Correlations of In Vitro Assays for Assessing Cytotoxicity and Biocompatibility of Contact Lens Multipurpose Solutions.

OBJECTIVES: To demonstrate correlations among in vitro assays used for assessing cytotoxicity of contact lens multipurpose solution (MPS) and propose the use of multiple assays as a part of preclinical evaluation for MPS biocompatibility assessment. METHODS: The effect of four different MPS on cell cytotoxicity, metabolic activity, and membrane integrity was performed by evaluating toxicity, expression of tight junction protein zonula occludens-1, and transepithelial electrical resistance in human corneal epithelial cells and Chinese hamster fibroblast cells. RESULTS: Cytotoxicity of four MPS was assayed with five different experimental systems at various concentrations. In vitro MPS-induced cytotoxicity was dependent on assay choice, concentration of MPS used, and duration of treatment. Overall, MPS-1 and MPS-2 were comparable to MPS-4 and better than MPS-3 in maintaining corneal barrier integrity and cell viability. CONCLUSIONS: In vitro cytotoxicity testing with MPS exposure to monolayer of cells in culture could be used as a tool to understand the potential cytotoxicity profiles of MPS and possibly a predictor of clinical outcome. Furthermore, MPS effects on in vitro cytotoxicity are best demonstrated by performing multiple assays to evaluate cell viability, metabolic activity, and membrane integrity during development.

Penetrating and Intrastromal Corneal Arcuate Incisions in Rabbit and Human Cadaver Eyes: Manual Diamond Blade and Femtosecond Laser-Created Incisions.

OBJECTIVES: To compare morphologic differences between freehand diamond or femtosecond laser-assisted penetrating and intrastromal arcuate incisions. METHODS: Freehand diamond blade, corneal arcuate incisions (180° apart, 60° arc lengths) and 150 kHz femtosecond laser (80% scheimpflug pachymetry depth corneal thickness) arcuate incisions were performed in rabbits. Intrastromal arcuate incisions (100 µm above Descemet's membrane, 100 µm below epithelium) were performed in rabbit corneas (energy 1.2 µJ, spot line separation 3 × 3 µm, 90° side cut angle). Eyes were examined by slit lamp and light microscopy up to 47 days post-procedure. Freehand diamond blade penetrating incisions, and femtosecond laser penetrating and intrastromal arcuate incisions (energy 1.8 µJ, spot line separation 2 × 2 µm) were performed in cadaver eyes. Optical coherence tomography was performed immediately after surgery and the corneas were fixed for light scanning and transmission electron microscopy. RESULTS: The rabbit model showed anterior stromal inflammation with epithelial hyperplasia in penetrating blade and laser penetrating wounds. The laser intrastromal and penetrating incisions showed localized constriction of the stromal layers of the cornea near the wound. In cadaver eyes, penetrating wound morphology was similar between blade and laser whereas intrastromal wounds did not affect the cornea above or below incisions. CONCLUSION: Penetrating femtosecond laser arcuate incisions have more predictable and controlled outcomes shown by less post-operative scarring than incisions performed with a diamond blade. Intrastromal incisions do not affect uncut corneal layers as demonstrated by histopathology. The femtosecond laser has significant advantages in its ability to make intrastromal incisions which are not achievable by traditional freehand or mechanical diamond blades.

Reduced myocilin expression in cultured monkey trabecular meshwork cells induced by a selective glucocorticoid receptor agonist: comparison with steroids.

PURPOSE: To assess in vitro myocilin (MYOC) expression in trabecular meshwork (TM) cells exposed to BOL-303242-X, a selective glucocorticoid receptor (GR) agonist (SEGRA), in comparison with dexamethasone (DEX), and prednisolone acetate (PA). METHODS: After drug treatment of monkey TM cultures, MYOC protein in conditioned media (CM) was measured by Western blot and densitometry. MYOC mRNA levels were analyzed by qRT-PCR. RU-486 was tested for antagonism of MYOC protein expression induced by DEX and BOL-303242-X. RESULTS: Baseline MYOC protein released into CM and MYOC mRNA were detected. DEX or PA elicited dose-dependent increases in MYOC in CM and also in MYOC mRNA. BOL-303242-X effects typified partial agonism, with significantly reduced MYOC protein and mRNA, compared with DEX. Maximum efficacy for BOL-303242-X was 53% of that for DEX. Mean EC(50) across all strains tested was lower, but not significantly different, for BOL-303242-X versus DEX. Compared with DEX, MYOC mRNA levels were significantly lower in BOL-303242-X-treated TM cells at the highest doses tested. EC(50)s for PA were higher than DEX, for both myocilin protein and mRNA. RU-486 displayed a dose-dependent antagonism to drug-induced increases in myocilin levels. CONCLUSIONS: In vitro quantitative assays of myocilin expression in TM cells can be used for characterizing anti-inflammatory drugs that are GR ligands. The results suggest that, compared with traditional ocular steroids, therapeutic doses of BOL-303242-X elicit a reduced myocilin expression profile in TM cells by virtue of the partial agonist properties of this compound.

BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells.

PURPOSE: BOL-303242-X is a novel selective glucocorticoid receptor agonist under clinical evaluation for the treatment of inflammatory skin and eye diseases. Data from in vitro and in vivo studies suggest an improved side-effect profile of this compound compared to classical glucocorticoids. The aim of this study was to determine the anti-inflammatory effect of BOL-303242-X in ocular cells. METHODS: Four primary human ocular cell cultures, including human conjunctival fibroblasts (HConFs), human corneal epithelial cells (HCEpiCs), human optic nerve astrocytes (HONAs), and human retinal endothelial cells (HRECs), as well as a human monocytic cell line, THP-1, were challenged with either lipopolysacharide (LPS) or interleukin-1ss (IL-1ss). Luminex technology was used to determine the effect of BOL-303242-X on LPS- or IL-1ss-induced cytokine release and intercellular adhesion molecule-1 (ICAM-1) levels. Effects of BOL-303242-X on nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) in HCEpiCs were also assessed by measuring inhibitory kappa B protein-alpha (IkappaB-alpha), phosphorylated p65 NFkappaB, and MAPK levels by western blotting. Dexamethasone (DEX) or triamcinolone acetonide (TA) was used as the control. RESULTS: LPS or IL-1ss induced multiple cytokine release in all cell types studied. BOL-303242-X significantly reduced LPS- or IL-1ss-induced inflammatory cytokine release in a dose-dependent manner, including granulocyte colony-stimulating factor (G-CSF), IL-1ss, IL-6, IL-8, IL-12p40, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha). BOL-303242-X showed activity and potency comparable to that observed for DEX or TA. A statistically significant inhibitory effect of BOL-303242-X was observed at doses ranging from 1 to 100 nM in HConFs, HCEpiCs, HONAs, and THP-1. The IC(50) values for these effects were in the low nM range. BOL-303242-X also significantly reduced LPS-induced IL-1ss release and ICAM-1 levels in HRECs. Furthermore, BOL-303242-X inhibited IL-1ss-induced decreases in IkappaB-alpha levels, as well as IL-1ss-induced phosphorylation of NFkappaB, p38, and c-Jun-N-terminal kinase (JNK) MAPKs in HCEpiCs. CONCLUSIONS: BOL-303242-X acts as a potent anti-inflammatory agent in various primary human ocular cells with similar activity and potency compared to classical steroids. Results also suggest that MAPK (p38 and JNK) and NFkappaB signaling pathways are involved in the anti-inflammatory properties of BOL-303242-X in HCEpiCs. An improved side effect profile of this novel SEGRA compound has been reported recently. Thus, BOL-303242-X may provide a new option for the treatment of ophthalmic conditions with an inflammatory component.

Retinoid processing proteins in the ocular ciliary epithelium.

PURPOSE: To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP). METHODS: Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells. RESULTS: CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers. CONCLUSIONS: The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.

The bovine iris-ciliary epithelium expresses components of rod phototransduction.

Earlier studies have documented that the iris in lower vertebrates is photosensitive. In the present work, we examined whether the bovine iris which exhibits a common embryonic origin with the ocular ciliary epithelium and the neural retina, expresses components of phototransduction. By Northern blot and RT-PCR amplification we detected in the iris, rhodopsin, rhodopsin kinase and arrestin transcripts and DNA products, respectively, of the same size as in the retina. By Western blot, antibodies to rhodopsin, rhodopsin kinase and arrestin detected low levels of protein with similar molecular masses as in the retina. Transient transfections of bovine iris cells in vitro with rhodopsin promoter-luciferase-reporter constructs (p130-Luc, p176-Luc, 1225-Luc and p2000-Luc) containing proximal and distal promoter elements led to a significant stimulation of promoter activity over the basal activity. In particular, the construct p225-Luc containing proximal promoter elements upstream of the transcription start site (-225 to +70 bp) led to 3.1-fold stimulation of activity over p176-Luc, 2.1-fold over p130 or p2000-Luc and 190-fold over the basal activity. These results suggested that the bovine iris cells contain factors that could either stimulate or attenuate rhodopsin transcription. The data also supported the view that components associated with non-visual phototransduction are expressed in extraretinal sites including the ciliary epithelium and the iris.

Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium.

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na(+)/H(+) exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pH(i)) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1-100 nM), the rate of pH(i) recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na(+)-dependent pH(i) recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 microM) completely abolished the pH(i) recovery by NHE. 18alpha-Glycyrrhetinic acid (18alpha-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pH(i) recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined.

Responses and signaling pathways in human optic nerve head astrocytes exposed to hydrostatic pressure in vitro.

In this study, we examined the effects of mechanical stress induced by elevated hydrostatic pressure (HP) on the migration of human optic nerve head (ONH) astrocytes, using an in vitro model that follows repopulation of a cell-free area (CFA) created on a monolayer of cultured astrocytes. alpha-Tubulin staining detected phenotypic changes in astrocytes exposed to HP. The influence of proliferation in closure of the CFA was determined by incorporation of BrdU under 1.5-cm H2O, control pressure (CP), and 10-cm H2O HP with or without 5-fluorouracil. Under control and experimental conditions, closure of the CFA occurred mostly by migration and less by proliferation. Exposure to 10-cm H2O HP induced faster closure of the CFA at 1, 3, and 5 days. The signaling pathways involved in responses to HP were determined using genistein, tyrphostin A25, AG1478, and AG1295, inhibitors of receptor tyrosine kinases; wortmannin and LY294002, inhibitors of phosphatidyl inositol 3-kinase (PI-3K); and SC58236, an inhibitor of inducible cyclooxygenase-2 (COX2). Genistein and tyrphostin A25 blocked HP-induced migration at 1, 3, and 5 days, but did not affect closure of the CFA under CP. AG1478 and AG1295 blocked HP-induced migration and partially inhibit closure of the CFA under CP. LY294002 blocked HP-induced migration. SC58236 markedly inhibited closure of the CFA under CP by inhibiting COX2 activity. Exposure to HP, a physical stress, induced faster closure of the CFA via activation of members of the epidermal growth factor receptor (EGFR) family and PI-3K pathways. Under CP, closure of the CFA in response to denudation, a form of injury, is due to activation of COX2 in ONH astrocytes.

DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure.

There is clinical and experimental evidence that elevated intraocular pressure (IOP), a mechanical stress, is involved in the pathogenesis of glaucomatous optic neuropathy. The mechanism by which astrocytes in the optic nerve head (ONH) respond to changes in IOP is under study. Gene transcription by ONH astrocytes exposed either to 60 mmHg hydrostatic pressure (HP) or control ambient pressure (CP) for 6, 24, and 48 h was compared using Affymetrix GeneChip microarrays to identify HP-responsive genes. Data were normalized across arrays within each gene. A linear regression model applied to test effect of time and HP on changes in expression level identified 596 genes affected by HP over time. Using GeneSpring analysis we selected genes whose average expression level increased or decreased more than 1.5-fold at 6, 24, or 48 h. Expression of selected genes was confirmed by real-time RT-PCR; protein levels were detected by Western blot. Among the genes highly responsive to HP were those involved in signal transduction, such as Rho nucleotide exchange factors, Ras p21 protein activator, tyrosine kinases and serine threonine kinases, and genes involved in transcriptional regulation, such as c-Fos, Egr2, and Smad3. Other genes that increased expression included ATP-binding cassettes, solute carriers, and genes associated with lipid metabolism. Among the genes that decreased expression under HP were genes encoding for dual activity phosphatases, transcription factors, and enzymes involved in protein degradation. These HP-responsive genes may be important in the establishment and maintenance of the ONH astrocyte phenotype under conditions of elevated IOP in glaucoma.

Differential gene expression in astrocytes from human normal and glaucomatous optic nerve head analyzed by cDNA microarray.

Recent advances in cDNA microarray technology have made it possible to analyze expression of several thousand genes at the same time. Using this technique, gene expression in human astrocytes cultured from glaucomatous and normal optic nerve heads (ONH) was compared. One hundred-fifty genes were differentially expressed more than 5-fold in glaucomatous cell cultures compared with normal. These genes are involved in a number of biological processes, including signal transduction, cell adhesion and proliferation, ECM synthesis, and degradation. Confirmation of differential gene expression was performed by quantitative RT-PCR. Western blots and immunohistochemistry demonstrated gene products in cell cultures or in human ONH tissues. Proliferation, adhesion and migration assays tested physiological responses suggested by differential gene expression. Our study suggests that cultured glaucomatous ONH astrocytes retain in culture many phenotypic characteristics that may be relevant to their role in the pathogenesis of glaucoma and, in general to reactive astrocytes in the CNS. Potential applications of these data include the identification and characterization of signaling pathways involved in astrocyte function, studies of the role of steroid-metabolizing enzymes in the glaucomatous ONH, and further exploration of the role of selected identified genes in experimental animal and in vitro models of glaucoma.