement membrane (arrowheads) is observed intermingled with irregular extracellular collagen fibers in the stromal fibrous tissue (FT). (C) Leading head of an epithelial indentation extending into the underlying stroma. PECs with high cytoplasmic electron density are shown with enlarged intercellular spaces. Surrounding stroma contains enlarged, fusiform fibroblast-like cells (Fb). (D) Adjacent section of (C). PECs no longer have intercellular adhesion complexes, and show enlarged intercellular spaces ( Image not available ). Fb, fibroblast-like cells; FT, fibrous tissue. Original magnification: (A) ×2000; (B) ×4000; (C) ×2500; (D) ×5000.
Figure 3.
 
Transmission electron micrographs of pterygial epithelium. (A) PECs slightly posterior to the pterygial head are small, cuboid basal cells with high cytoplasmic electron density resembling normal basal limbal epithelial cells. The basal cytoplasmic membrane shows indentations associated with the basement membrane (arrowheads). (B) High magnification of basal PECs. Numerous fibrils are observed in the cytoplasm. A multilayered basement membrane (arrowheads) is observed intermingled with irregular extracellular collagen fibers in the stromal fibrous tissue (FT). (C) Leading head of an epithelial indentation extending into the underlying stroma. PECs with high cytoplasmic electron density are shown with enlarged intercellular spaces. Surrounding stroma contains enlarged, fusiform fibroblast-like cells (Fb). (D) Adjacent section of (C). PECs no longer have intercellular adhesion complexes, and show enlarged intercellular spaces ( Image not available ). Fb, fibroblast-like cells; FT, fibrous tissue. Original magnification: (A) ×2000; (B) ×4000; (C) ×2500; (D) ×5000.
Figure 4.
 
Immunohistochemistry of α-SMA/K14 and vimentin/K14. (A) Double immunohistochemistry was performed for α-SMA and K14 in surgically excised pterygial tissue. K14 (red) was diffusely expressed throughout the epithelium. Epithelial cells positive for both α-SMA (green) and K14 were observed focally (arrow). (B) Pterygial epithelium in eye bank corneas also diffusely stained for K14. α-SMA was observed in basal PECs at the leading tip of epithelial indentations extending into the stroma. Epithelium at the pterygial surface was α-SMA negative. (C) Normal corneal epithelium shows basal/suprabasal immunoreactivity for K14, but not for α-SMA. (D) Basal PECs in eye bank tissue also expressed vimentin, another marker of EMT (arrowheads). (E) Normal corneal epithelial cells do not express vimentin. Original magnification: (A) ×100 (B–E) ×400. Scale bars: 50 μm.
Figure 4.
 
Immunohistochemistry of α-SMA/K14 and vimentin/K14. (A) Double immunohistochemistry was performed for α-SMA and K14 in surgically excised pterygial tissue. K14 (red) was diffusely expressed throughout the epithelium. Epithelial cells positive for both α-SMA (green) and K14 were observed focally (arrow). (B) Pterygial epithelium in eye bank corneas also diffusely stained for K14. α-SMA was observed in basal PECs at the leading tip of epithelial indentations extending into the stroma. Epithelium at the pterygial surface was α-SMA negative. (C) Normal corneal epithelium shows basal/suprabasal immunoreactivity for K14, but not for α-SMA. (D) Basal PECs in eye bank tissue also expressed vimentin, another marker of EMT (arrowheads). (E) Normal corneal epithelial cells do not express vimentin. Original magnification: (A) ×100 (B–E) ×400. Scale bars: 50 μm.
Figure 5.
 
Immunohistochemistry of E-cadherin and β-catenin pathway molecules. (A) E-cadherin in a surgically excised pterygium. Focal loss of membrane-associated E-cadherin was observed ( Image not available ). Arrowheads: membrane-associated staining for E-cadherin. (B) Immunohistochemistry for β-catenin in surgically excised pterygium. Similar to E-cadherin, both membrane-bound staining (arrowheads) and focal cytoplasmic staining ( Image not available ) were observed in the same field of view. (C, D) Nuclear translocation of β-catenin. Immunohistochemistry of intact pterygium in eye bank corneas revealed strong β-catenin staining in the nucleus of focal PECs (arrows) invading the underlying stroma (C). Nuclear transition of β-catenin was also observed in surgically excised pterygium (D). (E) LEF-1 was detected in the nuclei of PECs in areas similar to where nuclear β-catenin was positive (arrows). (F) Normal limbal control did not show nuclear staining of LEF-1 in epithelial cells. (G) RT-PCR for MMP-7 in PECs and LECs. MMP-7 was found in all four pterygium samples examined, while limbal epithelial samples were negative. LEC, limbal epithelial cells; PEC, pterygial epithelial cells. Original magnification: (A, B) ×200; (CF) ×400; scale bar: (A, B) 100 μm; (CF) 50 μm.
Figure 5.
 
Immunohistochemistry of E-cadherin and β-catenin pathway molecules. (A) E-cadherin in a surgically excised pterygium. Focal loss of membrane-associated E-cadherin was observed ( Image not available ). Arrowheads: membrane-associated staining for E-cadherin. (B) Immunohistochemistry for β-catenin in surgically excised pterygium. Similar to E-cadherin, both membrane-bound staining (arrowheads) and focal cytoplasmic staining ( Image not available ) were observed in the same field of view. (C, D) Nuclear translocation of β-catenin. Immunohistochemistry of intact pterygium in eye bank corneas revealed strong β-catenin staining in the nucleus of focal PECs (arrows) invading the underlying stroma (C). Nuclear transition of β-catenin was also observed in surgically excised pterygium (D). (E) LEF-1 was detected in the nuclei of PECs in areas similar to where nuclear β-catenin was positive (arrows). (F) Normal limbal control did not show nuclear staining of LEF-1 in epithelial cells. (G) RT-PCR for MMP-7 in PECs and LECs. MMP-7 was found in all four pterygium samples examined, while limbal epithelial samples were negative. LEC, limbal epithelial cells; PEC, pterygial epithelial cells. Original magnification: (A, B) ×200; (CF) ×400; scale bar: (A, B) 100 μm; (CF) 50 μm.
Figure 6.
 
Immunohistochemistry of E-cadherin repressors. (A) Composite micrograph of Snail immunohistochemistry. Snail was observed in PECs from the head to the body of the pterygium. Arrow: pterygial head. (B, C) Strong expression of Snail (B) and Slug (C) was observed in the nucleus of PECs. (D, E) Normal corneal (D) and limbal epithelium (E). Original magnification: (A) ×40; (BE) ×200; scale bar: (A) 200 μm; (BE) 50 μm.
Figure 6.
 
Immunohistochemistry of E-cadherin repressors. (A) Composite micrograph of Snail immunohistochemistry. Snail was observed in PECs from the head to the body of the pterygium. Arrow: pterygial head. (B, C) Strong expression of Snail (B) and Slug (C) was observed in the nucleus of PECs. (D, E) Normal corneal (D) and limbal epithelium (E). Original magnification: (A) ×40; (BE) ×200; scale bar: (A) 200 μm; (BE) 50 μm.
Figure 7.
 
Illustration showing the proposed role of β-catenin signaling and EMT in the pathogenesis of pterygium. Snail/Slug, possibly upregulated by inflammatory signals, represses the transcription of E-cadherin. Decrease in membrane-associated E-cadherin causes cytoplasmic elevation and intranuclear translocation of β-catenin, followed by the upregulation of MMP-7, which may be involved in the degradation of extracellular matrix in pterygium.
Figure 7.
 
Illustration showing the proposed role of β-catenin signaling and EMT in the pathogenesis of pterygium. Snail/Slug, possibly upregulated by inflammatory signals, represses the transcription of E-cadherin. Decrease in membrane-associated E-cadherin causes cytoplasmic elevation and intranuclear translocation of β-catenin, followed by the upregulation of MMP-7, which may be involved in the degradation of extracellular matrix in pterygium.
The authors thank Miyuki Yasuda, Akiko Kujira, and Naoko Okada (Department of Ophthalmology, School of Medicine, Keio University); Toshihiro Nagai and Kunio Fujita (Department of Pathology, Keio University); and Fumito Morito, (Department of Ophthalmology, Tokyo Dental College) for technical help. 
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Figure 1.
 
Histopathology of pterygium. (A) Representative pathologic appearance of an intact pterygium found in an eye bank cornea. A longitudinal section shows pterygial tissue invading centripetally from the conjunctiva toward the central cornea. The pterygial head (box) shows an epithelium of irregular thickness accompanied by fibrous tissue between basal epithelial cells and Bowman’s layer. Arrowheads: epithelial cells extending into the superficial stroma. In the pterygial body, massive basophilic amorphous material was observed in the stroma ( Image not available ). Hematoxylin-eosin staining. (B) High magnification of the area in the box in (A). Fibrotic tissue with occasional cells is observed between the epithelium and Bowman’s layer, which becomes discontinuous (arrow) and eventually continuous with the loose fibrous tissue of the pterygium body. HE staining. Original magnification: (A) ×40; (B) ×200. Scale bar: (A) 1 mm; (B) 100 μm.
Figure 1.
 
Histopathology of pterygium. (A) Representative pathologic appearance of an intact pterygium found in an eye bank cornea. A longitudinal section shows pterygial tissue invading centripetally from the conjunctiva toward the central cornea. The pterygial head (box) shows an epithelium of irregular thickness accompanied by fibrous tissue between basal epithelial cells and Bowman’s layer. Arrowheads: epithelial cells extending into the superficial stroma. In the pterygial body, massive basophilic amorphous material was observed in the stroma ( Image not available ). Hematoxylin-eosin staining. (B) High magnification of the area in the box in (A). Fibrotic tissue with occasional cells is observed between the epithelium and Bowman’s layer, which becomes discontinuous (arrow) and eventually continuous with the loose fibrous tissue of the pterygium body. HE staining. Original magnification: (A) ×40; (B) ×200. Scale bar: (A) 1 mm; (B) 100 μm.
Figure 2.
 
Transmission electron micrographs of normal limbal epithelium and pterygial epithelium. (A) Normal corneal epithelium. Epithelial cells form stratified layers above the Bowman’s layer. Basal corneal epithelial cells (CEC) are cuboid and have a relatively thin basal cytoplasmic membrane with numerous cytoplasmic organellae. Beneath the basal cells, Bowman’s lamellae (BL) are observed. (B) Normal corneal limbus. Basal limbal epithelial cells (LECs) are smaller in cell size and have an electron-dense cytoplasm. Basal cytoplasmic membrane shows prominent indentations compared with central corneal epithelial cells, which are attached to the underlying stroma (St). (C, D) Pterygial head. Massive fibrous tissue (FT) is observed between basal PECs (PEC) and Bowman’s layer (BL). Fibrous tissue contain irregularly arranged collagen fibers, enlarged fibroblasts (Fb), a few melanocytes (Me), and vessels (V). Bowman’s layer becomes thin and interrupted at the pterygial head. BL, Bowman’s layer; CEC, corneal epithelial cells; LEC, limbal epithelial cells; PECs, pterygial epithelial cells; Fb, fibroblast; FT, fibrous tissue; Me, melanocyte; St, stroma; V, vessel. Original magnification: (A) ×3000; (B) ×4000 (C, D) ×2000.
Figure 2.
 
Transmission electron micrographs of normal limbal epithelium and pterygial epithelium. (A) Normal corneal epithelium. Epithelial cells form stratified layers above the Bowman’s layer. Basal corneal epithelial cells (CEC) are cuboid and have a relatively thin basal cytoplasmic membrane with numerous cytoplasmic organellae. Beneath the basal cells, Bowman’s lamellae (BL) are observed. (B) Normal corneal limbus. Basal limbal epithelial cells (LECs) are smaller in cell size and have an electron-dense cytoplasm. Basal cytoplasmic membrane shows prominent indentations compared with central corneal epithelial cells, which are attached to the underlying stroma (St). (C, D) Pterygial head. Massive fibrous tissue (FT) is observed between basal PECs (PEC) and Bowman’s layer (BL). Fibrous tissue contain irregularly arranged collagen fibers, enlarged fibroblasts (Fb), a few melanocytes (Me), and vessels (V). Bowman’s layer becomes thin and interrupted at the pterygial head. BL, Bowman’s layer; CEC, corneal epithelial cells; LEC, limbal epithelial cells; PECs, pterygial epithelial cells; Fb, fibroblast; FT, fibrous tissue; Me, melanocyte; St, stroma; V, vessel. Original magnification: (A) ×3000; (B) ×4000 (C, D) ×2000.
Figure 3.
 
Transmission electron micrographs of pterygial epithelium. (A) PECs slightly posterior to the pterygial head are small, cuboid basal cells with high cytoplasmic electron density resembling normal basal limbal epithelial cells. The basal cytoplasmic membrane shows indentations associated with the basement membrane (arrowheads). (B) High magnification of basal PECs. Numerous fibrils are observed in the cytoplasm. A multilayered basement membrane (arrowheads) is observed intermingled with irregular extracellular collagen fibers in the stromal fibrous tissue (FT). (C) Leading head of an epithelial indentation extending into the underlying stroma. PECs with high cytoplasmic electron density are shown with enlarged intercellular spaces. Surrounding stroma contains enlarged, fusiform fibroblast-like cells (Fb). (D) Adjacent section of (C). PECs no longer have intercellular adhesion complexes, and show enlarged intercellular spaces ( Image not available ). Fb, fibroblast-like cells; FT, fibrous tissue. Original magnification: (A) ×2000; (B) ×4000; (C) ×2500; (D) ×5000.
Figure 3.
 
Transmission electron micrographs of pterygial epithelium. (A) PECs slightly posterior to the pterygial head are small, cuboid basal cells with high cytoplasmic electron density resembling normal basal limbal epithelial cells. The basal cytoplasmic membrane shows indentations associated with the basement membrane (arrowheads). (B) High magnification of basal PECs. Numerous fibrils are observed in the cytoplasm. A multilayered basement membrane (arrowheads) is observed intermingled with irregular extracellular collagen fibers in the stromal fibrous tissue (FT). (C) Leading head of an epithelial indentation extending into the underlying stroma. PECs with high cytoplasmic electron density are shown with enlarged intercellular spaces. Surrounding stroma contains enlarged, fusiform fibroblast-like cells (Fb). (D) Adjacent section of (C). PECs no longer have intercellular adhesion complexes, and show enlarged intercellular spaces ( Image not available ). Fb, fibroblast-like cells; FT, fibrous tissue. Original magnification: (A) ×2000; (B) ×4000; (C) ×2500; (D) ×5000.
Figure 4.
 
Immunohistochemistry of α-SMA/K14 and vimentin/K14. (A) Double immunohistochemistry was performed for α-SMA and K14 in surgically excised pterygial tissue. K14 (red) was diffusely expressed throughout the epithelium. Epithelial cells positive for both α-SMA (green) and K14 were observed focally (arrow). (B) Pterygial epithelium in eye bank corneas also diffusely stained for K14. α-SMA was observed in basal PECs at the leading tip of epithelial indentations extending into the stroma. Epithelium at the pterygial surface was α-SMA negative. (C) Normal corneal epithelium shows basal/suprabasal immunoreactivity for K14, but not for α-SMA. (D) Basal PECs in eye bank tissue also expressed vimentin, another marker of EMT (arrowheads). (E) Normal corneal epithelial cells do not express vimentin. Original magnification: (A) ×100 (B–E) ×400. Scale bars: 50 μm.
Figure 4.
 
Immunohistochemistry of α-SMA/K14 and vimentin/K14. (A) Double immunohistochemistry was performed for α-SMA and K14 in surgically excised pterygial tissue. K14 (red) was diffusely expressed throughout the epithelium. Epithelial cells positive for both α-SMA (green) and K14 were observed focally (arrow). (B) Pterygial epithelium in eye bank corneas also diffusely stained for K14. α-SMA was observed in basal PECs at the leading tip of epithelial indentations extending into the stroma. Epithelium at the pterygial surface was α-SMA negative. (C) Normal corneal epithelium shows basal/suprabasal immunoreactivity for K14, but not for α-SMA. (D) Basal PECs in eye bank tissue also expressed vimentin, another marker of EMT (arrowheads). (E) Normal corneal epithelial cells do not express vimentin. Original magnification: (A) ×100 (B–E) ×400. Scale bars: 50 μm.
Figure 5.
 
Immunohistochemistry of E-cadherin and β-catenin pathway molecules. (A) E-cadherin in a surgically excised pterygium. Focal loss of membrane-associated E-cadherin was observed ( Image not available ). Arrowheads: membrane-associated staining for E-cadherin. (B) Immunohistochemistry for β-catenin in surgically excised pterygium. Similar to E-cadherin, both membrane-bound staining (arrowheads) and focal cytoplasmic staining ( Image not available ) were observed in the same field of view. (C, D) Nuclear translocation of β-catenin. Immunohistochemistry of intact pterygium in eye bank corneas revealed strong β-catenin staining in the nucleus of focal PECs (arrows) invading the underlying stroma (C). Nuclear transition of β-catenin was also observed in surgically excised pterygium (D). (E) LEF-1 was detected in the nuclei of PECs in areas similar to where nuclear β-catenin was positive (arrows). (F) Normal limbal control did not show nuclear staining of LEF-1 in epithelial cells. (G) RT-PCR for MMP-7 in PECs and LECs. MMP-7 was found in all four pterygium samples examined, while limbal epithelial samples were negative. LEC, limbal epithelial cells; PEC, pterygial epithelial cells. Original magnification: (A, B) ×200; (CF) ×400; scale bar: (A, B) 100 μm; (CF) 50 μm.
Figure 5.
 
Immunohistochemistry of E-cadherin and β-catenin pathway molecules. (A) E-cadherin in a surgically excised pterygium. Focal loss of membrane-associated E-cadherin was observed ( Image not available ). Arrowheads: membrane-associated staining for E-cadherin. (B) Immunohistochemistry for β-catenin in surgically excised pterygium. Similar to E-cadherin, both membrane-bound staining (arrowheads) and focal cytoplasmic staining ( Image not available ) were observed in the same field of view. (C, D) Nuclear translocation of β-catenin. Immunohistochemistry of intact pterygium in eye bank corneas revealed strong β-catenin staining in the nucleus of focal PECs (arrows) invading the underlying stroma (C). Nuclear transition of β-catenin was also observed in surgically excised pterygium (D). (E) LEF-1 was detected in the nuclei of PECs in areas similar to where nuclear β-catenin was positive (arrows). (F) Normal limbal control did not show nuclear staining of LEF-1 in epithelial cells. (G) RT-PCR for MMP-7 in PECs and LECs. MMP-7 was found in all four pterygium samples examined, while limbal epithelial samples were negative. LEC, limbal epithelial cells; PEC, pterygial epithelial cells. Original magnification: (A, B) ×200; (CF) ×400; scale bar: (A, B) 100 μm; (CF) 50 μm.
Figure 6.
 
Immunohistochemistry of E-cadherin repressors. (A) Composite micrograph of Snail immunohistochemistry. Snail was observed in PECs from the head to the body of the pterygium. Arrow: pterygial head. (B, C) Strong expression of Snail (B) and Slug (C) was observed in the nucleus of PECs. (D, E) Normal corneal (D) and limbal epithelium (E). Original magnification: (A) ×40; (BE) ×200; scale bar: (A) 200 μm; (BE) 50 μm.
Figure 6.
 
Immunohistochemistry of E-cadherin repressors. (A) Composite micrograph of Snail immunohistochemistry. Snail was observed in PECs from the head to the body of the pterygium. Arrow: pterygial head. (B, C) Strong expression of Snail (B) and Slug (C) was observed in the nucleus of PECs. (D, E) Normal corneal (D) and limbal epithelium (E). Original magnification: (A) ×40; (BE) ×200; scale bar: (A) 200 μm; (BE) 50 μm.
Figure 7.
 
Illustration showing the proposed role of β-catenin signaling and EMT in the pathogenesis of pterygium. Snail/Slug, possibly upregulated by inflammatory signals, represses the transcription of E-cadherin. Decrease in membrane-associated E-cadherin causes cytoplasmic elevation and intranuclear translocation of β-catenin, followed by the upregulation of MMP-7, which may be involved in the degradation of extracellular matrix in pterygium.
Figure 7.
 
Illustration showing the proposed role of β-catenin signaling and EMT in the pathogenesis of pterygium. Snail/Slug, possibly upregulated by inflammatory signals, represses the transcription of E-cadherin. Decrease in membrane-associated E-cadherin causes cytoplasmic elevation and intranuclear translocation of β-catenin, followed by the upregulation of MMP-7, which may be involved in the degradation of extracellular matrix in pterygium.
Table 1.
 
Sequences of Primers for RT-PCR
Table 1.
 
Sequences of Primers for RT-PCR
GAPDH Forward primer GAA GGT GAA GGT CGG AGT C
Reverse primer GAA GAT GGT GAT GGG ATT TC
MMP-7 Forward primer GTG GTC ACC TAC AGG ATC GT
Reverse primer ACC ATC CGT CCA GCG TTC AT
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