A Small Molecule Inhibitor of VE-PTP Activates Tie2 in Schlemm's Canal Increasing Outflow Facility and Reducing Intraocular Pressure.

Purpose: Tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function. Methods: AKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC. Results: AKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/- mice. Conclusions: This is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

Chylothorax with Transudate: An Unusual Presentation of Tuberculosis.

INTRODUCTION: Twenty-five per cent of tuberculosis patients have pleural tuberculosis, which is the third most common form of presentation. Most cases present as an exudative pleural effusion with just few cases reported as chylothorax in the literature. All pleural effusions from confirmed cases, including tuberculous chylothorax, had exudate features. AIM: To describe a patient with Mycobacterium tuberculosis affecting the lungs and pleura, which laboratory testing demonstrated had features of transudate chylothorax. PATIENT AND METHODS: A 70-year-old man presented with constitutional symptoms, progressive exertional dyspnoea and right pleural effusion with fibrocavitary changes on chest imaging. Thoracentesis and pleural fluid analysis revealed chylous fluid with transudate features, high triglycerides, low cholesterol content and mononuclear cell predominance. Acid-fast sputum stains and pleural fluid were negative for Mycobacterium tuberculosis as was an adenosine deaminase test for pleural effusion. Tomography-directed lung biopsy sampling of a lung nodule revealed a chronic granulomatous inflammatory process associated with the presence of acid-fast bacilli. DISCUSSION: Tuberculosis-associated chylothorax is an uncommon presentation of the disease. A recent review found only 37 cases of confirmed tuberculous chylothorax had been reported in the literature. All cases had exudate characteristics. The diagnosis of pleural tuberculosis was made through culture or testing of sputum, pleural fluid or biopsy samples in 72.2% of cases, with the rest identified by histopathology. LEARNING POINTS: The main cause of non-traumatic chylothorax is malignancy, which is found in 39-72% of cases.Few cases of transudative chylothorax have been reported in the literature; the main aetiology is chronic hepatopathy.Tuberculosis-associated chylothorax is a rare presentation of infection caused by Mycobacterium tuberculosis, an uncommon aetiology.

Trabodenoson, an Adenosine Mimetic With A1 Receptor Selectivity Lowers Intraocular Pressure by Increasing Conventional Outflow Facility in Mice.

Purpose: To evaluate the relationship between the IOP-lowering effect of trabodenoson and the associated structural and functional changes in the trabecular meshwork (TM). Methods: Six independent cohorts of young and aged mice were exposed to three different topical once-a-day formulations of trabodenoson and eyes were compared to those treated with placebo drops. IOP was measured daily just before drug administration using rebound tonometry. Outflow facility was measured in enucleated eyes. Flow patterns and morphology of conventional outflow tissues were monitored using tracer beads and standard histology, respectively. In parallel, three-dimensional human TM tissue constructs (3D-HTM) were grown and used in experiments to test effect of trabodenoson on the expression of collagen IV, fibronectin, matrix metalloproteinase (MMP)-2 and MMP-14 plus MMP-2 activity. Results: Topical administration of trabodenoson significantly lowered IOP on every day tested, up to 7 days. After 2 days of treatment, outflow facility increased by 26% in aged mice and 30% overall (young and aged mice), which was significantly different from vehicle (P < 0.05). Outflow facility was 15% higher than controls after 7 days of treatment (P = 0.07). While gross morphology was not affected by treatment, the intensity of tracer bead distribution increased by day 7 (P = 0.05). Parallel experiments in 3D-HTM showed that trabodenoson treatment significantly increased MMP-2 activity and MMP-14 abundance, while decreasing fibronectin and collagen IV expression. Conclusions: Trabodenoson alters ECM turnover by TM cells and increases conventional outflow facility, which accounts for its ability to lower IOP in young and aged mice.

Human aqueous humor exosomes.

Aqueous humor (AH) is a dynamic intraocular fluid that supports the vitality of tissues that regulate intraocular pressure. We recently discovered that extracellular nanovesicles called exosomes are a major constituent of AH. Exosomes function in extracellular communication and contain proteins and small RNA. Our goal was to characterize the physical properties of AH exosomes and their exosomal RNA (esRNA) content. We isolated exosomes from human AH collected during cataract surgery from five patients using serial ultracentrifugation. We measured the size and concentration of AH exosomes in solution using nanoparticle tracking analysis. We found a single population of vesicles having a mean size of 121 ± 11 nm in the unprocessed AH. Data show that centrifugation does not significantly affect the mean particle size (121 ± 11 nm versus 124 ± 21 nm), but does impact the final number of exosomes in solution (87% loss from the unprocessed AH; n = 5). We extracted esRNA from the pooled human AH samples using miRCURY RNA isolation kit from Exiqon. The quality of extracted esRNA was evaluated using Agilent Bioanalyzer 2100 and was used to generate a sequencing library for small RNA sequencing with Illumina MiSeq sequencer. More than 10 different miRNAs were identified; abundant species included miR-486-5p, miR-204, and miR-184. We found that the majority of extracellular vesicles in the AH were in the exosome size range, suggesting that miRNAs housed within exosomes may function in communication between AH inflow and outflow tissues.

Resveratrol prevention of oxidative stress damage to lens epithelial cell cultures is mediated by forkhead box O activity.

PURPOSE: To evaluate the potential role that FoxO transcription factors play in modulating resveratrol's protective effects against oxidative stress in lens epithelial cells. METHODS: Primary human or porcine lens epithelial cells (LECs) were treated with resveratrol (RES) 25 µM and incubated under either physiologic (5%) or chronic hyperoxic (40%) oxygen conditions. Acute oxidative stress was applied using 600 µM H(2)O(2). Changes in expression of FoxO1A, FoxO3A, and FoxO4 were analyzed. The production of intracellular reactive oxygen species (iROS), SA-ß-galactosidase (SA-ß-gal) activity, and autofluorescence (AF) was assessed by flow cytometry. SiRNAs of FoxO1A, FoxO3A, and FoxO4 were used to study the roles that these transcription factors play in resveratrol's protective effects against cell death induced by oxidative stress. RESULTS: RES incubation under 40% oxygen increased the expression of FoxO1A, FoxO3A, and FoxO4. RES also increases mitochondrial membrane potential under 5% and/or 40% O(2) conditions and significantly decreased iROS, SA-ß-gal, and AF normally induced by hyperoxic conditions. While RES had a mild pro-apoptotic effect in nonstressed cells, it significantly prevented apoptosis induced by H(2)O(2) stress. SiRNA inhibition of FoxO1A, FoxO3A, and FoxO4 not only led to loss of the anti-apoptotic effects of RES in stressed cells but actually exhibited a mild pro-apoptotic effect. CONCLUSIONS: RES exerts a protective effect against oxidative damage in LEC cultures. The levels of expression of FoxO1A, FoxO3A, and FoxO4 appear to play a central role in determining the pro- or anti-apoptotic effects of RES. This has implications for future studies on oxidative stress-related lenticular disorders such as cataract formation.

LOXL1 expression in lens capsule tissue specimens from individuals with pseudoexfoliation syndrome and glaucoma.

PURPOSE: To study lysyl oxidase-like 1 (LOXL1) expression in freshly collected lens capsules from pseudoexfoliation syndrome (XFS), pseudoexfoliation glaucoma (XFG), and normal cataract control individuals. We also investigated the effects of four glaucoma drug medications on LOXL1 expression in primary human lens epithelial cell cultures to see if they could affect LOXL1 expression. METHODS: Lens capsules were collected at the time of cataract surgery. Controls were matched to age, sex, and ethnicity. Total RNA was isolated from individual lens capsule samples and real-time PCR was performed on each sample using primers flanking the sixth exon of the LOXL1 gene. Cell cultures were grown to confluence in four separate six-well plates at 37 °C in 5% CO2. Each plate was then treated with one of four different glaucoma drugs (brinzolamide 1%, brimonidine tartrate 0.1%, timolol maleate 0.5%, and latanoprost 0.005%) once daily for seven days (at both 1:1,000 and 1:100 concentrations relative to media). Controls were not treated with any drug but media was changed in the same manner. After one week of treatment, cells were harvested and total RNA isolated. Real-time PCR was performed on each group of cells. RESULTS: Seven XFS, seven XFG, and ten cataract control specimens were analyzed. LOXL1 expression was detected in the lens capsule specimens from each of the four groups. Significant expression differences were found between the control and XFG groups and XFS and XFG groups. No significant difference was observed between the control and XFS group. No significant decrease in LOXL1 expression was seen with drug incubation of the four medications (Brinzolamide, Timolol, Latanoprost, and Brimonidine) at the 1:1,000 drug:media concentrations versus controls. At 10-fold higher concentrations (1:100 drug:media), brinzolamide, timolol maleate, and latanoprost showed small increases in LOXL1 expression relative to controls. This effect was not observed with brimonidine tartrate. CONCLUSIONS: These results establish that LOXL1 expression is reduced in lens capsule specimens from XFG individuals but not XFS. The drug treatment incubation studies suggest that the change in LOXL1 expression observed in XFG is not attributable to glaucoma drug therapy. If a causative functional relationship can be validated, modification of LOXL1 expression in affected tissues may represent a novel treatment strategy for this disorder.