Real-life medium term follow-up data for intravitreal dexamethasone implant in retinal vein occlusion.

Macular edema (ME) is the most frequent vision threatening consequence after retinal vein occlusion (RVO). In this study, we evaluate the effect of dexamethasone intravitreal implants (DII, Ozurdex) in a real-life cohort of 99 patients with ME due to RVO. All patients who received DII for ME following RVO between 2011 and 2016 at the University Eye Hospital Freiburg, Germany and who had fully accessible electronic medical records were eligible for this study. Most of the patients included in this study were not treatment-naïve: 61 eyes had received prior anti-VEGF drugs, 6 eyes had received intravitreal corticosteroids (triamcinolone) and 15 had been treated with both; 17 eyes were treatment-naïve. Mean follow-up was 312 ± 310 days. Mean visual acuity (VA) was maintained throughout the observation period (mean VA at baseline: 66.7 ± 23.5 letters; at last observation 64.9 ± 28.3). Central retinal thickness (CRT) decreased from 526 ± 179 µm at baseline to 431 ± 199 µm. Mean intraocular pressure (IOP) increased from 14.4 ± 3.1 mmHg at baseline to 17.1 ± 6.3 mmHg. Cataract surgery was performed in 22% of phakic eyes. DII was used as second-line treatment in the majority of cases in this cohort. The fact that mean VA remained unchanged while mean CRT decreased illustrates that morphologic improvement does not always translate into functional gain. Mean IOP was maintained within normal limits and cataract formation was as expected in this age group.

Anti-VEGF injection frequency correlates with visual acuity outcomes in pro re nata neovascular AMD treatment.

Clinical trials report substantial gains in visual acuity (VA) for eyes treated with intravitreal anti-VEGF for neovascular AMD (nAMD). In clinical reality, VA outcomes are more variable. Here we investigate pro-re nata treatment frequencies and VA in a real-life cohort of 1382 eyes (1048 patients). Patients with nAMD and one year complete follow-up treated with pro-re nata anti-VEGF between 2009 and 2016 were included. Injection frequency and VA was analyzed clustered by year of first treatment. Baseline parameters were compared between years. Median injection frequency in the first year was 5 with an IQR (interquartile range) of 5 for patients treated in 2009 and 8 with an IQR of 3 for patients treated from 2012 onwards. Median VA outcomes at one year were -5 to ±0 letters for patients treated between 2009 and 2013 and ±0 to +2 letters for patients treated from 2013 onwards. This cohort comprises all severities and subtypes of nAMD. 39% of patients had baseline VA outside the range for the MARINA or ANCHOR clinical trials. Higher treatment frequency was associated with improved VA in our real-life nAMD cohort. With adequate injection frequency, almost 90% of eyes had stable or improved VA over one year. Median VA gains, however, were lower compared to clinical trials. This may be due to a wider range of baseline characteristics in real-life cohorts.

Long-term follow-up of intraocular pressure and pressure-lowering medication in patients after ab-interno trabeculectomy with the Trabectome.

BACKGROUND: The minimally invasive ab-interno trabeculectomy (AIT) via electro-ablation with the Trabectome has been on the European market since 2009. Many studies have proven the safety and efficacy of the procedure. Up until now, studies investigating the long-term effect of AIT have been sparse. In this study, we present long-term results of AIT in patients with primary and secondary open-angle glaucoma. METHODS: In a retrospective monocentric study, the data of all the patients having undergone the procedure in 2010 at our institution were recorded. Data was collected during routine examinations at our institution. In total, 81 eyes of 74 patients (46 patients with primary open-angle glaucoma (POAG), 28 patients with pseudoexfoliative glaucoma (PEXG)) were included. At every examination, the intraocular pressure (IOP) was measured using Goldmann applanation tonometry and the number of IOP-lowering medication was registered. Statistical analysis was done using the Kaplan-Meier analysis or Dunnett's t test, respectively. RESULTS: For both groups (POAG and PEXG), we found a significant lowering of the IOP (28% for POAG and 26% for PEXG) and a significant reduction of the number of IOP-lowering medication (32% for POAG and 29% for PEXG) after a median follow-up period of 3.5 years. CONCLUSION: In patients with open-angle glaucoma and especially pseudoexfoliative glaucoma, ab-interno trabeculectomy is an effective surgical procedure to significantly lower the intraocular pressure on a long-term basis.

Lipid Emulsion-Based OCT Angiography for Ex Vivo Imaging of the Aqueous Outflow Tract.

Purpose: Contrast agents applicable for optical coherence tomography (OCT) imaging are rare. The intrascleral aqueous drainage system would be a potential application for a contrast agent, because the aqueous veins are of small diameter and located deep inside the highly scattering sclera. We tested lipid emulsions (LEs) as candidate OCT contrast agents in vitro and ex vivo, including milk and the anesthetic substance Propofol. Methods: Commercial OCT and OCT angiography (OCTA) devices were used. Maximum reflectivity and signal transmission of LE were determined in tube phantoms. Absorption spectra and light scattering was analyzed. The anterior chamber of enucleated porcine eyes was perfused with LEs, and OCTA imaging of the LEs drained via the aqueous outflow tract was performed. Results: All LEs showed a significantly higher reflectivity than water (P < 0.001). Higher milk lipid content was positively correlated with maximum reflectivity and negatively with signal transmission. Propofol exhibited the best overall performance. Due to a high degree of signal fluctuation, OCTA could be applied for detection of LE. Compared with blood, the OCTA signal of Propofol was significantly stronger (P = 0.001). As a proof of concept, time-resolved aqueous angiography of porcine eyes was performed. The three-dimensional (3D) structure and dynamics of the aqueous outflow were significantly different from humans. Conclusions: LEs induced a strong signal in OCT and OCTA. LE-based OCTA allowed the ability to obtain time-resolved 3D datasets of aqueous outflow. Possible interactions of LE with inner eye's structures need to be further investigated before in vivo application.

Impact of angiogenic activation and inhibition on miRNA profiles of human retinal endothelial cells.

BACKGROUND: Human retinal microvascular endothelial cells (HRMVECs) are involved in the pathogenesis of retinopathy of prematurity. In this study, the microRNA (miRNA) expression profiles of HRMVECs were investigated under resting conditions, angiogenic stimulation (VEGF treatment) and anti-VEGF treatment. MATERIALS AND METHODS: The miRNA profiles of HRMVECs under resting and angiogenic conditions (VEGF treatment), as well as after addition of aflibercept, bevacizumab or ranibizumab were evaluated by analyzing the transcriptome of small non-coding RNAs. Differentially expressed miRNAs were validated using qPCR and classified using Gene Ontology enrichment analysis. RESULTS: Ten miRNAs were found to be significantly changed more than 2-fold. Seven of these miRNAs were changed between resting conditions and angiogenic stimulation. Four of these miRNAs (miR-139-5p/-3p and miR-335-5p/-3p) were validated by qPCR in independent experiments and were found to be associated with angiogenesis and cell migration in Gene Ontology analysis. In addition, analysis of the most abundant miRNAs in the HRMVEC miRNome (representing at least 1% of the miRNome) was conducted and identified miR-21-5p, miR-29a-3p, miR-100-5p and miR-126-5p/-3p to be differently expressed by at least 15% between resting conditions and angiogenic conditions. These miRNAs were found to be associated with apoptotic signaling, regulation of kinase activity, intracellular signal transduction, cell surface receptor signaling and positive regulation of cell differentiation in Gene Ontology analysis. No differentially regulated miRNAs between angiogenic stimulation and angiogenic stimulation plus anti-VEGF treatment were identified. CONCLUSION: In this study we characterized the miRNA profile of HRMVECs under resting, angiogenic and anti-angiogenic conditions and identified several miRNAs of potential pathophysiologic importance for angioproliferative retinal diseases. Our results have implications for possible miRNA-targeted angiomodulatory approaches in diseases like diabetic retinopathy or retinopathy of prematurity.

Quantitative evaluation of an optimized Time of Flight Magnetic Resonance Imaging procedure using a phantom setup to simulate aqueous humor flow.

Preclinical Magnetic Resonance Imaging at high field strength offers the great advantage of combining anatomical information and very high resolution of down to 25 µm in mice and even higher resolutions in ex vivo settings. The presented data is Time of Flight MR imaging data using a tube phantom and a given flow-rate to determine the lower limit of the flow rate that is detectable with an experimental set-up and a specifically optimized 2D TOF sequence. In this work we present data on a phantom study which shows the ability of Time of Flight MR Imaging to detect very low flow rates down to 25 µl/h at a velocity of 0.1 mm/s non-invasively in a phantom study.

Functional assessment of the aqueous humour distal outflow pathways in bovine eyes using time-of-flight magnetic resonance tomography.

The major part of the aqueous humor leaves the eye through the "conventional outflow pathway", consisting of the trabecular meshwork, Schlemm's canal, collector channels, an intrasceral plexus and the episcleral veins. While the trabecular meshwork is well characterized, little is known about anatomical and functional features of the peripheral outflow tract beyond Schlemm's canal. The emergence of minimally-invasive glaucoma surgery directly targeting the outflow resistance in the trabecular meshwork has elicited growing interest in these structures. We used time-of-flight magnetic resonance imaging in ex vivo bovine eyes to map fluid flow under physiological conditions. We were able to identify the peripheral outflow vessels solely by the signal detected from the fluid flow inside their lumina. A question of clinical relevance is, whether localized opening of the trabecular meshwork leads to only localized or to a 360° increase in intrascleral flow. To address this, a goniotomy ab interno was performed in 3 eyes and the flow signal intensity was quantified sectorially. A significant increase in fluid flow was observed in the sector distal to the goniotomy (p = 0.0005) but not in the other sectors (p = 0.1). As a proof of concept we demonstrated that TOF-MRI based detection of flow in the peripheral aqueous outflow tract is feasible. The functional link observed between trabecular meshwork sectors and their distal outflow tract sectors may be relevant for minimally-invasive glaucoma surgery in humans.

[Real-life Evidence on Treatment Outcomes of Neovascular Age-related Macular Degeneration in Germany]. / Real-Life-Evidenz zur Therapie der neovaskulären altersabhängigen Makuladegeneration in Deutschland.

Treatment of neovascular age-related macular degeneration (AMD) with anti-vascular endothelial growth factor agents has been improved since its introduction in Germany. Several observational studies have demonstrated an increased frequency of injections in parallel with improved visual outcomes during the first year of treatment over the last 7 years. These results are reflected in several case series. However, studies with a longer follow-up reported that visual function regressed to baseline vision following an initial improvement, e.g. during the first three years of treatment. A delay in treatment initiation has been reported mainly for rural areas and management models involving more than one ophthalmologist or institution. Encouragingly, recent data confirm a positive trend towards reducing treatment delay, increasing injection frequency and improving treatment outcomes. Still, further improvements are needed to attain optimal treatment outcomes achievable under routine clinical conditions in Germany.

Trabeculotomy opening size and IOP reduction after Trabectome® surgery.

BACKGROUND: Trabeculotomy with the Trabectome® is an effective surgical procedure to lower intraocular pressure (IOP). However, in some patients it does not lead to a significant IOP reduction despite a gonioscopically well visible opening of Schlemm's canal. This study investigated whether the size of the trabeculotomy opening and other parameters, including anterior chamber depth (ACD) are related to IOP reduction. METHODS: Retrospective observational case series with 93 eyes of 93 patients who underwent Trabectome surgery. Trabeculotomy opening and ACD were measured with an anterior segment swept source OCT. IOP was taken pre-operatively and at a single follow-up visit [follow-up time 125 ± 66 days (mean ± SD)]. The relationship between IOP reduction and OCT parameters and possible confounding factors was analyzed in a multiple linear regression model. RESULTS: The trabeculotomy opening size did not correlate with IOP reduction (slope of regression line = 0.0016; 95% confidence interval of slope: -0.025 to 0.028). The same applied for all other parameters tested, including ACD, which showed a tendency towards better IOP reduction with a deeper AC (slope = -1.9; 95% confidence interval: -5.54 to 1.73). Comparison between the 1st and 4th quartile of the trabeculotomy opening showed a significantly higher ACD in the largest trabeculotomy opening quartile (3.32 ± 0.05 mm vs. 3.16 ± 0.04 mm; p = 0.031). CONCLUSIONS: The fact that the trabeculotomy opening size did not correlate with IOP reduction points to the poorly understood role of the intrascleral aqueous outflow pathway in glaucomatous IOP elevation. A deeper AC might be a factor promoting a larger trabeculotomy opening.

Five-year visual acuity outcomes and injection patterns in patients with pro-re-nata treatments for AMD, DME, RVO and myopic CNV.

BACKGROUND: Anti vascular endothelial growth factor (VEGF) therapy is an established treatment for various retinal diseases. Long-term data on injection frequencies and visual acuity (VA), however, are still rare. METHODS: Five-year analysis of real-life VA developments and injection patterns from 2072 patients (2577 eyes; 33 187 injections) with chronically active disease undergoing pro-re-nata treatment for age-related macular degeneration (AMD), diabetic macular oedema (DME), retinal vein occlusion (RVO) and myopic choroidal neovascularisation (CNV). RESULTS: Maximum mean VA gain in year 1 was+5.2 letters in AMD, +6.2 in DME, +10 in RVO and+7.2 in myopic CNV. Over 5 years, however, VA in patients with AMD declined. By year 5, 34% of patients with AMD had experienced VA loss of >15 letters, 56% had remained stable and 10% had gained >15 letters. Long-term VA developments in DME and RVO were more favourable with 81% of DME and 79% of patients with RVO gaining or maintaining vision at 5 years. In AMD, median injection frequency was six in year 1 and between four and five in consecutive years. In DME and RVO, median injection frequency was six in year 1 but lower compared with AMD in consecutive years. Injection frequency in DME was weakly associated with patient age (rs=0.1; p=0.03). CONCLUSIONS: In AMD, the initial VA gain was not maintained long term despite higher injection numbers compared with DME, RVO and myopic CNV. The presented real-world data provide a peer-group-based estimate of VA developments and injection frequencies for counselling patients undergoing long-term anti-VEGF therapy.

Ab Interno Trabeculectomy With the Trabectome as a Valuable Therapeutic Option for Failed Filtering Blebs.

PURPOSE: Uncontrolled intraocular pressure (IOP) after glaucoma filtration surgery is a challenging problem in the management of glaucoma patients. The Trabectome is a device for selective electroablation of the trabecular meshwork through a clear cornea incision without affecting the conjunctiva. Minimally invasive glaucoma surgery using the Trabectome is safe and effective as primary glaucoma surgery. Here we investigate the results of ab interno trabeculectomy with the Trabectome for IOP control in patients with a failed filtering bleb. METHODS: A total of 60 eyes of 60 consecutive patients with primary open-angle glaucoma (POAG) or pseudoexfoliative glaucoma (PXG) were enrolled in this single center observational study. Trabectome surgery was performed alone or in combination with phacoemulsification by 2 experienced surgeons. IOP readings and number of IOP lowering medication as primary outcome parameters were taken by an independent examiner. Intraoperative and postoperative medication were recorded systematically. RESULTS: Mean IOP before surgery was 24.5±3.5 mm Hg and decreased to 15.7±3.4 (-36%) after mean follow-up of 415 days. The number of necessary IOP lowering medication dropped from 2.1±1.3 to 1.8±1.2 (14% reduction from baseline). A total of 25% (n=15) of cases reported here needed additional surgery after 517 days (range: 6 to 1563 d). No major complications were observed. After mean follow-up, we found a qualified success rate for PXG of 87% and 50% for POAG as revealed by the Kaplan-Meier analysis according to the definitions for success in advanced glaucoma cases according to the World Glaucoma Association (40% reduction from baseline IOP and maximum IOP of 15 mm Hg). DISCUSSION: Trabectome surgery for uncontrolled IOP after trabeculectomy is safe and effective especially in PXG patients. Given the demanding subgroup of patients studied here, it is not surprising that success rates are lower compared with previous studies investigating the Trabectome for primary glaucoma surgery. The number of necessary IOP lowering medication drops at first, but seems to reach preoperative values after 20 months of follow-up. Trabectome surgery should be considered as a valuable escape procedure for patients with failed filtering blebs and uncontrolled IOP.

MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing.

PURPOSE: Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 µL human aqueous humor. METHODS: Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. RESULTS: One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. CONCLUSIONS: Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-ß and Wnt, which are important in intraocular pressure regulation and glaucoma.

Trabeculectomy ab interno with the Trabectome® as a therapeutic option for uveitic secondary glaucoma.

PURPOSE: Treatment of secondary glaucoma in uveitis patients is challenging. Owing to the young age of these patients, sufficient lowering of the intraocular pressure (IOP) is essential to prevent progression of visual field loss. However, because of the chronic inflammatory stimulus, filtration surgery has an increased risk of failure, especially in patients who have previously undergone surgery. Therefore, minimally invasive glaucoma surgery is a valuable alternative. METHODS: The clinical records of 24 consecutive patients with uveitic secondary glaucoma who underwent trabeculectomy ab interno with the Trabectome® at the Eye Center of the Albert-Ludwigs University of Freiburg between June 2009 and June 2014 (registered in the Freiburg trabectome database) were retrospectively analyzed. The general baseline information for each patient included age, gender, glaucoma type, ocular medication and current IOP. The postoperative IOP and number of antiglaucomatous medications were recorded at each visit. Statistical analyses were performed using the Kaplan-Meier estimator and Dunnett's t-test. RESULTS: The mean IOP before surgery was 31 ± 6.7 mmHg (median 32 mmHg). Both the IOP and the number of medications significantly decreased over the various follow-up intervals after trabeculectomy ab interno with the Trabectome®. Patients with follow-ups continuing past one year showed an IOP-reduction of approximately 40 % and a medication number reduction from 2 to 0.67. The failure rate (necessitating further glaucoma surgery) was N = 3 (12.5 %) patients. CONCLUSIONS: Trabeculectomy ab interno with the Trabectome® is a minimally invasive and effective method for controlling IOP in uveitic secondary glaucoma.

Trabectome surgery for primary and secondary open angle glaucomas.

PURPOSE: In most forms of open angle glaucoma, the trabecular meshwork is the main barrier for aqueous humor outflow, causing elevated intraocular pressure (IOP). The Trabectome is a minimal invasive device for the surgical treatment of open angle glaucoma, particularly eliminating the juxtacanalicular meshwork. This study was conducted to compare the effectiveness and complication profile among different glaucoma subgroups. METHODS: Single center prospective observational study. There were 557 consecutive eyes of 487 patients included in this study. Trabectome surgery was performed either alone or in combination with cataract surgery. Intraoperative and postoperative complications were documented systematically. Main outcome measures were IOP reduction over time and the preoperative and postoperative number of IOP-lowering medications. Due to subgroup sizes, only data from eyes with primary open angle glaucoma and pseudoexfoliation glaucoma were processed for statistical analysis. RESULTS: For the 261 eyes classified as primary open angle glaucoma, preoperative IOP was 24 ± 5.5 mmHg (mean ± SD) under 2.1 ± 1.3 IOP-lowering medications. After a mean follow-up of 204 ± 238 days, IOP was reduced to 18 ± 6.1 mmHg, and medication was reduced to 1.2 ± 1.1. For the 173 eyes classified as pseudoexfoliation glaucoma, after a mean follow-up of 200 ± 278 days, IOP was reduced from 25 ± 5.9 mmHg to 18 ± 8.2 mmHg, and medication was reduced from 2.0 ± 1.2 to 1.1 ± 1.1. A Cox proportional hazards model hinted forward superiority of the combined surgery cases (Trabectome + Phaco + intraocular lens) in comparison to Trabectome surgery only in phakic or pseudophakic eyes. No serious complications were observed. CONCLUSIONS: Minimal invasive glaucoma surgery with the Trabectome seems to be safe and effective. The subgroup analysis of different kinds of open angle glaucomas presented in this study may help in first-line patient selection. The lack of ocular surface alterations makes it a valuable addition to glaucoma surgery.

Effects of TGF-ß2 on cadherins and ß-catenin in human trabecular meshwork cells.

PURPOSE: The effects of TGF-ß2 on cadherin-mediated cell-cell adhesion in human trabecular meshwork (HTM) cells were characterized, since TGF-ß-induced changes in the cytoskeleton, cell-cell, and cell-matrix interactions of HTM cells are suggested to have a significant role in primary open angle glaucoma. METHODS: The HTM cells were derived from donor cornea rings and treated with TGF-ß2 or vehicle, and protein expression was studied by Western Blot, while protein localization was studied by fractionation of lysates and by confocal immunofluorescence microscopy. Cell-cell adhesion was assessed functionally in dissociation experiments and N-cadherin-mediated cell contact formation in cell spreading experiments on cadherin-coated substrates. A rho-associated protein kinase (ROCK) inhibitor was used to evaluate the contribution of cytoskeletal tension to TGF-ß2-induced changes in protein expression and localization. RESULTS: TGF-ß2 activated Smad-2/3, serine-threonine kinase (AKT), and extracellular signal-regulated kinase (ERK) signaling, and enhanced expression of ß-catenin as well as N- and OB-cadherin. The nuclear fraction of ß-catenin was not enhanced by TGF-ß2. Immunofluorescence microscopy revealed an increased localization of N-cadherin and ß-catenin to cell-cell adhesions, and an increase in F-actin. The TGF-ß2 increased cell-cell adhesion strength and enhanced N-cadherin-mediated cell contact formation. This effect was blocked by inhibition of mitogen-activated protein kinase kinase (MEK) or AKT. Cytoskeletal relaxation by a ROCK inhibitor did not prevent a TGF-ß2-induced increase in cadherin and ß-catenin expression. CONCLUSIONS: The cytokine TGF-ß2 enhances cadherin-mediated cell-cell adhesion and ß-catenin expression in HTM cells. Increased cell-cell adhesion may contribute to biomechanical alterations in glaucomatous trabecular meshwork (TM), and changes in ß-catenin levels and its possible sequestration to cell adhesion sites may affect Wnt signaling. Thus, the crosstalk of TGF-ß2 and Wnt signaling in TM may deserve further investigation.

Elasticity-dependent modulation of TGF-ß responses in human trabecular meshwork cells.

PURPOSE: To gain further insight into a possible role of biomechanical cues in glaucoma, the authors assessed the influence of extracellular matrix (ECM) elasticity on TGF-ß2-induced changes in trabecular meshwork (TM) cells. METHODS: Human TM cells derived from donor cornea rings were plated on rigid collagen-coated tissue culture plastic or polyacrylamide gels of different elasticity and treated with vehicle or TGF-ß2. Activation of Smad-2/3, ERK, and AKT signaling and expression of α-SMA and PAI-1 proteins were assessed by Western blot analysis. Subcellular localization of α-SMA was determined by confocal immunofluorescence microscopy. Transcription of collagen-I, -IV, and -VI, α-SMA, PAI-1, fibronectin, fibrillin-1, cochlin, and MGP-1 was studied by RT-qPCR. The contribution of non-Smad signaling pathways to TGF-ß-induced α-SMA and PAI-1 expression was assessed using the small molecule inhibitors U0126 and LY294002. RESULTS: TGF-ß2-induced activation of Smad-2/3, ERK, and AKT signaling as well as expression of collagen-1, α-SMA, fibulin, and MGP-1 were attenuated with increasing elasticity. In contrast, TGF-ß2-induced collagen-6, fibronectin, PAI-1, and cochlin expression were enhanced on elastic substrates. The MEK-inhibitor U0126 blocked TGF-ß-induced PAI-1 expression, whereas α-SMA expression was enhanced. PI3K inhibition with LY294002 reduced α-SMA expression. CONCLUSIONS: ECM elasticity modulates TGF-ß-induced signaling and protein expression in human TM cells. Non-Smad signaling contributes to TGF-ß-induced alterations. Increasing ECM elasticity in vitro promotes protein expression patterns reminiscent of patterns reported in primary open-angle glaucoma. Therefore, changes in TM elasticity and mechanical load may have a significant role in primary open-angle glaucoma.

Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.

PURPOSE: Extracellular matrix (ECM) composition, tension, and rigidity modulate cell-ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma. METHODS: Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), alpha-smooth muscle actin (alpha-SMA), tubulin, alpha-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of alpha-SMA and fibronectin were examined by confocal immunofluorescence microscopy. RESULTS: ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of alpha-SMA and recruitment of alpha-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and alpha-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices. CONCLUSIONS: Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma.