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.

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.

The Soluble Guanylate Cyclase Stimulator IWP-953 Increases Conventional Outflow Facility in Mouse Eyes.

PURPOSE: The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. METHODS: Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. RESULTS: IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 µM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 µM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 µM (P = 0.002) and in magnitude comparable to DETA-NO at 100 µM (97.5% increase, P = 0.030). CONCLUSIONS: These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.

Pigment epithelium-derived factor decreases outflow facility.

PURPOSE: Pigment epithelium-derived factor (PEDF) regulates blood-retinal barrier function. As a constituent of aqueous humor, the role of PEDF in conventional outflow function is unknown. The goals of the study were to examine the effects of PEDF on barrier function of cultured Schlemm's canal (SC) endothelia and outflow facility in mouse eyes in situ. METHODS: To model the inner wall of SC, transendothelial electrical resistance (TEER) of human SC and porcine angular aqueous plexus (AAP) cells was monitored. To examine an intact conventional outflow pathway, enucleated eyes from culled C57BL/6 mice were perfused with PEDF using a computer-controlled system. Purified PEDF (0.1 and 1 µg/mL) was perfused at four different pressure steps (4, 8, 15, 20 mm Hg), measuring flow to determine outflow facility (slope of flow/pressure relationship). RESULTS: Pigment epithelium-derived factor increased TEER of porcine AAP cells in a dose-dependent fashion (0.3-3 µg/mL), and 1 µg/mL recombinant PEDF or conditioned media from pigmented retinal pigment epithelial monolayers stabilized TEER of human SC monolayers over time (0-48 hours). In perfusion experiments, we observed a 43.7% decrease in outflow facility (0.016 vs. 0.029 µL/min/mm Hg, P = 4.5 × 10⁻5) in eyes treated with 1 µg/mL PEDF compared to vehicle-perfused controls, and a 19.9% decrease (0.021 vs. 0.027 µL/min/mm Hg, P = 0.003) at 100 ng/mL PEDF. CONCLUSIONS: Pigment epithelium-derived factor increased barrier function in both the in vitro and in situ models of the inner wall of SC. Modification of PEDF signaling in SC cells may be therapeutically exploited to increase outflow facility in people with ocular hypertension or decrease outflow facility in those with hypotony.

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.

Management of methicillin-resistant Staphylococcus aureus pneumonia.

PURPOSE OF REVIEW: Staphylococcus aureus, and particularly methicillin-resistant Staphylococcus aureus (MRSA) has become an increasingly important etiology of pneumonia, both in healthcare and community settings. Associated with highest morbidity, mortality and costs in public health, it represents a major challenge for the management of this group of patients. RECENT FINDINGS: MRSA is one of the most common pathogens of ventilator associated pneumonia, whereas its estimated incidence for hospital acquired pneumonia, healthcare associated pneumonia and community acquired pneumonia has risen in the past decades. Although vancomycin at standard doses remains as the mainstay for its treatment, the increasing rate of treatment failure has prompted other strategies of use (more frequent administration, continuous infusion, combination therapy), and the use of newer antimicrobials, particularly linezolid, with pharmacokinetic and pharmacodynamic profiles which produce promisingly improved clinical results. SUMMARY: Overall, MRSA is an important cause of pneumonia; optimal management strategies for improving morbidity and mortality are still under development.