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Originally published In Press as doi:10.1167/iovs.08-1674 on May 9, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:3981-3987.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-1674
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Interactions between Trabecular Meshwork Cells and Lens Epithelial Cells: A Possible Mechanism in Infantile Aphakic Glaucoma

Inbal Michael,1 Michael Shmoish,2 David S. Walton,3 and Shulamit Levenberg1

1From the Faculty of Bio-Medical Engineering, and the 2Bioinformatics Knowledge Unit, The Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion-Israel Institute of Technology, Haifa, Israel; and the 3Glaucoma Service, Massachusetts Eye and Ear Infirmary, and Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Infantile aphakic glaucoma may develop as a postoperative complication of early childhood cataract surgery. It has been associated with risk factors including surgery in early life and retained lens material; however, its cause and mechanism are poorly understood. This study focused on the potential role of retained lens material (specifically, exposed lens epithelial cells [LECs]) in undesired changes of the trabecular meshwork (TM) structure and function.

METHODS. Interactions between LECs and TM cells were studied by analyzing structural changes and differential gene and protein expression in TM cells cocultured with LECs.

RESULTS. Subjecting normal TM cells to the presence of LECs resulted in changes in their structural features (such as increase in volume and size, and decrease in cell–cell interactions), as well as in their protein expression (mainly cytoskeletal) and gene expression (such as genes related to organ and cell morphogenesis, inflammatory response, response to stimulus, ion homeostasis, and several signaling pathways).

CONCLUSIONS. Many of the changes observed in TM cells after exposure to LECs resemble alternations seen in primary open-angle glaucoma. This strengthens the suspected role of LECs in the development of aphakic glaucoma.

Cataract is a major, but treatable, cause of visual disability in childhood.1 2 3 Early treatment of congenital cataracts present at birth or in infancy is one of the most important factors in determining the eventual level of visual development.4 Cataract poses a serious impediment to the developing infantile visual system and must be removed to clear the visual axis. The removal of infantile cataract may introduce postoperative complications. Secondary glaucoma is the commonest and is sight threatening.5 6 This kind of secondary glaucoma is referred to as infantile aphakic glaucoma. Glaucoma develops because of an imbalance between aqueous production and its outflow through the drainage angle tissue of the eye, the trabecular meshwork (TM). Infantile aphakic glaucoma is most frequently an open-angle glaucoma and develops after infantile cataract surgery with an incidence ranging up to 32%.7 8 The highest incidence is found when the surgery is performed on a child younger than 2 months; a much lower incidence is found when surgery is performed on children older than 1 year.8 9 10 11 The occurrence of this glaucoma has been associated with some suspected risk factors including a preexisting ocular abnormality, chronic postoperative inflammation, retained lens material, and factors found in the aqueous humor of the eye.7 8 However, the direct cause and mechanism of TM injury responsible for infantile aphakic glaucoma is to date poorly understood.

Surgical removal of the infantile cataractous lens is typically incomplete and exposes the internal surfaces of the equatorial and posterior lens capsule to the anterior chamber aqueous humor. These membranes are covered by a monolayer of lens epithelial cells (LECs), creating new conditions in the eye that lead to potential interactions between LECs and TM cells mediated by the aqueous humor or by being in direct contact with each other. Therefore, we focused on the potential of retained lens material (specifically, residual exposed LECs) negatively affecting TM cellular structure and function. These interactions were studied by analyzing differential gene and protein expression and structural changes in TM cells cocultured with LECs.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Coculture of TM Cells and LECs
Normal human TM cells which were generously provided by Debra Fleenor and Abbot Clark (Alcon Laboratories, Fort Worth, TX; NTM5 cells, SV40 transformed, originating from an 18-year-old), and B-3 human LECs from ATCC (Catalog No. CRL-11421; SV40 transformed, originating from infant less than 1 year old) were used for coculture.

Both cell types were grown in a medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), 2 mM L-glutamine and 4500 mg/L glucose (Invitrogen-Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (HyClone, Erembodegem-Aalst, Belgium) and 1% penicillin-streptomycin (pen-strep) solution (Invitrogen-Gibco). The cells were maintained in a 5% CO2 humidified incubator at 37°C. Culture medium was replaced every 2 to 3 days. Before coculture, both cell types were grown for 2 days in a medium that contained only 1% fetal bovine serum.

Plates with membrane inserts (25 mm inserts designed for six-well plates, polycarbonate membrane with pores of 0.4 µm; Nunc, Roskilde, Denmark) were used for coculturing the cells.

LECs were seeded onto the inserts (105 cells/well), and the NTM5 cells were grown in the six-well plates (105 cells/well). Low-serum medium (1%) was replaced every 2 to 3 days. After a maximum 2 weeks of coculture, the NTM5 cells were analyzed for changes.

Since transformed LECs and TM cells may behave differently than primary cells, additional coculture was performed with primary cells. Normal primary human TM cells were generously provided by Donna Pesciotta Peters (Department of Ophthalmology and Visual Sciences, Wisconsin University, Madison, WI; N17RM.2, from a 17-year-old donor). Primary LECs (HLEpiCs, from fetal lens; catalog number 6550), were purchased from ScienCell Research Laboratories (Carlsbad, CA).

The N17RM.2 cells were grown in a medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with 1000 mg/L glucose and 4 mM L-glutamine (Invitrogen-Gibco) supplemented with 15% defined fetal bovine serum (HyClone), amphotericin B (250 µg/mL solution; Sigma-Aldrich, Poole, UK) and gentamicin sulfate (50 mg/mL solution; Sigma-Aldrich). Fresh bFGF solution (1 ng/mL final concentration; Invitrogen) was added directly to the plate until confluence. The cells were maintained in an 8% CO2 humidified incubator at 37°C. Culture medium was replaced every day. At confluence, the cells were subcultured and replaced in a 5% CO2 humidified incubator at 37°C 48 hours before coculture.

HLEpiCs were grown in a medium consisting of Eagle’s minimum essential medium with Earle’s balanced salt solution (EMEM), 1000 mg/L glucose, and 4 mM L-glutamine (Invitrogen-Gibco) supplemented with 20% fetal bovine serum (HyClone), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, and 1% pen-strep solution (all from Invitrogen-Gibco). The cells were maintained in a 5% CO2 humidified incubator at 37°C. Culture medium was replaced every 2 to 3 days.

Coculturing was performed as just described. Before coculturing, both cell types were grown for 48 hours in a medium containing DMEM with 1000 mg/L glucose and 4 mM L-glutamine, supplemented with 1% defined fetal bovine serum, amphotericin B, gentamicin sulfate, and pen-strep solution. Cocultured cells were grown in 5% CO2 humidified incubator at 37°C.

Analysis of Changes in TM Cells after Coculture
Cocultured TM cells (NTM5) were analyzed for differential gene and protein expression and for structural changes. NTM5 cells grown in low-serum medium (1%) for the same period as the cocultured cells used as the control.

Cocultured primary TM cells (N17RM.2) were analyzed for differential gene expression (TaqMan real-time PCR; ABI). N17RM.2 cells grown in low-serum medium (1%) for the same period as the cocultured cells were used as the control.

Structural Changes
Structural changes in NTM5 cells were observed with SEM (scanning electron microscopy). Two replicates of NTM5 cells grown on coverslips were prepared for SEM with hexamethyldisilazane (HMDS; Sigma-Aldrich). Briefly, samples were washed with saline, transferred to a solution containing 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7), washed in distilled water, dehydrated with a series of ethanol washes (70%, 85%, 90%, and 100%), and immersed in HMDS for 5 to 15 minutes. The coverslips were then air dried, mounted on SEM stubs, and coated with a thin layer of gold.

Morphologic changes in the primary TM cells were observed by using light microscopy.

Differential Protein Expression
Two replicates of total proteins of both coculture and control cells were extracted by using TRI-reagent (Sigma-Aldrich). Total protein content in the samples was determined according to Bradford’s method (Bio-Rad, Hercules, CA). The standard calibration curve was produced from serially diluted bovine serum albumin (Sigma-Aldrich). Equal quantities of proteins from coculture and control cells were used to perform SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). The separated proteins were visualized after staining with Coomassie blue-250 (Bio-Rad). The most differentially appearing protein bands in the gel were excised, digested by trypsin, analyzed by mass spectrometry (LC-MS/MS on DECA/LCQ), and identified by computer (Pep-Miner12 and Sequest software13 ; Finnigan, San Jose, CA) against a database of human, mouse, rat, bovine and rabbit (performed by the Smoler Protein Research Center, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel). The NCBI Gi accession numbers of the proteins identified were converted to gene link IDs by using the DAVID gene ID conversion tool and analyzed for molecular functional categorization by the functional annotation tool of DAVID-EASE (david.abcc.ncifcrf.gov/ Database for Annotation, Visualization and Integrated Discovery, provided in the public domain by the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD).

To validate the results of the MS-MS analysis, Western blot was performed for two of the identified proteins: filamin 1 and myosin heavy chain (nonmuscle). Proteins samples of 15 µg were electrophoresed on SDS-PAGE, and the proteins transferred to nitrocellulose membrane (0.45 µm; Invitrogen). The blots were incubated in 7.5% nonfat dry milk in PBS containing 0.1% Tween-20 (PBS-T) for 1 hour at room temperature, rinsed three times with PBS-T, incubated for 1 hour at room temperature with primary antibodies (anti-myosin heavy chain, nonmuscle, 1:1000 dilution, and antifilamin 1, 1:200 dilution; both from Santa Cruz Biotechnology, Santa Cruz, CA), rinsed again three times with PBS-T, and incubated for 1 hour at room temperature with secondary antibodies (horseradish peroxidase–conjugated goat anti-mouse IgG, 1:5000 dilution, and horseradish peroxidase–conjugated donkey anti-goat IgG, 1:10,000 dilution, both from Jackson ImmunoResearch, West Grove, PA). After five washes in PBS, the bound antibodies were visualized with a chemiluminescence detection kit for horseradish peroxidase (EZ-ECL; Biological Industries, Kibbutz Beit Haemek, Israel). Quantitation of each protein was performed by densitometric analysis (Multi Gauge ver. 3.0 software; FujiFilm, Tokyo, Japan). GAPDH served as an internal control.

Differential Gene Expression
RNA Isolation, Amplification, and Labeling.
Total RNA from NTM5 cells cocultured with LECs and from control cells were isolated after 3 days of coculture (RNeasy Mini Kit; Qiagen Inc., Valencia, CA), and RNA yields were determined by spectrophotometry (model ND1000; NanoDrop Technologies, Rockland, DE).

Ten micrograms of each total RNA sample was amplified, fractionated, and labeled with biotin according to the manufacturer’s protocols (Affymetrix, Santa Clara, CA). Overall, three replicates of cocultured NTM5 cells and three replicates from control cells were isolated and used for the microarray analysis.

Microarray Analysis
Labeled cDNA targets were hybridized to gene chips (HG-U133A 2.0; Affymetrix; six RNA samples to six arrays, i.e., each RNA sample per one array) according to the manufacturer’s instructions (performed by the microarray unit of Weizmann Institute of Science, Rehovot, Israel). Quality control evaluation was performed for each of the six microarrays, and one was excluded due to low quality. Overall, the analysis was performed on three replicates of control and duplicate of coculture.

Microarray data were first processed by R/Bioconductor software14 to compute RMA gene expression values with background correction and quantile normalization.15 Then by using a LIMMA (linear models for microarray data) package16 the set of differentially expressed genes between coculture and control was identified at the level of nonadjusted P < 0.01.

Gene set enrichment analysis was performed with the gene set enrichment tool (http://genie.weizmann.ac.il/genomica_web/gene_sets.jsp/ provided by the Weizmann Institute of Science), and also by the GOrilla tool (http://cbl-gorilla.cs.technion.ac.il/ provided by the Technion-Israel Institute of Technology) for identifying and visualizing enriched gene ontology (GO) terms in ranked lists of genes.17

Real-Time PCR
Real-time PCR (TaqMan; Applied Biosystems [ABI], Foster City, CA) was used to confirm the results of the microarray analysis, and for theanalysis of the primary cocultured cells. cDNA was prepared from total RNA with random hexamer (GE Healthcare, Buckinghamshire, UK) and reverse transcriptase (PrimeScript; Takara Bio Inc., Shiga, Japan). Each cDNA sample (300 ng) was loaded on a low-density array (part no. 4342253; Taqman; ABI) containing 48 genes, and the reaction was performed on a genotyping system (model BI 7900HT; ABI) All procedures were performed according to the manufacturer’s instructions (performed at The Center for Genomic Technologies at the Institute of Life Sciences in the Hebrew University of Jerusalem, Israel). Human GAPDH (ID Hs99999905_m1; Assay-on-Demand; ABI), which exhibited stable expression in our experimental conditions, was used as the endogenous control. Each sample was run in duplicate, and the expression level of each gene was analyzed on computer (SDS2.3 software; ABI). Only the genes with reproducible curves of both duplicates were analyzed and presented.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Structural Changes
Significant differences are seen between the cocultured NTM5 cells and their control (Fig. 1A) , mainly in their volume, size, and cell–cell interactions. The cells exhibit enlargement in size and volume and fewer contacts between cells. In addition, they show accumulation of granules (black spots) and reduction in vesicles.


Figure 1
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  		FIGURE 1. (A) Scanning electron micrographs of NTM5 cells cocultured with LECs. NTM5 cells grown alone served as the control. (B) Light micrographs of primary TM cells cocultured with primary LECs (primary TM cells grown alone served as the control). Magnification, x100.

 
The cocultured primary TM cells show similar alternations in morphology compared with the control (Fig. 1B) , specifically in their enlarged volume and shape.

Differential Protein Expression
Changes in protein expression after coculture were observed (Fig. 2A) . The LC-MS-MS analysis of the differentially appearing bands identified the proteins and indicated the direction of their change, as the number of peptides of each identified protein was proportional to its concentration in the sample. The most differently expressed proteins were filamins, myosins, tropomyosins, and spectrin, which showed an increased concentration in cocultured cells compared with the control. Western blot analysis for filamin A and myosin heavy chain (Fig. 2B) confirmed these results, as their concentration in cocultured TM cells increased by 32% and 55%, respectively, compared with the control (according to densitometry analysis).


Figure 2
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  		FIGURE 2. SDS-PAGE of proteins from NTM5 cells cocultured with LECS for 2 weeks (Coculture) and from NTM5 cells grown alone (Control) (A). The marked bands were excised, digested by trypsin, and subjected to LC-MS-MS analysis. The identified proteins from the outlined upper and lower bands are listed in the top and bottom boxes, respectively. Increased (decreased) concentration of the protein is shown by the {uparrow} ({downarrow}) respectively. The corresponding gene symbol is mentioned in parentheses for future references. Western blot of filamin A (FLNA) and myosin heavy chain, nonmuscle, (MYH) of proteins from NTM5 cells (coculture and control) was performed to validate the results of the MS-MS analysis. GAPDH served as the internal control (B).

 
DAVID-EASE molecular functional analysis categorized the changed proteins as follows: binding (66.7%, P = 3.19E-02), protein binding (52.4%, P = 5.66E-04), calmodulin binding (14.3%, P = 4.49E-03), cytoskeletal protein binding (38.1%, P = 4.42E-09), and actin binding (38.1%, P = 3.73E-10).

Differential Gene Expression
The gene expression profile of cocultured NTM5 cells was compared to that of the control by using gene chips (Affymetrix). After coculture 400 genes were found to be upregulated, and 566 genes were downregulated at the level of P < 0.01. Of these, the most changed genes are given in Table 1 (upregulated) and Table 2 (downregulated). Real-time PCR (TaqMan; ABI) confirmed the changes observed by the microarray analysis. The relative quantity (RQ) and the ID of each gene (Assay-on-Demand; ABI) are denoted in Tables 1 and 2 .


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  		TABLE 1. Upregulated Genes Showing Significant Differences in Expression between Cocultured NTM5 Cells and the Control, with Confirmation by Real-Time PCR

 

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  		TABLE 2. Downregulated Genes Showing Significant Differences in Expression between Cocultured NTM5 Cells and the Control, with Confirmation by Real-Time PCR

 
Table 3 presents the functional distribution of all the differentially expressed genes, as analyzed by the gene set enrichment tool. The most affected cellular components were the extracellular space, vesicle, and actin cytoskeleton. The coculture conditions seem to alter biological processes, such as organ and cell morphogenesis, inflammatory response, vasculature development, taxis, cell proliferation and growth, response to stimulus, ion homeostasis, and several signaling pathways. Many of the changed genes are molecularly functioning in binding, extracellular matrix, cytokine activity, growth factor, and protease inhibitor activities. The more sophisticated enrichment analysis with the GOrilla tool17 yielded similar results (see the Supplementary Material online at http://www.iovs.org/cgi/content/full/49/9/3981/DC1).


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  		TABLE 3. Gene Enrichment Analysis of the Changed Genes in Cocultured NTM5 Cells

 
Differential gene expression analysis of the primary cocultured TM cells (N17RM.2) by real-time PCR (Table 4) revealed very high similarity to the transformed cells, emphasizing the possible role of cytoskeletal alternations inflammatory and acute phase response, the MAP kinase signal transduction pathway, the response to biotic stimulus, and the involvement of growth factors and their receptors in the development of the aphakic glaucoma.


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  		TABLE 4. Differential Gene Expression of Cocultured Primary Human TM Cells (N17RM.2) Compared with the Control, by Real-Time PCR

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Subjecting normal TM cells to the presence of LECs resulted in changes in their protein and gene expression, as well as in their structural features. Many of the changes resemble alternations seen in ocular tissues of patients with primary open-angle glaucoma.18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

We report that the most changed proteins were the cytoskeletal proteins myosin, filamin A and B, tropomyosin 3 and 4, and spectrin. Cytoskeletal changes in TM cells have been well studied.18 19 20 Glaucomatous TM cells present alternations in their microfilament structure of the cytoskeleton, and have more cross-linked actin networks, suggesting a major reorganization of the microfilament structure.20 The changes seen in the actin cytoskeleton proteins are supported both by the gene expression and structural analyses, as the cytoskeleton is involved with cell shape, volume, and adhesion to neighboring cells and to the ECM.18 Tropomyosin was found to be increased after treatment of TM cells with TGF-β,21 which is increased in the aqueous humor of patients with glaucoma.22 23 Spectrin helps maintain cell shape.29 Thus, when spectrin is increased, cells are less easily deformed. This increase observed in the cells could be a defense response to minimize the deformations caused by the coculture conditions, as seen by the SEM analysis. Coculture also led to alternations in calmodulin binding, which is known to affect the cytoskeletal interactions, hence providing additional support of the cytoskeletal changes seen in the TM cells after coculture.

Coculture conditions seem to create chemical and biotic stimulus, as indicated by the gene expression analysis, implying a potential role of factors secreted by LECs in the development of aphakic glaucoma.

Genes altered after coculture were involved in inflammatory response (upregulation of chemokine [C-X-C motif] ligands 5, 6, 3, and 1), and acute-phase response (upregulated TNF-{alpha}-induced proteins 3 and 6, IL-8, and complement component 3). These biological processes were found to be altered in a comparison of normal and glaucomatous TM tissue, which revealed an upregulation of chemokine (C-X-C motif) ligand 6, chemokine (C-X-C motif) ligand 5, and several additional genes coding for proteins involved in inflammation and acute-phase response.26 Of note, this comparison was made with TM tissues from eyes with primary open-angle glaucoma (POAG) originating from patients who also had cataracts.

Coculture led to a decrease in proenkephalin (PENK). PENK has been reported to be one of the genes downregulated in cultured human TM cells mapping in glaucoma loci (GLC1D).26 This finding suggests its importance in the development of aphakic glaucoma.

Coculture activated the I-{kappa}B kinase/NF-{kappa}B cascade, in accordance with a stress response model that suggests activation of a defense mechanism through NF-{kappa}B, followed by further activation of inflammatory cytokines that would then contribute to the cell damage observed in glaucoma.27 28

Coculture downregulated DUSP4 and DUSP5 which are thought to play a role in the MAP kinase signal transduction pathway.38 MAP kinase was shown to be downregulated in glaucomatous TM tissue.26 The gene expression analysis indicated changes in the ion homeostasis, di- and trivalent inorganic cation homeostasis, and iron ion binding of the TM cells, as a result of the coculture. It was shown that volume-regulatory ion flux pathways such as Na-K-Cl cotransport may be involved in the reduced outflow of glaucoma.30 It was also suggested that there is an involvement of iron metabolism in the pathogenesis of glaucoma.31

Growth factor activity was affected by the coculture conditions. It has been suggested that growth factors and growth factor receptors play an important role in the pathophysiology of POAG.32

Coculture seemed to trigger genes associated with vasculature development and angiogenesis. Previous reports have postulated the involvement of vascular factors in the pathogenesis of POAG.33 Moreover, TGF-β2, which can affect angiogenesis in the anterior segment, has been found to be elevated in the aqueous of patients with glaucoma.34

POAG correlates not only with biochemical but also with ultrastructural changes in the TM. Coculture led to two main structural changes: volume and size enlargement of the TM cells and altered cell–cell interactions. Previous SEM analysis suggested that swelling of the TM cells could significantly obstruct outflow through the trabecular beams.35 It was also shown that cell volume of glaucomatous TM cells is greater than that of normal TM cells.30 Furthermore, it was suggested that aqueous outflow could be impaired by changes in the connections between TM cells.36 Alternations in vesicles can be observed both by the SEM and gene expression analyses. Vesicles mark phagocytosis, which, in TM cells, plays an important role in the self cleaning of the outflow pathway.28 Reduction in TM cell phagocytosis is associated with certain types of glaucoma, such as steroid-induced glaucoma.37

Overall, observing all these changes implies that LECs affect the TM cells’ functionality, and may be responsible for the development of aphakic glaucoma. In future studies, the effect of young and adult LECs on TM cells will be compared, to examine the possibility that the age of the LECs is the critical factor leading to the development of the aphakic glaucoma. This possibility is supported by studies suggesting that LECs exhibit a loss in their functional physiology with age39 and that there is an age-related decrease in cell density. In addition, the interactions between infant TM cells, which were found to differ from adult cells,28 40 and LECs should be studied.


   Acknowledgements
 
The authors thank Orna Geyer and Alvit Wolf (Carmel Hospital, Haifa, Israel) for helpful advice and discussions.


   Footnotes
 
Supported by the Children’s Glaucoma Foundation, and in part by the Russell Berrie Nanotechnology Institute at the Technion-Israel Institute of Technology.

Submitted for publication January 3, 2008; revised April 16, 2008; accepted July 21, 2008.

Disclosure: I. Michael, None; M. Shmoish, None; D.S. Walton, None; S. Levenberg, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Shulamit Levenberg, Technion-Israel Institute of Technology, Silver Building, Room 265, Haifa 32000, Israel; shulamit{at}bm.technion.ac.il.


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