January 2010
Volume 51, Issue 1
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Physiology and Pharmacology  |   January 2010
Reduced Myocilin Expression in Cultured Monkey Trabecular Meshwork Cells Induced by a Selective Glucocorticoid Receptor Agonist: Comparison with Steroids
Author Affiliations & Notes
  • Bruce A. Pfeffer
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Charu A. DeWitt
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Mercedes Salvador-Silva
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Megan E. Cavet
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Francisco J. López
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Keith W. Ward
    From the Preclinical Pharmacology Department, Pharmaceutical Research and Development, Bausch & Lomb, Inc, Rochester, New York.
  • Corresponding author: Bruce A. Pfeffer, Preclinical Pharmacology, Pharmaceutical R&D, Bausch & Lomb, Inc, 1400 North Goodman Street, Rochester, NY 14609; bruce_pfeffer@bausch.com
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 437-446. doi:10.1167/iovs.09-4202
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      Bruce A. Pfeffer, Charu A. DeWitt, Mercedes Salvador-Silva, Megan E. Cavet, Francisco J. López, Keith W. Ward; Reduced Myocilin Expression in Cultured Monkey Trabecular Meshwork Cells Induced by a Selective Glucocorticoid Receptor Agonist: Comparison with Steroids. Invest. Ophthalmol. Vis. Sci. 2010;51(1):437-446. doi: 10.1167/iovs.09-4202.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To assess in vitro myocilin (MYOC) expression in trabecular meshwork (TM) cells exposed to BOL-303242-X, a selective glucocorticoid receptor (GR) agonist (SEGRA), in comparison with dexamethasone (DEX), and prednisolone acetate (PA).

Methods.: After drug treatment of monkey TM cultures, MYOC protein in conditioned media (CM) was measured by Western blot and densitometry. MYOC mRNA levels were analyzed by qRT-PCR. RU-486 was tested for antagonism of MYOC protein expression induced by DEX and BOL-303242-X.

Results.: Baseline MYOC protein released into CM and MYOC mRNA were detected. DEX or PA elicited dose-dependent increases in MYOC in CM and also in MYOC mRNA. BOL-303242-X effects typified partial agonism, with significantly reduced MYOC protein and mRNA, compared with DEX. Maximum efficacy for BOL-303242-X was 53% of that for DEX. Mean EC50 across all strains tested was lower, but not significantly different, for BOL-303242-X versus DEX. Compared with DEX, MYOC mRNA levels were significantly lower in BOL-303242-X–treated TM cells at the highest doses tested. EC50s for PA were higher than DEX, for both myocilin protein and mRNA. RU-486 displayed a dose-dependent antagonism to drug-induced increases in myocilin levels.

Conclusions.: In vitro quantitative assays of myocilin expression in TM cells can be used for characterizing anti-inflammatory drugs that are GR ligands. The results suggest that, compared with traditional ocular steroids, therapeutic doses of BOL-303242-X elicit a reduced myocilin expression profile in TM cells by virtue of the partial agonist properties of this compound.

Glucocorticoids (GCs) are often prescribed to treat a variety of ocular conditions that have an inflammatory component, such as macular edema, uveitis, and complications of surgery. 1,2 The therapeutic benefit of GCs is due to pleiotrophic modulation and mobilization of multiple intracellular signaling pathways, encompassing predominantly transrepressive effects of the steroid–nuclear receptor complex that interfere with elements governing transcription of selected genes. 3 One of the adverse events commonly associated with glucocorticoid therapy, regardless of route of administration, is an elevation of intraocular pressure (IOP) that may lead to glaucoma, 4 a side-effect assumed to result from transactivation of a gene or genes unrelated to the indication being treated. 5 Some patients receiving ocular GC therapy may exhibit no effect, whereas others, classified as responders, may demonstrate a range of documented increases in IOP, 6 attributed to several risk factors, including age, history of primary open-angle glaucoma (POAG), and genetic predisposition. 7,8  
POAG is a sight-threatening disease characterized by impaired efflux of aqueous fluid through the trabecular meshwork (TM), resulting in either elevated IOP, or, in its normotensive form, an ostensibly normal yet still deleterious IOP value, either of which results in progressive loss of vision if left untreated. 9 Since the juxtacanalicular region (JCT) of the TM abutting the inner wall endothelium of Schlemm's canal is the likely site of resistance to outflow under normal physiological conditions, 10,11 structural and biochemical changes in the JCT would be expected to affect IOP. 12 A feature shared by both POAG and steroid-induced glaucoma is the accumulation of extracellular matrix (ECM) and other material (plaque) in the JCT, consisting of abnormal aggregates of macromolecules obstructing the outflow pathway and raising IOP. 1315 As with many other nonocular cells and tissues that have been examined, 16,17 the TM is susceptible to GC-induced ECM changes, demonstrated experimentally 18,19 and in clinical samples. 20,21  
Myocilin is a protein normally detected in ocular tissues, with a constitutive expression that is most pronounced in the TM, both intra- and extracellularly. 2224 The discovery that mutations in the myocilin gene (MYOC) give rise to selected forms of POAG and juvenile open-angle glaucoma 25,26 eventually directed attention to the roles of wild-type myocilin in eye health and disease. An apparently unique, and diagnostic, property of TM cells in vitro and in situ is the overexpression of myocilin in response to GCs. 24 The precise functional role of myocilin is not understood, but GC-enhanced TM expression of myocilin has raised the possibility that this protein has an etiologic role in steroid-induced glaucoma. 27 Besides effects on TM cell internal structure and function, as assessed in organ cultured material, 28 GC treatment may induce excessive myocilin synthesis and secretion by these cells, culminating with deposition of the protein in the ECM of the outflow pathway, 29 and hence, elevating IOP. 30 Pharmacologic doses of dexamethasone (DEX) elicit elevated expression of myocilin in cultured TM cells from normal human donors, shown by analysis of myocilin mRNA or through immunochemical detection of soluble myocilin released into the culture medium. 27,31,32 Irrespective of whether these changes underlie a direct role for myocilin in the pathophysiology of any form of glaucoma, drug-induced elevations of this protein could be considered a surrogate indicator of risk for secondary glaucoma. 33  
There is considerable interest in novel GC receptor (GR) agonists that, by virtue of their structures and of the specific conformational changes they generate on binding to the GR, exhibit partial dissociation with respect to transactivation and transrepression of selected genes normally affected by GCs. Molecules with these distinct biochemical profiles may offer an improved clinical safety profile compared with steroidal GR agonists routinely used in the clinic. 34,35 Human TM cells have been widely used as an in vitro model to study responses to GCs. 36,37 In the experiments reported herein, we used cultured macaque TM cells, as an in vitro model of drug-induced expression of myocilin protein and mRNA, to characterize a newly developed selective glucocorticoid receptor agonist (SEGRA) that has demonstrated full agonist properties as an anti-inflammatory agent accompanied by minimal undesired systemic side effects. 38 The SEGRA compound was tested alongside DEX and also compared with prednisolone acetate (PA), another traditional GC used to treat ocular inflammation. 39 The results demonstrate that this novel SEGRA may provide a beneficial side effect profile for myocilin expression in the TM, behaving as a partial agonist in comparison with traditional GCs. 
Materials and Methods
TM Cells and Culture Medium
All animal procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyes were obtained from freshly killed, healthy rhesus monkeys (Macaca mulatta) obtained from Lonza (Walkersville, MD). These animals exhibited little variation in chronological age and general health. Monkey eyes were transported in CO2-independent medium on ice and processed approximately 40 hours after enucleation. After removal of iris, lens, and the bulk of the ciliary body, opercula (an anatomic feature of monkey TM 40 ) were stripped from anterior segment quadrants. Using fine scissors, we excised strips of TM and further subdivided them into smaller fragments. The latter were explanted to multiwell plates, containing growth medium (described later) and incubated with gelatin-coated beads (Cytodex-3; Sigma-Aldrich, St. Louis, MO). The beads attach to the explants within hours and provide additional substrate area for out-migration of cells. Proliferating TM cells colonize additional beads and also “spill” onto the tissue culture plastic and form colonies. After several days, the original TM explants and beads were transferred to new wells, generating additional primary cultures. Subconfluent monolayers of cells on tissue culture plastic were passed from 12-well plates to 35- or 60-mm dishes with collagenase-Dispase (Roche Applied Bioscience, Indianapolis, IN). This protease combination was also used to enzymatically harvest second- or third-passage subcultures, and cells thus obtained were then counted and cryopreserved in liquid nitrogen. 
In the preparation of monkey TM cell cultures for use in evaluating drug effects, we adopted a strategy suggested by the observations of Fautsch et al., 41 who called attention to the differences in TM cell phenotypes when exposed in vitro to aqueous fluid, compared with conventional cell culture media containing high concentrations of serum. At different stages of the process, we made successive changes in the media formulations to optimize expansion of cultures, to minimize proliferation for monolayer culture stabilization, and to permit modulation of myocilin expression by test compounds. The medium for initiating and expanding cultures of TM (proliferation medium) was human endothelial serum-free medium (HESFM; Invitrogen, Carlsbad, CA), containing the following supplements: fetal bovine serum (FBS; 1% [vol/vol]; Hyclone, Logan, UT), endothelial cell growth supplement (ECGS, 25 μg/mL; BD Biosciences, San Jose, CA), heparin (2.5 μg/mL; Sigma-Aldrich), taurine (3.2 μM; Sigma-Aldrich), fatty acid-albumin complex (200 mg/L; Invitrogen), ascorbic acid phosphate (0.1 mM; Wako Pure Chemicals, Richmond, VA), human transferrin (25 mg/L; Sigma-Aldrich), human fetuin (0.1 mg/mL; Sigma-Aldrich), supplemental glucose (1.32 g/L; Sigma-Aldrich), fructose (0.33 g/L; Sigma-Aldrich), glutathione (5 μg/mL; Sigma-Aldrich), hydrocortisone (14 nM; Sigma-Aldrich), and penicillin-streptomycin (Invitrogen) as an antibiotic additive. 
For each study, up to nine TM cell strains, each derived from an individual monkey, were tested separately. The cells were thawed and seeded into 12- or 48-well clusters (Falcon, BD Biosciences; 150,000 and 30,000 cells/well, respectively) in proliferation medium. When the cells were 75% to 90% confluent, the culture medium was modified, beginning with a 5:4 mixture of HESFM and Dulbecco's MEM (DMEM; Sigma-Aldrich), respectively, as the basal medium. This second-stage medium, now lacking ECGS and heparin, was supplemented with 10% FBS, with added taurine, ascorbic acid phosphate, glutathione, and antibiotic as for proliferation medium (above), and with 0.34 g/L supplemental glucose and 0.34 g/L fructose. At confluence, the cells were switched to DMEM, containing 10% FBS (shown to be permissive for DEX-induced increased myocilin expression 41 ), ascorbic acid phosphate, antibiotic, 0.17 g/L supplemental glucose and 0.17 g/L fructose. The cells were maintained as stable, confluent monolayers in this latter medium (the “base” medium for drug treatments) for 4 to 7 days before experimental treatments commenced. 
TM Cell Treatments with DEX, PA, and BOL-303242-X: A SEGRA Compound
For studies of myocilin protein, TM cell strains from nine monkeys were used to directly compare the responses to DEX and BOL-303242-X, a SEGRA compound, whereas a subset of four of these nine were used for studies comparing DEX with PA. For mRNA processing and determinations, two TM strains were selected. Cells in triplicate sample wells (24-well clusters) were incubated with DEX (Sigma-Aldrich) in individual studies alongside corresponding cell samples exposed to either BOL-303242-X or PA (Sigma-Aldrich); drug concentrations ranged from 3 to 300 nM. BOL-303242-X (ZK 245,186; R-1,1,1-trifluoro-4-(5-fluoro-2,3-dihydrobenzofuran-7-yl)-4-methyl-2-{[(2-methyl-5-quinolyl)amino]methyl}pentan-2-ol), provided by Bayer Schering Pharma, Berlin, Germany, is being clinically developed for topical treatment of atopic dermatitis. 38 For investigating the GR-mediated antagonistic effect of RU-486 on drug-induced myocilin protein expression, cells from a single TM strain, in triplicate sample wells (48-well clusters) were treated separately either with DEX or BOL-303242-X at 30 nM or with vehicle, either alone or in combination with RU-486 (BioMol, Plymouth Meeting, PA), the latter in a concentration range from 10 to 1000 nM. 
All treatments, including media for vehicle control samples, contained a final DMSO concentration of 0.1% (vol/vol) across the concentration ranges selected. Treatments lasted 96 hours, with one exchange of medium on the third treatment day. All treatment media exchanges were made with fresh drug and other additives from source stocks. The final 48-hour conditioned media (CM) samples were collected in their entirety (0.5 mL), centrifuged briefly to remove particulates, aliquoted, and stored at −20°C until thawed for analysis. 
Cell Metabolic Activity Assay
A modification of previously described methods 42 was used to evaluate cell metabolic activity, an index of cell viability. After collection of CM samples, cells were briefly rinsed in modified Hanks' balanced salt solution containing Ca2+ and Mg2+ (MHBSS), and then 0.0025% (wt/vol) resazurin (Sigma-Aldrich) in MHBSS was added to sample wells. Plates were incubated (37°C, 5% CO2, 95% humidity) for 90 minutes, after which fluorescence (excitation 560 nm, emission 590 nm) was read (Victor 3V Multilabel Counter; Wallac, Turku, Finland). As a positive control for decreased cellular metabolic reduction of resazurin, in each plate an additional well of vehicle control-treated cells was preincubated with 0.06% hydrogen peroxide (Fisher, Atlanta, GA) in MHBSS. 
Western Blot Analysis
Undiluted CM was combined with denaturing 4× sample buffer containing 2% SDS, and samples were loaded at equivalent protein content onto 4% to 20% Tris-HCl polyacrylamide gels (Bio-Rad, Hercules, CA). After electrophoresis, proteins underwent wet transfer to 0.2 mm nitrocellulose (Bio-Rad) for immunoblotting. The filters were blocked with 5% (wt/vol) nonfat dry milk (Bio-Rad) in Tris-buffered saline plus 0.02% (vol/vol) Tween-20 (TBST; Tween-20 from Calbiochem, La Jolla, CA), and incubated with a 1:2000 dilution (from 200 μg/mL) of goat anti-recombinant human myocilin antibody (R&D Systems, Minneapolis, MN) in blocking buffer, overnight at 4°C. After washing in TBST, the filters were incubated with a 1:25,000 dilution (from 0.8 mg/mL) of horseradish peroxide-conjugated mouse anti-goat IgG (H+L) (Pierce Biotechnology, Rockford, IL) in blocking buffer, for 90 minutes at room temperature. After washing in TBST, the blots were developed for detection by chemiluminescence (SuperSignal West Dura Extended Duration Substrate; Pierce). Bands corresponding to myocilin were digitally captured and stored with a fluorescence imager (FluorChem Imager; AlphaInnotech, San Leandro, CA), with all blots receiving equal exposure/capture times. The imager system software was then used to calculate pixel density for equivalent rectangular areas incorporating the bands. 
Quantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction (qRT-PCR)
After triplicate treatments with DEX, BOL-303242-X, PA, or vehicle control medium, cultured TM cells prepared in a selected subset of wells from which CM had been collected were lysed, and total RNA was isolated (RNeasy Plus MiniKit; Qiagen, Valencia, CA), according to the manufacturer's instructions. After quantification of purified total RNA (Quant-iT RNA Assay kit; Molecular Probes, Eugene, OR), equivalent amounts of this RNA were apportioned to generate first-strand cDNAs for each treatment sample, using random primers (Affinity Script; Stratagene, La Jolla, CA). Oligonucleotide myocilin primers, designed based on the cynomolgus MYOC gene, 43 and fluorescent probe (Taqman; Applied Biosystems, Foster City, CA) were used for PCR amplification. Equal amounts of total RNA-equivalent mass (approximate range, 250–1000 μg) reactant cDNA were added to the PCR master mix (Stratagene) and myocilin primers/fluorescent probe. Amplification was performed in a thermocycler (Mx3005P; Stratagene), with an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute for extension. Every run included standard controls (i.e., either without reverse transcriptase or lacking template). Relative quantities of myocilin mRNA abundance were determined by using differences in threshold cycles (Ct) between vehicle control and drug treatments. 44 Each sample was analyzed in triplicate wells, and the corresponding values averaged for further quantitative analysis. Myocilin mRNA abundance, expressed in proportion to vehicle control-treated samples, was calculated using the Mx3005P software. 
Data Analysis and Statistical Methods
All statistical evaluations of the data were conducted using a statistical software package (JMP; SAS Institute, Cary, NC). Data from each experiment were evaluated for normality and variance homogeneity using the Shapiro-Wilk and the Bartlett or Levene tests, respectively. If these tests indicated deviations from the assumptions required to conduct parametric statistics (i.e., data did not follow a normal, Gaussian population or the variances were not homogeneous), the data were transformed in accordance with the Box-Cox family of transformations. 45,46 Using Box-Cox procedures, the data were elevated to the power of a parameter (λ) which can take values from −2 to 2. When λ equaled zero, under Box-Cox provisions, the results were subjected to a logarithmic transformation. The λ value providing the smallest sum of square errors relative to the mean was defined by the statistical software as the best transformation, and this was used to complete the statistical analysis. Data were entered as the mean ± SEM or, in some cases, when the λ values ranged from −0.2 to 0.2, as the geometric mean ± SE, estimated by using the Taylor series expansion. Statistical analysis was conducted by either one- or two-way analysis of variance (ANOVA) followed by the Tukey-Kramer test on either raw or Box-Cox–transformed data. P < 0.05 was considered statistically significant. The specific transformations used for analyses are mentioned in the figure legends. 
For each set of triplicate samples from the individual monkey TM cell strains tested, Western blot densitometry values for myocilin protein detected in CM (as geometric means) and relative abundance of myocilin mRNA (as geometric means) were plotted as a function of drug concentrations. Dose–response curve data were fitted to a reparameterized four-parameter logistic equation using methodology similar to that previously described, 47 and these equations provided estimates of the EC50 and associated 95% confidence intervals for each drug treatment. Data summaries across experiments were computed using weighted averages. The weights used corresponded to the inverse of the variances obtained in each experiment. In this manner, all the information from a given experiment (central measures as well as variability) was taken into consideration for the computation of the weighted averages discussed in the Results section. 
Results
In Vitro Properties of Monkey TM Cells
Rhesus monkey TM cells demonstrated robust proliferation both in primary explants (Fig. 1A) and during early passage. General cellular morphology and the uniform cobblestone pattern of the monolayers, consistent with TM cells propagated from young human donors and cynomolgus monkeys, 48,49 were maintained in confluent subcultures used for these studies (Fig. 1B). There was no noticeable variation in in vitro growth properties between monkey TM cell strains, and all had equivalent, homogeneous morphology during proliferation and at confluence. Commonly used markers for TM cells in vitro, DiI-Acetylated LDL uptake, 50 and expression of factor VIII antigen (von Willebrand factor), 51 were documented in monkey TM cell cultures equivalent to those used in these experiments (results not shown). 
Figure 1.
 
(A) Monkey TM explants with attached gelatin-coated beads. After 4 days, the cells began to proliferate and migrate from the original TM tissue onto the surface of the beads (arrows). (B) Monkey TM cells in a second passage culture, at confluence.
Figure 1.
 
(A) Monkey TM explants with attached gelatin-coated beads. After 4 days, the cells began to proliferate and migrate from the original TM tissue onto the surface of the beads (arrows). (B) Monkey TM cells in a second passage culture, at confluence.
Effects of DEX, BOL-303242-X, and PA on Myocilin Protein in Monkey TM Cell CM
As predicted by other reports, 25,52 myocilin protein was released to CM by rhesus monkey TM cells, and was detected in Western blot analysis as a single thick band—probably a fused doublet 53 —at the expected molecular size, approximately 55 kDa (Fig. 2), previously noted in Western blot analysis of CM from DEX-treated human TM cells, and of human and monkey aqueous fluid. 25,27,54 With exposure to increasing concentrations of BOL-303242-X or DEX, immunoreactive bands of higher density could be discerned by visual inspection alone (Fig. 2); similar results were obtained with PA (not shown). It is important to note, moreover, based on relative band intensities, that BOL-303242-X induced lower expression of myocilin than DEX at high doses—an initial suggestion of a partial agonist effect for the former drug on myocilin gene expression. 
Figure 2.
 
Western immunoblot for myocilin, performed on monkey TM cell culture CM. Results shown are from one set of samples from an individual monkey TM cell strain, incubated with DEX or BOL-303242-X in a concentration range of 3 to 300 nM, or with vehicle control (C) lacking either drug. After polyacrylamide gel electrophoresis, Western transfer, and immunodetection via chemiluminescence, the density of the myocilin bands (closely spaced doublets at ∼55 kDa) was quantified for each sample.
Figure 2.
 
Western immunoblot for myocilin, performed on monkey TM cell culture CM. Results shown are from one set of samples from an individual monkey TM cell strain, incubated with DEX or BOL-303242-X in a concentration range of 3 to 300 nM, or with vehicle control (C) lacking either drug. After polyacrylamide gel electrophoresis, Western transfer, and immunodetection via chemiluminescence, the density of the myocilin bands (closely spaced doublets at ∼55 kDa) was quantified for each sample.
Figure 3 shows the effects of DEX and BOL-303242-X on the amount of accumulated myocilin protein released into the CM during the second 48-hour treatment period, for a single monkey TM cell strain, as representative of the nine strains in which these two drugs were compared. Whereas both compounds increased myocilin concentrations in a dose-dependent manner, the SEGRA compound BOL-303242-X behaved as a partial agonist in this respect, in comparison with the more fully efficacious DEX. For the particular TM cell strain depicted, the full range of DEX treatments from 3 to 300 nM gave statistically significant effects compared with vehicle control. (Note that 100 nM, corresponding to the topical dose routinely used for DEX in clinical applications, is also commonly invoked to assess steroid responsiveness in vitro. 55 ) Myocilin protein also increased after treatments with BOL-303242-X, accumulating above vehicle control levels in the CM of the monkey TM cells throughout the concentration range tested, as exemplified for a single TM cell strain in Figure 3. In this case, though, specific partial agonism with respect to myocilin protein expression was demonstrated by the statistically significant differences observed between BOL-303242-X and the model GC DEX at 3, 10, 100, and 300 nM (Fig. 3, daggers); in this example, at 300 nM, BOL-303242-X exhibited only 46% of the maximum efficacy displayed by DEX. 
Figure 3.
 
Comparison of effects of BOL-303242-X versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities at each dose of drug are shown for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data (from triplicate samples) were analyzed by two-way ANOVA, followed by the contrast procedure on logarithmically transformed data, and are presented as the geometric mean ± SE, estimated by using the Taylor series expansion. Solid and dashed lines: the calculated best fit four-parameter equation. *P < 0.05 versus vehicle control. †P < 0.05 versus the same dose of DEX.
Figure 3.
 
Comparison of effects of BOL-303242-X versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities at each dose of drug are shown for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data (from triplicate samples) were analyzed by two-way ANOVA, followed by the contrast procedure on logarithmically transformed data, and are presented as the geometric mean ± SE, estimated by using the Taylor series expansion. Solid and dashed lines: the calculated best fit four-parameter equation. *P < 0.05 versus vehicle control. †P < 0.05 versus the same dose of DEX.
At 300 nM, the individual TM cell strain results ranged from 1.3- to 5.27-fold increases over control levels for the SEGRA compound BOL-303242-X, as opposed to 2.79-fold to as high as 12.33-fold for the same TM cell strains treated with DEX. However, the combined maximum efficacy for BOL-3032542-X, computed across all nine TM cell strains using weighted averages, was approximately 50% of that observed after DEX treatment (Table 1); this difference was statistically significant, as indicated by the nonoverlapping 95% confidence intervals. Although not obviously apparent for DEX in the example illustrated in Figure 3, clear efficacy plateaus in the high concentration range were graphically identified for DEX by using the dose–response data for many of the TM cell strains tested. In addition, as with the one featured in Figure 3, for any of the nine individual monkey TM strains examined separately, there were no statistically significant differences in efficacies between the 100 and 300 nM doses, for either drug investigated (results not shown), indicating that maximum efficacy had been attained. The full dose–response results for both BOL-303242-X and DEX, compiled using the data from all nine TM cell strains, also indicated that a plateau at the upper end of the concentration range had been attained. 
Table 1.
 
Partial Agonism of BOL-303242-X in Comparison with DEX
Table 1.
 
Partial Agonism of BOL-303242-X in Comparison with DEX
Compound Efficacy ± SE* (Weighted Average, as %) 95% Confidence Interval for Efficacy (%) Coefficient of Variation (%)
DEX 100.00 ± 6.09 88.07–111.93 18.27
BOL-303242-X 53.12 ± 2.20 48.81–57.43 12.42
With respect to potency, using data collected from all TM cell strains tested, DEX and BOL-303242-X displayed EC50s of 14.58 and 20.96 nM, respectively (Study 1 in Table 2). These differences were not statistically significant, with overlapping 95% confidence intervals for the estimates (Table 2). In experiments conducted over a 3-month period, the responses of the nine monkey TM isolates were similar and very reproducible; the interisolate coefficients of variation for the EC50s were 18.20% and 20.40% for DEX and BOL-303242-X, respectively (Table 2). The results were consistent with those shown in Figure 3 for a single TM strain, and underscored that the partial agonism profile of the SEGRA compound BOL-303242-X was defined in terms of the efficacy, but not the potency, of this compound. 
Table 2.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin Protein by Cultured Monkey TM Cells
Table 2.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin Protein by Cultured Monkey TM Cells
Study Compound EC50 ± SE (nM)* 95% Confidence Interval for EC50 Coefficient of Variation (%)
1 DEX 14.58 ± 2.65 10.21–20.83 18.20
BOL-303242-X 20.96 ± 4.28 14.05–31.26 20.40
2 DEX 6.14 ± 3.29 2.15–17.53 53.55
PA 104.34 ± 66.79 29.76–365.86 64.01
An additional set of experiments on four TM cell strains evaluated similarly the responsiveness of the in vitro system to another GC, PA, in comparison to DEX. Representative results from an individual monkey TM strain are illustrated in Figure 4. Consistent with the results from the earlier experiments using DEX (exemplified in Fig. 3), statistically significant increases in myocilin protein expression were again observed here for DEX treatments at all concentrations, compared with vehicle control. Maximum efficacies for DEX, as 6.90- and 6.48-fold increases over control values, respectively, were attained at 100 and 300 nM. Values for myocilin protein induced by PA were significantly lower than those for DEX when tested at 10 and 30 nM (Fig. 4, daggers), but incubation with the two highest doses of PA did not result in responses that were significantly different from equivalent doses of DEX, a confirmation of the full agonist activity of PA in this model. 
Figure 4.
 
Comparison of effects of PA versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities are represented for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power −0.2. Data are presented as the geometric mean ± SE estimated by using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 versus same dose of DEX.
Figure 4.
 
Comparison of effects of PA versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities are represented for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power −0.2. Data are presented as the geometric mean ± SE estimated by using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 versus same dose of DEX.
In accord with its higher estimated EC50 (Table 2, Study 2), and evident from the fitted dose–response curves (e.g., the single TM strain plotted in Fig. 4), PA was less potent than DEX with respect to increasing myocilin protein release by monkey TM cells into CM. The estimated EC50 for DEX, 6.14 nM, was slightly lower than its value in the earlier study when tested against BOL-303242-X (Table 2, cf. studies 1 and 2), and was statistically different from the EC50 for PA, 104.34 nM, since there was no overlap in the 95% confidence intervals calculated for these two GCs (Table 2). This apparent shift in potency compared to DEX was accompanied by a range of individual estimated maximum efficacies when the four monkey TM strains were treated with PA, 4.8- to 8.9-fold above vehicle control, which would have corresponded to a drug concentration range beyond that tested (Fig. 4; Table 2, study 2). The responses across the four monkey TM strains in these DEX versus PA studies were not as reproducible as in the previous assessment of DEX versus BOL-303242-X; the interisolate coefficients of variation for the experiments with DEX and PA were 53.55% and 64.01%, respectively (Table 2). 
Effects of DEX, BOL-303242-X, and PA on Myocilin mRNA Expression
The effects of DEX, BOL-303242-X, and PA on myocilin mRNA expression in monkey TM are exemplified by the results shown in Figure 5; data are from the same cell strain as is depicted in Figures 3 and 4. For both experiments making comparisons to DEX, either with BOL-303242-X (Fig. 5, left) or with PA (Fig. 5, right), the patterns for expression of mRNA for myocilin, in terms of dose–response, were similar to those examining myocilin protein. They also showed statistical significances similar to those observed for the comparative protein levels. The qRT-PCR data for BOL-303242-X were again indicative of the partial agonist nature of this agent, with significantly lower mRNA abundances at all doses compared with DEX. Maximum efficacy for BOL-303242-X, demonstrated at 300 nM, was in the particular example shown approximately 67% of that for DEX (Fig. 5, left). Regarding estimated EC50s for all three drugs, using combined data generated from the two strains tested in the parallel experiments, there was excellent general correlation between the values both for mRNA abundance and the data for myocilin protein (cf. Tables 2, 3). Indeed, as previously shown for myocilin protein in Table 1, the average relative values for myocilin message were significantly lower for BOL-303242-X versus DEX over the full concentration range from 3 to 300 nM (evident in the graph for one TM cell strain in Fig. 5, left). In contrast, myocilin mRNA abundance in PA-treated TM cells was significantly lower versus DEX at 100 nM, but not at 300 nM, as exemplified in the results for one TM cell strain in Figure 5 (dagger, right). 
Figure 5.
 
Representative quantitative real-time RT-PCR results for a single strain of cultured monkey TM cells, from two independent dose–response experiments comparing the effects of BOL-303242-X with DEX or of PA with DEX on myocilin mRNA expression. Data, as multiples of vehicle control (open bar), were analyzed by two-way ANOVA followed by the contrast procedure on logarithmically transformed data (BOL-303242-X versus DEX), or transformed data elevated to the power 0.2 (PA versus DEX). Data are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 for either BOL-303242-X or PA, versus DEX at the same concentration tested.
Figure 5.
 
Representative quantitative real-time RT-PCR results for a single strain of cultured monkey TM cells, from two independent dose–response experiments comparing the effects of BOL-303242-X with DEX or of PA with DEX on myocilin mRNA expression. Data, as multiples of vehicle control (open bar), were analyzed by two-way ANOVA followed by the contrast procedure on logarithmically transformed data (BOL-303242-X versus DEX), or transformed data elevated to the power 0.2 (PA versus DEX). Data are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 for either BOL-303242-X or PA, versus DEX at the same concentration tested.
Table 3.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin mRNA in Cultured Monkey TM Cells
Table 3.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin mRNA in Cultured Monkey TM Cells
Study Compound EC50 ± SE (nM)* 95% Confidence Interval for EC50 Coefficient of Variation (%)
1 DEX 14.66 ± 1.27 12.37–17.38 8.68
BOL-303242-X 20.75 ± 2.74 16.02–26.88 13.21
2 DEX 17.14 ± 3.12 12.00–24.48 18.19
PA 131.64 ± 56.89 56.43–365.86 43.22
Effects of RU-486 on BOL-303242-X– and DEX-Induced Myocilin Protein Expression
RU-486 (RU 38486; mifepristone) is a steroid that antagonizes many of the documented in vitro and in vivo effects of GCs such as DEX, by competing for the ligand binding site on the GR and inducing a modification of the three-dimensional structure of the bound complex that prevents transactivation. 56,57 RU-486 inhibits DEX-induced myocilin expression in cultured human TM cells, 58 and we endeavored to replicate this result for myocilin protein release by cultured monkey TM, for both DEX and BOL-303242-X, and to establish that both of these compounds' effects in our model were mediated via the GR. Using one of the monkey TM cell strains used in the previous studies, and with DEX and BOL-303242-X each at 30 nM, i.e., within a log10 unit of their EC50s with respect to myocilin expression (Table 2), co-incubation with RU-486 in a concentration range from 10 to 1000 nM resulted in dose-dependent abrogation of both DEX- and BOL-303242-X–induced elevations in myocilin protein detected in CM (Fig. 6, left, middle). Myocilin protein values were reduced to vehicle control levels at the highest RU-486 concentration tested, 1000 nM (Fig. 6, left, middle). In the context of the concentration of either agonist compound tested, the estimated EC50s for RU-486 were not significantly different (15.48 ± 6.03 nM, with 95% confidence limits of 7.21 and 33.22 nM in the presence of DEX; 11.74 ± 4.37 nM, with 95% confidence limits of 5.66 and 24.37 nM for BOL-303242-X). Similar to previous findings by Shepard et al., 58 at a few concentrations intermediate in the dose range, RU-486 alone caused some slight but statistically significant increases in myocilin protein expression (Fig. 6, right), indicating low level partial agonism. These data demonstrated that RU-486 was a highly effective antagonist, with equivalent potency for inhibiting induction of myocilin protein by both BOL-303242-X and DEX, and suggested that these latter two compounds both exerted their effects in monkey TM cells via the classic GR. 
Figure 6.
 
RU-486 inhibits DEX- and BOL-303242-X–induced myocilin protein expression by cultured monkey TM cells. Levels of myocilin that accumulated in CM during the final 48 hours of a 96-hour treatment with either 30 nM BOL-303242-X plus a dose range of RU-486 (left), DEX at 30 nM plus a dose range of RU-486 (center), or RU-486 alone in treatment vehicle (right) were assessed by densitometric analysis of Western blot images. EC50s for RU-486 were 11.74 ± 4.37 nM and 15.48 ± 6.03 nM, against 30 nM BOL-303242-X and 30 nM DEX, respectively. (■) Vehicle control treatment, without drugs; open bar, left: 30 nM BOL-303242-X alone; gray bar, center: 30 nM DEX alone. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power 0.2, and are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus control (BOL-303242-X, DEX, or vehicle control with RU-486). †P < 0.05 for BOL-303242-X + RU-486, versus DEX + RU-486, at the same RU-486 concentration.
Figure 6.
 
RU-486 inhibits DEX- and BOL-303242-X–induced myocilin protein expression by cultured monkey TM cells. Levels of myocilin that accumulated in CM during the final 48 hours of a 96-hour treatment with either 30 nM BOL-303242-X plus a dose range of RU-486 (left), DEX at 30 nM plus a dose range of RU-486 (center), or RU-486 alone in treatment vehicle (right) were assessed by densitometric analysis of Western blot images. EC50s for RU-486 were 11.74 ± 4.37 nM and 15.48 ± 6.03 nM, against 30 nM BOL-303242-X and 30 nM DEX, respectively. (■) Vehicle control treatment, without drugs; open bar, left: 30 nM BOL-303242-X alone; gray bar, center: 30 nM DEX alone. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power 0.2, and are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus control (BOL-303242-X, DEX, or vehicle control with RU-486). †P < 0.05 for BOL-303242-X + RU-486, versus DEX + RU-486, at the same RU-486 concentration.
Effects of Drugs on Cultured Monkey TM Cells in the Resazurin Reduction Assay
There was no correlation of myocilin expression levels with general cell metabolic status, as a consequence of exposure to different concentrations of DEX, PA, BOL-303242-X, or RU-486, nor did any drug treatments result in a loss of cell viability compared with vehicle controls, as determined by measuring chemical reduction of resazurin at the conclusion of the treatment periods (results not shown). The results suggest, then, that any increases or decreases observed in myocilin expression relative to control, induced by any of the drug treatment regimens, were not due to compromise of functional cell integrity. 
Discussion
Underlying the unique pathophysiology of inflammatory eye diseases are processes driven by genes and transcription factors, the upregulated expression of which is common to all inflamed tissues and cells and which are targeted in the transrepression arm of GC therapy. 3 To avoid incurring the well-known undesirable side effects of systemic GC administration, believed to be mediated predominantly via transactivation of specific genes, novel, selective glucocorticoid receptor agonists (SEGRAs) have been developed. 34,35,38 Since GC-induced myocilin upregulation in TM may be due to a transactivation mechanism, 26,36 we hypothesized that BOL-303242-X, a SEGRA currently in development for treating inflammatory skin conditions, 38 and under consideration as a candidate drug to treat inflammatory ocular indications, would show a decreased activation profile for myocilin, compared to GCs such as DEX. This is the first report of the effects of a SEGRA compound on myocilin expression in TM cells tested in vitro. Our results indicate that BOL-303242-X, a drug exhibiting a full agonist profile as an anti-inflammatory agent, 38 may have a more favorable therapeutic index than conventional GCs when used for the treatment of ocular diseases with an inflammatory component. 
Although clinical evidence is lacking for a direct role of wild-type myocilin in any form of acquired open-angle glaucoma, 33 excess or overexpressed myocilin protein can disrupt the structure and function of cultured TM cells and affect the eye's outflow apparatus in vitro and in vivo. 5961 Furthermore, increased expression of myocilin in the TM may be a marker of derangements in ECM associated with glaucoma pathophysiology, including changes induced by steroids. 62,63 Microarray studies have repeatedly singled out myocilin as the gene most positively upregulated by DEX treatment of TM cells. 64,65 Full analysis suggests that myocilin, if not playing an immediate role in the etiology of drug-induced glaucoma, is at least a suitable marker for a subset of GC-responsive genes that interact when upregulated and, based on current understanding, would generate increased aqueous fluid outflow resistance and ocular hypertension. 66  
In the studies described herein, the EC50s calculated as elements of the curve-fitting parameters were not significantly different between DEX and BOL-303242-X, for either myocilin protein or mRNA, and our EC50 for monkey TM myocilin protein expression induced by DEX in vitro, calculated as a weighted average, was acceptably close to those values revealed for cultured human TM by both Polansky et al. (30 nM), 31 and Shepard et al. (10 nM). 58 Nevertheless, with respect to both individual TM cell strains as well as in composite results, treatment with BOL-303242-X induced significantly lower average myocilin expression values compared to DEX, at the highest doses tested, characterizing this SEGRA as a partial agonist in our in vitro model system. Such partial agonism has been described in a nonocular context for other SEGRAs, 34,67 and is attributed to a conformation of the ligand/GR complex that is different from what is induced by full agonists, engendering altered interactions with coactivators of gene expression. 68 Although not tested directly against it in the present studies, PA could be distinguished pharmacologically from BOL-303242-X when either drug was compared directly with DEX. In a separate experiment comparing the effects of PA with DEX, the differences in the values of both myocilin expression parameters were not statistically significant at the highest dose used, thereby revealing the corticosteroid PA to be a full agonist in this context. 
Results from in vitro models for cytokine expression, employing a variety of ocular cell types, support a transrepression mechanism of action for BOL-303242-X as a highly efficacious anti-inflammatory compound when tested side by side with DEX (Zhang JZ, et al. IOVS 2009;49:ARVO E-Abstract 5547). If it should be determined that in clinical application it either matches or exceeds the efficacy of conventional GCs in the treatment of inflammatory eye conditions, the therapeutic index of BOL-303242-X could be greatly augmented by a reduction in associated myocilin expression. In an optimal scenario, a more modest potential increase in expression of myocilin and/or associated ECM macromolecules induced in a treated eye by an effective dose regimen of BOL-303242-X, in contrast with GCs in current use, would be below a threshold necessary to cause a clinically significant elevation in IOP. 
There may be innate features of monkey TM, as expressed in vitro at least, that impart increased sensitivity, and hence utility, for testing the effects of GCs and SEGRAs. Although living monkeys, like humans, may be responders or nonresponders with respect to GC-induced ocular hypertension, 43 we found that all nine separate monkey TM strains tested responded with significant increases in myocilin expression as a result of DEX treatment, albeit distributed over an extensive range. Not surprisingly, the primers we used for real-time qRT-PCR, from the sequences designed by Fingert et al. 43 for cynomolgus monkey (Macaca fascicularis), performed as expected for the closely related rhesus macaque. Also, the commercial anti-human myocilin antibody retained cross-reactivity to the monkey protein (with a predicted 97% amino acid sequence shared between human and monkey 43 ), permitting sensitive detection of myocilin even in control CM samples via Western blot. In comparison with the human myocilin gene, differences in the proximal 5′-upstream promoter region sequence for macaque monkey myocilin 43 may also influence GC-induced transcriptional activity. Specific message abundance is arguably not predictive of any downstream events affecting protein expression levels, but it appears to be the case in our studies that the myocilin mRNA abundance profile in the drug-treated cultured monkey TM cells exhibited an excellent correlation with that of myocilin protein released into CM. 
In studies using nonocular systems, it has been reported previously that the SEGRA compound tested in the present study was a selective ligand for the classic GR and that the transrepressive efficacy of this compound was sensitive to antagonism by RU-486. 38 Consistent with its classification as a partial GR agonist, a statistically significant efficacy reduction in response to treatments with BOL-303242-X (∼62%–67%, and with EC50s comparable to those we calculated for monkey TM) was documented, using reporter- and activity-based in vitro transactivation assays, in direct comparison to two clinically relevant full GC agonists (i.e., these latter agents having efficacies close to 100% with respect to DEX). 38 The reduced efficacy in upregulating myocilin expression demonstrated by BOL-303242-X may be further emblematic of a dissociated pharmacologic profile for this SEGRA, compared with DEX. Our efficacy results for BOL-303242-X are not attributable to chemical or metabolic instability of this drug during the course of our experiments, as forced degradation studies indicate that it is stable in an aqueous environment, under conditions similar to what it would encounter in our culture system (results not shown). 
Transcriptional control of myocilin expression mediated by GR in TM cells was suggested by the effects of the GR antagonist RU-486 in combination with either DEX or BOL-303242-X. In agreement with Shepard et al. 58 for their cultured human TM cells, we, too, demonstrated classic inhibition of both DEX- and BOL-303242-X–induced myocilin protein release into monkey TM cell CM in response to RU-486, indicating that pharmacologic stimulation of myocilin expression was due to specific binding to the GR by both DEX and BOL-303242-X. Some partial agonism of myocilin expression by RU-486 alone was also evident for cultured monkey TM, confirming previous in vitro findings with human TM. 58 Although RU-486 can bind to and inactivate the progesterone receptor (PR), BOL-303242-X has low affinity for and shows little activity via the PR. 38 Besides the canonical ligand-binding (α) form of the GR that has been detected in both native and cultured human TM, 69,70 human TM does express message for the mineralocorticoid receptor (MR), 71 but neither BOL-303242-X nor DEX can form stable, active complexes with the MR. 38,72  
We have documented here that our two cell-based assay systems, using quantitative densitometry of Western immunoblots and qRT-PCR, provided enough accuracy, reproducibility, and statistical significance to distinguish between and rank two GCs and one SEGRA compound, BOL-303242-X, with respect to myocilin protein and mRNA expression. These results can be translated to preclinical studies using nonhuman primates, including a prediction of dosages for in vivo testing. In much the same way that a transactivation profile for systemic SEGRAs has been analyzed in vitro using a human tyrosine aminotransferase gene expression system, 73 we have identified specifically and endogenously expressed target- and cell-based quantitative assays that are predictive and may be used to characterize, distinguish between, and rank new candidate SEGRAs for ocular anti-inflammatory therapy. 
Although they display high potency and efficacy, the use of GCs as anti-inflammatory drugs carries with it the risk of side effects because of their nonselective activation profile. This especially holds true for GC therapy to treat eye diseases 1,4 ; there is also significant risk for ocular adverse events associated with GCs administered periorbitally for dermatologic conditions. 74 In preclinical testing, BOL-303242-X has been shown to have full agonist properties as a topical treatment for dermatologic conditions, owing to its dissociated transrepression profile, when compared with traditionally prescribed GCs. 38 With respect to potential for elevating IOP, our results comparing effects on in vitro expression of myocilin by cultured nonhuman primate TM cells indicate that BOL-303242-X, a SEGRA compound that is presently under development as a topical treatment for ocular inflammation, would be expected to have an ocular safety profile superior to GCs. 
Footnotes
 Disclosure: B.A. Pfeffer, Bausch & Lomb, Inc. (E, P); C.A. DeWitt, Bausch & Lomb, Inc. (E, P); M. Salvador-Silva, Bausch & Lomb, Inc. (E, P); M.E. Cavet, Bausch & Lomb, Inc. (E, P); F.J. López, Bausch & Lomb, Inc. (E, P); K.W. Ward, Bausch & Lomb, Inc. (E, P)
Footnotes
 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2008 and May 2009.
The authors thank Kristina Getman and Danielle Meyer for technical support in cell culture and immunochemistry, and also Mohannad Shawer and Ezra Lowe for stability data. 
References
Cunningham MA Edelman JL Kaushal S . Intravitreal steroids for macular edema: the past, the present, and the future. Surv Ophthalmol. 2008; 53: 139–149. [CrossRef] [PubMed]
Read RW . Uveitis: Advances in understanding of pathogenesis and treatment. Curr Rheumatol Reports. 2006; 8: 260–266. [CrossRef]
Barnes PJ . Corticosteroid effects on cell signaling. Eur Respir J. 2006; 27: 413–426. [CrossRef] [PubMed]
Kersey JP Broadway DC . Corticosteroid-induced glaucoma: a review of the literature. Eye. 2006; 20: 407–416. [CrossRef] [PubMed]
Tripathi RC Parapuram SK Tripathi BJ . Corticosteroids and glaucoma risk. Drugs Aging. 1999; 15: 439–450. [CrossRef] [PubMed]
Palmberg PF Mandell A Wilensky JT Podos SM Becker B . The reproducibility of the intraocular pressure response to dexamethasone. Am J Ophthalmol. 1975; 80: 844–856. [CrossRef] [PubMed]
Armaly MF . Effect of corticosteroids on intraocular pressure and fluid dynamics, I: the effect of dexamethasone in the normal eye. Arch Ophthalmol. 1963; 70: 482–491. [CrossRef] [PubMed]
Becker B Mills DW . Corticosteroids and intraocular pressure. Arch Ophthalmol. 1963; 70: 500–507. [CrossRef] [PubMed]
Shields MB . Normal-tension glaucoma: is it different from primary open-angle glaucoma?. Curr Opin Ophthalmol. 2008; 19: 85–88. [CrossRef] [PubMed]
Ethier CR Kamm RD Palaszewski BA Johnson MC Richardson TM . Calculations of flow resistance in the juxtacanalicular meshwork. Invest Ophthalmol Vis Sci. 1986; 27: 1741–1750. [PubMed]
Johnson M Shapiro A Ethier CR Kamm RD . Modulation of outflow resistance by the pores of the inner wall endothelium. Invest Ophthalmol Vis Sci. 1992; 33: 1670–1675. [PubMed]
Acott TS Kelley MJ . Extracellular matrix in the trabecular meshwork. Exp Eye Res. 2008; 86: 543–561. [CrossRef] [PubMed]
Rodrigues MM Spaeth GL Sivalingam E Weinreb S . Histopathology of 150 trabeculectomy specimens in glaucoma. Trans Ophthalmol Soc UK. 1976; 96: 245–255. [PubMed]
Rohen JW Lütjen-Drecoll E Flügel C Meyer M Grierson I . Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp Eye Res. 1993; 56: 683–692. [CrossRef] [PubMed]
Tawara A Tou N Kubota T Harada Y Yokota K . Immunohistochemical evaluation of the extracellular matrix in trabecular meshwork in steroid-induced glaucoma. Graefes Arch Clin Exp Ophthalmol. 2008; 246: 1021–1028. [CrossRef] [PubMed]
Kato Y Gospodarowicz D . Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro. J Biol Chem. 1985; 260: 2364–2373. [PubMed]
Vincenti MP White LA Schroen DJ Benbow U Brinckerhoff CE . Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability. Crit Rev Eukaryot Gene Express. 1996; 6: 391–411. [CrossRef]
Steely HT Browder SL Julian MB Miggans ST Wilson KL Clark AF . The effects of dexamethasone on fibronectin expression in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 1992; 33: 2242–2250. [PubMed]
Tane N Dhar S Roy S Pinheiro A Ohira A Roy S . Effect of excess synthesis of extracellular matrix components by trabecular meshwork cells: Possible consequence on aqueous outflow. Exp Eye Res. 2007; 84: 832–842. [CrossRef] [PubMed]
Johnson D Gottanka J Flügel C Hoffman F Futa R Lütjen-Drecoll E . Ultrastructural changes in the trabecular meshwork of human eyes treated with corticosteroids. Arch Ophthalmol. 1997; 115: 375–383. [CrossRef] [PubMed]
Rohen JW Linnér E Witmer R . Electron microscopic studies on the trabecular meshwork in two cases of corticosteroid-glaucoma. Exp Eye Res. 1973; 17: 19–31. [CrossRef] [PubMed]
Karali A Russell P Stefani FH Tamm ER . Localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein in the human eye. Invest Ophthalmol Vis Sci. 2000; 41: 729–740. [PubMed]
Swiderski RE Ross JL Fingert JH . Localization of MYOC transcripts in human eye and optic nerve by in situ hybridization. Invest Ophthalmol Vis Sci. 2000; 41: 3420–3428. [PubMed]
Lo WR Rowlette LL Caballero M Yang P Hernandez MR Borrás T . Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma. Invest Ophthalmol Vis Sci. 2003; 44: 473–485. [CrossRef] [PubMed]
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 cells. J Biol Chem. 1998; 273: 6341–6350. [CrossRef] [PubMed]
Fingert JH Stone EM Sheffield VC Alward WLM . Myocilin glaucoma. Surv Ophthalmol. 2002; 47: 547–561. [CrossRef] [PubMed]
Clark AF Steely HT Dickerson JEJr . Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest Ophthalmol Vis Sci. 2001; 42: 1769–1780. [PubMed]
Clark AF Brotchie D Read AT . Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue. Cell Motil Cytoskeleton. 2005; 60: 83–95. [CrossRef] [PubMed]
Lütjen-Drecoll E May CA Polansky JR Johnson DH Bloemendal H Nguyen TD . Localization of the stress proteins αB-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci. 1998; 39: 517–525. [PubMed]
Filla MS Liu X Nguyen TD . In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin. Invest Ophthalmol Vis Sci. 2002; 43: 151–161. [PubMed]
Polansky JR Fauss DJ Chen P . Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica. 1997; 211: 126–139. [CrossRef] [PubMed]
Tamm ER Russell P Epstein DL Johnson DH Piatigorsky J . Modulation of myocilin/TIGR expression in human trabecular meshwork. Invest Ophthalmol Vis Sci. 1999; 40: 2577–2582. [PubMed]
Tamm ER . Myocilin and glaucoma: facts and ideas. Prog Retin Eye Res. 2002; 21: 395–428. [CrossRef] [PubMed]
Rosen J Miner JN . The search for safer glucocorticoid receptor ligands. Endocrin Rev. 2005; 26: 452–464. [CrossRef]
Schäcke H Schottelius A Döcke W-D . Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci USA. 2004; 101: 227–232. [CrossRef] [PubMed]
Wordinger RJ Clark AF . Effects of glucocorticoids on the trabecular meshwork: towards a better understanding of glaucoma. Prog Ret Eye Res. 1999; 18: 629–667. [CrossRef]
Rozsa FW Reed DM Scott KM . Gene expression profile of human trabecular meshwork cells in response to long-term dexamethasone exposure. Mol Vis. 2006; 12: 125–141. [PubMed]
Schäcke H Zollner TM Döcke WD . Characterization of ZK 245186, a novel, selective glucocorticoid receptor agonist for the topical treatment of inflammatory skin diseases: characterization of ZK 245186. Br J Pharmacol. Published online May 6, 2009.
Paccola L Jorge R Barbosa JC Costa RA Scott IU . Anti-inflammatory efficacy of a single posterior subtenon injection of triamcinolone acetonide versus prednisolone acetate 1% eyedrops after pars plana vitrectomy. Acta Ophthalmol Scand. 2007; 85: 603–608. [CrossRef] [PubMed]
Lütjen-Drecoll E Kaufman PL Bárány EH . Light and electron microscopy of the anterior chamber angle structures following surgical disinsertion of the ciliary muscle in the cynomolgus monkey. Invest Ophthalmol Vis Sci. 1977; 16: 218–225. [PubMed]
Fautsch MP Howell KG Vrabel AM Charlesworth MC Muddiman DC Johnson DH . Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements. Invest Ophthalmol Vis Sci. 2005; 46: 2848–2856. [CrossRef] [PubMed]
Larson EM Doughman DJ Gregerson DS Obritsch WF . A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Invest Ophthalmol Vis Sci. 1997; 38: 1929–1933. [PubMed]
Fingert JH Clark AF Craig JE . Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension. Invest Ophthalmol Vis Sci. 2001; 42: 145–152. [PubMed]
Heid CA Stevens J Livak KJ Williams PM . Real time quantitative PCR. Genome Res. 1996; 6: 986–994. [CrossRef] [PubMed]
Box GEP Cox DR . An analysis of transformation. J Roy Statist Soc Ser B. 1964; 26: 211–252.
Box GEP Hill WH . Correcting inhomogeneity of variance with power transformation weighting. Technometrics. 1974; 16: 385–389. [CrossRef]
Ghosh K Shen ES Arey BJ López FJ . A global model to define the behavior of partial agonists (bell-shaped dose-response inducers) in pharmacological evaluation of activity in the presence of the full agonist. J Biopharm Statistics. 1998; 8: 645–665. [CrossRef]
Alvarado JA Wood I Polansky JR . Human trabecular cells II. Growth pattern and ultrastructural characteristics. Invest Ophthalmol Vis Sci. 1982; 23: 464–478. [PubMed]
Yue BYJT Kurosawa A Elvart JL Elner VM Tso MOM . Monkey trabecular meshwork cells in culture: growth, morphologic, and biochemical characteristics. Graefes Arch Clin Exp Ophthalmol. 1988; 226: 262–268. [CrossRef] [PubMed]
Chang IL Elner G Yue YJT Cornicelli A Kawa JE Elner VM . Expression of modified low-density lipoprotein receptors by trabecular meshwork cells. Curr Eye Res. 1991; 10: 1101–1112. [CrossRef] [PubMed]
Stamer WD Seftor REB Williams SK Samaha HAM Snyder RW . Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion. Curr Eye Res. 1995; 14: 611–617. [CrossRef] [PubMed]
Hardy KM Hoffman EA Gonzalez P McKay BS Stamer WD . Extracellular trafficking of myocilin in human trabecular meshwork cells. J Biol Chem. 2005; 280: 28917–28926. [CrossRef] [PubMed]
Jacobson N Andrews M Shepard AR . Non-secretion of mutant proteins of glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor. Hum Mol Genet. 2001; 10: 117–125. [CrossRef] [PubMed]
Rao PV Allingham RR Epstein DL . TIGR/myocilin in human aqueous humor. Exp Eye Res. 2000; 71: 637–641. [CrossRef] [PubMed]
Polansky JR Fauss DJ Zimmerman CC . Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells. Eye. 2000; 14: 503–514. [CrossRef] [PubMed]
Moguilewsky M Philibert D . RU 38486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J Steroid Biochem. 1984; 20: 271–276. [CrossRef] [PubMed]
Beck CA Estes PA Bona BJ Muro-Cacho CA Nordeen SK Edwards DP . The steroid antagonist RU486 exerts different effects on the glucocorticoid and progesterone receptors. Endocrinology. 1993; 133: 728–740. [PubMed]
Shepard AR Jacobson N Fingert JH Stone EM Sheffield VC Clark AF . Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2001; 42: 3173–3181. [PubMed]
Wentz-Hunter K Shen X Okazaki K Tanihara H Yue BYJT . Overexpression of myocilin in cultured human trabecular meshwork cells. Exp Cell Res. 2004; 297: 39–48. [CrossRef] [PubMed]
Fautsch MP Bahler CK Vrabel AM . Perfusion of His-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor. Invest Ophthalmol Vis Sci. 2006; 47: 213–221. [CrossRef] [PubMed]
Paper W Kroeber M Heersink S . Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression. Exp Eye Res. 2008; 87: 257–267. [CrossRef] [PubMed]
Tawara A Okada Y Kubota T . Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes. Curr Eye Res. 2000; 21: 934–943. [CrossRef] [PubMed]
MacKay EO Källberg ME Gelatt KN . Aqueous humor myocilin protein levels in normal, genetic carriers, and glaucoma beagles. Vet Ophthalmol. 2008; 11: 177–185. [CrossRef] [PubMed]
Ishibashi T Takagi Y Mori K . cDNA microarray analysis of gene expression changes induced by dexamethasone in cultured human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2002; 43: 3691–3697. [PubMed]
Fan BJ Wang DY Tham CCY Lam DSC Pang CP . Gene expression profiles of human trabecular meshwork cells induced by triamcinolone and dexamethasone. Invest Ophthalmol Vis Sci. 2008; 49: 1886–1897. [CrossRef] [PubMed]
Leung YF Tam POS Lee WS . The dual role of dexamethasone on anti-inflammation and outflow resistance demonstrated in cultured human trabecular meshwork cells. Mol Vis. 2003; 9: 425–439. [PubMed]
McDonnell DP Clemm DL Hermann T Goldman ME Pike JW . Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol. 1995; 9: 659–669. [PubMed]
Simons SSJr . The importance of being varied in steroid receptor transactivation. Trends Pharmacol Sci. 2003; 24: 253–259. [CrossRef] [PubMed]
Weinreb RN Bloom E Baxter JD . Detection of glucocorticoid receptors in cultured human trabecular cells. Invest Ophthalmol Vis Sci. 1981; 21: 403–407. [PubMed]
Zhang X Clark AF Yorio T . Regulation of glucocorticoid responsiveness in glaucomatous trabecular meshwork cells by glucocorticoid receptor-β. Invest Ophthalmol Vis Sci. 2005; 46: 4607–4616. [CrossRef] [PubMed]
Stokes J Noble J Brett L . Distribution of glucocorticoid and mineralocorticoid receptors and 11β-hydroxysteroid dehydrogenases in human and rat ocular tissues. Invest Ophthalmol Vis Sci. 2000; 41: 1629–1638. [PubMed]
Reul JMHM Gesing A Droste S . The brain mineralocorticoid receptor: greedy for ligand, mysterious in function. Eur J Pharmacol. 2000; 405: 235–249. [CrossRef] [PubMed]
Cato AC Schäcke H Sterry W Asadullah K . The glucocorticoid receptor as target for classic and novel anti-inflammatory therapy. Curr Drug Targets Inflamm Allergy. 2004; 3: 347–353. [CrossRef] [PubMed]
Garrott HM Walland MJ . Glaucoma from topical corticosteroids to the eyelids. Clin Exp Ophthalmol. 2004; 32: 224–226. [CrossRef]
Figure 1.
 
(A) Monkey TM explants with attached gelatin-coated beads. After 4 days, the cells began to proliferate and migrate from the original TM tissue onto the surface of the beads (arrows). (B) Monkey TM cells in a second passage culture, at confluence.
Figure 1.
 
(A) Monkey TM explants with attached gelatin-coated beads. After 4 days, the cells began to proliferate and migrate from the original TM tissue onto the surface of the beads (arrows). (B) Monkey TM cells in a second passage culture, at confluence.
Figure 2.
 
Western immunoblot for myocilin, performed on monkey TM cell culture CM. Results shown are from one set of samples from an individual monkey TM cell strain, incubated with DEX or BOL-303242-X in a concentration range of 3 to 300 nM, or with vehicle control (C) lacking either drug. After polyacrylamide gel electrophoresis, Western transfer, and immunodetection via chemiluminescence, the density of the myocilin bands (closely spaced doublets at ∼55 kDa) was quantified for each sample.
Figure 2.
 
Western immunoblot for myocilin, performed on monkey TM cell culture CM. Results shown are from one set of samples from an individual monkey TM cell strain, incubated with DEX or BOL-303242-X in a concentration range of 3 to 300 nM, or with vehicle control (C) lacking either drug. After polyacrylamide gel electrophoresis, Western transfer, and immunodetection via chemiluminescence, the density of the myocilin bands (closely spaced doublets at ∼55 kDa) was quantified for each sample.
Figure 3.
 
Comparison of effects of BOL-303242-X versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities at each dose of drug are shown for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data (from triplicate samples) were analyzed by two-way ANOVA, followed by the contrast procedure on logarithmically transformed data, and are presented as the geometric mean ± SE, estimated by using the Taylor series expansion. Solid and dashed lines: the calculated best fit four-parameter equation. *P < 0.05 versus vehicle control. †P < 0.05 versus the same dose of DEX.
Figure 3.
 
Comparison of effects of BOL-303242-X versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities at each dose of drug are shown for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data (from triplicate samples) were analyzed by two-way ANOVA, followed by the contrast procedure on logarithmically transformed data, and are presented as the geometric mean ± SE, estimated by using the Taylor series expansion. Solid and dashed lines: the calculated best fit four-parameter equation. *P < 0.05 versus vehicle control. †P < 0.05 versus the same dose of DEX.
Figure 4.
 
Comparison of effects of PA versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities are represented for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power −0.2. Data are presented as the geometric mean ± SE estimated by using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 versus same dose of DEX.
Figure 4.
 
Comparison of effects of PA versus DEX on myocilin protein detected in CM of cultured monkey TM cells. Myocilin protein band densities are represented for a single TM strain in one experiment. Open bar: vehicle control-treated cells. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power −0.2. Data are presented as the geometric mean ± SE estimated by using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 versus same dose of DEX.
Figure 5.
 
Representative quantitative real-time RT-PCR results for a single strain of cultured monkey TM cells, from two independent dose–response experiments comparing the effects of BOL-303242-X with DEX or of PA with DEX on myocilin mRNA expression. Data, as multiples of vehicle control (open bar), were analyzed by two-way ANOVA followed by the contrast procedure on logarithmically transformed data (BOL-303242-X versus DEX), or transformed data elevated to the power 0.2 (PA versus DEX). Data are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 for either BOL-303242-X or PA, versus DEX at the same concentration tested.
Figure 5.
 
Representative quantitative real-time RT-PCR results for a single strain of cultured monkey TM cells, from two independent dose–response experiments comparing the effects of BOL-303242-X with DEX or of PA with DEX on myocilin mRNA expression. Data, as multiples of vehicle control (open bar), were analyzed by two-way ANOVA followed by the contrast procedure on logarithmically transformed data (BOL-303242-X versus DEX), or transformed data elevated to the power 0.2 (PA versus DEX). Data are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus vehicle control. †P < 0.05 for either BOL-303242-X or PA, versus DEX at the same concentration tested.
Figure 6.
 
RU-486 inhibits DEX- and BOL-303242-X–induced myocilin protein expression by cultured monkey TM cells. Levels of myocilin that accumulated in CM during the final 48 hours of a 96-hour treatment with either 30 nM BOL-303242-X plus a dose range of RU-486 (left), DEX at 30 nM plus a dose range of RU-486 (center), or RU-486 alone in treatment vehicle (right) were assessed by densitometric analysis of Western blot images. EC50s for RU-486 were 11.74 ± 4.37 nM and 15.48 ± 6.03 nM, against 30 nM BOL-303242-X and 30 nM DEX, respectively. (■) Vehicle control treatment, without drugs; open bar, left: 30 nM BOL-303242-X alone; gray bar, center: 30 nM DEX alone. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power 0.2, and are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus control (BOL-303242-X, DEX, or vehicle control with RU-486). †P < 0.05 for BOL-303242-X + RU-486, versus DEX + RU-486, at the same RU-486 concentration.
Figure 6.
 
RU-486 inhibits DEX- and BOL-303242-X–induced myocilin protein expression by cultured monkey TM cells. Levels of myocilin that accumulated in CM during the final 48 hours of a 96-hour treatment with either 30 nM BOL-303242-X plus a dose range of RU-486 (left), DEX at 30 nM plus a dose range of RU-486 (center), or RU-486 alone in treatment vehicle (right) were assessed by densitometric analysis of Western blot images. EC50s for RU-486 were 11.74 ± 4.37 nM and 15.48 ± 6.03 nM, against 30 nM BOL-303242-X and 30 nM DEX, respectively. (■) Vehicle control treatment, without drugs; open bar, left: 30 nM BOL-303242-X alone; gray bar, center: 30 nM DEX alone. Data were analyzed by two-way ANOVA followed by the contrast procedure on transformed data elevated to the power 0.2, and are presented as the geometric mean ± SE estimated using the Taylor series expansion. *P < 0.05 versus control (BOL-303242-X, DEX, or vehicle control with RU-486). †P < 0.05 for BOL-303242-X + RU-486, versus DEX + RU-486, at the same RU-486 concentration.
Table 1.
 
Partial Agonism of BOL-303242-X in Comparison with DEX
Table 1.
 
Partial Agonism of BOL-303242-X in Comparison with DEX
Compound Efficacy ± SE* (Weighted Average, as %) 95% Confidence Interval for Efficacy (%) Coefficient of Variation (%)
DEX 100.00 ± 6.09 88.07–111.93 18.27
BOL-303242-X 53.12 ± 2.20 48.81–57.43 12.42
Table 2.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin Protein by Cultured Monkey TM Cells
Table 2.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin Protein by Cultured Monkey TM Cells
Study Compound EC50 ± SE (nM)* 95% Confidence Interval for EC50 Coefficient of Variation (%)
1 DEX 14.58 ± 2.65 10.21–20.83 18.20
BOL-303242-X 20.96 ± 4.28 14.05–31.26 20.40
2 DEX 6.14 ± 3.29 2.15–17.53 53.55
PA 104.34 ± 66.79 29.76–365.86 64.01
Table 3.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin mRNA in Cultured Monkey TM Cells
Table 3.
 
Comparison of the Potencies of DEX, SEGRA, and PA on Expression of Myocilin mRNA in Cultured Monkey TM Cells
Study Compound EC50 ± SE (nM)* 95% Confidence Interval for EC50 Coefficient of Variation (%)
1 DEX 14.66 ± 1.27 12.37–17.38 8.68
BOL-303242-X 20.75 ± 2.74 16.02–26.88 13.21
2 DEX 17.14 ± 3.12 12.00–24.48 18.19
PA 131.64 ± 56.89 56.43–365.86 43.22
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