The finding of mutations in the gene encoding the trabecular

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1 mrna In Situ Hybridization of TIGR/MYOC in Human Trabecular Meshwork Xiaofang Wang and Douglas H. Johnson PURPOSE. To determine the distribution of mrna expression of the trabecular meshwork induced glucocorticoid response protein/myocilin (TIGR/MYOC) in human trabecular meshwork. METHODS. In situ hybridization using a 1.25-kb probe obtained from reverse transcription polymerase chain reaction of TIGR/MYOC cdna was performed to determine the location of cell labeling within the different regions of the meshwork. The effect of dexamethasone on the pattern of labeling was studied in organ cultured meshwork. Trabecular meshwork from three sources was studied: enucleated eyes obtained at autopsy, trabeculectomy specimens obtained during filtration surgery, and meshworks from anterior segments in perfusion organ culture. Hybridization was performed on frozen sections, paraffin sections, and sections from JB-4 plastic embedded tissue. RESULTS. Labeling for TIGR/MYOC mrna was present in most trabecular cells of the uveal, corneoscleral, and juxtacanalicular regions but only variably present in the endothelial cells of Schlemm s canal. A similar pattern was found in the trabeculectomy specimens from eyes with primary open-angle or pseudoexfoliative glaucoma. Dexamethasone treatment increased the labeling intensity and number of labeled cells in meshwork, and also the number of labeled endothelial cells of Schlemm s canal. Fresh tissue processed within 12 hours postmortem gave more consistent labeling than older tissue, although some label was found up to 48 hours postmortem. Labeling was found in tissue from all three sources, and with all three embedding techniques; JB-4 sections provided the best morphologic resolution. CONCLUSIONS. In situ hybridization reveals that mrna expression for TIGR/MYOC is present in most cells in all regions of the meshwork but only variably present in the endothelial cells of Schlemm s canal. Dexamethasone treatment increased the number and intensity of labeled cells, and also increased the number of labeled cells in the endothelial lining of Schlemm s canal. (Invest Ophthalmol Vis Sci. 2000;41: ) The finding of mutations in the gene encoding the trabecular meshwork induced glucocorticoid response protein (TIGR) in some cases of juvenile and adult open-angle glaucomas has focused research on how this protein is involved in aqueous outflow. 1 TIGR, also known as myocilin (will be referred to as MYOC), is normally present in the trabecular meshwork (TM) and has been demonstrated with immunohistochemical and biochemical techniques. 2 6 MYOC is present both intracellularly and extracellularly. It may serve an intracellular cytostructural role, because it has been described in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. 7 Secretion of extracellular MYOC increases after dexamethasone treatment in a timedependent fashion in cultured cells, making it a candidate in the pathophysiology of corticosteroid-induced glaucoma. 2 5 From the Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Supported in part by National Institutes of Health Research Grant EY 07065; by Research to Prevent Blindness, New York, New York; and by the Mayo Foundation, Rochester, Minnesota. Submitted for publication October 1, 1999; revised December 29, 1999; accepted January 18, Commercial relationships policy: N. Corresponding author: Douglas H. Johnson, Department of Ophthalmology, Mayo Clinic, 200 First Street SW, Rochester, MN johnson.douglas@mayo.edu The TM actively synthesizes MYOC protein, as demonstrated with Northern blot analysis of mrna for MYOC from TM samples and trabecular cells in monolayer culture. 2 5 Which cells within the human meshwork synthesize MYOC is unknown. If the effect of MYOC on outflow occurs because of an intracellular role, it should be present in the cells most likely to control aqueous outflow, the inner wall endothelial cells of Schlemm s canal. If MYOC mainly serves an extracellular role in the meshwork, it could be secreted into the extracellular space, or secreted and subsequently bound to the glycocalyx of that cell or a downstream cell. It is possible that MYOC could be made preferentially in the upstream cells of the uveal and corneoscleral meshwork and wash into the outer corneoscleral and juxtacanalicular regions, as may occur with hyaluronic acid. 8 Thus, knowing the specific cells in the meshwork that synthesize MYOC may give an insight into the physiological role of MYOC. The present study was performed to determine the exact cellular sites of synthesis of MYOC, and whether they would be affected by dexamethasone treatment. METHODS MYOC was studied in a total of 32 eyes from 20 donors. TM from fresh eyes, surgical trabeculectomy specimens from eyes with glaucoma, and the anterior segments of perfusion organ Investigative Ophthalmology & Visual Science, June 2000, Vol. 41, No Copyright Association for Research in Vision and Ophthalmology

2 IOVS, June 2000, Vol. 41, No. 7 Localization of TIGR/MYOC mrna in Human TM 1725 cultured eyes were studied. Eyes were either normal, with no history of ocular disease, or had a known history of glaucoma, as will be noted below. This protocol was approved by the Mayo Institutional Review Board and met the tenets of the Declaration of Helsinki. Source of Tissue Fresh Tissue. Autopsy Eyes: Seven normal eyes from four donors (mean age, 77 7 years) were processed for frozen, paraffin, or JB-4 sections. Two of these pairs of eyes were used to determine the stability of mrna postmortem, by fixing tissue wedges of each eye from one pair at 4, 6, 8, and 12 hours postmortem and from the second pair of eyes at 7, 18, 24, 36, and 48 hours postmortem. To compare embedding techniques, two pairs of eyes had tissue processed in both paraffin and JB-4 plastic. Trabeculectomy Specimens: Seven trabeculectomy specimens were obtained at filtration surgery from seven donors (six primary open-angle glaucoma [POAG], one pseudoexfoliation; mean age, years) and immediately fixed in 10% formalin, then processed for either paraffin sections or JB-4 blocks. Cultured Meshwork. Human autopsy eyes obtained up to 40 hours postmortem were used. Eyes were bisected at the equator, and the iris, lens, and vitreous were removed. The anterior segment was clamped in a modified petri dish and perfused with Dulbecco s modified Eagle s medium with added antibiotics (penicillin, 10,000 U; streptomycin, 10 mg; amphotericin B, 25 mg; and gentamicin, 17 mg; all in 100 ml medium; Sigma, St Louis, MO) at the normal human flow rate (2.5 l/min). The anterior segments were cultured at 37 C in a 5% CO 2 atmosphere. 9,10 At the end of the culture period, the anterior segments were immediately fixed in 10% formalin. Effect of Culture: Anterior segments from both eyes of four normal donors (mean age, years) were placed into perfusion organ culture for 1 week, then processed for in situ hybridization. Postmortem times were 10, 16, 16, and 40 hours. Particular note was made of the number and location of labeled cells and the labeling intensity, in the shortest and longest postmortem-time eyes, to determine whether the culture process could revive meshworks obtained beyond 24 hours postmortem. An additional pair of eyes from a 97 year old donor with primary open angle glaucoma, obtained 22 hours postmortem, was cultured to determine the effect of culture on the labeling pattern of glaucomatous eyes. Dexamethasone Treatment: Anterior segments from both eyes of four normal donors (mean age, years) were placed into perfusion organ culture. Postmortem times were 7, 19, 22, and 23 hours. For each pair, one eye received the standard culture medium, and the fellow eye received the culture medium with the addition of dexamethasone 10 7 M for 21 days. All anterior segments were then processed for JB-4 sections. Embedding and Sectioning Frozen Sections. Tissue samples were fixed in 4% paraformaldehyde for 3 hours, soaked in 30% sucrose at 4 C overnight, and then frozen in OCT compound (Sakura Finetek USA, Torrance, CA) at 70 C. Sections 10- m thick were placed on glass slides, dried in an oven at 40 C overnight, and then dehydrated in a graded series of ethanol (75%, 85%, 95%, 100%) and stored at 70 C until hybridization. Paraffin Sections. Tissue samples were fixed in 10% neutral-buffered formalin for 24 hours, dehydrated in a graded series of ethanol (75%, 85%, 95%, 100%), soaked in Xylene, then embedded in paraffin. Sections 5- m thick were placed on glass slides, baked at 60 C for 2 hours, and stored at room temperature until hybridization. JB-4 Sections. Tissue was hybridized with probe before embedding and sectioning. After in situ hybridization, tissue wedges were processed into JB-4 plastic sections by dehydration in a graded series of ethanol (50%, 75%, 95%), soaking in JB-4 solution A C for 30 minutes at room temperature, then incubated in the same fresh solution at 4 C overnight. Tissue was embedded in JB-4 solution A B C, and sections 5- m thick were placed on glass slides. In Situ Hybridization RNA Probes. A 1.25-kb cdna clone encoding human MYOC was obtained by reverse transcription polymerase chain reaction (RT PCR) with the following primers: 5 -GC- CAGTC-CCAATGAATCCAG-3 (upstream primer) and 5 -CG- GTTCTTGAATGGGATGGT 3 (downstream primer). The template for this reaction was total RNA extracted from human TM. Reaction conditions were 95 C denaturation, 65 C annealing, and 72 C extension for 35 cycles. PCR consistently yielded a single product of the expected size (1.25 kb), which was then cloned into pgem-t easy vector (Amersham, Piscataway, NJ). The identity of this cdna was confirmed by sequencing. RNA transcripts were labeled with digoxigenin (DIG) UTP (Genius 4 RNA labeling kit; Boehringer Mannheim, Indianapolis, IN). The single-strand RNA probes were purified by ethanol precipitation. Application of Probe to Tissue. Before application of the probe, frozen sections were warmed to room temperature, and paraffin sections were deparaffinized. Because tissue to be processed for JB-4 required the hybridization step before JB4 embedding, it was treated similarly to the frozen and paraffin sections at this point. All sections and tissue wedges were then incubated in 0.2 N HCl, treated with proteinase K, washed in phosphate-buffered saline, immersed 0.5% acetic anhydride in 0.1 M triethanolamine at ph 8, prehybridized in mrna hybridization solution (DAKO, Carpinteria, CA), and hybridized in a solution containing 300 ng/ml of either the sense or antisense probe. All sections and tissue wedges were incubated in a closed moist chamber at 55 C overnight to allow hybridization. After hybridization, microscopic slides and tissue wedges washed in SSC, incubated in 4 g/ml RNase A to remove unbound probe, and washed in SSC at 50 C. All slides and wedges were then blocked with 5% bovine serum albumin in 150 mm NaCl, 100 mm Tris HCl (ph 7.50) for 30 minutes at room temperature and incubated in the same solution containing a 1:200 dilution of anti-dig alkaline phosphatase conjugate (Boehringer Mannheim) for 2 hours at 37 C. After washing in 150 mm NaCl, 100 mm Tris HCl (ph 7.5), the nitroblue tetracolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) color detection reaction was then performed according to the manufacturer s instructions (Boehringer Mannheim). The color detection reaction was monitored microscopically and interrupted after 2 hours by washing in 10 mm Tris, 1 mm EDTA. Sections were then mounted and coverslips applied, whereas the tissue wedges were processed into JB-4 plastic and sectioned at 5 m thickness. Nuclear fast red was used as a counterstain for some sections.

3 1726 Wang and Johnson IOVS, June 2000, Vol. 41, No. 7 FIGURE 1. TM: Comparison of labeling with frozen, paraffin, and JB-4 sections from fresh eyes. (A) Frozen section (CRYO). (B) Paraffin section. (C) JB-4 section. (D) JB-4 section with nuclear fast red staining. Note label can be localized to the cytoplasm of specific cells with JB-4 section. Schlemm s canal endothelial cells are not labeled. SC, Schlemm s canal. (Magnification for all, 400.) Control Sections. Two types of controls were run. Sense probe was used in parallel, simultaneous processing with the antisense probes for all eyes. In addition, sections were also run with no probe of either kind, but otherwise processed similarly to the other tissue. For frozen sections and paraffin sections, control sections were taken from the same tissue block, often as serial sections. Because JB-4 required hybridization before embedding, control tissue was taken from a tissue wedge adjacent to the experimental tissue wedge and processed with either the sense probe or with no probe. RESULTS Labeling for MYOC mrna was present in the meshwork of most eyes. Almost all meshwork cells of the uveal, corneoscleral, and JCT regions were labeled. In contrast, labeling of endothelial cells of Schlemm s canal was variable, with most eyes having only three or four labeled cells around the inner and outer walls of the canal. Multiple blocks and multiple sections from the same eye were similar in the amount and location of labeling. In control sections, no signal was detected when serial sections from the same sample (or an adjacent tissue wedge for JB-4 sections) were processed with the sense probe or with no probe. Labeling was found with all three embedding techniques. JB-4 sections provided the best cellular and morphologic resolution (Fig. 1). Precise location of the mrna for MYOC was possible using JB4 sections, and localization of the label to the cytoplasm was evident. Counterstaining with nuclear fast red was often helpful in defining nuclei and the overall anatomic structures. Paraffin sections gave adequate intensity of label, although it was not always possible to distinguish a faint label from no label in some cells. Paraffin control sections, again performed with sense probe and also with no probe, could have a faint background color that was felt to be nonspecific binding of the dye. Paraffin sections provided limited resolution of cellular details, and it was difficult at times to determine which specific cells were labeled, due to section thickness, less clear histologic resolution of detail, and a tendency for the dye to bleed or leach into adjacent tissue. Frozen sections gave poorest histologic resolution, and it was often difficult to determine cell boundaries and the exact location of labeled cells. Source of Tissue Fresh Eyes. Strong hybridization signal was noted in sections from 7 of 8 eyes. In the time-course study to determine stability of mrna with postmortem time, labeling was strong up to 24 hours postmortem but was decreased by 36 hours postmortem and only sporadically present in the mesh-

4 IOVS, June 2000, Vol. 41, No. 7 Localization of TIGR/MYOC mrna in Human TM 1727 FIGURE 2. Trabeculectomy specimens. (A) POAG, case 1. Label is found in many but not all cells of the meshwork, and in a few endothelial cells of the canal (JB-4 sections; magnification, 400). (B) Pseudoexfoliation glaucoma (PEX). Label is more widespread and more intense than in POAG specimens. (Magnification for paraffin section, 400.) work by 48 hours postmortem (data not shown). In eyes from one 71-year-old donor, the right and left eyes gave differing results. One eye showed moderately strong labeling, whereas the fellow eye, processed simultaneously under identical conditions, gave only faint label. Trabeculectomy Specimens. In the 6 specimens from eyes with POAG, light to moderate labeling intensity was found in many but not all cells of the uveal and corneoscleral meshwork and JCT. Labeling was not uniform throughout the meshwork. Labeling was neither as common nor as strong as in the normal eyes. Only a few cells of the inner wall endothelium of Schlemm s canal were labeled (Fig. 2). In the control sections, no signal was detected when serial sections from the same sample were hybridized with the sense riboprobe. The one specimen from an eye with pseudoexfoliation glaucoma demonstrated strong labeling of the uveal and corneoscleral meshwork and JCT cells but only faint labeling of the inner wall endothelium cells of Schlemm s canal (Fig. 2). Cultured Eyes. After 1 week in culture, labeling pattern and intensity resembled those of fresh eyes in 5 of 8 eyes (Fig. 3). Three eyes did not show any mrna MYOC label; two were from one pair obtained from a 61-year-old donor, 16 hours postmortem. The eyes obtained 40 hours postmortem, from a 77-year-old donor, demonstrated normal labeling when compared with fresh eyes. More Schlemm s canal cells appeared labeled in the cultured eyes than in fresh eyes, involving approximately 50% of the cells of the canal. Control sections were negative, with no label detected when serial sections from the same sample were hybridized with the sense riboprobe (data not shown). Labeling of the glaucomatous pair of eyes was similar in pattern to that of fresh eyes, but only of moderate intensity. Schlemm s canal endothelial cells did not label in this pair of eyes. Dexamethasone Treatment Strong hybridization signal was present in all cells of the meshwork and JCT in all four cultures receiving dexamethasone. Of interest, label of Schlemm s canal cells was prominent in three of the four treated eyes, with nearly all canal cells labeled. In control eyes, label throughout the meshwork was much less intense, and was less intense than in eyes cultured for only one week in the study above. In addition, fewer labeled cells were present in the meshwork and JCT, and only a few labeled endothelial cells were present in the canal (Fig. 4). No signal was detected when serial sections from the same sample were FIGURE 3. Cultured meshworks. (A) Normal eye, postmortem 16 hours, cultured 7 days. (B) Normal eye, postmortem 40 hours, cultured 7 days. Strong and widespread label evident. (Magnification for paraffin sections, 400.)

5 1728 Wang and Johnson IOVS, June 2000, Vol. 41, No. 7 FIGURE 4. Cultured meshworks, all with JB-4 sections. (A, B) Dexamethasone treatment for 21 days. Widespread labeling of meshwork cells (A; magnification, 400). Note cells lining canal are labeled (B; magnification, 1000). (C) Fellow control eye to (A). Label is less intense, canal cells not labeled (magnification, 400). (D) Dexamethasone treatment for 21 days. Sense probe. Note no label is found (magnification, 400). hybridized with the sense riboprobe for either the dexamethasone-treated or the control cultures (data not shown). DISCUSSION mrna expression of MYOC appears in trabecular cells of both normal and glaucomatous eyes. In normal fresh eyes, labeling was found in most cells of all three regions of the meshwork: uveal, corneoscleral, and JCT. In contrast, labeling of the endothelial cells of Schlemm s canal was variable, with only a few cells showing label. Trabeculectomy specimens from glaucoma patients demonstrated labeling in many, but not all, cells of the uveal and corneoscleral meshwork regions, and in the JCT. As in the normal eyes, only a few of the endothelial cells of the canal were labeled. In glaucomatous eyes, however, the labeling of MYOC mrna was neither as common nor as strong as in normal eyes. These results suggest that the mrna level of MYOC is not necessarily increased in POAG. Other factors could also affect MYOC label, however, including the chronic use of medications, effects of surgery, postmortem conditions in the normal eyes, and the fact that in situ hybridization is necessarily only a snapshot of MYOC mrna expression, and does not determine the life span of the mrna. The physiological function of MYOC is unknown, although its presence intracellularly, and its reported localization in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells, suggest a structural role. Kubota et al. suggested that we assume that myocilin is a membrane-associated cytoskeletal protein synthesized in the ciliary neuorepithelium and in skeletal and cardiac muscle. 7 How MYOC mutations cause glaucoma is also unknown. Because MYOC is an intracellular protein, one hypothesis suggests that abnormal MYOC, or excess normal MYOC, could make the meshwork cells become stiffer or less deformable. This could hinder the deformation of canal cells involved in any intercellular pathway, or intracellular pathway of aqueous entry into the canal. Our study showed that the mrna expression of MYOC was present in only a few cells of Schlemm s canal in both normal and glaucomatous eyes. This leads to the speculation that MYOC is not present in large amounts within the canal cells, especially in POAG, and thus may not act in a manner to alter intracellular elements of the canal cells. The role of extracellular MYOC cannot be ascertained from this study. Of interest, dexamethasone treatment increased the mrna expression of MYOC in both trabecular cells and in the endothelial cells of Schlemm s canal. Nearly all the canal cells demonstrated strong MYOC mrna label. Does increased MYOC production in the canal cells play a role in corticosteroid-induced glaucoma? Without knowing the potential role of extracellular MYOC in this condition, the question cannot be answered. The increase in label for mrna for MYOC found in the present study is in agreement with studies reporting in-

6 IOVS, June 2000, Vol. 41, No. 7 Localization of TIGR/MYOC mrna in Human TM 1729 creased MYOC gene expression in monolayer cultured meshwork cells treated with similar doses of dexamethasone. 2,3,5 Monolayer culture studies of isolated Schlemm s canal cells differ: one found no increased MYOC after dexamethasone treatment, whereas another did find an increase in MYOC. 4,11 Differences in culture technique may explain differences with our study. The perfusion organ-culture technique used in the present study keeps the canal cells in situ, attached to their usual extracellular matrix, and also maintains the cells exposure to an intraocular pressure and the directional flow of media. These conditions are not present in monolayer cultures of cells grown on a plastic dish. Our results demonstrate that MYOC mrna is present in the cells of the TM but is found less often in the endothelial-lining cells of Schlemm s canal. References 1. Stone EM, Fingert JH, Alward WLM, et al. Identification of a gene that causes primary open-angle glaucoma. Science. 1997;275: Polansky JR, Fauss DJ, Chen P, et al. Cellular pharmacology and molecular biology of the trabecular meshwork inducible Glucocorticoid response gene product. Ophthalmologica. 1997;211: Nguyen TD, Chen P, Huang WD, Chen H, Johnson DH, Polansky JR. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem. 1997;273: Stamer WD, Roberts BC, Howell DN, Epstein DL. Isolation, culture, and characterization of endothelial cells from Schlemm s canal. Invest Ophthalmol Vis Sci. 1998;39: 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: Lütjen Drecoll E, May CA, Polansky JR, Johnson DH, Bloemendal H, Nguyen TD. Localization of the stress proteins B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci. 1998;39: Kubota R, Noda S, Wang Y, et al. A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping. Genomics. 1997;41: Rittig M, Flügel C, Prehm P, Lütjen Drecoll E. Hyaluronan synthase immunoreactivity in the anterior segment of the primate eye. Graefes Arch Clin Exp Ophthalmol. 1993;231: Johnson DH, Tschumper RC. Human trabecular meshwork organ culture: a new method. Invest Ophthalmol Vis Sci. 1987;28: Johnson DH, Tschumper RC. The effect of organ culture on human trabecular meshwork. Exp Eye Res. 1989;49: O Brien E, Polansky JR, Metheney CD, Ren X. Quantification of TM and SC cell TIGR response to dexamethasone (Dex) [ARVO Abstract]. Invest Ophthalmol Vis Sci. 1999;40(4):S504. Abstract nr 2662.