Matrix Metalloproteinase-9 Knockout Confers Resistance to Corneal Epithelial Barrier Disruption in Experimental Dry Eye
2005; Elsevier BV; Volume: 166; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62232-8
ISSN1525-2191
AutoresStephen C. Pflugfelder, William J. Farley, Lihui Luo, Lucy Zhuo Chen, Cintia S. de Paiva, Lisa C. Olmos de Koo, De‐Quan Li, M. Elizabeth Fini,
Tópico(s)Corneal Surgery and Treatments
ResumoAltered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin. Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin. Corneal epithelial disease, termed keratitis sicca, is a severe and sight-threatening complication of dry eye.1Murillo-Lopez F Pflugfelder SC Dry eye.in: Krachmer J M Mannis E Holland The Cornea. Mosby, St. Louis1996: 663-686Google Scholar A key clinical feature of keratitis sicca is disruption of corneal epithelial barrier function.2Yokoi N Kinoshita S Clinical evaluation of corneal epithelial barrier function with the slit-lamp fluorophotometer.Cornea. 1995; 14: 485-489Crossref PubMed Scopus (89) Google Scholar, 3Gobbels M Spitznas M Corneal epithelial permeability of dry eyes before and after treatment with artificial tears.Ophthalmology. 1992; 99: 873-878Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 4Pflugfelder SC Tseng SC Sanabria O Kell H Garcia CG Felix C Feuer W Reis BL Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation.Cornea. 1998; 17: 38-56Crossref PubMed Scopus (431) Google Scholar This results in eye irritation, corneal surface irregularity, blurred and fluctuating vision, and increased risk for corneal ulceration.4Pflugfelder SC Tseng SC Sanabria O Kell H Garcia CG Felix C Feuer W Reis BL Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation.Cornea. 1998; 17: 38-56Crossref PubMed Scopus (431) Google Scholar, 5Rolando M Iester M Macri A Calabria G Low spatial-contrast sensitivity in dry eyes.Cornea. 1998; 17: 376-379Crossref PubMed Scopus (94) Google Scholar, 6de Paiva CS Lindsey JL Pflugfelder SC Assessing the severity of keratitis sicca with videokeratoscopic indices.Ophthalmology. 2003; 110: 1102-1109Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7de Paiva SC Pflugfelder SC Corneal epitheliopathy of dry eye induces hyperesthesia to mechanical air jet stimulation.Am J Ophthalmol. 2004; 137: 109-115Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Ormerod LD Fong LP Foster CS Corneal infection in mucosal scarring disorders and Sjogren's syndrome.Am J Ophthalmol. 1988; 105: 512-518Abstract Full Text PDF PubMed Scopus (43) Google Scholar Ocular surface inflammation has been implicated in the pathogenesis of keratitis sicca. Elevated levels of proinflammatory cytokines, such as interleukin (IL)-1, have been detected in the tear fluid of patients with this condition.9Barton K Monroy DC Nava A Pflugfelder SC Inflammatory cytokines in the tears of patients with ocular rosacea.Ophthalmology. 1997; 104: 1868-1874Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 10Solomon A Dursun D Liu Z Xie Y Macri A Pflugfelder SC Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry eye disease.Invest Ophthalmol Vis Sci. 2001; 42: 2283-2292PubMed Google Scholar, 11Afonso A Sobrin L Monroy DC Selzer M Lokeshwar B Pflugfelder SC Tear fluid gelatinase B activity correlates with IL-1β concentration and fluorescein tear clearance.Invest Ophthalmol Vis Sci. 1999; 40: 2506-2512PubMed Google Scholar Furthermore, the concentration and activity of matrix metalloproteinase (MMP)-9 in the tear fluid was found to be significantly increased in these eyes, with the highest concentrations observed in eyes with the severe corneal epithelial disease or sterile corneal ulcers.10Solomon A Dursun D Liu Z Xie Y Macri A Pflugfelder SC Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry eye disease.Invest Ophthalmol Vis Sci. 2001; 42: 2283-2292PubMed Google Scholar, 11Afonso A Sobrin L Monroy DC Selzer M Lokeshwar B Pflugfelder SC Tear fluid gelatinase B activity correlates with IL-1β concentration and fluorescein tear clearance.Invest Ophthalmol Vis Sci. 1999; 40: 2506-2512PubMed Google Scholar, 12Smith VA Rishmawi H Hussein H Easty DL Tear film MMP accumulation and corneal disease.Br J Ophthalmol. 2001; 85: 147-153Crossref PubMed Scopus (113) Google ScholarWe hypothesize that MMP-9 plays an important role in the disruption of corneal epithelial barrier function in dry eye. We previously reported an experimental murine model of dry eye that disrupts corneal epithelial barrier function similar to human dry eye disease.13Dursun D Wang M Monroy D Li DQ Lokeshwar BL Stern ME Pflugfelder SC A mouse model of keratoconjunctivitis sicca.Invest Ophthalmol Vis Sci. 2002; 43: 632-638PubMed Google Scholar The purpose of this study was to compare the effects of experimentally induced dry eye (EIDE) on corneal epithelial morphology and barrier function in MMP-9 knockout mice and their wild-type (WT) littermates.Materials and MethodsMiceThis research protocol was approved by the Baylor College of Medicine Center for Comparative Medicine and it conformed to the standards in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in ophthalmic and vision research. MMP-9 (gelatinase B) knockout mice (referred to as BKO mice) were created on a 129SvEv/CD-1 mixed background as previously reported.14Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1484) Google Scholar WT (GelB +/+) littermates were used as controls.Mouse genotypes were verified throughout the study by polymerase chain reaction (PCR) performed on tail genomic DNA. Genomic DNA was isolated from tails of 1-month-old mice using a Genomic DNA Isolation kit (Sigma, St. Louis, MO) according to the manufacturer's instructions. A specific primer pair including a sense primer (GCATACTTGTACCGCTATGGT) and an anti-sense primer (TGTGATGTTATGATGGTCCC) was designed from the sequence of MMP-9 gene exon 2 (accession no. X72794), which was knocked out and replaced in BKO mice by a cassette containing the neomycin phosphotransferase cDNA (neo′) driven by the phosphoglycerate kinase (PGK) promoter.15Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar PCR amplification was performed in a 96-well GeneAmp PCR System 9700 using a GeneAmp PCR kit (Applied Biosystems, Foster City, CA) in a 50-μl volume containing 1 μg of genomic DNA, dNTPs, Taq polymerase, and the specific primers.Creation of Dry EyeEIDE was created by subcutaneous injection of 0.5 mg/0.2 ml scopolamine hydrobromide four times a day (8:00 a.m., 12:00 p.m., 2:00 p.m., and 5:00 p.m.), alternating between the left and right flanks of 4- to 6-week-old WT and BKO mice as previously described.16Yeh S Song XJ Farley W Li DQ Stern ME Pflugfelder SC Apoptosis of ocular surface cells in experimentally induced dry eye.Invest Ophthalmol Vis Sci. 2003; 44: 124-129Crossref PubMed Scopus (236) Google Scholar Up to five mice were placed in a cage with a perforated plastic screen on one side to allow airflow from a fan (Cafrano, Wiarton, Ontario, Canada) placed 6 inches in front of it for 16 hours per day. Tear production was measured with phenol-red impregnated cotton threads (Zone-Quick; Oasis, Glendora, CA) placed into the tear meniscus of the lateral canthus for 30 seconds. Tear fluorescein clearance (the residual concentration of fluorescein dye in the tear fluid 15 minutes after instillation) was measured as previously reported.13Dursun D Wang M Monroy D Li DQ Lokeshwar BL Stern ME Pflugfelder SC A mouse model of keratoconjunctivitis sicca.Invest Ophthalmol Vis Sci. 2002; 43: 632-638PubMed Google ScholarGelatin ZymographyThe relative amount of MMP-9 in tear washings was measured by gelatin zymography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gelatin zymography was performed using a previously reported method.17Sobrin L Selzer MG Lokeshwar BL Pflugfelder SC Stromelysin (MMP-3) activates pro-MMP-9 secreted by corneal epithelial cells.Invest Ophthalmol Vis Sci. 2000; 41: 1703-1709PubMed Google Scholar Tear fluid was collected using 1.0-μl polished glass capillary pipettes (Drummond, Broomhall, PA) immediately after a 1.0-μl drop of Brij buffer was placed on the ocular surface. Pooled tear samples from treated or untreated WT and BKO mouse were fractionated in an 8% polyacrylamide gel containing gelatin (0.5 mg/ml). The gels were soaked in 0.25% Triton X-100 for 30 minutes at room temperature, and incubated in a digestion buffer containing 5 mmol/L phenylmethyl sulfonyl fluoride at 37°C overnight. They were stained with 0.25% Coomassie brilliant blue R-250 in 40% isopropanol for 2 hours, and destained overnight in 7% acetic acid. Gelatinolytic activities appeared as clear bands of digested gelatin against a dark blue background of stained gelatin.RNA Isolation and Semiquantitative Reverse Transcriptase (RT)-PCRMMP-9 RNA expression in the cornea epithelium was measured by RT-PCR. Total RNA was isolated from corneal epithelia (six eyes per group) of WT or BKO mice with or without EIDE by acid guanidium thiocyanate-phenol-chloroform extraction method. The 371 bp of the mouse MMP-9 gene was amplified by semiquantitative RT-PCR using the sense primer TGTACCGCTATGGTTACACCCG and anti-sense primer CGCGACACCAAACTGGATGAC, which was specifically designed to amplify a segment from exon 2 to exon 3 of the mouse MMP-9 sequence (accession no. NM_013599 and X72794), of which the part of exon 2 and all of intron 2 were replaced in BKO mouse. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was amplified as an internal control using the primer pair, GCCAAGGTCATCCATGACAAC and GTCCACCACCCTGTTGCTGTA, which was specifically designed from exon 7 and exon 9 of the mouse GAPDH sequence (accession no. M32599).Corneal Epithelial PermeabilityCorneal epithelial permeability to three different molecules [carboxyfluorescein, Alexa-Fluor-dextran (AFD), and horseradish peroxidase (HRP)] was assessed in six eyes of control mice and mice with EIDE for 2 weeks. One μl of 0.3% carboxyfluorescein (Holles Laboratory, Cohasset, MA) or AFD (Molecular Probes, Eugene, OR) (10 kd molecular weight) was placed on the ocular surface 15 minutes before euthanasia. Corneas were excised, rinsed four times in phosphate-buffered saline (PBS), and placed in wells of a 96-well plate (Costar-Corning, Corning, NY) containing 100 μl of PBS, covered, wrapped in aluminum foil, and placed on a shaker for 60 minutes. Carboxyfluorescein and AFD concentrations were measured with a Cytofluor II fluorometer (Perseptive Biosystems, Framingham, MA) as previously reported.13Dursun D Wang M Monroy D Li DQ Lokeshwar BL Stern ME Pflugfelder SC A mouse model of keratoconjunctivitis sicca.Invest Ophthalmol Vis Sci. 2002; 43: 632-638PubMed Google Scholar HRP uptake by the cornea was measured by placing 1 μl of a 1 U/ml HRP solution on the ocular surface 15 minutes before euthanasia. HRP was detected in excised corneas using an Amplex Red detection system (Molecular Probes) and measuring absorbance using a 530-nm absorbance and 590-nm emission filters at 10, 20, and 30 minutes.To assess the affects of topical application of MMP-9 on corneal epithelial permeability in BKO mice, 1 μl of a 1 μg/ml solution of active MMP-9 (Oncogene Research Products, Boston, MA) prepared in PBS was administered topically every 2 hours for 10 hours a day for 2 days to mice with and without EIDE. Control mice received 1 μl of PBS every 2 hours or no drops. Three eyes of three animals were evaluated in each treatment group.HistologyEyes from WT and BKO mice with and without EIDE were surgically excised, fixed in 10% formalin, and embedded in paraffin. Six-μm sections were stained with hematoxylin and eosin or periodic acid-Schiff (PAS) reagent. Sections from three different eyes in each group were examined and photographed with a Nikon Eclipse E400 (Garden City, NY) microscope equipped with a Nikon DXM 1200 digital camera.Transmission Electron MicroscopyAfter fixation, the corneal samples from WT and BKO mice with and without EIDE (n = 2) were rinsed in buffer and postfixed in PIPES-buffered osmium tetroxide (pH 7.2) for 1 hour at room temperature, then rinsed in several changes of distilled water, and dehydrated through a graded series of ethanol. The dehydrated tissues were incubated in two 45-minute changes of propylene oxide followed by a 1:1 mixture of propylene oxide and Spurr's resin for 1 hour and 30 minutes. The tissue pieces were then incubated in pure resin for 1 hour and 30 minutes, after which they were transferred to fresh resin in block molds and allowed to cure at 60°C overnight. Thick sections (1 μm) cut from the hardened blocks were mounted on glass slides, stained with an alcoholic solution of toluidine blue and basic fuchsin, and examined under the light microscope. Areas of interest were trimmed and 60-nm sections were cut and mounted on copper grids (300 mesh). The grids were stained with uranyl acetate and lead citrate and photographed with a Zeiss EM-900 transmission electron microscope (Peabody, MA). Photographs were taken with Kodak 4489 electron microscope film (Rochester, NY).Confocal MicroscopyWhole freshly harvested murine corneas from WT and BKO mice with and without EIDE (n = 3) were fixed in cold methanol for 10 minutes at −20°C. After fixation, they were permeabilized with PBS containing 0.1% Triton X for 10 minutes. The tissues were blocked with 20% goat serum in PBS for 1 hour at room temperature to reduce nonspecific labeling. The tissues were then incubated with polyclonal rabbit anti-occludin (1:25 dilution; Zymed, San Francisco, CA) diluted in 5% goat serum and PBS, overnight at 4°C. Tissues without primary antibody were used as negative controls. After extensive washing with PBS, Alexa Fluor-488-conjugated goat anti-rabbit antibody (1:300, Molecular Probes) was applied for 1 hour at room temperature. Tissues were rinsed and counterstained with propidium iodide (1 μg/ml in PBS) for 30 minutes. After washing with PBS, corneal tissues were flattened on microscope slides, mounted with anti-fade Gel/Mount (Fisher, Atlanta, GA) and coverslips were applied.Digital confocal images (512 × 512 pixels) were captured with a laser-scanning confocal microscope (LSM 510, Zeiss with krypton-argon and He-Ne laser; Zeiss, Thornwood, NY) with 488-excitation and 543-nm emission filters, LP505 and LP560, respectively. They were acquired with a 40/1.3× oil-immersion objective. Samples from treated and untreated animals were captured by using identical photomultiplier tube gain settings and processed using Zeiss LSM-PC software and Adobe Photoshop 6.0. The number of desquamating superficial epithelial cells was counted in three microscopic fields per cornea in paraffin-embedded histological sections from three corneas obtained from each of four groups of mice: control WT, control BKO, WT with EIDE, and BKO with EIDE.Corneal Epithelial Explant CulturesPrimary human corneal epithelial cells were grown from limbal explants taken from corneoscleral tissues provided by the Lions Eye Bank of Texas (Houston, TX) using a previously described method.18Li DQ Shang TY Kim HS Solomon A Lokeshwar BL Pflugfelder SC Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells.Invest Ophthalmol Vis Sci. 2003; 44: 2928-2936Crossref PubMed Scopus (92) Google Scholar Explants were grown in six-well culture plates for ∼20 days until they were near confluence. Groups of three wells were exposed to MMP-9 (0.5 and 1 μg/ml) for 8 and 24 hours, and another group of three wells served as a media control. The morphology of these cells was observed and photographed before and after MMP-9 treatment by phase and fluorescent microscopy after staining with calcein AM dye (10 μg/ml, Molecular Probes) for 30 minutes. Immunostaining for occludin was performed as described above.Western BlotTo make soluble and insoluble pools, mouse corneal epithelial cells obtained by scraping were lysed in a buffer containing 1% Triton X-100, 100 mmol/L NaCl, 10 mmol/L HEPES, 2 mmol/L ethylenediaminetetraacetic acid (EDTA) and a EDTA-free protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN), then centrifuged at 15,000 × g for 30 minutes at 4°C. This supernatant was considered the Triton-soluble pool. The pellet was solubilized in 1% SDS and referred to as the Triton-insoluble pool. The total protein concentrations of the cell extracts were measured by a Micro BCA protein assay kit (Pierce, Rockford, IL). The protein samples (50 μg) were mixed with 6× SDS-reducing sample buffer and boiled for 5 minutes before loading. Proteins were separated by SDS-polyacrylamide gel electrophoresis (4 to 15% Tris-HCl, gradient gels; Bio-Rad, Hercules, CA), and transferred electronically to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk in TTBS (50 mmol/L Tris, pH 7.5, 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature, and then incubated 2 hours at room temperature with a 1:160 dilution (1.56 μg/ml) of rabbit anti-occludin antibody (Zymed) with 5% nonfat milk in TTBS. After three washings with TTBS, the membranes were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody donkey anti-rabbit IgG (1:10,000 dilution; Promega, Madison, WI), or goat anti-rat IgG (1:5000 dilution; Pierce, Rockford, IL). After washing the membranes for four times, the signals were detected with an ECL advance chemiluminescence reagent (Amersham, Piscataway, NJ) and the images were acquired and analyzed by a Kodak image station 2000R (Eastman-Kodak, New Haven, CT). Band intensities from three different experiments were averaged. Lysates from control and MMP-9-treated human corneal epithelial cell cultures were also analyzed in a similar manner.ImmunprecipitationThe corneal epithelia of untreated WT mice were lysed in 150 μl of RIPA buffer containing 50 mmol/L Tris-HCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mmol/L NaCl, 10 mmol/L HEPES, pH 7.3, 2 mmol/L EDTA, 20 mmol/L sodium fluoride, and a EDTA-free protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN). The cell extracts were centrifuged at 15,000 × g for 30 minutes at 4°C and the supernatants were incubated with 20 μl of polyclonal anti-occludin antibody (Zymed) followed by rotation at 4°C overnight. Subsequently, 15 μL of protein A agarose (Sigma, St. Louis, MO) and 100 μl of ImmunoPure IgG-binding buffer (Pierce) were added with further incubation for 2 hours at 4°C. The immunoprecipitates were collected by centrifugation at 3000 × g, washed three times in wash buffer I (20 mmol/L Tris-HCl, pH 8.0, 400 mmol/L KCl, 0.5 mmol/L EDTA, 10% glycerol, and 0.25% Nonidet P-40) and one time in wash buffer II (20 mmol/L Tris-HCl, pH 8.0, 100 mmol/L KCl, 0.5 mmol/L EDTA, 10% glycerol, and 0.25% Nonidet P-40). Bound protein was eluted in 6× SDS-reducing sample buffer and boiled for 5 minutes for immunoblot analysis. Immunoblot were probed with 1:625 dilution (0.8 μg/ml) of monoclonal anti-occludin antibody (Zymed). Cell lysates from control and MMP-9-treated human corneal epithelial cell cultures were immunoprecipitated in a similar manner.StatisticsThe Student's t-test or the Mann-Whitney test were used where appropriate for between group statistical comparisons. Analysis of variance with Turkey posthoc analysis was used for the MMP-9 reconstitution experiment and comparison of corneal epithelial desquamation.ResultsGenotypeGenotyping was performed with PCR to confirm that BKO mice lacked a full-length MMP-9 gene. Using a primer pair spanning a sequence of MMP-9 gene exon 2 that was knocked out in BKO mice, a 225-bp fragment was generated from WT animals, but not from BKO mice (Figure 1).Induction of Dry EyeEIDE with scopolamine injections and a fan resulted in a significant decrease in tear production (Figure 2A) and worsening of tear fluorescein clearance (Figure 2B) within 4 days that was sustained throughout the treatment period in both groups. There was no difference in tear production between groups at any time point; however, clearance of fluorescein from the tear film was significantly more delayed in the BKO group at day 14 (P < 0.0001, Figure 2B).Figure 2A: Aqueous tear production measured in mm with a cotton thread in mice before (day 0) and after 4 and 14 days of EIDE. *, P < 0.001 compared to baseline. B: Measurement of clearance of fluorescein dye from the tear fluid 15 minutes after instillation of 1 μl of 2% sodium fluorescein in mice before (day 0) and 4 and 14 days after EIDE. *, P < 0.001 compared to baseline; ¥, P < 0.001 compared to WT at day 14. Values are mean ± SD from 14 eyes of seven mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tear Fluid MMP-9 Increases with Experimental Dryness in WT MiceMMP-9 could not be detected in pooled tear fluid washings of untreated (week 0) WT or BKO mice by gelatin zymography (Figure 3A). Distinct MMP-9 bands were noted in the tear fluid of WT mice after 2 weeks of EIDE, but not in BKO mice with EIDE. Mouse MMP-9 has been reported to have a higher molecular weight (107 kd) than human MMP-9 (92 kd).15Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar Western blot of tear MMP-9 could not be performed because of the low protein concentration in the pooled tear washings.Figure 3A: Gelatin zymography of pooled tear fluid washings obtained from WT and BKO mice before (wk 0) and after 2 weeks (wk 2) of EIDE. Mouse MMP-9 has a molecular weight of 107 kd.15Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar B: Expression of MMP-9 and GAPDH RNA in the corneal epithelium of WT and BKO mice by RT-PCR before (wk 0) and after 1 (wk 1) and 2 (wk 2) weeks of EIDE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that BKO mice do not produce a functional MMP-9 gene product, MMP-9 RNA expression in the corneal epithelium was evaluated by RT-PCR using primers that span the deleted region of the MMP-9 gene in BKO mice. A PCR product of the appropriate size (371 bp) was obtained in WT mice and there was a progressive increase in intensity throughout a 2-week period in response to EIDE. In contrast, no MMP-9 RNA expression was observed in BKO mice at any time point (Figure 3B).Disruption of the Corneal Epithelial Permeability Barrier Is Significantly Less in Dry Eye BKO MiceA hallmark of human keratoconjunctivitis sicca is increased corneal epithelial permeability to the diagnostic dye sodium fluorescein. Corneal epithelial permeability to molecules of three different sized molecules (carboxyfluorescein, molecular weight 750 kd; AFD, molecular weight, 10 kd; and HRP, molecular weight, 44 kd) was assessed in control and EIDE mice. Compared to baseline, permeability to all three molecules was significantly increased in WT mice, but not in BKO mice after 2 weeks of EIDE (Figure 4).Figure 4Corneal permeability to carboxyfluorescein (A, FL units = units of fluorescein emission at 530 nm), AFD (B, FL units = units of fluorescein emission at 530 nm), and HRP (C, FL units = units of fluorescein emission from activated peroxidase substrate Amplex Red at 590 nm) in untreated control and after 2 weeks of EIDE in WT and BKO mice Values are mean ± SD for four eyes. A: *, P = 0.002 WT with EIDE versus untreated control WT and BKO with EIDE; B: *, P = 0.048 WT with EIDE versus untreated control WT and BKO with EIDE; C: *, P < 0.03 WT with EIDE versus untreated control WT and treated BKO.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the addition of MMP-9 to the ocular surface of BKO mice would worsen corneal epithelial barrier function in response to EIDE, control BKO mice and BKO mice with EIDE received topical application of activated MMP-9 to their ocular surface every 2 hours for 10 hours per day for 2 days, then corneal permeability to HRP was measured. There was no difference in corneal epithelial barrier function in BKO mice treated with topical MMP-9 or saline compared to the untreated control group. In contrast, a significant increase (P < 0.05) in corneal epithelial permeability to HRP was noted in BKO mice with EIDE that received topical MMP-9 compared to mice receiving saline or nothing (Figure 5). Treatment with MMP-9 was not continued beyond 2 days because MMP-9-treated mice with EIDE began to develop frank corneal epithelial sloughing.Figure 5Corneal permeability to HRP in untreated controls and mice treated 10 hours per day for 2 days with PBS, MMP-9 (1 μl of 1 μg/ml), EIDE, EIDE plus PBS, EIDE plus MMP-9 (1 μl of 1 μg/ml). Drops were administered every 2 hours. Values are mean ± SD for 3 eyes. ¥, Between group difference was 0.002 ANOVA. Post-hoc analysis showed significant differences between EIDE + MMP-9 and controls (P < 0.05), EIDE (P < 0.01), and EIDE + PBS (P < 0.05). FL, fluorescent.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Corneal Histology and UltrastructureTo determine the mechanism by which MMP-9 increases corneal epithelial permeability in dry eye, corneal histology and ultrastructure were
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