Overexpression of Matrix Metalloproteinase-1 (MMP-1) and MMP-3 in Superior Limbic Keratoconjunctivitis

Abstract

Purpose.: To explore the presentations of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in superior limbic keratoconjunctivitis (SLK) with typical redundant superior conjunctiva.

Methods.: Eight surgical specimens from medically refractory SLK patients were examined. Another nine conjunctival specimens from patients who underwent cataract and retinal surgery served as controls. Expression of RNA and proteins of MMPs and TIMPs in conjunctival specimens and cultured conjunctival fibroblasts were determined by reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC).

Results.: The expression of mRNA of MMP-1 and -3 was detected in six and seven SLK patients, respectively, but was not detected in any case in the control group. IHC showed more prominent immunostaining of MMP-1 and -3 in the subepithelial stroma of the SLK patients than in the controls. After culturing, conjunctival fibroblasts of the SLK patients also shown apparent overexpression of MMP-1 and -3 compared with that in the controls. MMP-9 was not detected in both groups. MMP-2 and TIMP-1 and -2 were detected in some cases in both groups without a statistically significant difference between groups.

Conclusions.: Overexpression of MMP-1 and -3 was found in surgical specimens and cultured conjunctival fibroblasts from SLK patients. MMP imbalance may contribute to SLK pathogenesis. (ClinicalTrials.gov number, NCT00167050.)

Superior limbic keratoconjunctivitis (SLK), first described clinically by Frederick Theodore in 1963, ¹ is characterized by unilateral or bilateral redundant superior bulbar conjunctiva and inflammation of the superior tarsal and bulbar conjunctiva with dilatation of the superior bulbar conjunctival blood vessels, papillary reaction, punctate staining, and filaments of the superior cornea. ² Although the underlying cause remains unknown, redundant conjunctiva may contribute to disease progression, since SLK resolves after superior bulbar conjunctival resection. ^{3 –6}  

Redundant conjunctiva is a common characteristic of SLK and conjunctivochalasis, an aging process associated with redundant inferior bulbar conjunctiva. The exact etiology of conjunctivochalasis is also unknown. Overexpression of matrix metalloproteinase (MMP)-1 and MMP-3, demonstrated in conjunctival fibroblasts of conjunctivochalasis patients, may attribute to conjunctival redundancy. ⁷ MMPs are a family of zinc-dependent enzymes that modify or degrade the extracellular matrix. They are produced by a variety of cells, including fibroblasts, keratinocytes, chondrocytes, leukocytes, and malignant cells. ⁸ They can be divided into four classes on the basis of their preferred substrate: collagenases, gelatinases, stromelysins, and membrane type MMPs. ⁹ MMP-1 (collagenase), MMP-2 (gelatinase), and MMP-3 (stromelysin) are all found in normal conjunctival tissue. ¹⁰ The level of MMPs in normal tissues is very low, and their activity is highly regulated by several pathways, including transcriptional regulation, proenzyme regulation, and tissue inhibitors of metalloproteinases (TIMPs). TIMP function is controlled by its two binding sites; one is responsible for MMP inhibition, the other binds to pro-MMPs. Previous reports indicate that each TIMP can inhibit most MMPs. ^11,12 Only by highly regulating MMP activity can normal tissue avoid uncontrolled and excessive tissue destruction. The purpose of this study was to determine for the first time the presence of MMPs and TIMPs in SLK patients. 

Methods

Specimen Collection

Eight specimens were collected from eight patients visiting the Department of Ophthalmology at National Taiwan University Hospital and Buddhist Tzu-Chi General Hospital, Taipei Branch, from 2006 to 2008 with suspected symptoms of SLK. They were also examined by impression cytology for a definite diagnosis. The patients with advanced squamous metaplasia (grades 3–5, according to Tseng ¹³ ) who were unresponsive to medical treatment underwent superior bulbar conjunctival resection. All surgeries were performed by two surgeons (FRH, YCS) in patients under topical anesthesia. The area of involved superior bulbar conjunctiva (hyperproliferating and keratinized epithelium with dilated episcleral vessel) was marked with light cauterization, and the marked conjunctiva and underlying Tenon's tissue were excised. In another nine patients, the redundant conjunctiva noted after peritomy during cataract or retinal surgery was excised to serve as a normal control. Each of the surgical specimens in both groups was separated into three pieces: one for paraffin-embedded sections, another for primary conjunctival fibroblast culture, and the other for storage in liquid nitrogen. The research adhered to the tenets of the Declaration of Helsinki, and written informed consent was obtained from all subjects. This study was approved by the Institutional Review Board of the National Taiwan University Hospital (NTUH-REC 9461700634) and the Taipei Tzu-Chi General Hospital (96-IRB-10). 

RNA Isolation and Reverse Transcription–Polymerase Chain Reaction

All surgical specimens in both groups were kept in liquid nitrogen for reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was extracted from the conjunctiva of SLK and control groups (TRIzol reagent; Invitrogen-Life Sciences, Gaithersburg, MD). One microgram of total RNA from each sample was annealed for 5 minutes at 70°C with 500 ng oligo(dT) (Fermentas, Hanover, MD) and reverse transcribed to cDNA (200u RevertAid M-MuLV Reverse Transcriptase; Fermentas, Hanover, MD) per 20-μL reaction for 1 hour at 42°C. The reaction was stopped by heating for 10 minutes at 70°C. PCR was performed on the resultant cDNA from each sample with specific primers for human MMP-1, -2, -3, and -9; TIMP-1 and -2; and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), which served as an internal control. Amplification was performed with a thermocycler. The 25-μL reaction mixture consists of 2 μL cDNA, 1 μL sense and antisense primer, and 12.5 μL 2× PCR mix. The annealing temperature and cycle for MMP-1, -2, -3, and -9 and TIMP-1 and -2 were all 55°C and 40 cycles. At the end of amplification, the reaction mixture was heated for 10 minutes at 72°C and then cooled to 4°C. A 10-μL sample of each PCR product was separated by performing gel electrophoresis on 2% agarose containing ethidium bromide (Sigma, St Louis, MO). The 2% agarose gel was analyzed under ultraviolet light against the DNA molecular length markers. 

Real-Time Quantitative PCR

Paraffinized block were used in this part of experiment. RNA was extracted (RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues; Ambion Diagnostics, Austin, TX). Total RNA was reverse transcribed into cDNA with a kit (High Capacity cDNA Reverse Transcription; Applied Biosystems [ABI], Inc., Foster City, CA). Real-time quantitative PCR was performed by a commercial apparatus (Prism 7900HT; ABI). MMP-1, -2, -3, and -9; TIMP-1 and -2; and GAPDH gene expression was studied (TaqMan Gene Expression Assay; ABI). Cycling conditions were as follows: after incubation for 2 minutes at 50°C and 10 minutes at 95°C, the reaction continued for 30 cycles at 95°C for 15 seconds and 60°C for 1 minute. At the end of each run, melting point analysis was performed to validate the specificity of the PCR transcripts. All samples were analyzed in triplicate. The collected data were analyzed by commercial software (SDS, ver. 2.21; ABI). 

Immunohistochemistry (IHC) for MMP-1, -2, and -3 and TIMP-1 and -2

All the SLK and control specimens were placed into 4% paraformaldehyde at 4°C for 1 to 2 days and then stored in 1% sodium phosphate buffer at 4°C. They were then embedded in paraffin. To enhance tissue adhesion, 5-μm-thick sections were mounted on pretreated glass slides (Vectabond; Vector Laboratories, Burlingame, CA). After drying, the paraffin was removed with xylene, and the sections rehydrated by passing them through graded alcohols to distilled water. Antigen retrieval in 10 mM sodium citrate buffer was performed by autoclaving for 10 minutes and quenching of endogenous peroxidase activity in 3% hydrogen peroxidase. After this, paraffin sections (5 μm) were incubated with anti-human MMP-1, -2, and -3 and TIMP-1 and -2 antibodies (Laboratory Vision, Fremont, CA) overnight at 4°C. Washes in PBS were followed by incubation with biotinylated secondary antibody for 30 minutes at room temperature. Antibody detection was achieved with aminol-ethyl carbazole (AEC) peroxidase substrate solution for 15 minutes. Sections were counterstained with hematoxylin to facilitate tissue orientation, and then mounted. For negative controls, the primary antibody was omitted. The conjunctival subepithelial stroma immunostaining was graded 0 to 3 under 100× light microscopy according to the following scale: grade 0, no staining; grade 1, one third of the area with positive staining; grade 2, one third to two thirds of the area with positive staining; and grade 3, more than two thirds of the area with positive staining. 

Conjunctival Fibroblast Culture from SLK and Normal Subjects

Each specimen was collected for primary conjunctival fibroblast culture. Fibroblasts at the third or fourth passage were used. In brief, each tissue specimen was cut into explants of approximately 2 mm² and placed onto 100-mm tissue culture dishes. After 10 to 20 minutes, each explant was covered with a drop of DMEM containing 10% FBS (DMEM-FBS; Sigma, St. Louis, MO), 50 mg/mL gentamicin, and 1.25 mg/mL amphotericin B and placed overnight in an incubator at 37°C with 95% humidity and 5% CO₂. On the next day, 10 mL of the same media was added and the media were changed three times weekly thereafter. These fibroblasts were subcultured with 0.1% trypsin and 0.02% EDTA in calcium-free minimum essential medium (MEM) at 80% to 90% confluence with a 1:2 to 1:3 split for several passages. Immunostaining for vimentin and α-smooth muscle actin was performed to characterize the cultured conjunctival fibroblasts. Total RNA was extracted from the conjunctival fibroblasts of SLK and control groups. The methods of RNA isolation and RT-PCR were similar to those described above. 

Statistical Analysis

The RT-PCR and Q-PCR results were analyzed by Fischer's exact test and Student's t-test, respectively. The differences of MMPs and TIMPs from the immunohistochemical stain between the study and control groups were analyzed by the Wilcoxon signed-rank test. Values for P < 0.05 were considered statistically significant. 

Results

PCR of Resected Conjunctival Specimens: MMP-1, -2, -3, and -9 and TIMP-1 and -2

In the eight conjunctival specimens of SLK patients and nine control specimens, the mRNAs of MMP-1, -2, -3, and -9 and TIMP-1 and -2 were identified through RT-PCR. Expression of the mRNAs of MMP-1 and -3 was detected in six and seven SLK patients, respectively. In contrast, the mRNAs of MMP-1 and -3 were not detected in any case in the control group. mRNAs of MMP-2 and TIMP-1 and -2 were detected in some cases in both groups. Meanwhile, the expression of mRNA of MMP-9 was not detected in both groups. Statistical analysis showed a significant difference in the expression of mRNAs of MMP-1 and -3 and no difference in the expression of mRNAs of MMP-2 and TIMP-1 and -2 between the SLK and control groups (Table 1). 

Table 1.

 

RT-PCR Results of MMP-1, -2, -3, -9 and TIMP-1 and -2 in Superior Limbic Keratoconjunctivitis and Control Conjunctival Specimens

	MMP-1		MMP-2		MMP-3		MMP-9		TIMP-1		TIMP-2
	+	−	+	−	+	−	+	−	+	−	+	−
SLK (n = 8)	6	2	5	3	7	1	0	8	5	3	4	4
Control (n = 9)	0	9	8	1	0	9	0	9	3	6	2	7
P	0.002		0.294		0.0004		1.000		0.347		0.335

 Bold P values indicate significant results. +, positive expression in RT-PCR; −, no expression in RT-PCR.

In parallel to RT-PCR, Q-PCR demonstrated consistent results. MMP-1 and -3 mRNA level was higher in SLK conjunctival specimens than in control conjunctival tissue (P = 0.020 and P = 0.032). No significant difference in MMP-2 and -9 and TIMP-1 and -2 mRNA level between SLK and control groups was observed (MMP-1, -2, -3 and -9 data shown in Fig. 1, TIMP-1 and -2 data not shown). 

Figure 1.

 

Q-PCR showed overexpression of MMP-1 and -3 in SLK surgical specimens.

IHC of MMP-1, -2, and -3 and TIMP-1 and -2

Based on the RT-PCR results (no MMP-9 RNA transcription shown), IHC staining for MMP-1, -2, and -3 and TIMP-1 and -2 was performed. In both groups, the basal layer of conjunctival epithelium always showed weakly positive for MMP-1, -2, and -3 and TIMP-1 and -2. In the SLK group, the layer of subepithelial stroma was moderately positive for MMP-1 (Fig. 2A) and MMP-3 and weakly positive for MMP-2, TIMP-1, and TIMP-2. Control specimens stained undetectable to weakly positive for MMP-1 (Fig. 2B); MMP -2, MMP -3, TIMP-1, and TIMP-2 in the subepithelial stroma. Statistical analysis revealed a significant overexpression of MMP-1 and -3 in the subepithelial stroma in the SLK group (P < 0.000) and no significant difference in the expression of MMP-2, TIMP-1 and TIMP-2 by group (Fig. 2C). 

Figure 2.

 

Light microscopy of SLK and control conjunctival sections stained by IHC method for MMP-1 in SLK (A) and control (B) specimens. The results of IHC grading of conjunctival subepithelial stroma for (C) MMP-1, -2, and -3 and TIMP-1 and -2. (A, B) Immunostaining of MMP-1 appeared dark-brown. The basal layer of conjunctival epithelium always showed positivity in both groups (black arrows). The immunostaining of MMP-1 in SLK patient showed more prominent than in control specimen (white arrows). (C) The grading of subepithelial stroma revealed only MMP-1 and -3 with statistically significances between SLK and control groups (P < 0.0001).

RT-PCR of Cultured Conjunctival Fibroblasts for MMP-1, -2, -3, and -9 and TIMP-1 and -2

Eight conjunctival specimens of SLK patients and nine control conjunctival specimens were sent for primary fibroblast culture (Table 2). The cultured conjunctival fibroblasts showed positive staining for vimentin and negative staining for α-smooth muscle that indicated that the cultured cells were fibroblasts instead of myofibroblasts. The mRNA of MMP-1 was detected in six of eight SLK specimens, and none of the specimens in the control group expressed MMP-1 mRNA (P = 0.002). Eight patients in the SLK group, compared to 0 in control group, also showed a statistically significant difference in the expression of mRNA of MMP-3 in cultured conjunctival fibroblasts (P < 0.000). The mRNAs of MMP-2, TIMP-1 and TIMP-2 were detected in some cases in both groups with no significant difference. Meanwhile, MMP-9 mRNA was not detected in either group. 

Table 2.

 

RT-PCR Results of MMP-1, -2, -3, and -9 and TIMP-1 and -2 in Cultured Conjunctival Fibroblasts from SLK and Control Patients

	MMP-1		MMP-2		MMP-3		MMP-9		TIMP-1		TIMP-2
	+	−	+	−	+	−	+	−	+	−	+	−
SLK (n = 8)	6	2	8	0	8	0	0	8	6	2	6	2
Control (n = 9)	0	9	8	1	0	9	0	9	4	5	4	5
P	0.002		1.000		0.000		1.000		0.335		0.335

 Bold P values indicate significant results. +, positive expression in RT-PCR; −, no expression in RT-PCR.

Discussion

MMPs and their TIMPs have been implicated in normal matrix remodeling in embryonic development, wound-healing, and tissue homeostasis. ^9,14 TIMPs are specific endogenous MMP inhibitors that bind MMPs in a 1:1 stoichiometry. ¹⁵ Overall, all MMPs are inhibited by TIMPs once they are activated. An imbalance between MMP and TIMP activity has been associated with several pathologic conditions, including cancer, ^{16 –18} cardiovascular diseases, ^19,20 inflammatory diseases, ^{21 –23} skin disorders such as cutis laxa, ²⁴ and several eye diseases, such as keratoconus, ²⁵ corneal inflammation, ²⁶ conjunctivochalasis, ^6,27 glaucoma, ²⁸ and floppy eyelid syndrome. ²⁹  

Superior conjunctival redundancy is one of the specific characteristics of SLK. ³⁰ The redundant phenomenon of peripheral skin, ²⁴ eyelid, ²⁹ and conjunctiva ⁶ is a prominent feature of the relationship of MMPs and those disease entities. In cutis laxa, a connective tissue disorder in which the skin becomes inelastic and hangs loosely in folds, diseased skin showed more MMP-1, -3 and -9 than control samples. ²⁴ Floppy eyelid syndrome (FES), an ocular condition characterized by flaccid and easily everted upper eyelids, showed an increased immunoreactivity of MMP-7 and -9 in FES skin and conjunctiva compared with controls. ²⁹ Conjunctivochalasis, an age-related ocular condition with loosened bulbar conjunctiva lying along the lateral or central lower eyelid margin, showed MMP-1 and -3 overexpression in cultured fibroblasts. ⁶ Because both SLK and conjunctivochalasis are characterized by redundant conjunctiva, we searched for MMP and TIMP transcripts in surgical specimens by PCR. We found an obvious imbalance in MMPs in the study group. The results showed that MMP-1 and -3 were significantly increasingly expressed in the study group comparing to the control group. We also performed IHC stain to recognize MMP and TIMP expression in the protein level, finding significantly higher levels of MMP-1 and -3 in the study group. Although the immunoreactivity of MMPs and TIMPs was detected in basal epithelium and subepithelial stroma in both groups, the intensity was equivalent in basal epithelium but differed in subepithelial stroma. This observation suggests that the cells of subepithelial stroma contributed to the overexpression of MMP-1 and -3 in the SLK group. 

Of the interstitial collagenases, MMP-1 (collagenase-1) is the most ubiquitously expressed. It is produced by a wide variety of normal cells (e.g., stromal fibroblasts, macrophages, endothelial cells, and epithelial cells), as well as by numerous tumors, suggesting a broad role in biology. ³¹ MMP-3, also known as stromelysin, has a broader substrate specificity, degrading collagens III, IV, IX, and X; laminin; proteoglycans; and fibronectin. It may be expressed in fibroblasts, chondrocytes, endothelial cells, macrophages, vascular smooth muscle cells, osteoblasts, and keratinocytes in response to appropriate stimuli. MMP-3 therefore may participate in physiological matrix turnover and pathologic destruction of tissue. ³² The conjunctival fibroblast is one of the most important cellular components of the conjunctiva and may be important in SLK pathogenesis through MMP-1 and -3 activities. We cultured conjunctival fibroblasts to ascertain whether the conjunctival redundancy of SLK resulted from diseased conjunctival fibroblasts. Apparent overexpression of MMP-1 and -3 transcripts in the SLK group was found in comparison to the control group and was also parallel to the IHC staining of the resected conjunctiva. These findings suggest that conjunctival resection should remove not only the redundant conjunctiva but also the underlying Tenon's layer of abnormal fibroblasts, to prevent SLK recurrence. 

Petersen et al. ³³ and Brenneisen et al. ³⁴ discovered that cultured normal dermal fibroblasts increase MMP-1 and -3 expression after UV irradiation. Pterygium is an ocular surface disorder highly correlated with UV radiation. ³⁵ Li et al. ³⁶ in 2001 found MMP-1 and -3 overexpression in pterygium head fibroblasts. The diseased conjunctiva of SLK was distributed near the superior limbus, which was covered by the upper eyelid and with limited UV exposure; thus, further studies are needed to investigate which factors besides UV exposure cause overexpression of MMP-1 and -3 in SLK patients. 

In conclusion, we found an imbalance of MMPs and TIMPs in SLK. Overexpression of MMP-1 and -3 in SLK may have come from the diseased conjunctival fibroblasts. This MMP imbalance resulted in conjunctival redundancy in SLK, which advanced the ocular condition. In the future, these results may suggest a treatment strategy for SLK patients. 

Footnotes

 Supported by Grant TPE-96-06 from the Buddhist Tzu-Chi General Hospital, Taipei Branch.

Footnotes

 Disclosure: Y.-C. Sun, None; C.-H. Hsiao, None; W.-L. Chen, None; F.-R. Hu, None