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(Investigative Ophthalmology and Visual Science. 2001;42:1781-1786.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Reduced TIGR/Myocilin Protein in the Monkey Ciliary Muscle after Topical Prostaglandin F2{alpha} Treatment

James D. Lindsey1, Dan D. Gaton1, Takeshi Sagara1, Jon R. Polansky2, Paul L. Kaufman3 and Robert N. Weinreb1

1 From the Glaucoma Center and the Department of Ophthalmology, University of California San Diego, La Jolla; the 2 Department of Ophthalmology, University of California San Francisco; and the 3 Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Mutations in the trabecular meshwork inducible glucocorticoid response (TIGR) gene, also known as myocilin, have recently been linked to some forms of glaucoma. Recent studies have shown that TIGR protein also is expressed in the ciliary muscle. Because uveoscleral outflow, which traverses the ciliary muscle, is increased by prostaglandins (PGs), the present study assessed whether topical PGs alter the amount of TIGR protein within the ciliary muscle.

METHODS. Vehicle was topically applied to one eye, and 2 µg PGF2{alpha}-isopropyl ester (PGF2{alpha}-IE) was applied to the other eye of cynomolgus monkeys twice daily for 5 days. Pressure reductions of 5 mm Hg in the PGF2{alpha}-IE–treated eyes were confirmed. The eyes were then fixed and paraffin sections were cut from each eye. The distribution of TIGR protein in the ciliary muscle was determined by confocal scanning laser microscopy. Additional sections were immunostained with a polyclonal antibody to recombinant TIGR protein or with a polyclonal antibody to a synthetic peptide corresponding to the leucine zipper region within the TIGR protein. Staining intensity in the ciliary muscle was assessed by measuring optical density (OD) along two line segments overlying the ciliary muscle, by using a high-resolution imaging densitometer.

RESULTS. TIGR protein immunoreactivity was observed in ciliary muscle fibers throughout the ciliary muscle. Extracellular TIGR immunoreactivity colocalized with collagen type IV immunoreactivity. Intracellular staining also was present. Immunoreactivity was less intense in the sections from the PGF2{alpha}-IE–treated eyes compared with the vehicle-treated eyes. This was reflected in the reduction of mean OD scores in each monkey. Overall, the reduction of mean OD scores in the treated eyes was 42.1% ± 9.9% (P < 0.005) with the anti-recombinant TIGR antibody and 27.3% ± 10.4% with the anti-TIGR peptide antibody (P < 0.005).

CONCLUSIONS. TIGR protein immunoreactivity was present both intracellularly and extracellularly in the ciliary muscle of the cynomolgus monkey. This suggests that extracellular TIGR protein is in contact with aqueous humor in the uveoscleral outflow pathway. Moreover, IOP-lowering topical PGF2{alpha}-IE treatment decreases the amount of TIGR protein in the ciliary muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The trabecular meshwork inducible glucocorticoid response (TIGR) protein was first identified in cultures of human trabecular meshwork cells as a glycoprotein that was induced by prolonged exposure to dexamethasone.1 2 3 It was subsequently noted that this gene mapped to the same genomic site as GLC1A, a locus that had been linked to certain forms of early-onset familial glaucoma. When this gene was sequenced from a group of patients with primary open-angle glaucoma, it was found that 3% to 4% had exon mutations in the TIGR gene.4 5 More than 20 different mutations of this gene have been linked to glaucoma.3 6 7 8 9 Subsequent cloning of a gene from the photoreceptor connecting cilium, termed myocilin (MYOC), showed that it had strong sequence homology with TIGR and was later found to be identical.10 11 Northern blot analysis, immunohistochemistry, and in situ hybridization experiments have detected transcription of the TIGR/MYOC gene and the biosynthesis of TIGR protein in many different eye structures.3 12 13 14 15 In addition, there appear to be multiple forms of the TIGR protein distributed both intracellularly and extracellularly.2 15 16 17 These observations have created substantial interest in the role of the TIGR/MYOC gene and its expression in normal eye function and in the pathophysiology of primary open-angle glaucoma. Although the function of TIGR protein is presently unknown, structural evidence has led to the proposal that extracellular TIGR protein could contribute to outflow resistance.2 15

Recent studies have shown that topical treatment with prostaglandin (PG)F2{alpha}, which facilitates uveoscleral outflow, is associated with reductions in the amount of collagen types I, III, and IV in the ciliary muscle.18 Because these collagens are contained in the extracellular space through which uveoscleral outflow passes, it is possible that such reductions contribute to increased uveoscleral outflow by reducing hydraulic resistance to aqueous flow.19 20 In view of the presence of TIGR protein within the ciliary muscle and evidence that, in other cell systems, TIGR protein is secreted,1 2 3 it is possible that topical PG treatment also influences TIGR protein expression in the ciliary muscle. The present study was undertaken to investigate this possibility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study took advantage of paraffin tissue blocks available from our previous study in which cynomolgus monkeys had 2 µg topical PGF2{alpha}-IE applied unilaterally for 5 days before quantitative immunohistochemical evaluation of collagen changes in the ciliary muscle.18 All procedures were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

The monkeys initially were evaluated by biomicroscopy to confirm normal appearance and by fluorophotometry to confirm blood–aqueous barrier integrity. Briefly, intravenous fluorescein, 10 mg/kg of 10% solution, was administered through the saphenous vein under intramuscular ketamine hydrochloride anesthesia (10 mg/kg). After a wash with 2 ml lactated Ringer solution, corneal and anterior chamber (AC) fluorescence was measured (Fluorotron Master Scanning Ocular Fluorophotometer equipped with an anterior segment adapter; Coherent Medical, Palo Alto, CA) at 20, 40, 60, 90, 120, 180, and 240 minutes after injection. For inclusion in the present study, AC fluorescence levels and time course of appearance and decay in both eyes had to be similar and within the range of values obtained for well-characterized control eyes. The following week, each of the qualifying monkeys received 2 µg PGF2{alpha}-IE (in 5 µl) in the morning and in the evening for 5 days. On the fourth and fifth days, IOP was measured at 3, 3.5, and 4 hours after the morning treatment to confirm IOP reduction. On the fifth day, the eyes were evaluated by biomicroscopy. A monkey was excluded from further study if it had less than 5 mm Hg reduction in the treated eye (compared with pretreatment ipsilateral baseline and simultaneous contralateral control) or if AC cells or flare were observed. Eight eyes were obtained from four monkeys that met the criteria.

Quantitative Analysis
Each animal was killed, its vascular bed cleared by perfusion, and the two eyes collected, fixed, and processed into paraffin, as previously described.18 Each anterior segment was embedded in a single block. For histopathologic analysis, several sections from each eye were stained with hematoxylin and eosin. For the present study, five midsagittal sections from each eye were cut at 10 µm, a thickness determined by previous studies to be optimal for our quantitative immunohistochemistry protocol. These sections were cleared, rehydrated, subjected to antigen retrieval solution and melanin extraction, and blocked with 0.1% bovine serum albumin, as previously described.18 The sections were then exposed overnight to a rabbit polyclonal antibody raised against the recombinant human 55-kDa form of the TIGR protein, diluted to 1:500. The usefulness of this antibody has been demonstrated by Western blot analysis and immunoprecipitation assays of dexamethasone-induced human trabecular meshwork cells and in tissue studies of the human trabecular meshwork.12 Parallel experiments were conducted with a polyclonal antibody generated in rabbits that had been raised against a synthetic polypeptide corresponding to the amino acid sequence DKSVLEEEKKRLRQ and characterized by Western blot analysis (diluted 1:500).15 This sequence is found within the leucine zipper portion of the TIGR protein.16 Subsequently, the sections were rinsed, exposed to biotinylated goat anti-rabbit antibody for exactly 20 minutes, rinsed, exposed to horseradish peroxidase–conjugated streptavidin for exactly 20 minutes, rinsed, and then developed with 3,3'-diaminobenzidine chromogen for 10 minutes, as previously described. To facilitate comparability, sections from the control vehicle-treated and PG-treated eye of each monkey were immunostained at the same time.21 22 To serve as control samples for nonspecific staining, sections from each eye were simultaneously processed by the same protocol but without the primary antibody.

Immunohistochemical staining intensity was directly measured using a high-resolution imaging densitometer, as previously described.18 23 Measurements from multiple sections stained at the same time facilitated assessment of measurement precision and permitted statistical comparison of differences among control and experimental eyes. Immunostained sections were scanned by placing the slides directly on the platen of an imaging densitometer at a resolution of 1200 dots per inch (50-µm-wide pixels), as previously described. The scanned digital data were displayed in a masked fashion and analyzed using an image analysis program (Molecular Analyst, ver. 2.1; Bio-Rad, Hercules, CA). The optical density (OD) along two line segments positioned perpendicular to the long axis of the ciliary body and near the widest region of the ciliary muscle was measured in each section, by two-dimensional–imaging densitometry. The positioning of these line segments over the anterior segment tissues avoided any remaining cluster of pigment granules.18 23

Confocal Scanning Laser Microscopy
To determine the distribution of the TIGR protein within ciliary muscle tissue, sections were immunostained with the anti-peptide antibody. Other sections were immunostained using mouse monoclonal antibodies to human collagen type IV (1:200; Biogenix, San Rafael, CA) or with both antibodies. After washing, the sections were incubated with either goat antibody to rabbit IgG conjugated to a rhodamine derivative that fluoresces at 510 nm when illuminated by 488 nm light (Alexa488, diluted 1:200; Molecular Probes, Eugene, OR), goat antibody to mouse IgG conjugated to a rhodamine derivative that fluoresces at 610 nm when illuminated with 568 nm light (Alexa568, 1:200; Molecular Probes), or both antibodies. To minimize nonspecific staining, the secondary antibodies were preabsorbed to a homogenate of human ciliary muscle tissue before use. The sections were washed and then a coverslip was mounted using an aqueous mounting medium. Examination was performed using a two-channel confocal scanning laser microscope (Fluoview; Olympus, Lake Success, NY) fitted with a krypton-argon laser (Melles-Griot, Carlsbad, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TIGR Distribution in the Ciliary Muscle
Ciliary smooth muscle cells were strongly immunoreactive for TIGR protein throughout the ciliary body of the vehicle-treated eyes (Fig. 1) . Among the longitudinal, circular, and radial ciliary muscle cells, the intensity of staining is similar. Nonspecific immunostaining was minimal in sections for which incubation with primary antibody had been omitted from the protocol (Fig. 1D) . A similar staining pattern was also observed with the antibody to the peptide (data not shown).



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  		Figure 1. Ciliary body from monkey eye immunostained with antibodies to TIGR protein (A). Labeled are the longitudinal region of the ciliary muscle (lcm) and the trabecular meshwork (tm). High-magnification view of longitudinal ciliary muscle from vehicle-treated eye (B) shows staining of ciliary muscle fiber bundles (mb) and minimal staining of the interstitial extracellular matrix (im). TIGR protein immunoreactivity was reduced in the longitudinal ciliary muscle of eyes that had 5 days’ treatment with PGF2{alpha}-IE (C). Immunostaining control produced without primary antibody showed minimal nonspecific staining (D). Magnification, (A) x190; (B) x750.

 
To determine cellular distribution of TIGR protein within the ciliary muscle, immunostained sections were examined by confocal scanning laser microscopy. For reference, sections were double stained with antibody to human collagen type IV. Figure 2 shows collagen type IV immunoreactivity within the basement membranes surrounding the smooth muscle cells in the longitudinal portion of the ciliary muscle. In the same section, TIGR immunoreactivity was present within the bundles of ciliary smooth muscle cells, but was largely absent from the interstitial spaces between the smooth muscle bundles (Fig. 2B) . Comparison of the TIGR protein image with the collagen type IV image revealed moderate TIGR protein immunoreactivity within the ciliary muscle cytoplasm, as well as extracellularly in association with collagen type IV. This was more easily seen when the images were superposed (Fig. 2C) and overlapping staining appeared yellow. Areas were observed where the confocal plane passed through the middle of the cell and the cross-sectioned basement membrane appeared as a red line with yellow areas. Adjacent areas were observed where the confocal plane was tangential to the plasma membrane and contained basement membrane en face. In the single images, these en face areas contained either bright red collagen type IV immunoreactivity or bright green TIGR protein immunoreactivity. In the superposed image, this staining typically overlapped and appeared yellow. These results indicate that TIGR protein was present in ciliary muscle cytoplasm, as well as extracellularly in association with the basement membrane.



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  		Figure 2. Collagen type IV and TIGR protein immunoreactivity in the longitudinal portion of the ciliary muscle. (A) Collagen type IV immunoreactivity within the basement membranes surrounding the smooth muscle cells. Interstitial extracellular matrix (im) did not contain collagen type IV. Areas were observed where the confocal plane passed through the middle of the cell and the cross-sectioned basement membrane appeared as a discrete red line (cs). In adjacent areas, the confocal plane passed tangential to the cell membrane and contained the basement membrane (t). (B) In the same section, TIGR immunoreactivity was present within the bundles of ciliary smooth muscle cells but was largely absent from the interstitial spaces between the smooth muscle bundles. (C) Composite image of (A) and (B) shows many areas where TIGR immunoreactivity overlapped the collagen type IV immunoreactivity and appeared yellow. Magnification, x1500.

 
Changes in TIGR Immunoreactivity Associated with Topical PGF2{alpha}-IE
Ciliary smooth muscle cells in the contralateral PGF2{alpha}-IE–treated eyes were less intensely stained than in the corresponding vehicle-treated eyes (Figs. 1B 1C) . The pattern of immunostaining was similar in the treated and control eyes–that is, intense staining in the ciliary muscle cell bundles and less intense staining in the interstitial extracellular matrix.

Quantitative analysis using imaging densitometry revealed that for all four monkeys analyzed using the antibody to recombinant TIGR protein, immunoreactivity was less in the PGF2{alpha}-IE–treated eye than in the vehicle-treated eye. Specifically, there was less intense immunostaining in the ciliary muscle of the treated eye than in that of the control eye (Fig. 3A ). Combining these data showed that the average mean OD score reduction in the treated eyes was 42.1% ± 9.9% (SEM; Fig. 3B , P < 0.005, paired Student’s t-test).



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  		Figure 3. TIGR immunoreactivity in the ciliary muscle measured using antibody raised against recombinant TIGR protein (A, B) or against the leucine zipper region peptide (C, D). Data shown include the mean OD measurements of sections from each eye (A, C) and the percentage reduction of OD in sections from each treated eye compared with the mean OD in sections the contralateral vehicle-treated eye (B, D). Ten ciliary muscle sections were analyzed from each eye. The average reduction of mean OD score with the anti-recombinant TIGR protein antibody was 42.1% ± 9.9% (P < 0.005, paired Student’s t-test). The average reduction of mean OD score with the anti-peptide antibody was 27.3% ± 10.4% (P < 0.005, paired Student’s t-test).

 
Similar quantitative analysis was performed using the anti-peptide antibody. For one of the eyes from K453, insufficient tissue in the tissue block was present for this analysis. Therefore, the analysis evaluated K91, K344, and K454. As was the case with the antibody to recombinant TIGR protein, there was less intense immunostaining in the treated eye ciliary muscle than in the control eye ciliary muscle stained with anti-peptide antibody (Fig. 3C) . Combining these data showed that the average mean OD score reduction in the treated eyes was 27.3% ± 10.4% (Fig. 3D ; P < 0.005, paired Student’s t-test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results indicate that repeated topical PGF2{alpha}-IE treatments were associated with a reduction in the amount of TIGR-protein expression within the monkey ciliary muscle. Control experiments indicate that this effect was due to the drug treatment.

As in human ciliary muscle,13 TIGR protein immunoreactivity was not present in the interstitial extracellular matrix of monkey ciliary muscle. However, TIGR protein immunoreactivity was present inside and adjacent to the outside surface of ciliary muscle cells. This extracellular TIGR immunoreactivity exactly coincided with immunoreactivity for collagen type IV, which typically is found in the basement membrane of normal tissues.24 25 Moreover, it appeared to be a substantial fraction of the total TIGR protein present. Supporting its physiological association with basement membrane is the observation that a molecular binding site is present in TIGR protein with affinity for an artificial extracellular matrix containing normal basement membrane components such as collagen type IV (Matrigel; Becton-Dickinson, Bedford, MA).16 This may indicate that PGF2{alpha}-IE treatment leads to reduction of TIGR protein associated with basement membrane extracellular matrix in the ciliary muscle.

Two antibodies were used in the present investigation. The first was raised against recombinant human TIGR protein, which is unglycosylated. It recognizes an epitope, or epitopes, that appear to be masked for certain extracellular forms of the molecule.16 In contrast, the present confocal microscopy results and prior immunoelectron microscopy results indicate that the anti-peptide antibody recognizes both intracellular and extracellular TIGR protein.15 Therefore, the measurements obtained using this antibody provided an overview of both intracellular and extracellular changes in TIGR protein with treatment. The results obtained with both antibodies were consistent with each other within the sensitivity limits of the present study.

A reduction in TIGR protein associated with basement membrane could be of significance in uveoscleral outflow. Tracer studies indicate that uveoscleral flow passes through extracellular spaces among ciliary muscle bundles.26 If TIGR protein represents a substantial volume fraction of basement membrane–associated proteins in the ciliary muscle, it would be plausible that specific reduction of extracellular TIGR protein would reduce the thickness of the stationary boundary to the ciliary muscle uveoscleral flow compartment. Such thickness reduction may produce an enlargement of the uveoscleral flow compartment and perhaps thereby reduce uveoscleral flow resistance. Alternatively, if TIGR protein retention in ciliary muscle extracellular matrix reflects only its binding to collagen type IV, then the reduction of collagen type IV (that has been previously documented in the same eyes used in the present study18 may explain the presently observed reduction of TIGR protein. In either case, the result would be an expansion of the extracellular spaces within the ciliary muscle that could facilitate increased uveoscleral outflow. This link between enlargement of the extracellular space in ciliary muscle and increased uveoscleral outflow facility was first suggested by early tracer studies27 28 and was subsequently supported by observation of increases in the size of these spaces in monkey eyes that had received topical PGF2{alpha}.29 30 Because uveoscleral flow appears to be predominantly through the interstitial spaces in the ciliary muscle,26 the significance of TIGR protein and collagen type IV reduction may be minimal.

A possible explanation for reduction of extracellular TIGR protein with PGF2{alpha}-IE is that it reflects matrix metalloproteinase (MMP) induction. Previous experiments have shown that topical PGF2{alpha}-IE treatment of monkey eyes induces reduction of collagen types I, III, and IV in the ciliary muscle.18 PG treatment of cultured ciliary muscle cells also leads to reduced collagen types I, III, and IV in the cell layer.20 Concomitantly, there is increased secretion of MMPs,31 32 enzymes that can initiate the degradation of extracellular matrix molecules.33 In view of the close association of extracellular TIGR protein with collagen type IV in the ciliary muscle, it is possible that either as a result of MMP action on collagen type IV, or directly on TIGR protein, there is mobilization of TIGR protein within the basement membranes facing the interstitial compartment and that it is subsequently washed out of the ciliary muscle. Therefore, PGF2{alpha}-IE–mediated induction of MMP secretion may explain the reduction of both collagens and extracellular TIGR protein in the ciliary muscle. These reductions each could contribute to increased uveoscleral outflow.

Both intracellular and extracellular TIGR protein changes also may reflect reduced TIGR/MYOC gene expression. PGF2{alpha} treatment of cultured ciliary muscle cells can increase intracellular messenger signals such as cAMP.34 Moreover, activation of the FP prostanoid receptor may trigger other signals associated with G-protein activation.35 36 TIGR/MYOC gene expression in trabecular meshwork cells has been shown to be regulated by several intracellular signals, including steroid receptor activation, oxidative signals, phorbol ester–related signals, and various hormonal signals.2 In astrocytes, several different DNA-binding elements as well as an upstream regulatory factor have been shown to modulate TIGR/MYOC gene expression.14 In view of these observations, it is possible that PGF2{alpha}-IE treatments lead to altered TIGR/MYOC gene expression in ciliary muscle cells that lead to reduced TIGR protein biosynthesis. Further work is needed to determine the regulatory mechanisms specific to ciliary muscle cells.

It also is possible that PGF2{alpha}-IE treatment influences TIGR protein destined for intracellular targets within ciliary muscle cells. Ultrastructural immunohistochemical analysis of TIGR protein distribution in trabecular meshwork in situ and in cultured trabecular meshwork cells found immunoreactivity in association with mitochondria and cytoplasmic filaments.15 In the present study, some TIGR protein immunoreactivity was present intracellularly in ciliary muscle cells. Thus, the reduced TIGR protein occurring with IOP-lowering PGF2{alpha}-IE treatment, detected using the antibody to recombinant protein, may have consequences for as yet undefined cytoplasmic functions in ciliary muscle cells. These considerations support the appropriateness of further investigations addressing the possible role of TIGR protein in uveoscleral outflow as well as in other ciliary muscle functions.


   Acknowledgements
 
The authors thank B’Ann T. Gabelt for assistance with all aspects of the live monkey work.


   Footnotes
 
Supported in part by National Eye Institute Grants EY05990 (RNW), EY02477 (JRP), and EY02698 (PLK); a Senior Scientific Investigator Award from Research to Prevent Blindness (RNW); and a grant from the Foundation For Eye Research (DDG).

Submitted for publication November 2, 2000; revised February 26, 2001; accepted April 6, 2001.

Commercial relationships policy: N.

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

Corresponding author: Robert N. Weinreb, Glaucoma Center, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0946. weinreb{at}eyecenter.ucsd.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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