Application of Cell Impedance as a Screening Tool to Discover Modulators of Intraocular Pressure.

Purpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay.

Regulation of Endothelin-1-Induced Trabecular Meshwork Cell Contractility by Latanoprostene Bunod.

PURPOSE: Previous in vivo studies demonstrated that latanoprostene bunod (LBN), a nitric oxide (NO)-donating prostaglandin F2α receptor agonist, results in greater intraocular pressure (IOP) lowering than latanoprost. The present series of investigations compared the effects of LBN and latanoprost on primary human trabecular meshwork cell (HTMC) contractility and underlying signaling pathways to determine whether LBN might mediate this additional IOP lowering via the conventional outflow pathway. METHODS: The effect of LBN (1-100 µM) on HTMC cGMP levels was determined by ELISA with or without the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Endothelin-1 (ET-1) was used to induce HTMC contractility. To determine the effect of LBN on myosin light chain-2 (MLC-2) phosphorylation, HTMCs were pretreated with 10 to 60 µM LBN for 1 hour and then ET-1 for 5 minutes. MLC-2 phosphorylation was determined by Western blotting. Effects of LBN (30 and 45 µM) on ET-1-induced filamentous (F)-actin cytoskeletal stress fibers and the focal adhesion associated protein vinculin were determined by confocal microscopy. ET-1-induced HTMC monolayer resistance in the presence of LBN (45 µM) was determined by electrical cell substrate impedance sensing, as an indicator of cell contractility. Latanoprost and SE 175 (an NO donor which releases NO on reductive transformation within the cells) were used as comparators in all studies. RESULTS: LBN (1-100 µM) significantly increased cGMP levels in a dose-dependent manner, with a half maximal effective concentration (EC50) of 1.5 ± 1.3 µM, and with maximal effect similar to that of 100 µM SE 175. In contrast, latanoprost caused a minimal increase in cGMP levels at 100 µM only. The cGMP elevation induced by LBN or SE 175 was abolished by ODQ and was therefore sGC-dependent. The two NO donors SE 175 and LBN elicited a reduction in ET-1-induced MLC-2 phosphorylation that was significantly greater than that mediated by latanoprost in HTMCs. SE 175 (100 µM) and LBN (30 or 45 µM) caused a dramatic reduction in ET-1-induced actin stress fibers and vinculin localization at focal adhesions, whereas 45 µM latanoprost was without observable effect. SE 175 reduced ET-1-induced increases in HTMC resistance in a dose-dependent manner. A synergistic effect on reduction of HTMC resistance was observed when latanoprost and SE 175 doses were given together. LBN significantly reduced ET-1-induced HTMC monolayer resistance increases to a greater extent than latanoprost, indicating a greater reduction in cell contractility with LBN. CONCLUSIONS: LBN, SE 175, and latanoprost caused relaxation of ET-1-contracted HTMCs. The effect on HTMC relaxation observed with LBN was significantly greater in magnitude than that observed with latanoprost or SE 175. Data indicate that the NO-donating moiety of LBN mediates HTMC relaxation through activation of the cGMP signaling pathway and a subsequent reduction in MLC-2 phosphorylation. These findings suggest that increased conventional outflow facility may mediate the additional IOP-lowering effects of LBN over that of latanoprost observed in in vivo studies.

Anti-inflammatory effects of mapracorat, a novel selective glucocorticoid receptor agonist, is partially mediated by MAP kinase phosphatase-1 (MKP-1).

Mapracorat is a novel selective glucocorticoid receptor agonist (SEGRA), structurally distinct from corticosteroids. In preclinical studies, mapracorat potently inhibits the production of a variety of inflammatory mediators including cytokines and prostaglandin E2 (PGE(2)), with limited side effects associated with traditional corticosteroids. The objective of this study was to delineate the mechanisms underlying the anti-inflammatory properties of mapracorat. We found that mapracorat potently inhibited the production of GM-CSF and TNF-α in LPS-stimulated Raw 264.7 macrophages. Mapracorat also substantially attenuated the expression of COX-2 and the production of PGE(2). The inhibition of mapracorat on the inflammatory response was dose-dependent, and substantially inhibitory effects were observed at concentrations in the 10-100 nm range. Examination of the activation kinetics of p38 and its downstream target MAPK-activated protein kinase-2 (MK-2) revealed a shortened activation course after LPS stimulation in cells pretreated with mapracorat. Supporting the notion that mapracorat augments a feedback control mechanism restraining the p38 pathway, we found that mapracorat enhanced the expression of MAPK phosphatase-1 (MKP-1), a critical negative regulator of MAPKs that drive the production of cytokines and other inflammatory mediators. While mapracorat alone did not stimulate MKP-1 expression, it markedly enhanced the expression of MKP-1 in cells stimulated by LPS, in a similar manner and potency to the augmenting effect of dexamethasone. Blocking MKP-1 expression by triptolide also abolished the accelerating effects of mapracorat on p38 and MK-2 deactivation, further supporting a role of MKP-1 in the anti-inflammatory mechanism of mapracorat. Taken together, these results indicate that mapracorat exerts its anti-inflammatory effects, at least in part, by augmenting MKP-1 expression.

Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells.

PURPOSE: To determine the anti-inflammatory and anti-oxidant effects of epigallocatechin gallate (EGCG), the major polyphenol component of green tea, in human corneal epithelial cells (HCEpiC). METHODS: HCEpiC were challenged with interleukin-1ß (IL-1ß) for 18 h or hyperosmolarity (440 mOsm) for 24 h. Luminex technology was used to determine the effects of EGCG (0.3-30 µM) on IL-1ß- or hyperosmolar-induced cytokine release into the medium. Cell metabolic activity was measured using the alamarBlue assay. Effects of EGCG on mitogen-activated protein kinase (MAPK) phosphorylation were determined by cell-based enzyme-linked immunosorbent assay (ELISA) and western blotting. Effects of EGCG on nuclear factor kappa B (NFκB) and activator protein-1 (AP-1) transcriptional activity were assessed by reporter gene assay. The effects of EGCG on glucose oxidase (GO)-induced reactive oxygen species (ROS) production was determined using the ROS probe CM-H2DCFDA. RESULTS: Treatment of HCEpiC with 1 ng/ml IL-1ß for 18 h significantly increased release of the cytokines/chemokines granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1), while hyperosmolarity-induced release of IL-6 and MCP-1. When cells were treated with IL-1ß and EGCG or hyperosmolarity and EGCG there was a dose-dependent reduction in release of these cytokines/chemokines, with significant inhibition observed at 3-30 µM. There was no effect of EGCG on cell metabolic activity at any of the doses tested (0.3-30 µM). EGCG significantly inhibited phosphorylation of the MAPKs p38 and c-Jun N-terminal kinase (JNK), and NFκB and AP-1 transcriptional activities. There was a significant dose-dependent decrease in GO-induced ROS levels after treatment of HCEpiC with EGCG. CONCLUSIONS: EGCG acts as an anti-inflammatory and anti-oxidant agent in HCEpiC and therefore may have therapeutic potential for ocular inflammatory conditions such as dry eye.