Effect of TIMP-1 and MMP in Pterygium Invasion

Abstract

Purpose.: The migration and invasion of tumor cells correlate with the interaction between MMP and TIMP. Therefore, the purpose of this study was to determine the role of MMP-9, MMP-10, and TIMPs in pterygium formation and progression.

Methods.: MMP-9, MMP-10, and TIMP proteins were studied using immunohistochemistry on 82 pterygial specimens and 30 normal conjunctivas. Pterygium epithelial cells (PECs), cultured in a serum-free culture medium, and siRNA were used to knock down TIMP gene expression to understand the role of TIMP in pterygium invasion.

Results.: Among the 82 pterygial samples, 29 specimens (35.4%) were positive for MMP-9 expression, 28 were positive for MMP-10 (34.1%), and 59 were positive for TIMP1 (72.0%). Staining for MMPs was limited to the cytoplasm of the epithelial layer. The TIMP staining was detected in the pterygium epithelium, fibroblasts and corneal epithelium. In the cell model, cell invasion and migration ability increased in TIMP knockdown PECs compared with the parental control.

Conclusions.: MMP-9 and MMP-10 may each play a role in pterygium formation, and TIMPs may contribute to pterygium invasion inhibition.

Pterygium is an uncontrolled cellular proliferation and a highly vascularized growth of the conjunctiva, thought to arise from activated and proliferating limbal epithelial stem cells. ¹  

Extracellular matrix remodeling is a prominent feature in pterygium, and this includes elastosis and the breakdown of Bowman's layer. ¹ Some of these changes are attributed to the actions of matrix metalloproteinases (MMPs), which contribute to the local invasive nature of this disease. ²  

MMPs are a family of enzymes that act to modify or degrade the extracellular matrix components in normal and remodeled tissues (e.g., collagens, laminin, fibronectin, and other glycoproteins), and they are found in many conditions such as arthritis, coronary artery disease, tumor invasion, metastasis, and angiogenesis. ^3–6 MMPs are ubiquitous proteolytic enzymes important in physiologic and pathologic processes. These enzymes are secreted by a variety of cell types, including fibroblasts, and they are categorized into five groups: collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -21, -22), membrane type MMPs, and others. ⁷ Elevations of MMP-1, MMP-2, MMP-3, MMP-7 (matrilysin), MMP-8, and MMP-9 in pterygia have been reported (Liu YP, et al. IOVS 1998;39:ARVO Abstract 3485). ^8–13 In cutaneous tumorigenesis, it has been proposed that the mechanism involving MMPs is induced by UV irradiation, ¹⁴ especially MMP-10 (stromelysin-2) and MMP-1 (interstitial collagenase). ¹⁴ MMPs are among the proteins whose gene expression is regulated by UVA and UVB irradiation, and our previous study identified that UV light induced ROS and oxidative stress in pterygium ¹⁵ ; hence, if pterygium is a UV-related, uncontrolled, cell proliferation-like cutaneous tumorigenesis, it is logical to assume that MMP-10 may be found in pterygium. In addition, MMP-9 is regarded as important in the degradation of the basement membrane and the extracellular matrix during cancer invasion and other tissue remodeling events, ¹⁶ which is compatible with the character of pterygium. 

MMPs are regulated by their specific inhibitors, called tissue inhibitors of MMPs (TIMPs). ¹⁷ The balance between the levels of activated enzymes and free TIMPs determines the overall MMP activity. Maintenance of this equilibrium is essential, and any disturbance in the balance is a critical determinant of proteolysis and tissue invasion. ¹⁷  

We hypothesize that MMP and TIMP molecules may be active participants in the extensive matrix turnover and infiltration that characterize pterygia. The goal of this study was to determine the expression of MMP-9, MMP-10, and TIMPs in pterygium and to understand the potential mechanism involved in pterygium invasion. 

Patients and Methods

Pterygial samples were harvested from 82 patients who underwent pterygium surgery at China Medical University Hospital and other institutions. All specimens were taken from the head, which meant the pterygium invaded the cornea. Normal conjunctival samples to be used as controls were collected from the conjunctiva of 30 patients without pterygium and pinguecula who underwent cataract surgery. This study was carried out with the approval of the Human Study Committee at China Medical University Hospital and adhered to the tenets of the Declaration of Helsinki. 

Immunohistochemistry

All specimens were fixed in 4% neutral buffered formalin and embedded in paraffin. Three-micrometer sections were cut, mounted on glass, and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated with alcohol, and washed in phosphate-buffered saline. This buffer was used for all subsequent washes. Immunohistochemical study was conducted using the streptavidin-biotin-peroxidase method, which was performed on paraffin-embedded tissues with anti–MMP-9, anti–MMP-10, and anti–TIMP-1 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The cutoff value for immunohistochemical analysis was set at 10%, which meant that more than 10% of the cell stained positive in the tissues. The first antibody replaced by IgG was used us a negative control, and a pulmonary carcinoma with strong immunoreactivity to MMP-9, MMP-10, and TIMP-1 was used as a positive control. 

Establishment of Pterygium Cell Lines from Patients

Fresh pterygial specimens were cut into small pieces (1–2 mm in diameter) under a stereomicroscope, washed in Dulbecco's modified Eagle's medium (DMEM) solution, and placed in a culture dish. Serum-free DMEM was added to cover the explants. The culture dish was put in a CO₂-regulated incubator with a 5% CO₂ atmosphere overnight. Culture medium was replaced three times a week after the appearance of an outgrowth of cells from the explants. Cell type was further confirmed using p63 and pan cytokeratin antibodies. 

TIMP-1 Protein Expression Checked by Western Blot Analysis

Total proteins were extracted from PECs with a lysis buffer (100 mM Tris, pH 8.0, 1% SDS), and recovered protein concentrations were determined using the Bio-Rad (Hercules, CA) protein assay kit followed by separation with SDS-PAGE (12.5% gel, 1.5-mm thick). After electrophoretic transfer to a polyvinylidene difluoride membrane, nonspecific binding sites were blocked with 5% nonfat milk in TBS-Tween 20. The detection of HPV 16 or 18 E6 and β-actin were conducted by incubating the membrane with anti–TIMP-1 (Santa Cruz Biotechnology; Chemicon International, Inc., Temecula, CA) and β-actin antibodies (Sigma, St. Louis, MO) for 60 minutes at room temperature, followed by subsequent incubation with a peroxidase-conjugated secondary antibody (1:5000 dilution). Extensive washings with TBS-Tween 20 were performed between incubations to remove nonspecific binding. The protein bands were visualized using enhanced chemiluminescence (NEN Life Science Products Inc., Boston, MA). 

Silencing of Endogenous TIMP Expression by RNAi

To suppress the transcription of the endogenous TIMP gene, PECs were transiently transfected with synthetic siRNAs (Santa Cruz Biotechnology) against TIMP using reagent (Oligofectamine; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, 24 hours before transfection, 1.4 × 10⁵ cells were seeded in each well of a six-well plate. Reagent (Oligofectamine, 3 mL; Invitrogen) was added to 12 mL medium (Opti-Mem; Invitrogen). After 5 minutes, 60 pmol each siRNA in 175 mL medium (Opti-Mem; Invitrogen) was combined with the reagent (Oligofectamine; Invitrogen) mixture. After 20 minutes of incubation at 25°C, siRNA/reagent (Oligofectamine; Invitrogen) mixtures were added to the cells. After 48 hours of incubation at 37°C, the cells were harvested and subjected to Western blot analysis, cell migration, and invasion assay. 

Effects of TIMP RNAi on the Migration and Invasion Efficiency of PECs

For transwell migration assays, 5 × 10⁴ cells were plated into the top chamber with the noncoated membrane (24-well insert; pore size, 8 mm; BD Biosciences, Bedford, MA). For transwell invasion assays, each well insert was layered with 80 mL of a 1:5 mixture of basement membrane matrix (Matrigel; BD Biosciences)/DMEM. A total of 10 × 10⁴ cells were added to the top of the Matrigel layer. In both assays, cells were plated in medium without serum; medium containing 10% serum was used as a chemoattractant in the lower chamber. The cells were incubated for 48 hours, and those that did not migrate or invade through the pores were removed with a cotton swab. Cells on the lower surface of the membrane were stained with Giemsa and counted. 

Statistical Analysis

Statistical analysis was performed with a statistical software program (SPSS Inc., Chicago, IL). The χ² test was applied for statistical analysis. P < 0.05 was considered statistically significant. 

Results

There were 48 men and 34 women in the pterygium group (age range, 50–78 years; mean age, 62.3 years) and 20 men and 10 women in the control group (age range, 55–75 years; mean age, 66.8 years). 

MMP-9 and MMP-10 Expression in Pterygium

To understand the association between MMP-9 and MMP-10 protein expression and pterygium, immunohistochemistry was used to analyze MMP-9 and MMP-10 protein expression in 82 pterygial and 30 conjunctival samples. In the pterygium group, 29 samples (35.4%; 29 of 82) were positive for MMP-9, and 28 (34.2%) were positive for MMP-10. MMP-9 and MMP-10 staining was distributed in the epithelium and stroma of the epithelial cells (Table 1; Figs. 1A, 1C). In the normal conjunctiva group, all specimens were negative for MMP-9 and MMP-10 immunostaining. The frequency of MMP-9 and MMP-10 expression was higher in the pterygium group (35.4% vs. 0% for MMP-9, P < 0.0001; 34.2% vs. 0% for MMP-10, P < 0.0001) than in the control group. 

Table 1.

 

Differences in MMP-9, MMP-10, and TIMP-1 Protein Expression between Pterygium Tissues and Conjunctiva Controls

Protein	Patients (n = 82)	Control (n = 30)	P
MMP-9
Negative	53	30
Positive	29	0	<0.0001
MMP-10
Negative	54	30
Positive	28	0	<0.0001
TIMP
Negative	23	27
Positive	59	3	<0.0001

Figure 1.

 

Representative positive and negative immunostaining for MMP-9, MMP-10, and TIMP-1 proteins in paraffin sections of pterygium. (A, C, E) Representative positive MMP-9, MMP-10, and TIMP-1 immunostaining (200×). (B, D, F) Representative negative MMP-9, MMP-10, and TIMP-1 immunostaining (400×).

TIMP Expression in Pterygium

To understand the association between TIMP protein expression and pterygium, TIMP protein expression in pterygial and conjunctiva samples was also analyzed. In the pterygium group, 59 (72%) patients had positive staining for TIMP, and staining was distributed in the epithelium and stroma of the epithelial cells and fibroblasts (Table 1; Fig. 1E). In the normal conjunctiva group, only 3 (10%) patients had positive staining. There was a higher frequency of TIMP expression in the pterygium group than in the control group (72.0% vs. 10%; P < 0.0001). 

MMP-9 and MMP-10 Protein Expression Correlate with Pterygium Type

The expression of MMP-9 and MMP-10 protein in the intermediate type of pterygium was significantly higher than in the atrophic and fleshy types of pterygium (P = 0.001 for MMP-9; P = 0.002 for MMP-10). No significant correlation was observed between pterygium type and TIMP-1 protein. In addition, there was no association between MMP-9, MMP-10, and TIMP-1 expression and other clinical parameters, including age and sex (Table 2). 

Table 2.

 

Correlation of MMP-9, MMP-10 and TIMP-1 Protein Expression and Clinical Parameters of Pterygium

Parameters	MMP-9		MMP-10		TIMP-1
	Negative (n = 53)	Positive (n = 29)	Negative (n = 54)	Positive (n = 28)	Negative (n = 23)	Positive (n = 59)
Age, (y)
Mean ± SD	65.2 ± 9.7	61.9 ± 9.9	65.2 ± 10.5	61.7 ± 8.2	65.3 ± 9.9	63.5 ± 9.8
P	0.103		0.158		0.482
Sex
Female	27	11	25	13	10	28
Male	26	18	29	15	13	31
P	0.355		1.000		0.890
Type
Atrophic	25	2	25	2	7	20
Intermediate	12	20	12	20	9	23
Fleshy	10	4	11	3	2	12
P	<0.0001		<0.0001		0.567
Missing data	6	3	6	3	5	4

Correlation of MMP-9, MMP-10, and TIMP Expression in Pterygium

The associations between MMP-9, MMP-10, and TIMP protein expression in the pterygium tissues are shown in Table 3. In the 29 MMP-9–positive tissues, the frequency of MMP-10–positive expression was higher than was negative expression (19 of 29 [65.5%] vs. 10 of 29 [34.5%]; Table 3). A significant association was seen between MMP-10 expression and MMP-9–positive staining (Table 3; P < 0.0001). All the MMP-9– and MMP-10–positive patients were also positive for TIMP-1 (MMP-9 vs. TIMP-1, P < 0.0001; MMP-10 vs. TIMP-1, P < 0.0001; Table 4). In the 53 MMP-9– and 54 MMP-10–negative pterygium groups, 30 (56.7%) and 31 (57.4%), respectively, were positive for TIMP-1 staining. 

Table 3.

 

Relationship between MMP-9 and MMP-10 Expression in Pterygium

	MMP-9 Protein		P
	Negative (n = 53)	Positive (n = 29)
MMP-10 Protein
Negative (n = 54)	44	10
Positive (n = 28)	9	19	<0.0001

Table 4.

 

Relationships among MMP-9, MMP-10, and TIMP-1 Protein Expression in Pterygium

Protein	TIMP-1		P
	Negative (n = 23)	Positive (n = 59)
MMP-9
Negative (n = 53)	23	30
Positive (n = 29)	0	29	<0.0001
MMP-10
Negative (n = 54)	23	31
Positive (n = 28)	0	28

Correlation of TIMP Protein Expression and the Ability of Migration and Invasion of PECs

To verify whether a minor invasion of pterygium correlated with high TIMP expression, we established primary cultured epithelial cell lines (PECs) from pterygium patients. Cell type was confirmed by p63 and by pan-cytokeratin staining to demonstrate that they were epithelial cells. TIMP protein expression of these cell lines was evaluated by Western blot analysis. Results showed that 5 of 7 PECs had higher TIMP-1 protein expression and that 2 of 7 had low or no TIMP protein expression (Fig. 2A). All PECs with higher TIMP expression were used to knock down TIMP expression by TIMP-specific siRNA (Fig. 2B). Cell migration and invasion were compared in low TIMP-1 expression, high TIMP-1 expression and TIMP-knockdown cells. There was an increase in the ability of migration (Fig. 2C) and invasion (Fig. 2D) ranging from 20% to 70% and 42% to 78% in TIMP-1 knockdown PECs compared with the parental and negative controls. Additionally, the migration and invasion ability in PECs with low TIMP expression was an intermediary between PECs with higher TIMP expression and si-TIMP. Therefore, we suggest that minor invasion of pterygium correlates with high TIMP expression. 

Figure 2.

 

Effect of TIMP-1 in cell migration and invasion. (A) TIMP-1 protein expression in PECs analyzed by Western blot. (B) TIMP-1 protein expression by si-TIMP-1 knockdown. (C) Migration and (D) invasion ability of PECs increased after transfection of si-TIMP-1. PC, parental control; NC, si-negative control.

Discussion

Our present results show that the invasion and migration ability of PECs increased after the knockdown of TIMP-1 expression. To the best of our knowledge, this is the first study to demonstrate that the overexpression of TIMP-1 protein correlates with a minor invasion and migration of pterygium. In addition, emerging evidence indicates that a shift in the MMPs/TIMPs balance toward increased proteolytic activity and extracellular matrix degradation occurs within the first few days after myocardial infarction. ¹⁸ TIMP is the prototype inhibitor for most MMP family members, which also, through Ras activation, binds to a cell surface protein complex consisting of CD63 and β-1 integrin, causing cell proliferation and inhibition of apoptosis. ¹⁹ Several studies have indicated that MMP-9 and TIMP1 can be detected in pterygium tissues and correlated with disease progression. ^2,20 Our present study also shows that 72% of the pterygium patients had TIMP-1–positive expression. These results indicate that the expressions of MMP and TIMP are involved in pterygium progression. 

Several types of MMPs have been detected in pterygium, including MMP-1, MMP-2, MMP-3, MMP-7 (matrilysin), MMP-8, and MMP-9. ^2,20,21 This is the first report to evaluate the correlation between MMP-10 and pterygium. With regard to human epidermal wound healing, MMP-9 (gelatinaseB) and MMP-10 (stromelysin-2) have been shown to be expressed by migrating keratinocytes in normally healing skin wounds. ^22,23 Because the histologic feature of pterygium overgrowth is excessive fibrovascular proliferation, basement membrane (Bowman's membrane) degradation occurs, and the superficial corneal stroma is invaded by the fibrovascular tissue. The degradation of the basement membrane or the extracellular matrix and cellular proliferation might be related to MMP-9 and MMP-10 expression. 

MMPs have been localized at the cell surface and at extracellular matrix components. In our series, MMP-9 expression occurred in 35.4% of the pterygial specimens though no MMP-9 expression occurred in the normal conjunctiva and limbal specimens, which is compatible with previous reports. ^9,11 In addition, strong MMP-10 expression in the pterygial specimens, but not in the normal conjunctiva and limbus, indicates active involvement of MMP-10 in the pathogenesis of pterygia. Moreover, the function of MMPs in cutaneous tumor formation—such as photocarcinogenesis and cleavage of types I and IV collagen—have also been reported in pterygium ¹⁴ and induce basic fibroblast growth factor and vascular endothelial growth factor gene expression. Kamat et al. ²⁴ discovered positive MMP immunostaining of the cytoplasm and membranes in 97% of ovarian cancer patients. In our study, a similar staining pattern was also found: MMP-9 and MMP-10 had positive immunoreaction in the cytoplasm and membranes of pterygial tissues but not in the normal conjunctiva and limbus. Hence, we suggest that MMPs may play a role in pterygium formation similar to that found in cutaneous tumorigenesis. 

Although many MMPs have been identified, it is still unknown which, if any, is involved in pterygium pathogenesis. MMP-9 was the focus of this study because collagen type IV, the major component of the basal membrane of corneal epithelial cells, is degraded by MMP-9. ¹¹ High levels of MMP-9 may be responsible for the dissolution of basement membrane components such as hemidesmosomes, which may lead to the migration and invasion of tumor cells. ¹⁰ MMP-10 is another important group of enzymes because of its ability to denature basement membrane components such as type IV collagen, fibronectin, vitronectin, and proteoglycans, thereby activating MMP-1 and other MMPs by proteolytic cleavage of their prodomains. Even though it has been recently shown that MMP-10 is involved in the invasion and metastasis of head and neck carcinomas and of esophageal squamous cell carcinomas, ^25,26 knowledge of alterations in the induction of MMP-10 in pterygium is limited. In this study, we discovered positive MMP-10 immunostaining in the cytoplasm and membranes of pterygial cells, which gives us direction for further investigation of the association between MMP-10 and pterygial pathogenesis. 

In this study, we used pterygium tissues and primary PECs to demonstrate that MMP-9 and MMP-10 may play a role in pterygium formation and that TIMPs may contribute to the inhibition of pterygium invasion. The role of TIMP in cell proliferation and inhibition of apoptosis in pterygium warrants further study. These results provide an alternative for pterygium therapy. 

Footnotes

 Supported by grants from the National Science Council (NSC 96–2314-B-039–009-MY2) of Taiwan, Republic of China.

Footnotes

 Disclosure: Y.-Y. Tsai, None; C.-C. Chiang, None; K.-T. Yeh, None; H. Lee, None; Y.-W. Cheng, None