Effects of Latanoprost and Bimatoprost on the Expression of Molecules Relevant to Ocular Inflow and Outflow Pathways

<div><p>Background and Purpose</p><p>The intraocular pressure (IOP)-lowering and side effects in response to different prostaglandin F₂α analogues can be variable, but, the underlying basis for this difference remains unknown. This study investigated the differential changes of cellular proteins relevant to IOP-lowering effects of latanoprost and bimatoprost.</p><p>Methods</p><p>The human T lymphoblast (MOLT-3) cell line and immortalized human trabecular meshwork (iHTM) cells were studied by quantitative PCR and by immunofluorescence after treatment with either latanoprost or bimatoprost. New Zealand white rabbit eyes were treated topically with each agent and, following euthanasia, anterior segment tissues were studied with immunostaining.</p><p>Results</p><p>In cultured MOLT-3 cells, mRNA expression of both c-fos and matrix metalloproteinase 9 increased significantly in response to each agent. In addition, there was little change in tissue inhibitor of metalloproteinase (TIMP)-3 mRNA, but a significant decrease in TIMP-4. Fibronectin mRNA in MOLT-3 cells was down-regulated with bimatoprost, but was up-regulated with latanoprost. Immunofluorescence analysis of iHTM cells showed that intracellular fibronectin was significantly decreased by bimatoprost, but was increased by latanoprost. Both latanoprost and bimatoprost increased mRNA expression of NF-кB p65 and decreased that of IкBα. Aquaporin-1 mRNA expression was significantly down-regulated by bimatoprost. Immunostaining also revealed a significant decrease of aquaporin-1 in the ciliary epithelium of New Zealand white rabbits after bimatoprost treatment.</p><p>Conclusions</p><p>Similarities in protein expression produced by latanoprost and bimatoprost in vitro may be relevant to the mechanism for their IOP-lowering effects in vivo. Differences in fibronectin expression and in aquaporin-1 expression in response to each agent may contribute to variability in the IOP-lowering efficacy in some studies.</p></div

Effects of PGAs on NF-кB p65, IкBα and AQP1 mRNA expression in MOLT-3 cells after 5 days of continuous treatment.

<p>Control, MOLT-3 cells with 0.1% DMSO; Latan, MOLT-3 cells with 10 μg/ml lantanoprost; Bima, MOLT-3 cells with 10 μg/ml bimatoprost. Total RNA extracted from MOLT-3 cells was subjected to qPCR analysis and compared to that of the reference gene GAPDH as described in “Methods”. The expression value of control was considered as 1. Error bars represent <i>SD</i>, <i>n</i> = 3. *, <i>p</i><0.05, **, <i>p</i><0.01, versus control.</p

Effects of topical PGA medication on rabbit IOP.

<p>Thirty μl of Lumigan (Bima, A) or Xalatan (Latan, B) was administered unilaterally to rabbit eyes (qd) for 5 consecutive days, i.e. at 0, 24, 48, 72, and 96 h; NS (control) was administered contralaterally. IOP was measured in a masked fashion using a TonoVet^™ rebound tonometer immediately before each dosing, as well as 4 h and 8 h after dosing. The results were expressed as <i>means±SD</i> (<i>n</i> = 3). *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001, compared with control by two-tailed paired Student’s <i>t</i>-test.</p

Changes in intracellular fibronectin levels in iHTM cells following PGA treatment.

<p>Before grown to confluence, cultures were treated with 0.1% DMSO (Control), 10 μg/ml bimatoprost (Bima) or 10 μg/ml latanoprost (Latan) continuously for 5 days. Cells were permeabilized and then labeled with antibody against fibronectin and IgG conjugated with FITC successively. A: Phase-contrast micrographs showing the morphology of iHTM cells treated with PGA or DMSO vehicle. B: Representative photomicrographs showing fibronectin immunostaining. Scale bar, 20 μm. C: Quantification of fluorescence intensity. Fluorescence intensity was measured using IPP 6.0 System to determine the means integrated option density to express the relative protein level. The results were expressed as <i>means±SD</i> (<i>n</i> = 3). *, <i>p</i><0.05, compared with control.</p

Univariate hazard ratios from Cox proportional hazards regression models.

<p>Abbreviation: HR, Hazard Ratio; CRT, Concurrent chemoradiotherapy; RT+CT, Radiotherapy+Chemotherapy.</p

Decreased deposition of extracellular fibronectin in iHTM cells after PGA treatment.

<p>Confluent cell cultures were treated with 0.1% DMSO (Control), 10 μg/ml bimatoprost (Bima) or 10 μg/ml latanoprost (Latan) continuously for 5 days. Cells were then labeled with antibody against fibronectin and IgG conjugated with FITC successively. A: Phase-contrast micrographs showing the morphology of iHTM cells treated with PGA or vehicle DMSO. B: Representative photomicrographs showing fibronectin immunostaining. Scale bar, 20 μm. C: Quantification of fluorescence intensity. Fluorescence intensity was measured using IPP 6.0 System to determine the means integrated option density to express the relative protein level. The results were expressed as <i>means±SD</i> (<i>n</i> = 3). *, <i>p</i><0.05, **, <i>p</i><0.01, compared with control.</p

Kaplan-Meier plots of the influence of various lifestyle behaviors on survival in NPC patients.

<p>(A) Smoking status; (B) Lifetime cigarette consumption; (C) Alcohol intake; (D) Duration of alcohol intake; (E) Frequency of fruit intake; (F) Body-mass index.</p

Patient characteristics.

<p>Patient characteristics.</p

Effects of PGAs on the mRNA expression of fibronectin and ECM degradation related proteins in MOLT-3 cells after 5-day consecutive treatment.

<p>Control, MOLT-3 cells with 0.1% DMSO; Latan, MOLT-3 cells with 10 μg/ml lantanoprost; Bima, MOLT-3 cells with 10 μg/ml bimatoprost. Total RNA extracted from MOLT-3 cells was subjected to qPCR analysis and compared to that of the reference gene GAPDH as described in “Methods”. The expression value of control was considered as 1. Error bars represent <i>SD</i>, <i>n</i> = 3. *, <i>p</i><0.05, **, <i>p</i><0.01, versus control.</p

Primers of the specific genes for qPCR analysis.

<p>Primers of the specific genes for qPCR analysis.</p