Relationships between Intraocular Pressure, Effective Filtration Area, and Morphological Changes in the Trabecular Meshwork of Steroid-Induced Ocular Hypertensive Mouse Eyes.

We investigated whether an inverse relationship exists between intraocular pressure (IOP) and effective filtration area (EFA) in the trabecular meshwork (TM) in a steroid-induced ocular hypertensive (SIOH) mouse model and the morphological changes associated with the reduction of EFA. C57BL/6 mice (n = 15 per group) received either 0.1% dexamethasone (DEX) or saline eye drops twice daily for five weeks. IOP was measured weekly. Fluorescent tracers were injected into the anterior chamber to label EFA at the endpoint. Injected eyes were fixed and processed for confocal microscopy. EFA in the TM was analyzed. Light and electron microscopy were performed in high- and low-tracer regions of six eyes per group. The mean IOP was ~4 mm Hg higher in DEX-treated than saline-treated control eyes (p < 0.001) at the endpoint. EFA was reduced in DEX-treated eyes compared to controls (p < 0.01) and negatively correlated with IOP (R2 = 0.38, p = 0.002). Reduced thickness of juxtacanalicular tissue (JCT) and increased abnormal extracellular matrix in the JCT were found to be associated with reduced EFA. Our data confirm the inverse relationship between EFA and IOP, suggesting that morphological changes in the JCT contribute to the reduction of EFA, thus elevating IOP in SIOH mouse eyes.

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles.

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.

Morphological factors associated with giant vacuoles with I-pores in Schlemm's canal endothelial cells of human eyes: A serial block-face scanning electron microscopy study.

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm's canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2-6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV's maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.

ROCK inhibitors beneficially alter the spatial configuration of TGFß2-treated 3D organoids from a human trabecular meshwork (HTM).

To elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFß2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFß2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFß2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFß2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFß2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFß2-induced HTM 3D organoids.

Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating ß-catenin and YAP/TAZ Signaling Pathways.

Purpose: The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating ß-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways. Methods: Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/ß-catenin and YAP/TAZ pathways were determined. Results: At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total ß-catenin was unchanged, pß-catenin was elevated, and pGSK3ß was suppressed. Although 10% XCDM had no effect on cytoplasmic ß-catenin levels, it reduced nuclear ß-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear ß-catenin. Conclusions: Increased cytoplasmic TAZ may inhibit ß-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.

3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy.

PURPOSE: The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. METHODS: After perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. RESULTS: RSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm's canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. CONCLUSION: In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma.

The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress.

The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. ABBREVIATION: AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.

Rho-associated protein kinase inhibitor induced morphological changes in type VI collagen in the human trabecular meshwork.

AIMS: To investigate morphological changes in type VI collagen in the human trabecular meshwork associated with the rho kinase inhibitor ripasudil. METHODS: This cross-sectional study evaluated the effects of ripasudil eye drop administration (RA) or no ripasudil eye drop administration (NRA) in patients with primary open-angle glaucoma (POAG; age range 60-80 years) who underwent conventional outflow reconstruction between December 2015 and September 2016 at Tokai University Hachioji Hospital. The juxtacanalicular tissue was removed and imaged using transmission electron microscopy. Type VI collagen comprises cross-banded aggregates with transverse bands 30 nm apart repeating every 105 nm. The transverse bands are called the outer rod-like region (ORR) and the intervals are called the inner rod-like region (IRR). The waveform intensity in the type VI collagen was analysed in electron micrographs using Fourier transformation to detect the IRR and ORR borders. RESULTS: Ten eyes of 10 patients were included (n=5/group). The baseline characteristics did not differ significantly between groups. ORR width was significantly smaller in the RA group (37.85±3.43 nm) than in the NRA group (50.62±5.23 nm, p<0.05), whereas IRR width was significantly greater in the RA group (70.68±10.84 nm) than in the NRA group (58.19±5.34 nm, p<0.05). Morphological changes in the type VI collagen total width tended to correlate with the duration of ripasudil administration (r=0.9, p=0.08). CONCLUSIONS: Ripasudil administration in patients with POAG induced morphological changes in type VI collagen. Patients with POAG administered RA had a significantly smaller ORR width and a significantly greater IRR width than patients with POAG not administered RA.

Mitochondria and Autophagy Dysfunction in Glucocorticoid-Induced Ocular Hypertension/Glaucoma Mice Model.

Purpose: This study is aimed to investigate the effects of periocular steroids induction on intraocular pressure (IOP), retinal ganglion cells (RGCs) and trabecular meshwork (TM) ultrastructure in glucocorticoid-induced ocular hypertension mice model.Materials and Methods: Dexamethasone-21-acetate (Dex-Ace) was administered through periocular conjunctival fornix injection every 3 days in C57BL/6J mice. Intraocular pressure was measured weekly by rebound tonometry. RGCs were examined with immunofluorescent staining of BRN3a at week 1, 4, and 8. TM morphology was visualized with electron microscopy. Autophagy was evaluated with immunoblotting in TM tissues.Results: Dex-Ace rapidly and significantly induced IOP, which peaked at week 4. The absolute increase in IOP in the Dex-Ace-treated mice was 8.1 ± 1.4 mmHg, a 60% induction (p < .0001) compared with that in the vehicle-treated mice. The IOP sustained a higher level in the Dex-Ace group from week 4 to week 8. Dex-Ace treatment decreased the number of RGCs in a time-dependent manner, suggesting that high IOP resulted in optic neuropathy. In addition, Dex-Ace thickened trabecular beams and decreased intertrabecular spaces, with marked accumulation of fibrillar and amorphous granular extracellular material. Moreover, Dex-Ace induced swollen and elongated mitochondria in TM cells. The average mitochondria area was 0.090 ± 0.044 µm2 in the vehicle-treated mice, and increased to 0.161 ± 0.094 µm2 (p < .0001), 0.121 ± 0.029 µm2 (p = .0223) and 0.171 ± 0.076 µm2 (p < .0001) in the Dex-Ace-treated mice at weeks 1, 4 and 8, respectively. Autophagy was also increased by Dex-Ace treatment, indicating by the upregulation of LC3-I, LC3-II and beclin-1, and downregulation of p62.Conclusion: Dex-Ace administration decreased RGCs and changed TM ultrastructure, mimicking hallmarks of human glucocorticoid-induced glaucoma (GIG). In addition, mitochondria and autophagy dysfunction suggested abnormal energy metabolism in TM cells, which warranted further study to fully elucidate the pathogenesis of GIG.

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.

Electron Microscopic Evidence of Photoreceptor Outer-segments in the Trabecular Meshwork in a Case of Schwartz-Matsuo Syndrome.

PURPOSE: To describe a case of photoreceptor outer segment glaucoma (Schwartz-Matsuo syndrome) with electron microscopic evidence of photoreceptor outer segments in the trabecular meshwork (TM). DESIGN: This is a clinicopathologic case report. PARTICIPANT: A 48-year-old Filipino man. METHODS: Specimens of aqueous humor and TM in a clinical case of Schwartz-Matsuo syndrome were examined by electron microscopy. MAIN OUTCOME MEASURES: Electron photomicroscopy. RESULTS: Electron microscopy showed evidence of retinal photoreceptor outer-segments in both an aqueous humor and a TM specimen. CONCLUSION: Schwartz-Matsuo syndrome is associated with the presence of photoreceptor outer segments in the TM.

Microarchitecture of Schlemm Canal Before and After Cataract Extraction Surgery.

PRECIS: Schlemm canal (SC) expands after cataract extraction (CE), both in the area and in volume by 25% as was measured using enhanced-depth imaging optical coherent tomography (EDI-OCT) in patients before and 1 week after CE. PURPOSE: This study aims to characterize the structural and volume changes on the microstructure of SC in patients before and after uneventful phacoemulsification CE by using EDI-OCT. MATERIALS AND METHODS: Forty-one serial horizontal EDI-OCT B-scans (interval between B-scans, 69 µm) were obtained in the nasal corneoscleral limbus before and 1 week after CE. The structure of aqueous channels, conjunctival blood vessels and iris anatomy in each scan were used as landmarks to select for overlapping scans taken before and following CE. The SC cross-section area was measured in each of the selected scans and SC volume was determined following a 3-dimensional reconstruction. RESULTS: Eleven eyes (6 females and 5 males) were imaged successfully before and after CE. Mean age was 70.54±11.38 years. The mean axial length was 23.10±0.87 mm. After CE, the mean best-corrected visual acuity in logMAR improved from 0.4±0.13 to 0.2±0.13 (P=0.028). There was no significant change in the mean intraocular pressure before and after CE (15.09±1.33 to 15.0±2.16 mm Hg; P=0.39). The mean SC cross-section area increased by 25%, from 4744±376 to 5941±1048 µm (P<0.001). SC volume in the analyzed region increased by 25% from 6,641,473±585,954 to 8,317,909±1,328,809 µm (P<0.001). CONCLUSION: CE expands SC dimensions in healthy eyes. EDI-OCT imaging of SC may prove useful in the evaluation of the SC dimensions in vivo before and after CE.

An In-Depth View of the Porcine Trabecular Meshwork Proteome.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

The Role of Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole Formation: A 3D Electron Microscopy Study.

Purpose: We investigated whether cellular connectivity between Schlemm's canal (SC) inner wall (IW) endothelium, and juxtacanalicular connective tissue (JCT), and between IW endothelial cells, plays a role in giant vacuole (GV) and pore formation by comparing perfusion- and immersion-fixed eyes. Methods: Normal human donor eyes (n = 4) were either immersion-fixed (0 mm Hg) or perfusion-fixed (15 mm Hg). Trabecular meshwork near SC was imaged using serial block-face scanning electron microscopy. A total of 12 IW cells from each group were 3D-reconstructed from ∼7040 electron micrographs and compared. In each cell, connections between IW cells and JCT cells/matrix were quantified; IW/IW connectivity was measured by cell border overlap length. GV volume, density, shape, and intracellular and paracellular pores were analyzed. Results: The mean number of IW/JCT cell-cell connections per cell significantly decreased (P < 0.01) while the summed GV volume per cell significantly increased (P < 0.01) in perfusion-fixed eyes compared to immersion-fixed eyes. Intracellular pores were observed in 14.6% of GVs in perfusion-fixed eyes and not observed in immersion-fixed eyes. The mean IW/IW overlap length per cell decreased (P < 0.01), and paracellular pores were found only in regions where IW/IW connectivity was minimal (overlap length = 0 µm) in perfusion-fixed eyes and not observed in immersion-fixed eyes. Conclusions: Our data suggest that changes in IW/JCT connectivity may be an important factor in the formation of larger GVs, and decreased IW/IW connectivity may promote paracellular pore formation. Targeting the IW/JCT and IW/IW connectivity may therefore be a potential strategy to regulate outflow resistance and IOP. .

Impact of pigment dispersion on trabecular meshwork cells.

PURPOSE: Dysfunction of the trabecular meshwork (TM) in pigmentary glaucoma contributes to increased aqueous humor outflow resistance and intraocular pressure. In this study, we investigated the effect of pigment dispersion on trabecular meshwork cells. METHODS: Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then, analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose migrate, and contraction was quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. RESULTS: Stress fiber formation was increased in the pigment dispersion group (P) (60.1 ± 0.3%, n = 10) compared to control (C) (38.4 ± 2.5%, n = 11, p < 0.001). Phagocytosis declined (number of cells with microspheres in P = 37.0 ± 1.1% and in C = 68.7 ± 1.3%, n = 3, p < 0.001) and migration was reduced after 6 h (cells within the visual field over 6 h in P = 28.0.1 ± 2.3 (n = 12) and in C = 40.6 ± 3.3 (n = 13), p < 0.01). Pigment induced contraction at 24 h onwards (p < 0.01). Microarray analysis revealed that Rho signaling was central to these responses. CONCLUSION: Exposure of TM cells to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigmentary glaucoma.

Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway.

Purpose: This study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers. Methods: Human TM cells and TMSCs were exposed to tunicamycin, brefeldin A, or thapsigargin. Cell apoptosis was evaluated by flow cytometry. ER stress markers were detected by quantitative PCR, Western blotting, and immunostaining. Morphologic changes were evaluated by transmission electron microscopy. Cells were treated with the PERK inhibitor GSK2606414 or the elF2α dephosphorylation inhibitor Salubrinal together with tunicamycin to evaluate their effects on ER stress. Results: Both TMSCs and TM cells underwent apoptosis after 48- and 72-hour treatment with ER stress inducers. ER stress triggered the unfolded protein response (UPR) with increased expression of GRP78, sXBP1, and CHOP, which was significantly lower in TMSCs than TM cells. Swollen ER and mitochondria were detected in both TMSCs and TM cells. Neither GSK2606414 nor salubrinal alone activated UPR. GSK2606414 significantly reduced cell survival rates after tunicamycin treatment, and salubrinal increased cell survival rates. The increased expression of GRP78, sXBP1, CHOP, and GADD34 peaked at 6 or 12 hours and lasted longer in TM cells than TMSCs. Salubrinal treatment dramatically increased OCT4 and CHI3L1 expression in TMSCs. Conclusions: In response to ER stress inducers, TMSCs activated a lower level of UPR and lasted shorter than TM cells. Inhibition of elF2α dephosphorylation had a protective mechanism against cell death. Stem cells combined with salubrinal may be a more effective way for TM regeneration in glaucoma.

Mutant myocilin impacts sarcomere ultrastructure in mouse gastrocnemius muscle.

Myocilin (MYOC) is the gene with mutations most common in glaucoma. In the eye, MYOC is in trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. To date, the function of wild-type MYOC remains unknown and how mutant MYOC causes high intraocular pressure and glaucoma is ambiguous. By investigating mutant MYOC in a non-ocular tissue we hoped to obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse expressing human mutant MYOC Y437H protein and we examined its skeletal (gastrocnemius) muscle phenotype. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates from transgenics indicated a decrease in two M-band proteins, myomesin 1 (MYOM1) and muscle creatine kinase (CKM). Immunoprecipitation identified CKM as a MYOC binding partner. Our results suggest that binding of mutant MYOC to CKM is changing sarcomere ultrastructure and this may adversely impact muscle function. We speculate that a person carrying the mutant MYOC mutation will likely have a glaucoma phenotype and may also have undiagnosed muscle ailments or vice versa, both of which will have to be monitored and treated.

Infection of endotheliotropic human cytomegalovirus of trabecular meshwork cells.

PURPOSE: Human cytomegalovirus (HCMV) infections can cause endotheliitis which is associated with an elevation in the intraocular pressure (IOP). However, the mechanism of the IOP elevation has not been established. The purpose of this study was to determine whether HCMV strains which are capable of infecting corneal endothelial cells can also replicate and induce anti-viral responses, and can reorganize the actin cytoskeleton in trabecular meshwork cells. STUDY DESIGN: Experimental study design. METHODS: Cultured primary human trabecular meshwork cells (HTMCs) were infected with the Towne or TB40/E strains of HCMV. TB40/E is trophic for vascular endothelial and corneal endothelial cells. Real-time PCR, western blot, and fluorescent immunostaining have been used to determine whether HCMV-infected HTMCs will support the expression of viral mRNA and protein, allow viral replication, and elicit anti-viral host responses. We also determined whether lytic replication was present after HCMV infection. RESULTS: HCMV infection led to the expression of viral mRNA and proteins of IE1, glycoprotein B(gB), and pp65. TB40/E infection induced interferon-ß, a sign of host anti-viral immune response and monocyte chemotactic protein-1 (MCP-1) as IOP-related chemokine. Together with the induction of the regulators of actin cytoskeleton, myosin phosphatase Rho interacting protein (MPRIP) and MCP-1, TB40/E induced a high level of expression of viral proteins, including IE1, gB, and pp65 as well as actin stress fiber formation, and achieved pathogenically high viral titers. CONCLUSIONS: Human trabecular meshwork cells support the replication of endotheliotropic TB40/E strain of HCMV which indicates that this strain may have high virulence for trabecular meshwork.

Deep tissue analysis of distal aqueous drainage structures and contractile features.

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm's canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.

Differentiation of mesenchymal stem cells -derived trabecular meshwork into dopaminergic neuron-like cells on nanofibrous scaffolds.

Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by the damage to dopaminergic neurons. Stem cell transplantation with a nanofibrous scaffold is one of the encouraging strategies for Parkinson's disease therapy. In this study, human mesenchymal stem cells (MSCs) from eye trabecular meshwork (TM) were differentiated into dopaminergic neurons on nanofibrous scaffold. After Trabecular meshwork biopsy, MSCs were isolated, cultivated on Poly-l-Lactide Acid (PLLA) nanofibrous scaffold (fabricated by electrospinning methods) and treated with medium containing DMEM supplemented with RA, IBMX and forskolin for 7 days. Scanning electron microscopy imaging, qPCR and immunostaining were used to analyze differentiated TM-MSCs on scaffold and their expression of dopaminergic-specific markers such as TH and Nurr-1. qPCR analysis revealed the expression of dopaminergic neuron genes such as TH, Nurr-1 on fibrous scaffold as well as TCPS. Immunostaining revealed that the differentiated TM-MSCs on TCPS and Scaffold not only express TH and Nurr-1 genes, but also express TH protein. In conclusion, the results indicate that TM-MSCs might be a suitable source for cell transplantation therapy. In addition the nanofibrous scaffold reported herein could be used as a potential cell carrier for the central system diseases such as PD.