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TO study mouse trabecular meshwork (TM) and to develop a murine TM cell line. METHODS. Mouse TM in situ was studied by light and electron microscopy ...
Development and Characterization of an Immortal and Differentiated Murine Trabecular Meshwork Cell Line Ernst R. Tamm,1'2 Paul Russell? and Joram Piatigorsky1 PURPOSE. TO

study mouse trabecular meshwork (TM) and to develop a murine TM cell line.

Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was isolated from the H-2Kb-tsA58 transgenic mouse strain in which promoter sequences of the major histocompatibility complex H-2K0 class 1 gene are fused to sequences of the SV40 mutant temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-y, and the tsA58 gene product is active at 33°C (permissive conditions), but not at 37°C (nonpermissive conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcriptionpolymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1 cells were transfected with plasmid DNA.

METHODS.

The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin. MUTM-NEI/1 cells were immunoreactive for vimentin, aB-crystallin, and neural cell adhesion molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for osmooth muscle actin, whereas after 3 days of treatment with transforming growth factor-/3, almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular meshwork glucocorticoid-inducible protein, and aB-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA. RESULTS.

CONCLUSIONS. The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important tool for study of the expression of TM genes. (Invest Ophthalmol Vis Sci. 1999;4O:1392-l4O3)

P

rimary open-angle glaucoma (POAG) is commonly associated with intraocular pressure GOP) that is too high for the health of the optic nerve head. The reason for elevated IOP in POAG is an increase in outflow resistance in the trabecular meshwork (TM), the major site of aqueous humor outflow.1 A broad variety of structural changes have been described in the TM of glaucomatous eyes. Still, the major

From the 'Laboratory of Molecular and Developmental Biology and the 'Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland. Supported by Grants Ta 115/8-1 and Ta 115/11-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany; and the American Health Assistance Foundation, Rockville, Maryland (all to ERT). Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1997. Submitted for publication August 20, 1998; revised January 22, 1999; accepted February 16, 1999. Proprietary interest category: N. 2 Present address: Department of Anatomy II, University of Erlangen-Niirnberg, Universitatsstrasse 19, D-91054 Erlangen, Germany. Reprint requests: Ernst R. Tamm, Department of Anatomy II, University of Erlangen-Niirnberg, Universitiitsstrasse 19, D-91054 Erlangen, Germany.

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reason for the increase in trabecular resistance is unknown.2 A possible candidate in some cases of POAG is myocilin/trabecular meshwork-induced glucocorticoid response protein (MYOC/TIGR) that is produced in large amounts by cultured human TM cells after long-term treatment with dexamethasone.3 Mutations in the coding sequences of MYOC/TIGR have been described in patients with juvenile glaucoma, an autosomal dominant hereditary form of POAG, and in a small percentage of patients with nonhereditary late-onset POAG.4"6 Until now, other TM genes that are causatively involved in the pathogenesis of POAG have not been isolated. A major obstacle for die identification and the characterization of such genes has been the lack of a suitable animal model; species with a disease similar to POAG in humans have not been identified.7 A possibly appropriate animal model is one using the transgenic or gene knockout technique and the development of animals that express modified forms of candidate genes or that are deficient in such genes. Because such studies are usually performed in mice, we were interested in whether the structure and the cell biology of the murine TM is comparable to that in humans. Although some aspects of the biology of the murine TM have been studied,89 a detailed analysis of its morphology is lacking. Investigative Ophthalmology & Visual Science, June 1999, Vol. 40, No. 7 Copyright © Association for Research in Vision and Ophthalmology

IOVS, June 1999, Vol. 40, No. 7 In the present study, we investigated the structure of the murine outflow tissues by light microscopy and EM. In addition, to have an experimental tool for studies on gene expression in the TM, we developed and characterized a clonal, immortal, and differentiated murine TM cell line (MUTM-NEI/ 1). TM was isolated from the H-2Kb-tsA58 transgenic mouse strain in which the 5'-flanking sequences of the mouse major histocompatibility complex H-2Kb class 1 gene are fused to the early-region coding sequences of the simian virus 40 (SV 40) mutant temperature-sensitive (ts) strain tsA58, which encodes a thermolabile large tumor (T) antigen.10 The promoter is inducible by interferon (TFN)-y, and the tsA58 gene product is active at 33°C (permissive conditions) but not at 37°C (nonpermissive conditions). Our results indicate that the murine TM shared essential structural elements with the TM of primates and that MUTM-NEI/1 cells, when grown in nonpermissive conditions express typical characteristics of cultured human primary TM cells.

MATERIALS AND METHODS

EM of Mouse TM Normal FVB/N and C57BL/6J mice (age, 2-4 months) were killed by cervical dislocation. The eyes were enucleated and fixed overnight in a mixture of 2% glutaraldehyde, 4% paraformaldehyde, and 20% sucrose in cacodylate buffer (pH 7.4). After fixation, the eyes were dehydrated through graded alcohols and embedded in JB-4 glycol methacrylate (Piano, Wetzlar, Germany) or in Epon (Roth, Karlsruhe, Germany). Meridional semithin sections were cut through the pupillary- optic nerve plane on a microtome and stained with hematoxylin and eosin or with Richardson's stain.11 Ultrathin sections were treated with lead citrate and uranyl acetate and viewed using an electron microscope (model 902; Zeiss, Oberkochen, Germany). Cell Culture of Mouse TM H-2Kb-tsA48 heterozygote mice (genetic background CBA/ ca X C57B1/10) were obtained from Charles River (Wilmington, MA) and mated to FVB/N mice. Tail DNA from the resultant hybrid mice was isolated and assayed for the presence of the transgene using sense and antisense primers (5-CCATCTCCACAGTTTCACTTCTGCA-3' and 5'-GCAGTACATTGCATCAACACCAGGA-3') and the following reaction conditions: denaturation at 94°C for 90 seconds, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a final extension step at 72°C for 5 minutes. Mice harboring the transgene were killed at 2 months of age by cervical dislocation, and the eyes were enucleated. Methods for securing animal tissue were humane, included proper consent and approval, and complied with the National Institutes of Health Guidelines on the Care and Use of Animals in Research and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The eyes were bisected along the equator and the posterior half removed. The anterior half was placed on a sterile petri dish, corneal side down. Under a dissecting microscope (X40), the zonule was cut with fine scissors and the lens removed. One branch of a fine forceps was inserted in the suprachoroidal space of the specimen. The ciliary body was grasped and gently pulled away from the sclera while its attachments to the sclera were simultaneously cut with a fine knife. After removing of the ciliary body and the attached iris,

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the outflow tissue could be easily distinguished against the white background of the sclera as a pigmented circumferentially oriented band. This band was cut free, placed in a 35-mm sterile, laminin-coated plastic petri dish, and covered with a coverslip and 1 ml Dulbecco's modified Eagle's medium (DMEM). The medium, which was changed every second day, was supplemented with 20% fetal bovine serum (FBS), 20 /Ag/ml gentamicin (all Life Technologies, Gaithersburg, MD) and recombinant murine IFN-y (Sigma, St. Louis, MO) at a final concentration of 20 U/ml. Coating was performed by adding laminin from basement membrane of Engelbreth-HolmSwarm sarcoma (Sigma) to the culture medium at a final concentration of 10 /ng/ml. Cultures were incubated at 33°C in humidified air enriched with 10% CO2. Confluent cultures were trypsinized with 0.25% trypsin for a few minutes and transferred to uncoated culture flasks at split ratios of 1:4 to 1:10. For experimental assays, confluent cultures grown at 33°C (permissive conditions) were transferred to 37°C in the absence of IFN-y (nonpermissive conditions). For cell cloning, 96-well microtiter plates were used. Into the wells, 0.2 ml of a cell suspension was added, so that only approximately one in three wells received a viable cell. When colonies were seen in phase-contrast optics, they were examined to ensure only single colonies were present. When these single colonies grew to a sufficient size, they were trypsinized and transferred to larger culture vessels. Certain of these cell clones were selected because they showed a cellular phenotype comparable to that of human primary TM cell cultures. These cell clones were again cloned using the same procedure. For assessing growth rates, 2 X 104 cells were plated in 96-well microtiter plates (6 wells for each time point) and incubated at either 33°C or 37°C. At each time point (1 hour, 23 hours, 62 hours, and 73 hours), the number of viable cells in each well was determined using the protocol of Brasaemle and Attie.12 The number of viable cells at each time point (mean ± SD) was plotted against time. For EM, cells were grown in uncoated plastic petri dishes, fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epon. Polymerization was performed at 60°C. For treatment with transforming growth factor-/3, (TGFj3,), confluent cultures were treated for 3 days with 1 ng/ml human recombinant TGF-/3, (Sigma) which was added to DMEM supplemented with 20% FBS and 20 jixg/ml gentamicin (37°C).

Immunohistochemistry Cells were grown to confluence at 33°C in tissue culture chambers mounted on microslides (Permanox; Laboratory Tek; Nunc, Naperville, IL) and transferred to 37°C for 5 to 7 clays. For the detection of collagen types I, III, IV, and VI; laminin; fibronectin; and neural cell adhesion molecule (NCAM), the cells were fixed with a mixture of ether-ethanol (1:1, 10 minutes at — 20°C), for vimentin, desmin, and cytokeratins; with methanol (5 minutes at — 20°C); and with 4% phosphate-buffered saline (PBS)-paraformaldehyde for aB-crystallin (room temperature for 20 minutes). After fixation, the slides were washed three times (5 minutes each) in PBS and preincubated with SuperBlock (Pierce, Rockford, IL, 30 minutes). After preincubation, the slides were incubated overnight at room temperature with the primary antibodies listed in Table 1. After three washings in PBS, biotinylated secondary antibodies

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TABLE

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1. Antisera Used for Immunocytochemistry

Primary Antisera Collagen type I Collagen types III, IV, VI Laminin Fibronectin (cellular) Vimentin Desmin Pan-cytokeratin a-Smooth muscle actin aB-Crystallin Neural cell adhesion molecule

Source

Host Species

Dilution

Chemicon (Temecula, CA) Research Diagnostics (Flanders, NJ) Research Diagnostics Dako (Carpinteria, CA) Sigma (St. Louis, MO) Sigma Boehringer Mannheim (Mannheim, Germany) Sigma Sigma Sigma J. Horwitz (UCLA, CA) Chemicon

Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Mouse Mouse Mouse Mouse Rabbit

1:50 1:50 1:50 1:50 1:30 1:400 1:10 1:20 1:100 1:200 1:1000 1:100

Rat

Rabbit antibodies were polyclonal; mouse or rat antibodies were monoclonal.

against rabbit, rat, or mouse (depending on the origin of the primary antibody) immunoglobulins (Vector, Burlingame, CA) were added for 1 hour. The slides were washed again, covered with streptavidin-fluorescein isothiocyanate (Vector) for 1 hour, mounted in fluorescent mounting medium (Dako, Carpinteria, CA), and viewed with a microscope (Zeiss). Control experiments were performed using either PBS or preimmune serum instead of the primary antibody.

RNA Analysis Total RNA was isolated from cultured MUTM-NEI/1 cells using RNAzol (Tel Test, Friendswood, TX). For reverse transcription-polymerase chain reaction (RT-PCR), a one-step RT-PCR kit (PCR-Superscript, Life Technologies) was used according to the manufacturer's protocol. The RT-PCR reaction was performed in a total volume of 50 /xl for 30 minutes at 44°C (the RT step), followed by melting at 94°C for 2 minutes, then 30 cycles of 50 seconds' melting at 94°C, 75 seconds' annealing at 55°C, and 2 minutes' extension at 72°C. After the last cycle, the polymerization step was extended for 10 minutes so that all strands were completed. The primers were designed according to the published structures of the mouse genes encoding for aquaporin-1/ CHIP28,13 NCAM,14 myoc/tigr 1516 and glyceraldehyde-3phosphate dehydrogenase (G3PDH)17 and were added to a final concentration of 0.2 micromole. The sequences of the primer pairs were 5'-TGGAGGAGTGAAAGTGAG-3' and 5'CAAACACACACACACCAG-3' (position 1113-1793; product size 680 bp) for aquaporin-l/CHIP28, 5'-ACTCCTCTACCCTCACCATC-3' and 5'-GCCTCGTCGTTTTTATCC-3' (position 391-1012, product size 621 bp) for NCAM, 5'-AGCCTATAACAATCTCCTTC-3' and 5'-TCTCATCCACACTCCATAC-3' (position 472-869, product size 397 bp) for myoc/tigr, and 5'-CCCTTCATTGACCTCAAC-3' and 5'-TTCACACCCATCACAAAC-3' (position 146-447, product size 301 bp) for G3PDH, respectively. Control experiments were performed for each primer pair and RNA sample by omitting the RT step (30 minutes at 44°C). The primer sequences for myoc/ tigr crossed exon-intron boundaries. The RT-PCR amplification products were gel purified and sequenced with fluorescent dideoxynucleotides on an automated sequencer (Applied Biosystems model 310; Perkin Elmer, Hayward, CA).

For northern blot experiments, RNA was separated on a 2.2-M formaldehyde-1.2% agarose gel and blotted onto a membrane (Duralon; Stratagene, La Jolla, CA). After the transfer, the blot was cross linked (UV Stratalinker; Stratagene). Blots were hybridized with a 244-bp BamHl-Hindlll restriction fragment isolated from the mouse aB-crystallin gene (+2849 to +3092) containing much of exon III.18 The probe was labeled with 32P-dCTP using a random prime kit (Life Technologies). Prehybridizations were performed at 55°C for 1 hour and hybridizations at 55°C overnight using Hybrisol (Oncor, Gaithersburg, MD), according to the manufacturer's instructions (including 10% formamide in the hybridization solution). Membranes were washed twice for 15 minutes each with 2X SSC-0.1% SDS at 55°C and twice with 0.2X SSC-0.1% SDS at room temperature and autoradiographed (XAR5 film; Eastman Kodak; Rochester, NY) at — 80°C with an intensifying screen (1-2 days). To monitor the integrity of RNA, the relative amounts of RNA loaded on the gel, and the efficiency of transfer, membranes were hybridized to a cDNA probe for guinea pig 18S rRNA. mRNA size was estimated by reference to the mobility of RNA size markers (Life Technologies) stained with methylene blue. For oxidative stress experiments, confluent cultures were washed three times with serum-free medium and subsequently incubated with serum-free medium containing 200 (xM H2O2 for 1 hour. After this time the medium was changed and regular culture medium was added. Control cultures were incubated for 1 hour in serum-free medium without H2O2. Transfection Experiments Cells grown at 33°C were transfected with 2 fxg to 6 /xg pCMV/3 plasmid (Clontech) using lipofectAMINE (Life Technologies) according to the manufacturer's protocol. The cells were transferred to 37°C, and after 48 hours, cellular extract was prepared and a j3-galactosidase assay performed as described previously.19'20

RESULTS

Mouse TM In Situ In all sections examined, a circumferential outflow vessel, equivalent to Schlemm's canal in primates, was visible (Fig.

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FIGURE 1. Morphology of mouse TM in situ. (A) Light microscopy shows that a circumferential outflow vessel (asterisk), equivalent to Schlemm's canal in primates, is present in the chamber angle. The vessel is covered by a thin layer of TM (arrows; semithin section, hematoxylin-eosin stain). (B). Electron microscopy of mouse TM consists of two to four trabecular lamellae that are covered by a single layer of flat endothelial-like cells (arrows'). Between Schlemm's canal (asterisk) and the trabecular lamellae, there is a 2- to-5-jnm thick layer of loose connective tissue equivalent to the juxtacanalicular or cribriform meshwork in primate eyes (arrowheads). (C, D) Electron microscopy of the cribriform meshwork. Flat polygonal tnibecular cells (arrows) are embedded in afinefilamentousmatrix (arrowheads). The inner wall of the endothelium (E) of Schlemm's canal (asterisk) expresses distinct giant vacuoles (V). Scale bar, (A) 28 /urn; 5H a water channel; and myoc/tigr, 3 a protein of unknown function that is mutated in hereditary juvenile glaucoma and in some cases of late-onset POAG. In contrast to

kb

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NCAM, aquaporin-1 and myoc/tigr mRNAs are only expressed in nonpermissive conditions, a fact that may indicate that these genes are markers for differentiated TM cells. It has been reported that TM cells have some contractile properties in situ and in vitro. 3 2 ' 5 9 ' 6 1 In accordance with this, some cells of the human and bovine TM express a-sm-actin. 2 5 3 2 * 5 2 "^ In primary human TM cultures, the expression of ct-sm-actin can be induced on treatment with TGF-/3,.f"' Our results show that this is also the case for MUTM-NET/1 cells.

0.6



0.5

•-

0.4

-

Co 8h 12h 24h

2.37 — 1.35 —

0.3 •

0.2 0.1 •

18S — 1 0 fig FIGURE 8. Northern blot for cdJ-crystallin mRNA in MUTM-NEI/1 cells grown at 37°C without INI7-7 after oxidative stress (1 hour serum-free medium with 200 ptM hydrogen peroxide) and under control conditions. Twenty micrograms total RNA were loaded on each lane. The size of molecular markers is expressed in kilobases. Integrity and relative amounts of RNA were controlled by reprobing the membrane with a cDNA probe specific for 18S ribosomal RNA.

2.5 ug

6.0

pCMV[3 FIGURE 9. Transient transfection of MUTM-NEI/1 cells with plasmid pCMVfJ. /3-Galactosidase activities in transfected cells were determined using 50 (i\ cell extract and measuring optical density ( 0 0 ) at a wavelength of 460 A. Transfections were carried out three times, and the mean ± SD is shown.

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MUTM-NEI/1 cells express vimentin, the typical intermediate filament of TM cells,65 but not desmin the characteristic intermediate filament of ciliary muscle cells or cytokeratins, which are found in ciliary epithelial cells.66"69 Although all the MUTM-NEI/1 proteins that were investigated in this study are typical for TM cells, we are aware of the fact that none of these proteins is specific for TM per se. Nevertheless, we believe that the combined expression of all these TM markers necessarily supports the idea that the MUTM-NEI/1 cell line, when grown in nonpermissive conditions, consists of well differentiated TM cells. Furthermore, our findings indicate that the H-2kb-tsA58 transgenic mouse strain may be very useful in establishing immortal and differentiated cell lines from other ocular cell populations that are difficult to cultivate, such as corneal epithelium or endothelium, lens epithelium or retinal pigmented epithelium. The fact that MUTM-NEI/1 cells can be transfected by plasmid DNA indicates that this cell line may be valuable in studying the transcriptional regulation of TM genes. Such studies might involve the identification of DNA regulatory sequences important for the control of these genes. Possible candidates might be genes that show an increase in expression in POAG, such as type VI collagen,45 aB-crystallin, and myoc/ tigr.70 Because MUTM-NEI/1 cells are of murine origin, the regulation of TM genes and their products can be directly studied both in MUTM-NEI/1 cells and in transgenic mice using a single homologous system. The MUTM-NEI/1 cell line will be an invaluable tool for elucidating the complex pattern of TM gene expression including that of potential candidate genes for POAG. Acknowledgments The authors thank Elke Kretzschmar (Department of Anatomy, University of Erlangen-Niirnberg, Germany) for excellent help with electron microscopy; Rick Dreyfuss (Visual Arts and Photography, National Institutes of Health, Bethesda, Maryland) and Marco Gosswein (Department of Anatomy, University of Erlangen-Niirnberg, Germany) for expert assistance with photography; Joseph Horwitz (University of California, Los Angeles) for providing the antibodies for aB-crystallin, and Terete Borras (Duke University Eye Center, Durham, North Carolina) for providing the guinea pig 18S ribosomal RNA cDNA probe; and Ales Cvekl, Marc Kantorow, and Todd Kays for helpful discussions. References 1. Bill A, Maepea O. Mechanisms and routes of aqueous humor drainage. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmology: Basic Sciences. Philadelphia: WB Saunders; 1994:206-225. 2. Lutjen-Drecoll E, Rohen JW. Functional morphology of the trabecular meshwork. In: Tasman W, Jaeger EA, eds. Duane's Foundations of Clinical Ophthalmology. Philadelphia: JB Lippincott; 1992:1-33. 3. Nguyen TD, Chen P, Huang WD, Chen H, Johnson D, Polansky JR. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork ceUs. J Biol Chem. 1998;273:634l-6350. 4. Stone EM, Fingert JH, Alward WLM, et al. Identification of a gene that causes primary open angle glaucoma. Science. 1997;275:668670. 5. Adam MF, Belmouden A, Binisti P, et al. Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedinhomology domain of TIGR in familial open-angle glaucoma. Hum MolGen. 1997;12:2091-2097. 6. Alward WL, Fingert JH, Coote MA, et al. Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene. N Englf Med. 1998;338:1022-1027.

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