13 research outputs found
CXCR3 Antagonism of SDF-1(5-67) Restores Trabecular Function and Prevents Retinal Neurodegeneration in a Rat Model of Ocular Hypertension
Glaucoma, the most common cause of irreversible blindness, is a neuropathy commonly initiated by pathological ocular hypertension due to unknown mechanisms of trabecular meshwork degeneration. Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed. Here we show that the chemokine CXCL12, its truncated form SDF-1(5-67), and the receptors CXCR4 and CXCR3 are expressed in human glaucomatous trabecular tissue and a human trabecular cell line. SDF-1(5-67) is produced under the control of matrix metallo-proteinases, TNF-α, and TGF-ÎČ2, factors known to be involved in glaucoma. CXCL12 protects in vitro trabecular cells from apoptotic death via CXCR4 whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase activation. Ocular administration of SDF-1(5-67) in the rat increases intraocular pressure. In contrast, administration of a selective CXCR3 antagonist in a rat model of ocular hypertension decreases intraocular pressure, prevents retinal neurodegeneration, and preserves visual function. The protective effect of CXCR3 antagonism is related to restoration of the trabecular function. These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration
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The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant CellsBenzalkonium Chloride Inhibits Mitochondrial Function
PurposeBenzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects.MethodMitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3).ResultsBenzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 ÎŒM) and O2 consumption (IC50, 10.9 ÎŒM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 ÎŒM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 ÎŒM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 ÎŒM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect.ConclusionsBenzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients
The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells
CX3CL1 is involved in the ocular surface inflammation induced by benzalkonium chloride
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NaCl-induced hyperosmotic stress as an in vitro dry eye model potentiated by synergic toxic effects of Benzalkonium chloride on conjunctival epithelial cells
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Blockade of the chemokine receptor CXCR3 lowers intraocular pressure and prevents retinal degeneration in an animal model of glaucoma
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CXCL12 protects trabecular cells from apoptosis <i>via</i> CXCR4, whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase-3 activation.
<p><i>(A)</i> 24-h incubation with CXCL12 (10 ng/mL [1.3 nM]) protects HTM3 cells from apoptotic stress induced by 15-min exposure to 0.01% benzalkonium chloride (BAC), whereas SDF-1(5-67) (10 ng/mL [1.3 nM]) increases apoptosis as assessed by microplate cytometry using Hoechst dye. ** <i>P</i><0.05 vs. unstressed cells, § <i>P</i><0.05 and §§ <i>P</i><0.01 vs. BAC-exposed cells. <i>(B)</i> The protective effect of CXCL12 (10 ng/mL, 24 h) is reversed by CXCR4 antagonist (AMD-3100, 1 ”M), whereas the apoptotic effect of SDF-1(5-67) (10 ng/mL, 24 h) is inhibited by CXCR3 antagonist (NBI-74330, 1 ”M). CXCL10 (10 ng/mL [1.1 nM], 24 h), a conventional ligand for CXCR3, mimics the apoptotic effect of SDF-1(5-67). ** <i>P</i><0.01. <i>(C)</i> Dose-dependent effect of 24-h incubation with SDF-1(5-67) or with CXCL12. ** <i>P</i><0.01 vs. CXCL12. <i>(D)</i> SDF-1(5-67) (10 ng/mL) increases caspase-3 activation as assessed by immunoflowcytometry. CXCR3 antagonist (NBI-74330, 1 ”M) inhibits SDF-1(5-67)-induced caspase 3 activation. ** <i>P</i><0.01 vs. unstimulated, §§ <i>P</i><0.01 vs. SDF-1(5-67)-stimulated. Data in graphs are presented as means ± SEM.</p
Ophthalmic administration of CXCR3 antagonist restores trabecular filtrating function and protects trabecular cells from apoptosis in a rat model of ocular hypertension.
<p><i>(A)</i> Aqueous humor outflow impairment in hypertensive eyes is counteracted by treatment with CXCR3 antagonist as measured <i>in vivo</i> by fluorophotometry (nâ=â10 each). <i>(B,C,D)</i> Trabecular filtrating function is restored by treatment with CXCR3 antagonist as assessed by trabecular trapping of fluorescent microspheres (red), and quantitatively measured as percent of effective filtration length (PEFL), which is more important in treated eyes <i>(B)</i> than in untreated hypertensive eyes <i>(C)</i> (nâ=â10 each). <i>(E,F,G)</i> Density of apoptotic trabecular cells is lower in eyes treated with the CXCR3 antagonist <i>(E)</i> than in untreated hypertensive eyes <i>(F),</i> as assessed by TUNEL (green) and DAPI (blue) nuclear staining (nâ=â10 each). ** <i>P</i><0.01. (scale bar, 50 ”m). Data in bar graphs are presented as means ± SEM.</p
Novel in situ gelling ophthalmic drug delivery system based on gellan gum and hydroxyethylcellulose: Innovative rheological characterization, in vitro and in vivo evidence of a sustained precorneal retention time
International audienceAchieving drug delivery at the ocular level encounters many challenges and obstacles. In situ gelling delivery systems are now widely used for topical ocular administration and recognized as a promising strategy to improve the treatment of a wide range of ocular diseases. The present work describes the formulation and evaluation of a mucoadhesive and ion-activated in situ gelling delivery system based on gellan gum and hydroxyethylcellulose for the delivery of phenylephrine and tropicamide. First, physico-chemical characteristics were assessed to ensure suitable properties regarding ocular administration. Then, rheological properties such as viscosity and gelation capacity were determined. Gelation capacity of the formulations and the effect of hydroxyethylcellulose on viscosity were demonstrated. A new rheological method was developed to assess the gel resistance under simulated eye blinking. Afterward, mucoadhesion was evaluated using tensile strength test and rheological synergism method in both rotational and oscillatory mode allowing mucoadhesive properties of hydroxyethylcellulose to be point out. Finally, residence time on the ocular surface was investigated in vivo, using cyanine 5.5 dye as a fluorescent marker entrapped in the in situ gelling delivery systems. Residence performance was studied by non-invasive optical imaging on vigilant rabbits, allowing eye blinking and nasolacrimal drainage to occur physiologically. Fluorescence intensity profiles pointed out a prolonged residence time on the ocular surface region for the developed formulations compared to conventional eye drops, suggesting in vitro/in vivo correlations between rheological properties and in vivo residence performances
CXCL12, CXCR3, and CXCR4 expression by human glaucomatous trabecular tissue and a trabecular cell line.
<p><i>(AâC)</i> The chemokine CXCL12 <i>(A)</i> and receptors CXCR3 <i>(B)</i> and CXCR4 <i>(C)</i> are detected in unstimulated human glaucomatous trabecular cells HTM3 by indirect immunofluorescence (secondary antibody in green, propidium iodide in red, scale bar: 50 ”m, magnification Ă200). <i>(D)</i> Chemokine receptor CXCR4 appears as distinct spots located at the cell membrane surface (scale bar: 5 ”m, mag. Ă800). Representative images of three independent experiments are depicted. <i>(D)</i> Cell expression of CXCL12 and receptors is also detected and quantified by immunoflowcytometry. Representative results obtained over 6 independent experiments, mean ± SEM of positive cells. <i>(E)</i> Chemokine and receptor mRNAs are detected in human glaucomatous trabecular tissues (nâ=â15) and in the HTM3 trabecular cell line. Data in the bar graph are presented as means ± SEM.</p