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Characterization of a spontaneously immortalized bovine trabecular meshwork cell line
Experimental Eye Research 105 (2012) 53e59
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Characterization of a spontaneously immortalized bovine trabecular meshwork cell line Weiming Mao a, *, Yang Liu a, Avani Mody a, Michela Montecchi-Palmer a, b, Robert J. Wordinger a, Abbot F. Clark a a
Department of Cell Biology and Anatomy, North Texas Eye Research Institute, University of North Texas Health Science Center, CBH449, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, USA Alcon Research, Ltd., Fort Worth, Texas, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2012 Accepted in revised form 18 October 2012 Available online 29 October 2012
Trabecular meshwork (TM) cells have widely been used as an in vitro model for glaucoma research. However, primary TM cells suffer the disadvantages of limited cell numbers and slow rates of proliferation. We discovered a spontaneously transformed bovine TM (BTM) cell line, BTM-28T. This cell line proliferated rapidly in low-glucose culture medium but also demonstrated contact inhibition in highglucose culture medium. BTM-28T cells expressed key TM cell markers including a-smooth muscle actin (a-SMA), laminin and collagen IV (col IV). Also, 100 nM dexamethasone (DEX) enhanced the formation of cross-linked actin networks (CLANs) in confluent BTM-28T cell cultures. Transforming growth factor beta 2 (TGFb2) induced the expression of fibronectin (FN), plasminogen activator inhibitor1 (PAI-1), and connective tissue growth factor (CTGF) in our cell cultures. This cell line will be helpful to better understand the aqueous humor outflow pathway as related to the pathophysiology of glaucoma. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: glaucoma glaucoma model trabecular meshwork spontaneously immortalized cells cross-linked actin networks TGFb2
1. Introduction Primary open angle glaucoma (POAG) is a major subtype of glaucoma that leads to visual impairment in millions of people worldwide (Resnikoff et al., 2004). Although the precise disease mechanism(s) of POAG is still not clear, elevated intraocular pressure (IOP) is the most important risk factor for the development and progression of glaucoma (AGIS, 2000; Heijl et al., 2002; Kass et al., 2002; Lichter, 2002). In POAG patients, IOP elevation results from altered TM function, which leads to increased aqueous humor outflow resistance (Tektas and Lutjen-Drecoll, 2009). Glaucomatous changes of the TM consist of loss of TM cells, increased extracellular matrix deposition and fusion of TM beams (Tektas and Lutjen-Drecoll, 2009). Therefore, cultured TM cells are frequently
Abbreviations: a-SMA, alpha-smooth muscle actin; BTM, bovine trabecular meshwork; CLANs, cross-linked actin networks; col IV, collagen IV; CTGF, connective tissue growth factor; DEX, dexamethasone; GAPDH, glyceraldehyde 3phosphate dehydrogenase; HTM, human trabecular meshwork; HRP, horseradish peroxidase; FN, fibronectin; IOP, intraocular pressure; PAI-1, plasminogen activator inhibitor-1; POAG, primary open angle glaucoma; RT, room temperature; TGFb2, transforming growth factor beta 2; TM, trabecular meshwork. * Corresponding author. Tel.: þ1 817 735 0564; fax: þ1 817 735 2637. E-mail address: [email protected] (W. Mao). 0014-4835/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2012.10.007
used as a model to better understand aqueous outflow biology and glaucoma. TM cells from human, monkey, porcine and bovine eyes have been isolated for glaucoma research. Although primate TM cells are an ideal choice, their application is limited by the lack of donors, slow proliferation rate, as well as limited passage numbers. An alternative to primate TM cells is BTM cells. Studies have shown that BTM behaves similarly to human TM (HTM) (O’Reilly et al., 2011). For example, we found that like human eyes, a subpopulation of bovine eyes can develop ocular hypertension upon DEX treatment (Mao et al., 2011). Also, DEX and TGFb2 induce the formation of CLANs in both HTM (Clark et al., 1994) as well as BTM cells (O’Reilly et al., 2011; Wade et al., 2009). In contrast to primate TM cells, BTM cells are easy to collect and they proliferate rapidly. However, like all the primary cells, BTM cells will eventually become senescent, and using different cell strains may affect the consistency of experiments. We discovered, for the first time, a spontaneously immortalized cell line (BTM-28T) derived from primary BTM cell cultures (BTM28). We characterized this cell line in terms of its morphology, proliferation capacity, TM cell markers, DEX-induced CLAN formation, and TGFb2-induced expression of ECM-related molecules. This cell line will be a useful tool for the study of the role of the TM in POAG.
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2. Materials and methods 2.1. BTM cell culture Calf eyes were obtained from a local abattoir and transported to the laboratory on ice. Eyes were processed within 6 h after death. The BTM tissue was carefully dissected, cut into small pieces, and placed in a 12-well plate containing DMEM-low glucose medium supplemented with 10% fetal bovine serum (Atlas, Fort Collins, CO) as well as glutamine and antibiotics (Thermoscientific, Worcester, MA). After several days, BTM cells migrated onto the plate and the tissue was removed. Culture medium was changed every other day. When cells were w90% confluent, they were trypsinized and passaged at a ratio of 1:2. In some experiments, BTM-28T cells were cultured in DMEM-high glucose medium (Thermoscientific) supplemented with either 10% or 0.5% fetal bovine serum. 2.2. Karyotyping BTM-28T cells were cultured in 100 mm dishes until they were 50% confluent. Cells were treated with 10 mg/ml Colcemid (Invitrogen, Carlsbad, CA) for 12e18 h to synchronize and facilitate karyotyping. Colcemid treated cells were washed with PBS, trypsinized, and pelleted at 2000 rpm for 2 min. After an additional PBS wash, cells were treated with 0.56% KCl at room temperature (RT) for 15 min. Cells were pelleted at 2000 rpm for 2 min, re-suspended in cold methanoleacetic acid mixture (3:1 mixed, respectively), and incubated on ice for 10 min. After pelleting the cells at 3000 rpm for 2 min, cells were re-suspended in 200 ml ice-old methanoleacetic acid mixture. About 50e100 ml cell suspension was dropped onto glass slides from a height of 1.5 m. Spread chromosomes were stained with Giemsa (Invitrogen) or DAPI, and were examined by microscopy. 2.3. Immunocytochemistry BTM cells were cultured on coverslips in 24-well plates. Cells were fixed in 4% paraformaldehyde at 4 C for 30 min. After a PBS wash, cells were incubated with 0.5% Triton X-100 in PBS at RT for 30 min, and then blocked with Superblock (Thermoscientific). Cells were incubated with the primary antibody at RT for 2 h or 4 C overnight and the corresponding secondary antibody at RT for 1 h. After three PBS washes, cells were mounted with Anti-fade Prolong Gold with DAPI (Invitrogen). Primary antibodies included: rabbit anti-laminin (1:100, Sigma, St. Louis, MO), rabbit anti-col IV (1:100, Sigma), mouse anti-a-SMA conjugated with FITC (1:100, Sigma), and rabbit anti-Ki67 (1:100, Abcam, Cambridge, MA). The secondary antibody was goat antirabbit Alexa-488 (1:200, Invitrogen).
100 nM DEX (Sigma) in DMEM-high glucose medium for 10 days. Cells were processed as described in “Immunocytochemistry” except that phalloidin-Alexa-568 (1:100, Invitrogen) was used to stain actin stress fibers at RT for 41 h or 4 C overnight. CLANs were defined as web-like structures containing at least three triangles (O’Reilly et al., 2011; Wade et al., 2009). CLAN formation rate was expressed as a ratio of CLAN positive cells (i.e. CLAN positive cells/ total number of cells as shown by DAPI staining). For each coverslip, 5 regions were counted with each region containing 80e200 cells. CLANs were counted in a masked manner. CLAN formation was compared using the Student’s t-test. P values less than 0.050 are considered significant. 2.6. Western immunoblotting (WB) BTM-28T cells were cultured in the 12-well plate in DMEM-low glucose medium with serum until they were confluent. Culture medium was then changed with serum free medium. After 24 h incubation, cells were treated with or without 5 ng/ml TGFb2 (R&D systems, Minneapolis, MN) for an additional 24 h. Conditioned medium and whole cell lysate were collected. For whole cell lysate collection, cells were lysed in M-PER lysis buffer with proteinase inhibitors (Thermoscientific) after a PBS wash. Protein concentrations were measured by using the DC protein assay kit (Bio-rad, Hercules, CA). 10 ml conditioned medium or 20 mg whole cell lysate were used for electrophoresis on the 4e15% SDS-PAGE gradient gel (Bio-rad). Proteins were transferred to the PVDF membrane. After blocking with 5% dry milk, the blots were incubated with the primary and secondary antibodies for each target protein. Signals were developed by using the SuperSignal West Femto Substrate kit (Thermoscientific), and were detected using the FluoroChem imaging system (Cell Biosciences, Santa Clara, CA). 2.6.1. Primary antibodies Rabbit anti-FN (1:500, Millipore, Billerica, MA) Mouse anti-PAI-1 (1:200, Santa Cruz, Santa Cruz, CA) Goat anti-CTGF (1:200, Santa Cruz) Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAP DH) (1:2000, Cell signaling, Danvers, MA) 2.6.2. Secondary antibodies Donkey anti-goat conjugate with horseradish peroxidase (HRP) (1:10,000, Santa Cruz) Goat anti-rabbit or anti-mouse conjugate with HRP (1:2000, Thermoscientific) 3. Results 3.1. Proliferation of spontaneously transformed BTM cells
2.4. Cell number counting BTM cells stained with Ki 67 and DAPI were counted in a masked manner using a Nikon epifluorescent microscope (Nikon, Melville, NY). For BTM-28 or BTM-28T cells, 6 coverslips of cells were prepared (n ¼ 6). For each coverslip, five representative regions were counted. For each region, we counted at least 200 to 400 cells. The percentage of Ki 67 positive cells was calculated (Ki 67 positive cells/DAPI positive cells), and subjected to Student’s t-test. P value less than 0.050 was considered significant. 2.5. Induction of CLANs in the BTM Confluent BTM cells cultured on coverslips in 24-well culture plates were treated with 0.1% ethanol (ETH) as a vehicle control or
During the culture of BTM-28 cells, we noticed that the cells in one culture dish demonstrated a sudden acceleration in growth. These cells had a doubling time of 2 days prior to reaching a passage number of 42. After p42, the doubling time increased to 3 days. However, other BTM-28 cells originating from the same bovine eye grew much slower with a doubling time ranging from 4 to 7 days and frequently became senescent, even at low passage numbers (
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Fig. 1. Elevated Ki67 expression in BTM-28T cells. There were more Ki67 positive cells in p49 BTM-28T cells (C) compared to non-transformed BTM-28 cells (A). (A) and (B): nontransformed primary BTM-28 cells stained with Ki67 (A) and DAPI (B). (C) and (D): transformed BTM-28T cells stained with Ki67 (C) and DAPI (D). Magnification: 200. Scale bar ¼ 200 mm. (E) Statistical analysis of Ki67 positive cells. Columns and error bars represent means and standard deviations, respectively. ***p < 0.001.
“longevity” of mammalian primary cells indicated spontaneous immortalization of these BTM cells. Therefore, we named the spontaneously transformed cells “BTM-28T” derived from the nontransformed BTM cell strain “BTM-28”. 3.2. The karyotype of BTM28-T cells Cell contamination can occur when more than one cell line/ strain are cultured in a laboratory. Since our laboratory routinely uses transformed HTM cells (Pang et al., 1994), we determined whether the immortalization might be due to contamination by transformed HTM cells. Because all our transformed cell lines are of human origin, we performed a karyotyping study to confirm the cell source. We observed a karyotype of 2n ¼ 60 in BTM-28T cells (Fig. 2), showing that these cells are of bovine but not human origin.
3.3. BTM-28T cells are morphologically similar to BTM-28 cells BTM-28T cells are oval to elongated in shape with overlapping processes, which is similar to BTM-28 cells. However, BTM-28T cells appear to be a little smaller and thinner than BTM-28 cells (Fig. 3). Also, neither BTM-28T or BTM-28 resembled fibroblast cells, which have a characteristic growth pattern of forming superimposed layers (Tripathi and Tripathi, 1982). 3.4. BTM-28T cells can be maintained in high-glucose medium One common problem often encountered with transformed cells is the loss of contact inhibition. Their unlimited growth makes the maintenance of those cells at confluent or nearly confluent status very difficult. This changes the underlying biology of contact
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these characteristics, we studied the expression of these three proteins in cultured BTM-28T cells. We found that similar to BTM28 cells, all three proteins were highly expressed in BTM-28T cells (Fig. 4).
3.6. DEX induces CLAN formation in BTM28-T cells
Fig. 2. The karyotype of BTM-28T cells. The chromosomes of a P49 BTM-28T cell were Giemsa-stained. A representative image with 2n ¼ 60 chromosomes is shown. Magnification: 1000. Scale bar ¼ 25 mm.
inhibition and limits the application of transformed cells in experiments that require long-term treatment. Different from virally transformed HTM cells, confluent BTM28T cells can be maintained in DMEM-high glucose medium supplemented with fetal bovine serum (0.5% or 10%) for more than 10 days without additional proliferation or noticeable cell loss. This appears to be due to cellecell contact inhibition because BTM-28T cells can still proliferate in high glucose medium when plated at low confluency. 3.5. BTM-28T cells express TM cell markers TM cells express a-SMA (Clark et al., 1994), laminin and col IV (Tamm et al., 1999). More importantly, laminin and col IV are not expressed in ocular fibroblast cells (Hernandez et al., 1987), which could be a major source of contamination due to their anatomical proximity to the TM. To determine whether BTM-28T cells retain
Another important criterion for TM cell identification is the induction of CLANs by glucocorticoids, including DEX, in confluent TM cell cultures (Clark et al., 1994). CLANs are web-like structures consisting of hubs and spokes, and these special structures are observed in confluent TM cells as well as TM tissues. The formation of CLANs can be increased by DEX in HTM and BTM cells (Clark et al., 1994; Wade et al., 2009). We treated confluent BTM-28T cells with 100 nM DEX or 0.1% ETH as a vehicle control for 10 days. CLANs were visualized by phalloidin staining (Fig. 5). To quantitate CLAN formation, BTM28T cells containing CLANs (CLAN positive cells) as well as total cell numbers were counted in a masked manner. The ratios of CLAN positive cells over total cells were compared using the Student’s ttest. We found there was a statistically significant increase of CLANs in DEX treated BTM-28T cells (n ¼ 5), compared to ETH control (24.46 6.72% vs. 15.06 5.61%, p < 0.050; n ¼ 5 or 6). 3.7. TGFb2 induces the expression of glaucoma-associated proteins in BTM-28T cells TGFb2 is the key activator of the TGFb signaling pathway and is elevated in the aqueous humor, TM cells and TM tissues from glaucoma patients (Fuchshofer and Tamm, 2012). It induces the expression of ECM molecules (such as FN) (Wordinger et al., 2007), decreases ECM turnover by the up-regulation of PAI-1 (Fleenor et al., 2006), as well as enhances ECM cross-linking (TovarVidales et al., 2008), while CTGF is the key mediator downstream of TGFb2 (Junglas et al., 2009). All these changes contribute to the clogging of the outflow pathway and IOP elevation. Therefore,
Fig. 3. Morphology of BTM-28 and BTM-28T cells. (A) and (C): BTM-28. (B) and (D): BTM-28T. (A) and (B) are higher magnification images (400 vs. 200) of (C) and (D), respectively. Images were taken from live cell cultures using the Nikon inverted microscope with Hoffman modulation contrast optics (Nikon). Scale bar ¼ 100 mm.
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Fig. 4. BTM-28T cells expressed TM cell markers. BTM28 and BTM-28T cells both expressed TM cell markers including a-SMA (A and B), col IV (C and D), as well as laminin (E and F), respectively. A no primary antibody control was performed as a negative control, showing no non-specific staining (data not shown). In order to display more cells and more representative images, two magnifications were used with 200 for BTM-28 (A, C and E) and 400 for BTM-28T (B, D, and F) cells. Scale bar ¼ 100 mm.
TGFb2-induced expression of FN, PAI-1 as well as CTGF are often used as “behavioral tests” to identify TM cells. We treated BTM-28T cells with or without 5 ng/ml TGFb2 for 24 h and collected conditioned medium and whole cell lysate for analysis. We found that FN, PAI-1, and CTGF were significantly elevated in TGFb2-treated BTM-28T cells (n ¼ 3) (Fig. 6). 4. Discussion Most of our understanding about glaucoma pathogenesis derives from the use of glaucoma models. Glaucoma models can be divided into three categories: in vivo, ex vivo, and in vitro. In vivo
models use animals including mice, rats, rabbits, dogs, cats, cows, sheep and monkeys. They closely mimic certain aspects of the disease process such as pressure-induced damage to the optic nerve and retina, but these models can be time-consuming and expensive. Most of these models do not mimic glaucomatous damage to the TM. Ex vivo models use perfusion cultured eyes to study aqueous humor outflow mechanisms. Human (Johnson and Tschumper, 1987), monkey (Erickson-Lamy et al., 1990), porcine (Keller et al., 2008), and bovine (Mao et al., 2011) anterior segment/ eye perfusion cultures have been reported. These ex vivo models can mimic some aspects of glaucomatous damage to the outflow pathway (Clark et al., 1994; Wang et al., 2008; Wordinger et al.,
Fig. 5. DEX induced CLAN formation in BTM-28T cells. (A): Morphology of CLANs (circled area). Green (pseudocolor): phalloidin staining; blue: DAPI staining. (B): DEX elevated CLAN formation in BTM-28T cells, compared to ETH vehicle control. Columns and error bars: means and standard deviations, respectively. *p < 0.050. Scale bar ¼ 20 mm.
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Fig. 6. TGFb2 induced expression of FN, PAI-1, and CTGF. BTM-28T cell cultures were treated with 5 ng/ml TGFb2 for 24 h. Conditioned medium and whole cell lysate were collected for WB. GAPDH served as an internal loading control for whole cell lysate. Experiments were performed in triplicates (n ¼ 3).
pathogenesis of glaucoma. For example, elevated TGFb2 in glaucomatous eyes and its associated pathway activation lead to compromised ECM turnover and ocular hypertension (Fuchshofer and Tamm, 2012); the RhoA/ROCK pathway is one of the targets for the development of next generation of IOP lowering agents (Tian et al., 2009); the glucocorticoid receptor pathway mediates glucocorticoid-induced glaucoma (Clark and Wordinger, 2009); and elevated sFRP1 (the Wnt pathway inhibitor) and inhibition of the Wnt pathway cause IOP elevation (Mao et al., 2012; Wang et al., 2008). In summary, we established a spontaneously immortalized BTM cell line that is similar to primary BTM cells morphologically and biologically. We anticipate that this cell line will become a useful tool for glaucoma research as related to the TM. References
2007). In vitro models include TM cell cultures, which require minimum maintenance. TM cell cultures are also easier to manipulate than animals and perfusion cultured eyes with respect to over-expression and/or knockdown of genes. Therefore, cultured TM cells are often used as a tool for molecular biology and cell biology studies. Although primary TM cells can be passaged for a limited number of times, cell senescence is inevitable. This limited proliferation capability may cause problems in glaucoma research, not only because time and effort have been invested in establishing these cell strains, but even more importantly, the consistency of experiments may be compromised since cell strains can respond to stimuli differently. In order to achieve an “unlimited” source of TM cells, transformed TM cells have been constructed using the SV40 large T antigen. Two well-known examples of transformed TM cells are the GTM-3 and HTM-5 cell lines (Pang et al., 1994). SV40-transformed cell lines are biologically distinct from primary cells. These transformed cells usually show low requirement for serum, loss of contact inhibition, and tumor cell-like morphology (Bryan and Reddel, 1994). In glaucoma research, the widely-used GTM-3 and HTM-5 cells, which were transformed by SV-40, also “inherited” these drawbacks. For example, GTM-3 cells are cobblestone like with large nuclei, proliferate rapidly, and are prone to grow into multiple layers (Pang et al., 1994). Their protein expression profiles as well as intracellular messenger systems are also distinct from their non-transformed parent cells (Pang et al.,1994). In contrast, our spontaneously transformed BTM-28Tcells are oval to elongated in shape with overlapping processes, smaller nuclei, and more cytoplasm. It is very interesting that BTM-28T cells demonstrated contact inhibition when they were cultured in DMEM high-glucose medium, which was not observed in GTM-3 or HTM-5 cell lines. Biologically, BTM-28T cells seem to retain primary BTM cell properties. They expressed TM markers including a-SMA, laminin, and col IV. Hernandez and colleagues reported that the latter two proteins were not expressed in fibroblast cells (Hernandez et al., 1987). Besides the expression of TM markers, CLAN formation could also be induced by DEX in the BTM-28T cell line, although the induction rate (w60%) is somehow lower than that in certain primary BTM cell strains (up to w3 fold) (Wade et al., 2009). We believe that this difference is due to decreased cytoplasmic and microfibrillar content in BTM-28T cells. In addition to TM markers and CLAN formation, TGFb2 induced the expression of FN, PAI-1, and CTGF in BTM-28Tcells. These findings suggest that BTM-28Tcells behave similarly to primary TM cells. Although not examined in this study, we speculate that cell signaling mechanisms in these cells would also be similar to primary TM cells, which would make them suitable for the study of glaucomaassociated cell signaling pathways, such as the TGFb/BMP pathway, the RhoA/ROCK pathway, the glucocorticoid receptor pathway, and the Wnt pathway. These pathways all play important roles in the
AGIS, 2000. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am. J. Ophthalmol. 130, 429e440. Bryan, T.M., Reddel, R.R., 1994. SV40-induced immortalization of human cells. Crit. Rev. Oncog. 5, 331e357. Clark, A.F., Wilson, K., McCartney, M.D., Miggans, S.T., Kunkle, M., Howe, W., 1994. Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 35, 281e294. Clark, A.F., Wordinger, R.J., 2009. The role of steroids in outflow resistance. Exp. Eye Res. 88, 752e759. Erickson-Lamy, K., Schroeder, A.M., Bassett-Chu, S., Epstein, D.L., 1990. Absence of time-dependent facility increase (“washout”) in the perfused enucleated human eye. Invest. Ophthalmol. Vis. Sci. 31, 2384e2388. Fleenor, D.L., Shepard, A.R., Hellberg, P.E., Jacobson, N., Pang, I.H., Clark, A.F., 2006. TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Invest. Ophthalmol. Vis. Sci. 47, 226e234. Fuchshofer, R., Tamm, E.R., 2012. The role of TGF-beta in the pathogenesis of primary open-angle glaucoma. Cell Tissue Res. 347, 279e290. Heijl, A., Leske, M.C., Bengtsson, B., Hyman, L., Hussein, M., 2002. Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial. Arch. Ophthalmol. 120, 1268e1279. Hernandez, M.R., Weinstein, B.I., Schwartz, J., Ritch, R., Gordon, G.G., Southren, A.L., 1987. Human trabecular meshwork cells in culture: morphology and extracellular matrix components. Invest. Ophthalmol. Vis. Sci. 28, 1655e1660. Johnson, D.H., Tschumper, R.C., 1987. Human trabecular meshwork organ culture. A new method. Invest. Ophthalmol. Vis. Sci. 28, 945e953. Junglas, B., Yu, A.H., Welge-Lussen, U., Tamm, E.R., Fuchshofer, R., 2009. Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells. Exp. Eye Res. 88, 1065e1075. Kass, M.A., Heuer, D.K., Higginbotham, E.J., Johnson, C.A., Keltner, J.L., Miller, J.P., Parrish 2nd, R.K., Wilson, M.R., Gordon, M.O., 2002. The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch. Ophthalmol. 120, 701e713. discussion 829e730. Keller, K.E., Bradley, J.M., Kelley, M.J., Acott, T.S., 2008. Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture. Invest. Ophthalmol. Vis. Sci. 49, 2495e2505. Lichter, P.R., 2002. Impact of intraocular pressure reduction on glaucoma progression. JAMA 288, 2607e2608. Mao, W., Millar, J.C., Wang, W.H., Silverman, S.M., Liu, Y., Wordinger, R.J., Rubin, J.S., Pang, I.H., Clark, A.F., 2012. Existence of the canonical Wnt signaling pathway in the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 53, 7043e7051. Mao, W., Tovar-Vidales, T., Yorio, T., Wordinger, R.J., Clark, A.F., 2011. Perfusioncultured bovine anterior segments as an ex vivo model for studying glucocorticoid-induced ocular hypertension and glaucoma. Invest. Ophthalmol. Vis. Sci. 52, 8068e8075. O’Reilly, S., Pollock, N., Currie, L., Paraoan, L., Clark, A.F., Grierson, I., 2011. Inducers of cross-linked actin networks in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 52, 7316e7324. Pang, I.H., Shade, D.L., Clark, A.F., Steely, H.T., DeSantis, L., 1994. Preliminary characterization of a transformed cell strain derived from human trabecular meshwork. Curr. Eye Res. 13, 51e63. Resnikoff, S., Pascolini, D., Etya’ale, D., Kocur, I., Pararajasegaram, R., Pokharel, G.P., Mariotti, S.P., 2004. Global data on visual impairment in the year 2002. Bull. World Health Organ. 82, 844e851. Tamm, E.R., Russell, P., Piatigorsky, J., 1999. Development of characterization of a immortal and differentiated murine trabecular meshwork cell line. Invest. Ophthalmol. Vis. Sci. 40, 1392e1403. Tektas, O.Y., Lutjen-Drecoll, E., 2009. Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp. Eye Res. 88, 769e775. Tian, B., Gabelt, B.T., Geiger, B., Kaufman, P.L., 2009. The role of the actomyosin system in regulating trabecular fluid outflow. Exp. Eye Res. 88, 713e717. Tovar-Vidales, T., Roque, R., Clark, A.F., Wordinger, R.J., 2008. Tissue transglutaminase expression and activity in normal and glaucomatous human trabecular meshwork cells and tissues. Invest. Ophthalmol. Vis. Sci. 49, 622e628.
W. Mao et al. / Experimental Eye Research 105 (2012) 53e59 Tripathi, R.C., Tripathi, B.J., 1982. Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy. Exp. Eye Res. 35, 611e624. Wade, N.C., Grierson, I., O’Reilly, S., Hoare, M.J., Cracknell, K.P., Paraoan, L.I., Brotchie, D., Clark, A.F., 2009. Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells. Exp. Eye Res. 89, 648e659.
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Wang, W.H., McNatt, L.G., Pang, I.H., Millar, J.C., Hellberg, P.E., Hellberg, M.H., Steely, H.T., Rubin, J.S., Fingert, J.H., Sheffield, V.C., Stone, E.M., Clark, A.F., 2008. Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure. J. Clin. Invest. 118, 1056e1064. Wordinger, R.J., Fleenor, D.L., Hellberg, P.E., Pang, I.H., Tovar, T.O., Zode, G.S., Fuller, J.A., Clark, A.F., 2007. Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. Invest. Ophthalmol. Vis. Sci. 48, 1191e1200.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Characterization of a spontaneously immortalized bovine trabecular meshwork cell line Weiming Mao a, *, Yang Liu a, Avani Mody a, Michela Montecchi-Palmer a, b, Robert J. Wordinger a, Abbot F. Clark a a
Department of Cell Biology and Anatomy, North Texas Eye Research Institute, University of North Texas Health Science Center, CBH449, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, USA Alcon Research, Ltd., Fort Worth, Texas, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2012 Accepted in revised form 18 October 2012 Available online 29 October 2012
Trabecular meshwork (TM) cells have widely been used as an in vitro model for glaucoma research. However, primary TM cells suffer the disadvantages of limited cell numbers and slow rates of proliferation. We discovered a spontaneously transformed bovine TM (BTM) cell line, BTM-28T. This cell line proliferated rapidly in low-glucose culture medium but also demonstrated contact inhibition in highglucose culture medium. BTM-28T cells expressed key TM cell markers including a-smooth muscle actin (a-SMA), laminin and collagen IV (col IV). Also, 100 nM dexamethasone (DEX) enhanced the formation of cross-linked actin networks (CLANs) in confluent BTM-28T cell cultures. Transforming growth factor beta 2 (TGFb2) induced the expression of fibronectin (FN), plasminogen activator inhibitor1 (PAI-1), and connective tissue growth factor (CTGF) in our cell cultures. This cell line will be helpful to better understand the aqueous humor outflow pathway as related to the pathophysiology of glaucoma. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: glaucoma glaucoma model trabecular meshwork spontaneously immortalized cells cross-linked actin networks TGFb2
1. Introduction Primary open angle glaucoma (POAG) is a major subtype of glaucoma that leads to visual impairment in millions of people worldwide (Resnikoff et al., 2004). Although the precise disease mechanism(s) of POAG is still not clear, elevated intraocular pressure (IOP) is the most important risk factor for the development and progression of glaucoma (AGIS, 2000; Heijl et al., 2002; Kass et al., 2002; Lichter, 2002). In POAG patients, IOP elevation results from altered TM function, which leads to increased aqueous humor outflow resistance (Tektas and Lutjen-Drecoll, 2009). Glaucomatous changes of the TM consist of loss of TM cells, increased extracellular matrix deposition and fusion of TM beams (Tektas and Lutjen-Drecoll, 2009). Therefore, cultured TM cells are frequently
Abbreviations: a-SMA, alpha-smooth muscle actin; BTM, bovine trabecular meshwork; CLANs, cross-linked actin networks; col IV, collagen IV; CTGF, connective tissue growth factor; DEX, dexamethasone; GAPDH, glyceraldehyde 3phosphate dehydrogenase; HTM, human trabecular meshwork; HRP, horseradish peroxidase; FN, fibronectin; IOP, intraocular pressure; PAI-1, plasminogen activator inhibitor-1; POAG, primary open angle glaucoma; RT, room temperature; TGFb2, transforming growth factor beta 2; TM, trabecular meshwork. * Corresponding author. Tel.: þ1 817 735 0564; fax: þ1 817 735 2637. E-mail address: [email protected] (W. Mao). 0014-4835/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2012.10.007
used as a model to better understand aqueous outflow biology and glaucoma. TM cells from human, monkey, porcine and bovine eyes have been isolated for glaucoma research. Although primate TM cells are an ideal choice, their application is limited by the lack of donors, slow proliferation rate, as well as limited passage numbers. An alternative to primate TM cells is BTM cells. Studies have shown that BTM behaves similarly to human TM (HTM) (O’Reilly et al., 2011). For example, we found that like human eyes, a subpopulation of bovine eyes can develop ocular hypertension upon DEX treatment (Mao et al., 2011). Also, DEX and TGFb2 induce the formation of CLANs in both HTM (Clark et al., 1994) as well as BTM cells (O’Reilly et al., 2011; Wade et al., 2009). In contrast to primate TM cells, BTM cells are easy to collect and they proliferate rapidly. However, like all the primary cells, BTM cells will eventually become senescent, and using different cell strains may affect the consistency of experiments. We discovered, for the first time, a spontaneously immortalized cell line (BTM-28T) derived from primary BTM cell cultures (BTM28). We characterized this cell line in terms of its morphology, proliferation capacity, TM cell markers, DEX-induced CLAN formation, and TGFb2-induced expression of ECM-related molecules. This cell line will be a useful tool for the study of the role of the TM in POAG.
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2. Materials and methods 2.1. BTM cell culture Calf eyes were obtained from a local abattoir and transported to the laboratory on ice. Eyes were processed within 6 h after death. The BTM tissue was carefully dissected, cut into small pieces, and placed in a 12-well plate containing DMEM-low glucose medium supplemented with 10% fetal bovine serum (Atlas, Fort Collins, CO) as well as glutamine and antibiotics (Thermoscientific, Worcester, MA). After several days, BTM cells migrated onto the plate and the tissue was removed. Culture medium was changed every other day. When cells were w90% confluent, they were trypsinized and passaged at a ratio of 1:2. In some experiments, BTM-28T cells were cultured in DMEM-high glucose medium (Thermoscientific) supplemented with either 10% or 0.5% fetal bovine serum. 2.2. Karyotyping BTM-28T cells were cultured in 100 mm dishes until they were 50% confluent. Cells were treated with 10 mg/ml Colcemid (Invitrogen, Carlsbad, CA) for 12e18 h to synchronize and facilitate karyotyping. Colcemid treated cells were washed with PBS, trypsinized, and pelleted at 2000 rpm for 2 min. After an additional PBS wash, cells were treated with 0.56% KCl at room temperature (RT) for 15 min. Cells were pelleted at 2000 rpm for 2 min, re-suspended in cold methanoleacetic acid mixture (3:1 mixed, respectively), and incubated on ice for 10 min. After pelleting the cells at 3000 rpm for 2 min, cells were re-suspended in 200 ml ice-old methanoleacetic acid mixture. About 50e100 ml cell suspension was dropped onto glass slides from a height of 1.5 m. Spread chromosomes were stained with Giemsa (Invitrogen) or DAPI, and were examined by microscopy. 2.3. Immunocytochemistry BTM cells were cultured on coverslips in 24-well plates. Cells were fixed in 4% paraformaldehyde at 4 C for 30 min. After a PBS wash, cells were incubated with 0.5% Triton X-100 in PBS at RT for 30 min, and then blocked with Superblock (Thermoscientific). Cells were incubated with the primary antibody at RT for 2 h or 4 C overnight and the corresponding secondary antibody at RT for 1 h. After three PBS washes, cells were mounted with Anti-fade Prolong Gold with DAPI (Invitrogen). Primary antibodies included: rabbit anti-laminin (1:100, Sigma, St. Louis, MO), rabbit anti-col IV (1:100, Sigma), mouse anti-a-SMA conjugated with FITC (1:100, Sigma), and rabbit anti-Ki67 (1:100, Abcam, Cambridge, MA). The secondary antibody was goat antirabbit Alexa-488 (1:200, Invitrogen).
100 nM DEX (Sigma) in DMEM-high glucose medium for 10 days. Cells were processed as described in “Immunocytochemistry” except that phalloidin-Alexa-568 (1:100, Invitrogen) was used to stain actin stress fibers at RT for 41 h or 4 C overnight. CLANs were defined as web-like structures containing at least three triangles (O’Reilly et al., 2011; Wade et al., 2009). CLAN formation rate was expressed as a ratio of CLAN positive cells (i.e. CLAN positive cells/ total number of cells as shown by DAPI staining). For each coverslip, 5 regions were counted with each region containing 80e200 cells. CLANs were counted in a masked manner. CLAN formation was compared using the Student’s t-test. P values less than 0.050 are considered significant. 2.6. Western immunoblotting (WB) BTM-28T cells were cultured in the 12-well plate in DMEM-low glucose medium with serum until they were confluent. Culture medium was then changed with serum free medium. After 24 h incubation, cells were treated with or without 5 ng/ml TGFb2 (R&D systems, Minneapolis, MN) for an additional 24 h. Conditioned medium and whole cell lysate were collected. For whole cell lysate collection, cells were lysed in M-PER lysis buffer with proteinase inhibitors (Thermoscientific) after a PBS wash. Protein concentrations were measured by using the DC protein assay kit (Bio-rad, Hercules, CA). 10 ml conditioned medium or 20 mg whole cell lysate were used for electrophoresis on the 4e15% SDS-PAGE gradient gel (Bio-rad). Proteins were transferred to the PVDF membrane. After blocking with 5% dry milk, the blots were incubated with the primary and secondary antibodies for each target protein. Signals were developed by using the SuperSignal West Femto Substrate kit (Thermoscientific), and were detected using the FluoroChem imaging system (Cell Biosciences, Santa Clara, CA). 2.6.1. Primary antibodies Rabbit anti-FN (1:500, Millipore, Billerica, MA) Mouse anti-PAI-1 (1:200, Santa Cruz, Santa Cruz, CA) Goat anti-CTGF (1:200, Santa Cruz) Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAP DH) (1:2000, Cell signaling, Danvers, MA) 2.6.2. Secondary antibodies Donkey anti-goat conjugate with horseradish peroxidase (HRP) (1:10,000, Santa Cruz) Goat anti-rabbit or anti-mouse conjugate with HRP (1:2000, Thermoscientific) 3. Results 3.1. Proliferation of spontaneously transformed BTM cells
2.4. Cell number counting BTM cells stained with Ki 67 and DAPI were counted in a masked manner using a Nikon epifluorescent microscope (Nikon, Melville, NY). For BTM-28 or BTM-28T cells, 6 coverslips of cells were prepared (n ¼ 6). For each coverslip, five representative regions were counted. For each region, we counted at least 200 to 400 cells. The percentage of Ki 67 positive cells was calculated (Ki 67 positive cells/DAPI positive cells), and subjected to Student’s t-test. P value less than 0.050 was considered significant. 2.5. Induction of CLANs in the BTM Confluent BTM cells cultured on coverslips in 24-well culture plates were treated with 0.1% ethanol (ETH) as a vehicle control or
During the culture of BTM-28 cells, we noticed that the cells in one culture dish demonstrated a sudden acceleration in growth. These cells had a doubling time of 2 days prior to reaching a passage number of 42. After p42, the doubling time increased to 3 days. However, other BTM-28 cells originating from the same bovine eye grew much slower with a doubling time ranging from 4 to 7 days and frequently became senescent, even at low passage numbers (
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Fig. 1. Elevated Ki67 expression in BTM-28T cells. There were more Ki67 positive cells in p49 BTM-28T cells (C) compared to non-transformed BTM-28 cells (A). (A) and (B): nontransformed primary BTM-28 cells stained with Ki67 (A) and DAPI (B). (C) and (D): transformed BTM-28T cells stained with Ki67 (C) and DAPI (D). Magnification: 200. Scale bar ¼ 200 mm. (E) Statistical analysis of Ki67 positive cells. Columns and error bars represent means and standard deviations, respectively. ***p < 0.001.
“longevity” of mammalian primary cells indicated spontaneous immortalization of these BTM cells. Therefore, we named the spontaneously transformed cells “BTM-28T” derived from the nontransformed BTM cell strain “BTM-28”. 3.2. The karyotype of BTM28-T cells Cell contamination can occur when more than one cell line/ strain are cultured in a laboratory. Since our laboratory routinely uses transformed HTM cells (Pang et al., 1994), we determined whether the immortalization might be due to contamination by transformed HTM cells. Because all our transformed cell lines are of human origin, we performed a karyotyping study to confirm the cell source. We observed a karyotype of 2n ¼ 60 in BTM-28T cells (Fig. 2), showing that these cells are of bovine but not human origin.
3.3. BTM-28T cells are morphologically similar to BTM-28 cells BTM-28T cells are oval to elongated in shape with overlapping processes, which is similar to BTM-28 cells. However, BTM-28T cells appear to be a little smaller and thinner than BTM-28 cells (Fig. 3). Also, neither BTM-28T or BTM-28 resembled fibroblast cells, which have a characteristic growth pattern of forming superimposed layers (Tripathi and Tripathi, 1982). 3.4. BTM-28T cells can be maintained in high-glucose medium One common problem often encountered with transformed cells is the loss of contact inhibition. Their unlimited growth makes the maintenance of those cells at confluent or nearly confluent status very difficult. This changes the underlying biology of contact
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these characteristics, we studied the expression of these three proteins in cultured BTM-28T cells. We found that similar to BTM28 cells, all three proteins were highly expressed in BTM-28T cells (Fig. 4).
3.6. DEX induces CLAN formation in BTM28-T cells
Fig. 2. The karyotype of BTM-28T cells. The chromosomes of a P49 BTM-28T cell were Giemsa-stained. A representative image with 2n ¼ 60 chromosomes is shown. Magnification: 1000. Scale bar ¼ 25 mm.
inhibition and limits the application of transformed cells in experiments that require long-term treatment. Different from virally transformed HTM cells, confluent BTM28T cells can be maintained in DMEM-high glucose medium supplemented with fetal bovine serum (0.5% or 10%) for more than 10 days without additional proliferation or noticeable cell loss. This appears to be due to cellecell contact inhibition because BTM-28T cells can still proliferate in high glucose medium when plated at low confluency. 3.5. BTM-28T cells express TM cell markers TM cells express a-SMA (Clark et al., 1994), laminin and col IV (Tamm et al., 1999). More importantly, laminin and col IV are not expressed in ocular fibroblast cells (Hernandez et al., 1987), which could be a major source of contamination due to their anatomical proximity to the TM. To determine whether BTM-28T cells retain
Another important criterion for TM cell identification is the induction of CLANs by glucocorticoids, including DEX, in confluent TM cell cultures (Clark et al., 1994). CLANs are web-like structures consisting of hubs and spokes, and these special structures are observed in confluent TM cells as well as TM tissues. The formation of CLANs can be increased by DEX in HTM and BTM cells (Clark et al., 1994; Wade et al., 2009). We treated confluent BTM-28T cells with 100 nM DEX or 0.1% ETH as a vehicle control for 10 days. CLANs were visualized by phalloidin staining (Fig. 5). To quantitate CLAN formation, BTM28T cells containing CLANs (CLAN positive cells) as well as total cell numbers were counted in a masked manner. The ratios of CLAN positive cells over total cells were compared using the Student’s ttest. We found there was a statistically significant increase of CLANs in DEX treated BTM-28T cells (n ¼ 5), compared to ETH control (24.46 6.72% vs. 15.06 5.61%, p < 0.050; n ¼ 5 or 6). 3.7. TGFb2 induces the expression of glaucoma-associated proteins in BTM-28T cells TGFb2 is the key activator of the TGFb signaling pathway and is elevated in the aqueous humor, TM cells and TM tissues from glaucoma patients (Fuchshofer and Tamm, 2012). It induces the expression of ECM molecules (such as FN) (Wordinger et al., 2007), decreases ECM turnover by the up-regulation of PAI-1 (Fleenor et al., 2006), as well as enhances ECM cross-linking (TovarVidales et al., 2008), while CTGF is the key mediator downstream of TGFb2 (Junglas et al., 2009). All these changes contribute to the clogging of the outflow pathway and IOP elevation. Therefore,
Fig. 3. Morphology of BTM-28 and BTM-28T cells. (A) and (C): BTM-28. (B) and (D): BTM-28T. (A) and (B) are higher magnification images (400 vs. 200) of (C) and (D), respectively. Images were taken from live cell cultures using the Nikon inverted microscope with Hoffman modulation contrast optics (Nikon). Scale bar ¼ 100 mm.
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Fig. 4. BTM-28T cells expressed TM cell markers. BTM28 and BTM-28T cells both expressed TM cell markers including a-SMA (A and B), col IV (C and D), as well as laminin (E and F), respectively. A no primary antibody control was performed as a negative control, showing no non-specific staining (data not shown). In order to display more cells and more representative images, two magnifications were used with 200 for BTM-28 (A, C and E) and 400 for BTM-28T (B, D, and F) cells. Scale bar ¼ 100 mm.
TGFb2-induced expression of FN, PAI-1 as well as CTGF are often used as “behavioral tests” to identify TM cells. We treated BTM-28T cells with or without 5 ng/ml TGFb2 for 24 h and collected conditioned medium and whole cell lysate for analysis. We found that FN, PAI-1, and CTGF were significantly elevated in TGFb2-treated BTM-28T cells (n ¼ 3) (Fig. 6). 4. Discussion Most of our understanding about glaucoma pathogenesis derives from the use of glaucoma models. Glaucoma models can be divided into three categories: in vivo, ex vivo, and in vitro. In vivo
models use animals including mice, rats, rabbits, dogs, cats, cows, sheep and monkeys. They closely mimic certain aspects of the disease process such as pressure-induced damage to the optic nerve and retina, but these models can be time-consuming and expensive. Most of these models do not mimic glaucomatous damage to the TM. Ex vivo models use perfusion cultured eyes to study aqueous humor outflow mechanisms. Human (Johnson and Tschumper, 1987), monkey (Erickson-Lamy et al., 1990), porcine (Keller et al., 2008), and bovine (Mao et al., 2011) anterior segment/ eye perfusion cultures have been reported. These ex vivo models can mimic some aspects of glaucomatous damage to the outflow pathway (Clark et al., 1994; Wang et al., 2008; Wordinger et al.,
Fig. 5. DEX induced CLAN formation in BTM-28T cells. (A): Morphology of CLANs (circled area). Green (pseudocolor): phalloidin staining; blue: DAPI staining. (B): DEX elevated CLAN formation in BTM-28T cells, compared to ETH vehicle control. Columns and error bars: means and standard deviations, respectively. *p < 0.050. Scale bar ¼ 20 mm.
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Fig. 6. TGFb2 induced expression of FN, PAI-1, and CTGF. BTM-28T cell cultures were treated with 5 ng/ml TGFb2 for 24 h. Conditioned medium and whole cell lysate were collected for WB. GAPDH served as an internal loading control for whole cell lysate. Experiments were performed in triplicates (n ¼ 3).
pathogenesis of glaucoma. For example, elevated TGFb2 in glaucomatous eyes and its associated pathway activation lead to compromised ECM turnover and ocular hypertension (Fuchshofer and Tamm, 2012); the RhoA/ROCK pathway is one of the targets for the development of next generation of IOP lowering agents (Tian et al., 2009); the glucocorticoid receptor pathway mediates glucocorticoid-induced glaucoma (Clark and Wordinger, 2009); and elevated sFRP1 (the Wnt pathway inhibitor) and inhibition of the Wnt pathway cause IOP elevation (Mao et al., 2012; Wang et al., 2008). In summary, we established a spontaneously immortalized BTM cell line that is similar to primary BTM cells morphologically and biologically. We anticipate that this cell line will become a useful tool for glaucoma research as related to the TM. References
2007). In vitro models include TM cell cultures, which require minimum maintenance. TM cell cultures are also easier to manipulate than animals and perfusion cultured eyes with respect to over-expression and/or knockdown of genes. Therefore, cultured TM cells are often used as a tool for molecular biology and cell biology studies. Although primary TM cells can be passaged for a limited number of times, cell senescence is inevitable. This limited proliferation capability may cause problems in glaucoma research, not only because time and effort have been invested in establishing these cell strains, but even more importantly, the consistency of experiments may be compromised since cell strains can respond to stimuli differently. In order to achieve an “unlimited” source of TM cells, transformed TM cells have been constructed using the SV40 large T antigen. Two well-known examples of transformed TM cells are the GTM-3 and HTM-5 cell lines (Pang et al., 1994). SV40-transformed cell lines are biologically distinct from primary cells. These transformed cells usually show low requirement for serum, loss of contact inhibition, and tumor cell-like morphology (Bryan and Reddel, 1994). In glaucoma research, the widely-used GTM-3 and HTM-5 cells, which were transformed by SV-40, also “inherited” these drawbacks. For example, GTM-3 cells are cobblestone like with large nuclei, proliferate rapidly, and are prone to grow into multiple layers (Pang et al., 1994). Their protein expression profiles as well as intracellular messenger systems are also distinct from their non-transformed parent cells (Pang et al.,1994). In contrast, our spontaneously transformed BTM-28Tcells are oval to elongated in shape with overlapping processes, smaller nuclei, and more cytoplasm. It is very interesting that BTM-28T cells demonstrated contact inhibition when they were cultured in DMEM high-glucose medium, which was not observed in GTM-3 or HTM-5 cell lines. Biologically, BTM-28T cells seem to retain primary BTM cell properties. They expressed TM markers including a-SMA, laminin, and col IV. Hernandez and colleagues reported that the latter two proteins were not expressed in fibroblast cells (Hernandez et al., 1987). Besides the expression of TM markers, CLAN formation could also be induced by DEX in the BTM-28T cell line, although the induction rate (w60%) is somehow lower than that in certain primary BTM cell strains (up to w3 fold) (Wade et al., 2009). We believe that this difference is due to decreased cytoplasmic and microfibrillar content in BTM-28T cells. In addition to TM markers and CLAN formation, TGFb2 induced the expression of FN, PAI-1, and CTGF in BTM-28Tcells. These findings suggest that BTM-28Tcells behave similarly to primary TM cells. Although not examined in this study, we speculate that cell signaling mechanisms in these cells would also be similar to primary TM cells, which would make them suitable for the study of glaucomaassociated cell signaling pathways, such as the TGFb/BMP pathway, the RhoA/ROCK pathway, the glucocorticoid receptor pathway, and the Wnt pathway. These pathways all play important roles in the
AGIS, 2000. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am. J. Ophthalmol. 130, 429e440. Bryan, T.M., Reddel, R.R., 1994. SV40-induced immortalization of human cells. Crit. Rev. Oncog. 5, 331e357. Clark, A.F., Wilson, K., McCartney, M.D., Miggans, S.T., Kunkle, M., Howe, W., 1994. Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 35, 281e294. Clark, A.F., Wordinger, R.J., 2009. The role of steroids in outflow resistance. Exp. Eye Res. 88, 752e759. Erickson-Lamy, K., Schroeder, A.M., Bassett-Chu, S., Epstein, D.L., 1990. Absence of time-dependent facility increase (“washout”) in the perfused enucleated human eye. Invest. Ophthalmol. Vis. Sci. 31, 2384e2388. Fleenor, D.L., Shepard, A.R., Hellberg, P.E., Jacobson, N., Pang, I.H., Clark, A.F., 2006. TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Invest. Ophthalmol. Vis. Sci. 47, 226e234. Fuchshofer, R., Tamm, E.R., 2012. The role of TGF-beta in the pathogenesis of primary open-angle glaucoma. Cell Tissue Res. 347, 279e290. Heijl, A., Leske, M.C., Bengtsson, B., Hyman, L., Hussein, M., 2002. Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial. Arch. Ophthalmol. 120, 1268e1279. Hernandez, M.R., Weinstein, B.I., Schwartz, J., Ritch, R., Gordon, G.G., Southren, A.L., 1987. Human trabecular meshwork cells in culture: morphology and extracellular matrix components. Invest. Ophthalmol. Vis. Sci. 28, 1655e1660. Johnson, D.H., Tschumper, R.C., 1987. Human trabecular meshwork organ culture. A new method. Invest. Ophthalmol. Vis. Sci. 28, 945e953. Junglas, B., Yu, A.H., Welge-Lussen, U., Tamm, E.R., Fuchshofer, R., 2009. Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells. Exp. Eye Res. 88, 1065e1075. Kass, M.A., Heuer, D.K., Higginbotham, E.J., Johnson, C.A., Keltner, J.L., Miller, J.P., Parrish 2nd, R.K., Wilson, M.R., Gordon, M.O., 2002. The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch. Ophthalmol. 120, 701e713. discussion 829e730. Keller, K.E., Bradley, J.M., Kelley, M.J., Acott, T.S., 2008. Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture. Invest. Ophthalmol. Vis. Sci. 49, 2495e2505. Lichter, P.R., 2002. Impact of intraocular pressure reduction on glaucoma progression. JAMA 288, 2607e2608. Mao, W., Millar, J.C., Wang, W.H., Silverman, S.M., Liu, Y., Wordinger, R.J., Rubin, J.S., Pang, I.H., Clark, A.F., 2012. Existence of the canonical Wnt signaling pathway in the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 53, 7043e7051. Mao, W., Tovar-Vidales, T., Yorio, T., Wordinger, R.J., Clark, A.F., 2011. Perfusioncultured bovine anterior segments as an ex vivo model for studying glucocorticoid-induced ocular hypertension and glaucoma. Invest. Ophthalmol. Vis. Sci. 52, 8068e8075. O’Reilly, S., Pollock, N., Currie, L., Paraoan, L., Clark, A.F., Grierson, I., 2011. Inducers of cross-linked actin networks in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 52, 7316e7324. Pang, I.H., Shade, D.L., Clark, A.F., Steely, H.T., DeSantis, L., 1994. Preliminary characterization of a transformed cell strain derived from human trabecular meshwork. Curr. Eye Res. 13, 51e63. Resnikoff, S., Pascolini, D., Etya’ale, D., Kocur, I., Pararajasegaram, R., Pokharel, G.P., Mariotti, S.P., 2004. Global data on visual impairment in the year 2002. Bull. World Health Organ. 82, 844e851. Tamm, E.R., Russell, P., Piatigorsky, J., 1999. Development of characterization of a immortal and differentiated murine trabecular meshwork cell line. Invest. Ophthalmol. Vis. Sci. 40, 1392e1403. Tektas, O.Y., Lutjen-Drecoll, E., 2009. Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp. Eye Res. 88, 769e775. Tian, B., Gabelt, B.T., Geiger, B., Kaufman, P.L., 2009. The role of the actomyosin system in regulating trabecular fluid outflow. Exp. Eye Res. 88, 713e717. Tovar-Vidales, T., Roque, R., Clark, A.F., Wordinger, R.J., 2008. Tissue transglutaminase expression and activity in normal and glaucomatous human trabecular meshwork cells and tissues. Invest. Ophthalmol. Vis. Sci. 49, 622e628.
W. Mao et al. / Experimental Eye Research 105 (2012) 53e59 Tripathi, R.C., Tripathi, B.J., 1982. Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy. Exp. Eye Res. 35, 611e624. Wade, N.C., Grierson, I., O’Reilly, S., Hoare, M.J., Cracknell, K.P., Paraoan, L.I., Brotchie, D., Clark, A.F., 2009. Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells. Exp. Eye Res. 89, 648e659.
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Wang, W.H., McNatt, L.G., Pang, I.H., Millar, J.C., Hellberg, P.E., Hellberg, M.H., Steely, H.T., Rubin, J.S., Fingert, J.H., Sheffield, V.C., Stone, E.M., Clark, A.F., 2008. Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure. J. Clin. Invest. 118, 1056e1064. Wordinger, R.J., Fleenor, D.L., Hellberg, P.E., Pang, I.H., Tovar, T.O., Zode, G.S., Fuller, J.A., Clark, A.F., 2007. Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. Invest. Ophthalmol. Vis. Sci. 48, 1191e1200.