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Anatomy and Pathology/Oncology  |   February 2012
Involvement of SPARC and MMP-3 in the Pathogenesis of Human Pterygium
Author Affiliations & Notes
  • Li-Fong Seet
    From the Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore;
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore;
  • Louis Tong
    From the Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore;
    Singapore National Eye Center, Singapore;
    Duke-NUS Graduate Medical School, Singapore; and
  • Roseline Su
    From the Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore;
  • Tina T. Wong
    From the Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore;
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore;
    Singapore National Eye Center, Singapore;
    School of Materials Science and Engineering, Nanyang Technological University, Singapore.
  • *Each of the following is a corresponding author: Li-Fong Seet, Singapore Eye Research Institute, 11 Third Hospital Avenue, 06-00 SNEC Building, Singapore 168751; seet.li.fong@seri.com.sg. Tina T. Wong, Glaucoma Service, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751; ttl_wong@hotmail.com
Investigative Ophthalmology & Visual Science February 2012, Vol.53, 587-595. doi:10.1167/iovs.11-7941
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      Li-Fong Seet, Louis Tong, Roseline Su, Tina T. Wong; Involvement of SPARC and MMP-3 in the Pathogenesis of Human Pterygium. Invest. Ophthalmol. Vis. Sci. 2012;53(2):587-595. doi: 10.1167/iovs.11-7941.

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

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Abstract

Purpose.: To investigate the expression of SPARC and matrix metalloproteinases (MMPs) in normal conjunctiva and pterygium tissues.

Methods.: This study involved paired control or uninvolved conjunctiva and pterygium tissue from 21 patients. Quantitative real-time PCR was performed to assess SPARC and MMP mRNA expression, whereas Western blot analysis was performed to assess SPARC protein levels in normal conjunctiva and pterygium tissue. Tissue localization of SPARC, extracellular matrix proteins, and MMPs were determined by immunofluorescence analyses.

Results.: SPARC transcript and protein levels were upregulated in pterygium compared with normal conjunctiva. Immunofluorescence analyses showed localization of SPARC to the epithelial basement membrane and stroma of normal conjunctiva tissue. Increased SPARC in the pterygium stroma colocalized partially with elevated collagen I, fibronectin, α-SMA, and MMP-3. SPARC and MMP-3 also colocalized in the pterygium epithelium.

Conclusions.: SPARC was upregulated in pterygium and may collaborate with increased MMP-3 in some patients to account for many of the phenotypic properties characteristic of pterygium.

Pterygium is a disease characterized by fibrovascular proliferation on the ocular surface. It affects a significant proportion of people, especially those living in equatorial and sun-exposed areas. 1 Typically, the lesion has a wing-shaped morphology that gradually encroaches centripetally onto the cornea, causing irritation and affecting visual function by disturbing the tear film, inducing astigmatism, or occluding the visual axis. 2 Currently, the only known treatment of this condition is surgical resection, which can fail because of postsurgical recurrence. An understanding of the underlying molecular events in pterygium is critical to devise strategies for the nonsurgical treatment of pterygium or the prevention of disease recurrence after surgery. 3  
Aberrant extracellular matrix (ECM) remodeling seems to be a major feature of pterygium, as evidenced by a recent study 4 that ECM genes including fibronectin, collagen III, and versican were upregulated in pterygium. In particular, matrix metalloproteinases (MMPs), a family of structurally related zinc-dependent, ECM-degrading proteinases, are elevated in pterygia. 5 MMPs are collectively capable of degrading essentially all components of the ECM 6 and have been implicated in a variety of normal and pathologic cellular processes such as embryogenesis, angiogenesis, wound healing, and cancer. 7 Members of the MMP family can be classified into gelatinases (e.g., MMP-2, MMP-9), stromelysins (e.g., MMP-3), collagenases (e.g., MMP-1), and membrane type-MMPs (MT-MMPs), depending on their substrate specificity and structural properties. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-8, and MMP-14 have all been reported to be upregulated in pterygium. 8 Given that many studies have now shown a tight association between MMPs and tumor progression/invasion, 9 the invasive nature of the pterygial lesion is thought to involve altered MMP activities. 
SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin or BM-40, is a matrix protein with roles in many biological processes such as cell-cell and cell-matrix adhesion, cell motility, angiogenesis, wound remodeling, and fibrosis. 10 The role of SPARC in modifying the ECM is well known, particularly its effects on collagen production and assembly. 11,12 Furthermore, there is increasing evidence for the involvement of SPARC in regulating the tumor microenvironment and affecting tumor development, invasion, metastases, angiogenesis, and inflammation. 13 Incidentally, SPARC is also known to regulate MMP activity in normal fibroblasts and cancer cell lines. 14 16  
We have previously shown in a gene microarray analysis that SPARC expression was significantly upregulated in pterygium compared with uninvolved conjunctiva tissue. 17 To date, there have been no studies examining the relationship between SPARC and MMPs and the aberrant ECM phenotype of pterygium. We show in this report that SPARC was overexpressed in the pterygia of all patients examined at both the mRNA and the protein levels. We also demonstrate that SPARC, typically expressed in the basement membrane of the conjunctival epithelium, was elevated in the entire pterygium epithelia of some patients and in the conjunctiva stroma of all patients in association with aberrant ECM deposition. Notably, the increased expression of SPARC may be associated with the upregulation of MMP-3 expression in the pterygium, epithelium, and stroma. Hence, we postulate that elevated SPARC and MMP-3 may collaborate with and contribute to the pathogenesis of pterygium by modulating various aspects of the disease phenotype, including ECM deposition, angiogenesis, inflammation, and epithelial-mesenchymal transition. 
Materials and Methods
Patients and Specimens
The method of collection of patient specimens has been published previously. 18 The tissues used in the current report were collected under the Pterygium Etiology and Conjunctival Evaluation study, which is ongoing. The procurement and use of human tissues in this study was in compliance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of the Singapore Eye Research Institute, with informed written consent obtained from all participating patients. Paired tissues were used for comparison. For example, the pterygium tissue from a patient was compared with the uninvolved conjunctival tissue of the same patient that was harvested from the superior-temporal limbal conjunctiva of the conjunctival autograft as part of the surgical procedure of pterygium excision. Only primary pterygia were collected for this study, and patients were not known to have undergone any other intervention before pterygium excision. Ten pairs of tissues were analyzed by quantitative PCR, three pairs by immunoblotting, and eight pairs by immunofluorescence analyses. 
RNA Isolation and Real-Time PCR
Total RNA recovery, first-strand cDNA synthesis, and quantitative real-time PCR (qPCR) were performed as described previously. 12 Briefly, the qPCR program consisted of an initial denaturation step of 95°C for 10 minutes followed by 45 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds, and then a final extension step at 72°C for 7 minutes. Melt curves between 40°C and 95°C were examined to ascertain the specificity of the PCR products. All PCR reactions were performed in triplicate, with each measured three times. All mRNA levels were measured as CT threshold levels and were normalized with the corresponding β-actin CT values. Values were expressed as fold increases over the values for uninvolved conjunctiva of the same patient by the 2-ΔΔCT method. The primers used were SPARC forward (5′-TGTTTGCAGTGTGGTTCTG-3′), SPARC reverse (5′-GTGCAGAGGAAACCGAAGAG-3′), MMP-1 forward (5′-GCTAACCTTTGATGCTATAACTACGA-3′), MMP-1 reverse (5′-TTTGTGCGCATGTAGAATCTG-3′), MMP-2 forward (5′-ATAACCTGGATGCCGTCGT-3′), MMP-2 reverse (5′-AGGCACCCTTGAAGAAGTAGC-3′), MMP-3 forward (5′-GGAGTTCCTGATGTTGGTCAC-3′), MMP-3 reverse (5′- ATCTGGTGTATAATTCACAATCCTGTA-3′), MMP-9 forward (5′-CGGTGATTGACGACGCCTTT-3′), MMP-9 reverse (5′-ACCAAACTGGATGACGATGTCTG-3′), β-actin forward (5′-CCAACCGCGAGAAGATGA-3′), and β-actin reverse (5′-CCAGAGGCGTACAGGGATAG-3′). 
Immunoblot Analysis
Total tissue extracts were prepared by homogenization and lysis in a solution containing 20 mM Tris-buffer, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 2 mM MgCl2, 1 mM dithiothreitol, and 1× Complete Protease Inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) followed by SDS-polyacrylamide gel electrophoresis and immunoblotting, as previously described. 12 Antibodies against SPARC and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase (HRP)–conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Densitometric quantitation was performed using advanced imaging technology (Kodak Image Station 4000R; Carestream Molecular Imaging, New Haven, CT). Variations in loading were corrected to levels of β-actin, which was used as the housekeeping protein. 
Immunofluorescence Analysis
Pterygium or normal conjunctiva tissue was cryosectioned, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 before incubation with specific antibodies. For coimmunostaining with mouse collagen VII antibody (clone 4D2, sc-33710; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), SPARC staining was performed using the goat polyclonal antibody (AF941, 1:20) from R&D Systems (Minneapolis, MN). The other coimmunostaining experiments were performed with mouse SPARC antibody (sc-59703) from Santa Cruz Biotechnology or mouse CD31 antibody from Abcam (AB9498; Cambridge, UK) and rabbit antibodies against collagen I (NB600–408; Novus Biologicals, Littleton, CO), fibronectin (1574–1; Epitomics Inc., Burlingame, CA), α-SMA (ab5694; Abcam), MMP-2 (250752; Abbiotec, San Diego, CA), and MMP-3 (SA-104; Biomol International, Plymouth Meeting, PA). Control coimmunostaining with isotype antibodies was performed with normal goat IgG from Santa Cruz Biotechnology (sc-2028) paired with mouse IgG (015–000-003) from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA) or with mouse IgG paired with rabbit IgG (011–000-003, Jackson ImmunoResearch Laboratories Inc.), followed by the appropriate fluorescence-conjugated secondary antibodies. Labeling by the goat or mouse antibodies was detected using anti–goat or anti–mouse secondary antibodies conjugated to Alexa Fluor-488 (Invitrogen, Eugene, OR), respectively, with the exception that labeling by the collagen VII antibody was detected using anti–mouse secondary antibodies conjugated to Alexa Fluor-568. The rabbit primary antibodies were detected with secondary antibodies conjugated to Alexa Fluor-594 (Invitrogen). All primary antibodies were used at 1:100 dilution unless otherwise indicated, whereas secondary antibodies were used at 1:1000 dilution. Mounting medium containing DAPI (4,6-diamidino-2-phenylindole) was purchased from Vector Laboratories (Burlingame, CA). Labeled cells were visualized using an upright microscope (Axio Imager.Z1; Carl Zeiss Inc., Thornwood, NY). 
Statistical Analysis
Data are expressed as mean ± SD where appropriate. The significance of differences among groups was determined by the two-tailed Student's t-test (Excel 5.0; Microsoft, Redmond, WA), with significance at P ≤ 0.05. 
Results
Expression of SPARC in Pterygium
To determine whether SPARC is overexpressed in pterygium tissue compared with its paired uninvolved conjunctival counterpart, we analyzed both the mRNA and protein expression levels of SPARC in these tissues. qPCR showed that SPARC transcripts were elevated in pterygium tissue compared with the respective unaffected conjunctiva in all six patients (Fig. 1A). The significant increase in SPARC mRNA expression in pterygium over normal conjunctiva ranged from two-fold to five-fold (P = 0.006). To verify that the increase in SPARC expression extends to the protein level, we performed immunoblot assays on the paired pterygial and uninvolved conjunctival tissues from another three patients. As evident in Figure 1B, SPARC protein expression was also elevated in all three pterygia compared with their corresponding uninvolved conjunctival tissues, though densitometric analyses of the immunoblot data did not show an overall statistical increase (Fig. 1C; P = 0.09). Nonetheless, the data suggest an upward trend for SPARC protein expression in pterygium. 
Figure 1.
 
Expression of SPARC in pterygium relative to paired uninvolved conjunctiva. (A) mRNA expression levels of SPARC in the pterygia from six patients were determined by real-time PCR. Values shown indicate fold expression of SPARC relative to that in the respective paired uninvolved conjunctiva. The β-actin transcript was used for normalization in all calculations. (B) Protein expression of SPARC in the pterygia and normal conjunctiva from three patients was determined by immunoblotting with antibodies specific for SPARC (top) and β-actin (loading control, bottom). (C) Densitometric analysis of the immunoblot shown in B. Data for both conjunctiva and pterygium were normalized against the respective β-actin level and presented as fold increase in SPARC expression in pterygium relative to the respective paired uninvolved conjunctiva control.
Figure 1.
 
Expression of SPARC in pterygium relative to paired uninvolved conjunctiva. (A) mRNA expression levels of SPARC in the pterygia from six patients were determined by real-time PCR. Values shown indicate fold expression of SPARC relative to that in the respective paired uninvolved conjunctiva. The β-actin transcript was used for normalization in all calculations. (B) Protein expression of SPARC in the pterygia and normal conjunctiva from three patients was determined by immunoblotting with antibodies specific for SPARC (top) and β-actin (loading control, bottom). (C) Densitometric analysis of the immunoblot shown in B. Data for both conjunctiva and pterygium were normalized against the respective β-actin level and presented as fold increase in SPARC expression in pterygium relative to the respective paired uninvolved conjunctiva control.
SPARC Localization in Normal Conjunctiva
To determine the tissue localization of SPARC in normal conjunctiva and pterygium, we performed immunofluorescence analysis of the cryosections using antibody specific for SPARC. In the conjunctival epithelium, SPARC expression overlapped significantly with that of collagen VII, a marker for the epithelial basal lamina 19 (Fig. 2A). Hence, it appears that SPARC expression is intimately associated with the basement membrane of conjunctival epithelium. Immunofluorescence analysis of the paired pterygium tissue also revealed the presence of SPARC protein throughout the pterygium epithelium in addition to the intense basement membrane accumulation (Fig. 2B). 
Figure 2.
 
Localization of SPARC in normal conjunctival and pterygial epithelium. Immunofluorescence images showing distribution of SPARC (green) and collagen VII (red) in normal uninvolved conjunctiva (A) and pterygium tissue (B). Nuclei were visualized by staining with DAPI (blue). The basement membrane is delineated by the white arrowheads. Coimmunostaining with the respective normal IgGs was also performed for normal conjunctiva (C) and pterygium (D) to reveal levels of background fluorescence. Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 2.
 
Localization of SPARC in normal conjunctival and pterygial epithelium. Immunofluorescence images showing distribution of SPARC (green) and collagen VII (red) in normal uninvolved conjunctiva (A) and pterygium tissue (B). Nuclei were visualized by staining with DAPI (blue). The basement membrane is delineated by the white arrowheads. Coimmunostaining with the respective normal IgGs was also performed for normal conjunctiva (C) and pterygium (D) to reveal levels of background fluorescence. Merged images are shown in the overlay. Scale bar, 50 μM.
SPARC and ECM Protein Expression in Pterygium
As SPARC is known to regulate ECM production, particularly that of collagen, and pterygium is known to feature an altered ECM, we next investigated the relationship between SPARC and ECM protein expression. In normal conjunctiva, collagen I appeared to be present in greater intensity in the stroma than in the epithelium, and focal areas of higher collagen expression coincided with higher SPARC expression (Fig. 3A, arrowheads). Fibronectin expression was hardly detectable in normal conjunctiva (Fig. 3B). The α-SMA antibody appeared to specifically label structures that resembled vascular vessels in the stroma of the conjunctiva, which overlapped well with SPARC expression (Fig. 3C, arrowheads). α-SMA is a marker for perivascular mural cells or pericytes. 20 Indeed, when the conjunctiva tissue was also interrogated with antibodies specific for CD31, a pan marker for vascular endothelial cells, we observed that α-SMA was expressed in the mural cell layer adjacent to the CD31+ endothelial cell layer (Fig. 3D, arrowheads). Hence, the SPARC/α-SMA/CD31+ structures represent blood vessels. 
Figure 3.
 
Colocalization of SPARC and ECM proteins in normal conjunctiva. Normal conjunctiva tissue was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and α-SMA antibodies to reveal the subconjunctiva vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. CE, conjunctiva epithelium; S, stroma. Scale bar, 50 μM.
Figure 3.
 
Colocalization of SPARC and ECM proteins in normal conjunctiva. Normal conjunctiva tissue was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and α-SMA antibodies to reveal the subconjunctiva vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. CE, conjunctiva epithelium; S, stroma. Scale bar, 50 μM.
In comparison, pterygium tissue appeared to label for collagen I more intensely, which showed significant overlap with SPARC distribution (Fig. 4A, arrowheads). In contrast to the low fibronectin expression in normal conjunctiva, pterygium sections contained high reactivity to the fibronectin antibody, which partially colabeled with the SPARC antibody in areas that appeared to contain aggregated proteins (Fig. 4B, arrows). The pterygium section also seemed to display higher vascularity, as disclosed by the appearance of more vascular-like structures expressing both α-SMA and CD31 in the pterygium stroma (Figs. 4C, 4D, arrowheads). Notably, these structures, which were also positive for SPARC, appeared to accumulate close to the epithelium of the pterygium tissue. Moreover, we observed CD31/α-SMA+ cells in the pterygium stroma (Fig. 4D, arrow). We believe these represent fibrogenic myofibroblasts, which may be derived from differentiation of fibroblasts in the subconjunctiva. 
Figure 4.
 
Colocalization of SPARC and ECM proteins in pterygium. Pterygium tissue from the same patient as depicted in Figure 3 was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and a-SMA antibodies to reveal the pterygium vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. PE, pterygium epithelium; S, stroma. Scale bar, 50 μM.
Figure 4.
 
Colocalization of SPARC and ECM proteins in pterygium. Pterygium tissue from the same patient as depicted in Figure 3 was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and a-SMA antibodies to reveal the pterygium vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. PE, pterygium epithelium; S, stroma. Scale bar, 50 μM.
Colocalization of SPARC and MMP-3 in Pterygium
Immunofluorescence analysis indicated an upregulation in the expression of both SPARC and MMP-3 in pterygium (see Supplementary Material and Supplementary Fig. S1). Hence, we next investigated the potential colocalization of SPARC and MMP-3 in pterygium in greater detail. As observed before, SPARC was expressed at the basement membrane of the normal conjunctival epithelium in the patient sample shown in Figure 5A. Interestingly, MMP-3 expression was clearly distinguished from that of SPARC; it was detected in the epithelial basal and suprabasal cell layers but not in the basement membrane (Fig. 5A). In the corresponding pterygium section, which showed nonhyperplastic epithelium, we observed intense, elevated SPARC expression throughout the epithelium, stretching from the basement membrane to the superficial cell layer (Fig. 5B). Importantly, there was a coincidence in the expression of MMP-3 that was similarly elevated and could be detected throughout the epithelium (Fig. 5B). In another pair of patient samples in which there were signs of epithelial hyperplasia in the pterygium, the clear limitation of SPARC expression to the basement membrane abutting the basal cell layer containing MMP-3 expression was even more conspicuous in the normal conjunctiva (Fig. 5C, white arrowheads). Although there was some correlation in the uneven expression of both SPARC and MMP-3 in the superficial cell layer of the normal conjunctiva (Fig. 5C, yellow arrowheads), the corresponding pterygium section exhibited much more elevated and even distribution of SPARC and MMP-3 expressions throughout the superficial cell layer (Fig. 5D, yellow arrowheads). In the basal cell layer, SPARC distribution in the basement membrane was more diffuse and less distinctly segregated from that of MMP-3 (Fig. 5D, white arrowheads). 
Figure 5.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium epithelium. Sections of normal conjunctiva (A) and pterygium (B) from the same patient showing no epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). The basement membrane stained weakly for SPARC in the normal conjunctiva epithelium in this sample (A, delineated by white arrowheads). Goblet cells in this pterygium epithelium are indicated (B, arrows). Sections of normal conjunctiva (C) and pterygium (D) from another patient showing epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red), as before. SPARC and MMP-3 colocalized in the superficial cell layer (yellow arrowheads) of both normal conjunctiva (C) and pterygium epithelium (D). Immunoreactivity for SPARC in the basement membrane (white arrowheads) segregated clearly from MMP-3 immunopositivity in the basal cell layer in the normal conjunctiva (C). Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 5.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium epithelium. Sections of normal conjunctiva (A) and pterygium (B) from the same patient showing no epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). The basement membrane stained weakly for SPARC in the normal conjunctiva epithelium in this sample (A, delineated by white arrowheads). Goblet cells in this pterygium epithelium are indicated (B, arrows). Sections of normal conjunctiva (C) and pterygium (D) from another patient showing epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red), as before. SPARC and MMP-3 colocalized in the superficial cell layer (yellow arrowheads) of both normal conjunctiva (C) and pterygium epithelium (D). Immunoreactivity for SPARC in the basement membrane (white arrowheads) segregated clearly from MMP-3 immunopositivity in the basal cell layer in the normal conjunctiva (C). Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. Scale bar, 50 μM.
In the stroma of the normal conjunctiva, SPARC expression was detectable at low levels, as was MMP-3 (Fig. 6A). In the corresponding pterygium sections, SPARC expression was increased and appeared to label protein aggregates intensely (Fig. 6B, arrowheads). MMP-3 expression was also increased and partially colocalized with SPARC in the aggregates in the pterygium stroma (Fig. 6B, arrowheads). Hence, SPARC and MMP-3 expressions were both elevated and partially overlapped in the pterygium tissue. 
Figure 6.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium stroma. Sections of normal conjunctiva (A) and pterygium (B) from the same patient were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). No significant colocalization was observed in the stroma of normal conjunctiva (A). Increased SPARC expression in the pterygium stroma partially colocalized with MMP-3 (B, arrowheads). Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 6.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium stroma. Sections of normal conjunctiva (A) and pterygium (B) from the same patient were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). No significant colocalization was observed in the stroma of normal conjunctiva (A). Increased SPARC expression in the pterygium stroma partially colocalized with MMP-3 (B, arrowheads). Merged images are shown in the overlay. Scale bar, 50 μM.
Overexpression of SPARC and MMP-3 Transcripts in Pterygium
Finally, we investigated whether SPARC and MMP-3 mRNAs are both overexpressed in pterygium compared with the uninvolved conjunctival counterpart. Tissues were collected from four patients and were subjected to qPCR analysis. Our data indicated that the pterygia from all four patients demonstrated a significant increase in SPARC mRNA expression compared with their respective uninvolved conjunctival counterpart (Table 1; P = 0.05) but only 2 of these patients exhibited an increase in MMP-3 mRNA expression (Table 1; P = 0.38). There did not seem to be any correlation between MMP-1 or MMP-2 mRNA expression and either SPARC or MMP-3 expression, whereas MMP-9 and MMP-14 mRNA expression levels in both pterygium and normal conjunctiva were generally too low or undetectable to be measured accurately (data not shown). 
Table 1.
 
Correlations between SPARC and MMP-3 Overexpression in Pterygium Relative to the Corresponding Uninvolved Conjunctival Counterpart of Four Patients
Table 1.
 
Correlations between SPARC and MMP-3 Overexpression in Pterygium Relative to the Corresponding Uninvolved Conjunctival Counterpart of Four Patients
Case Fold Change in SPARC mRNA Expression Fold Change in MMP-3 mRNA Expression Fold Change in MMP-1 mRNA Expression Fold Change in MMP-2 mRNA Expression Fold Change in MMP-9 mRNA Expression
1 1.99 ± 0.20 535.49 ± 28.50 ND 1.00 ± 0.05 ND
2 3.38 ± 0.08 6.80 ± 0.37 0.61 ± 0.19 99.59 ± 15.60 ND
3 2.79 ± 0.23 1.08 ± 0.12 23.62 ± 2.50 1.00 ± 0.02 1.01 ± 0.21
4 1.39 ± 0.06 1.18 ± 0.14 3.94 ± 1.57 0.63 ± 0.04 ND
Discussion
We show in this study the unique localization of SPARC in the human conjunctiva and its upregulation in pterygium. These observations may shed light on some of the phenotypic features of this disease as we examine the potential collaboration between SPARC and MMP-3 in causing the major biological changes reported in pterygium. 
The similarity in the staining pattern of various markers, including SPARC, collagen I, collagen VII, MMP-2, and MMP-3, between normal conjunctival and pterygial epithelia and stroma suggest that pterygium has a conjunctival origin. The location of SPARC in the normal conjunctival epithelial basement membrane is particularly intriguing. Because SPARC is a well-established mediator of collagen production and deposition, 11,12 the suggestion is that this matricellular protein may have a role in the production and assembly of anchoring collagen fibrils in the conjunctiva epithelial basal lamina. This function may be critical because the structural stability and mechanical integrity of the conjunctiva is dependent on the network of anchoring fibril interactions between the overlying epithelial cells and the underlying stroma. The segregation of MMP-3 expression to the basal and suprabasal cell layers and not the basement membrane in the normal conjunctiva is also interesting. Previous reports suggested that the conjunctival epithelial basal cell layer contains proliferative capacity, 21 with the potential to regenerate a multilayered epithelium. 22 Moreover, epithelial basal cells of various tissues are thought to be important for mediating cell migration in response to injury. 23,24 Whether MMP-3 plays a role in these processes remains to be investigated; nevertheless, its location suggests a potential involvement for this protein in the execution of these properties. 
The development of pterygium is strongly associated with exposure to ultraviolet (UV) irradiation. 5,8 A study exposing hairless SPARC-null mice to chronic UV irradiation demonstrated that these mice developed fewer papillomas compared with wild-type controls, suggesting that SPARC plays a role in mediating tumor formation in response to UV irradiation. 25 Importantly, the authors reported an increase in SPARC expression in the underlying dermis of the irradiated wild-type mice whereas it was undetectable in the nonirradiated control. Therefore, it is possible that SPARC expression may be induced in the conjunctiva after chronic exposure to UV irradiation and thence contribute to the formation of pterygium. 
Elevated SPARC expression may account for many of the phenotypic manifestations commonly associated with pterygium. Increased SPARC in the pterygium stroma may be the cause of greater deposition of collagen, as uncovered in previous global gene expression analyses of pterygium. 4,17 In addition to its profound influence on collagen production, SPARC is known to regulate other ECM proteins, including fibronectin and laminin as well as ECM-modifying proteins such as plasminogen activator inhibitor-1 and MMPs. 26 Taken together, it may be surmised that the altered ECM characteristic of pterygium, containing what appeared to be aggregated proteinaceous deposits containing SPARC and fibronectin, could arise from elevated SPARC expression. 
Increased SPARC may also account for the increased angiogenesis commonly observed in pterygium. SPARC expression has been noted to predominate in newly formed or immature blood vessels. 27,28 Our finding here that pterygium exhibited more SPARC/α-SMA+ or CD31/α-SMA+ blood vessels close to the pterygium epithelium may suggest active angiogenesis in this locale. Several studies have certainly shown a prominent role for SPARC in angiogenesis, 29 which may in part contribute to the increased vascularity observed in pterygium. 8 Increased vascularity, in turn, may account for the inflammatory component described in pterygium. 5 Pertaining to this, it has been reported that SPARC can mediate actin fiber reorganization and the appearance of intercellular gaps that increase endothelial barrier permeability. 30 It is possible that the increased transmigration of leukocytes to the pterygium stroma through the expanded vasculature could potentially act synergistically to elevate the inflammatory phenotype typical of pterygium. 
MMP-3 is a highly attractive candidate for the pathogenesis and progression of pterygium. First, MMP-3 has broad-spectrum enzymatic activity with the ability to degrade collagen, proteoglycans, fibronectin, laminin, and elastin, 31 and its expression and activity were increased in pterygium fibroblasts, which dominate the stroma. 32 The increase in MMP-3 expression in the pterygium stroma may account for the elastotic degeneration of collagen often observed in the disease tissue. Second, MMP-3 can activate other MMPs, such as MMP-1, MMP-7, and MMP-9, indicating that MMP-3 is upstream of the activation of many latent MMPs and is therefore crucial in ECM remodeling. 31 We hypothesize that the increased expression of MMP-3 in the pterygium epithelium may enable the disease cells to invade and cause the dissolution of the corneal Bowman's layer, leading to invasion by the lesion on the corneal surface. 5 Third, and of particular relevance, MMP-3 has been shown to cleave SPARC into bioactive peptides with the capacity to induce vascular endothelial growth or migration. 33 We predict that where there is increased coexpression of MMP-3 and SPARC in the pterygium, the vascularity and inflammatory phenotype of pterygium may be enhanced. In other words, SPARC and MMP-3 may collaborate in an intimate synergistic fashion to result in many of the phenotypes associated with pterygium. 
In the present study, we observed enhanced SPARC and MMP-3 expressions in the pterygium stroma compared with the corresponding uninvolved normal conjunctiva in the patient samples analyzed. Our next step was to determine whether there are differential expression patterns between the pterygium head and body. Future work will involve measuring and staging of the pterygia in conjunction with molecular analysis of SPARC and MMP-3 expression to correlate expression levels with disease phenotype. The data shown here suggest that the increased expressions of SPARC and MMP-3 in pterygium may be involved in the pathogenesis of pterygium and are therefore potential therapeutic targets for preventing or delaying progression of the disease. 
Supplementary Materials
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Footnotes
 Supported by a Biomedical Research Council Grant (BMRC/03/1/35/19/231) and a Singapore Eye Research Institute Grant (R502/51/2006) (LT), a research grant from the National Medical Research Council (NMRC/EDG/0019/2008) (TTW), and a National Research Foundation Council Translational and Clinical Research (TCR) Programe Grant (NMRC/TCR/002-SERI/2008) (L-FS).
Footnotes
 Disclosure: L.-F. Seet, None; L. Tong, None; R. Su, None; T.T. Wong, None
The authors thank Sharon Finger and Stephanie Chu for assisting in the revision of this study. 
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Figure 1.
 
Expression of SPARC in pterygium relative to paired uninvolved conjunctiva. (A) mRNA expression levels of SPARC in the pterygia from six patients were determined by real-time PCR. Values shown indicate fold expression of SPARC relative to that in the respective paired uninvolved conjunctiva. The β-actin transcript was used for normalization in all calculations. (B) Protein expression of SPARC in the pterygia and normal conjunctiva from three patients was determined by immunoblotting with antibodies specific for SPARC (top) and β-actin (loading control, bottom). (C) Densitometric analysis of the immunoblot shown in B. Data for both conjunctiva and pterygium were normalized against the respective β-actin level and presented as fold increase in SPARC expression in pterygium relative to the respective paired uninvolved conjunctiva control.
Figure 1.
 
Expression of SPARC in pterygium relative to paired uninvolved conjunctiva. (A) mRNA expression levels of SPARC in the pterygia from six patients were determined by real-time PCR. Values shown indicate fold expression of SPARC relative to that in the respective paired uninvolved conjunctiva. The β-actin transcript was used for normalization in all calculations. (B) Protein expression of SPARC in the pterygia and normal conjunctiva from three patients was determined by immunoblotting with antibodies specific for SPARC (top) and β-actin (loading control, bottom). (C) Densitometric analysis of the immunoblot shown in B. Data for both conjunctiva and pterygium were normalized against the respective β-actin level and presented as fold increase in SPARC expression in pterygium relative to the respective paired uninvolved conjunctiva control.
Figure 2.
 
Localization of SPARC in normal conjunctival and pterygial epithelium. Immunofluorescence images showing distribution of SPARC (green) and collagen VII (red) in normal uninvolved conjunctiva (A) and pterygium tissue (B). Nuclei were visualized by staining with DAPI (blue). The basement membrane is delineated by the white arrowheads. Coimmunostaining with the respective normal IgGs was also performed for normal conjunctiva (C) and pterygium (D) to reveal levels of background fluorescence. Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 2.
 
Localization of SPARC in normal conjunctival and pterygial epithelium. Immunofluorescence images showing distribution of SPARC (green) and collagen VII (red) in normal uninvolved conjunctiva (A) and pterygium tissue (B). Nuclei were visualized by staining with DAPI (blue). The basement membrane is delineated by the white arrowheads. Coimmunostaining with the respective normal IgGs was also performed for normal conjunctiva (C) and pterygium (D) to reveal levels of background fluorescence. Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 3.
 
Colocalization of SPARC and ECM proteins in normal conjunctiva. Normal conjunctiva tissue was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and α-SMA antibodies to reveal the subconjunctiva vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. CE, conjunctiva epithelium; S, stroma. Scale bar, 50 μM.
Figure 3.
 
Colocalization of SPARC and ECM proteins in normal conjunctiva. Normal conjunctiva tissue was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and α-SMA antibodies to reveal the subconjunctiva vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. CE, conjunctiva epithelium; S, stroma. Scale bar, 50 μM.
Figure 4.
 
Colocalization of SPARC and ECM proteins in pterygium. Pterygium tissue from the same patient as depicted in Figure 3 was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and a-SMA antibodies to reveal the pterygium vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. PE, pterygium epithelium; S, stroma. Scale bar, 50 μM.
Figure 4.
 
Colocalization of SPARC and ECM proteins in pterygium. Pterygium tissue from the same patient as depicted in Figure 3 was coimmunostained with antibodies for SPARC (green) and collagen (A), fibronectin (B), or α-SMA (C). (D) The tissue was coimmunostained with CD31 and a-SMA antibodies to reveal the pterygium vasculature. (C, D) Insets are enlarged images of the selected dotted boxes. (E) Coimmunostaining with the respective normal IgGs was also performed to demonstrate levels of background fluorescence. Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. PE, pterygium epithelium; S, stroma. Scale bar, 50 μM.
Figure 5.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium epithelium. Sections of normal conjunctiva (A) and pterygium (B) from the same patient showing no epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). The basement membrane stained weakly for SPARC in the normal conjunctiva epithelium in this sample (A, delineated by white arrowheads). Goblet cells in this pterygium epithelium are indicated (B, arrows). Sections of normal conjunctiva (C) and pterygium (D) from another patient showing epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red), as before. SPARC and MMP-3 colocalized in the superficial cell layer (yellow arrowheads) of both normal conjunctiva (C) and pterygium epithelium (D). Immunoreactivity for SPARC in the basement membrane (white arrowheads) segregated clearly from MMP-3 immunopositivity in the basal cell layer in the normal conjunctiva (C). Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 5.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium epithelium. Sections of normal conjunctiva (A) and pterygium (B) from the same patient showing no epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). The basement membrane stained weakly for SPARC in the normal conjunctiva epithelium in this sample (A, delineated by white arrowheads). Goblet cells in this pterygium epithelium are indicated (B, arrows). Sections of normal conjunctiva (C) and pterygium (D) from another patient showing epithelial hyperplasia were coimmunostained with antibodies for SPARC (green) and MMP-3 (red), as before. SPARC and MMP-3 colocalized in the superficial cell layer (yellow arrowheads) of both normal conjunctiva (C) and pterygium epithelium (D). Immunoreactivity for SPARC in the basement membrane (white arrowheads) segregated clearly from MMP-3 immunopositivity in the basal cell layer in the normal conjunctiva (C). Nuclei were visualized by staining with DAPI (blue). Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 6.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium stroma. Sections of normal conjunctiva (A) and pterygium (B) from the same patient were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). No significant colocalization was observed in the stroma of normal conjunctiva (A). Increased SPARC expression in the pterygium stroma partially colocalized with MMP-3 (B, arrowheads). Merged images are shown in the overlay. Scale bar, 50 μM.
Figure 6.
 
Colocalization of SPARC and MMP-3 in the normal conjunctiva and pterygium stroma. Sections of normal conjunctiva (A) and pterygium (B) from the same patient were coimmunostained with antibodies for SPARC (green) and MMP-3 (red). No significant colocalization was observed in the stroma of normal conjunctiva (A). Increased SPARC expression in the pterygium stroma partially colocalized with MMP-3 (B, arrowheads). Merged images are shown in the overlay. Scale bar, 50 μM.
Table 1.
 
Correlations between SPARC and MMP-3 Overexpression in Pterygium Relative to the Corresponding Uninvolved Conjunctival Counterpart of Four Patients
Table 1.
 
Correlations between SPARC and MMP-3 Overexpression in Pterygium Relative to the Corresponding Uninvolved Conjunctival Counterpart of Four Patients
Case Fold Change in SPARC mRNA Expression Fold Change in MMP-3 mRNA Expression Fold Change in MMP-1 mRNA Expression Fold Change in MMP-2 mRNA Expression Fold Change in MMP-9 mRNA Expression
1 1.99 ± 0.20 535.49 ± 28.50 ND 1.00 ± 0.05 ND
2 3.38 ± 0.08 6.80 ± 0.37 0.61 ± 0.19 99.59 ± 15.60 ND
3 2.79 ± 0.23 1.08 ± 0.12 23.62 ± 2.50 1.00 ± 0.02 1.01 ± 0.21
4 1.39 ± 0.06 1.18 ± 0.14 3.94 ± 1.57 0.63 ± 0.04 ND
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