ABCB1 transporter and Toll-like receptor 4 in trabecular meshwork cells.

PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 µM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 µM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 µM verapamil and 50 µM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. ß-amyloid significantly reduced ABCB1 activity, and the ß-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.

[Factors Affecting Uncorrected Visual Acuity following Implantation of Apodized Diffractive Intraocular Lenses].

PURPOSE: To investigate the preoperative factors affecting postoperative uncorrected visual acuity (UCVA) equal to or more than 20/20. SUBJECTS AND METHODS: One hundred sixty seven eyes receiving apodized diffractive multifocal intraocular lenses (SN6AD1) were included in this study. In the eyes with corneal astigmatism of less than 1.0 D, a 2.4 mm clear temporal corneal incision was created. In those equal to or more than 1.0 D, 4.1 mm steepest meridian clear corneal incisions and/or limbal relaxing incisions were conducted. Preoperative factors affecting postoperative UCVA equal to or more than 20/20 were assessed by univariate analysis and multiple logistic regression analysis. RESULTS: Patient's age and preoperative corneal astigmatism were significant in both univariate analysis and multiple logistic regression analysis. The ratio of postoperative UCVA equal to or more than 20/20 was significantly lower in the patients 70 years or older and in the eyes with corneal astigmatism equal to or more than 1.5 D. CONCLUSION: Multifocal toric intraocular lenses provide better surgical results in eyes with corneal astigmatism, however patient's age needs to be considered as a factor affecting UCVA.

sCD44 internalization in human trabecular meshwork cells.

PURPOSE: To determine whether soluble CD44 (sCD44), a likely biomarker of primary open-angle glaucoma (POAG), is internalized in cultured human trabecular meshwork (TM) cells and trafficked to mitochondria. METHODS: In vitro, 32-kD sCD44 was isolated from human sera, biotinylated, and dephosphorylated. TM cells were incubated for 1 hour at 4°C with biotinylated albumin (b-albumin), biotin-labeled sCD44 (b-sCD44), or hypophosphorylated biotin-labeled sCD44 (-p b-sCD44) in the presence or absence of unlabeled sCD44, hyaluronic acid (HA), and a selected 10-mer HA binding peptide. The slides were warmed for 1 or 2 hours at 37°C, and 125 nM MitoTracker Red was added for the last 20 minutes of the incubation. The cells were washed, fixed, incubated with anti-biotin antibody and FITC-labeled goat anti-mouse antibody, and examined under a confocal microscope. RESULTS: TM cell membranes were positive for b-sCD44 after 4°C incubation. When the temperature was raised to 37°C, b-sCD44 or -p b-sCD44 appeared in the cytoplasm. The internalization of b-sCD44 was blocked by excess unlabeled sCD44, HA, and a 10-mer HA-binding peptide. Double label experiments with b-sCD44 or -p b-sCD44 and MitoTracker Red indicated partial overlap. The percent co-localization of MitoTracker Red at 2 hours and FITC -p b-sCD44 was 17.4% (P < 0.001) and for FITC b-sCD44 was 11.7% (P < 0.001) compared with b-albumin. The influence of putative CD44 phosphorylation sites on mitochondrial trafficking was determined by TargetP 1.1. CONCLUSIONS: sCD44 is internalized by TM cells and trafficked in part to mitochondria, which may be a factor in the toxicity of sCD44 in the POAG disease process.

Lactate treatment causes NF-kappaB activation and CD44 shedding in cultured trabecular meshwork cells.

PURPOSE: To challenge human trabecular meshwork (TM) cells using lactate to mimic cell stress and observe the effects on cell viability, NF-kappaB, and membrane type 1 matrix metalloproteinase (MT1-MMP) expression and the ectodomain shedding of soluble (s)CD44. METHODS: Human TM cells grown in 10% fetal calf serum (FCS) were incubated in 0.1% FCS with 1, 10, or 40 mM lactate or PBS for 5 and 30 minutes and 1, 3, and 6 hours. Cell viability was determined with trypan blue staining. NF-kappaB and MT1-MMP expression was evaluated through Western blot analysis of medium and the cytoplasmic and nuclear fractions. Media sCD44 concentration was determined by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: The TM cell viability was significantly decreased after incubation for 3 hours with 40 mM lactate (P < 0.01) and 6 hours with 10 and 40 mM lactate (P < 0.001). Western blot analysis showed an increased NF-kappaB p50 and MT1-MMP expression and activity by 5 minutes in lactate-treated TM cells compared with that of control cells. At 6 hours, NF-kappaB p65 was increased in nuclear fraction of lactate-treated compared with control cells. Treatment with 1 mM lactate caused an increase in the media concentration of both the 32 and 55 kDa sCD44 at 3 (P < 0.05) and 6 hours (P < 0.01). CONCLUSIONS: Lactate treatment resulted in dose- and time-dependent effects on human TM cell viability, translocation of NF-kappaB, and activation of MT1-MMP. Increased shedding of sCD44 occurred with the l mM dose of lactate. Lactate treatment of human TM cells in culture offers a useful cell model to examine the stress responses that occur in glaucoma.

Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells.

We investigated the roles of Rho-associated protein kinase (ROCK) in regulating activities such as adhesion, contraction and migration in cultured human trabecular meshwork (TM) cells. Human TM cells in culture were treated with Y-27632, a specific ROCK inhibitor. Trypan blue exclusion test and TUNEL staining showed little or no direct toxicity of Y-27632 on TM cells. By MTT assay, Y-27632 did not significantly affect the proliferation of TM cells. The cell adhesion assay showed that Y-27632 promoted the cell adhesiveness to both fibronectin and collagen type I in a dose-dependent manner. Collagen gel contraction activity of TM cells was significantly inhibited by the treatment of Y-27632 in a dose-dependent manner. The addition of Y-27632 accelerated motility of TM cells in wound healing assay. Phosphorylated LIM kinase 2 and cofilin, related to actin bundling and integrin clustering, were dephosphorylated (activated) by Y-27632. In conclusion, Y-27632 elicits profound effects on TM cell activities including adhesion, gel contraction, and cell motility. These Y-27632-induced changes of TM cells may be relevance to the physiology of the aqueous outflow system.

The effect of subtenon triamcinolone acetonide injection for diabetic macular edema on retinal and choroidal circulation.

PURPOSE: To evaluate changes in retinal and choroidal circulation after subtenon triamcinolone acetonide (TA) injection for diabetic macular edema. DESIGN: Prospective interventional case series. METHODS: Thirteen eyes of 13 patients with diabetic macular edema were studied. Fluorescein and indocyanine green angiograms were performed at three periods: before the injection and 1 week and 6 months after subtenon injection of TA (40 mg). Retinal arteriovenous passage time (as an indicator of retinal circulation) and choroidal tau (as an indicator of early filling velocity of choroid) were obtained with image analysis software. RESULTS: Choroidal tau values before and 1 week after subtenon TA injection were, respectively, 3.2 +/- 0.4 and 4.0 +/- 0.7 seconds, which showed a significant delay (P = .01, Wilcoxon signed-rank test). The delayed choroidal tau values returned to pretreatment level at 6 months after TA injection. In contrast, the arteriovenous passage time remained unchanged. CONCLUSION: Subtenon TA injection transiently influences choroidal blood flow.

Trans-Tenon's retrobulbar triamcinolone acetonide infusion for refractory diabetic macular edema after vitrectomy.

PURPOSE: To evaluate the efficacy and safety of trans-Tenon's retrobulbar triamcinolone acetonide (TA) infusion for the treatment of refractory diabetic macular edema (DME) after vitrectomy. METHODS: After topical anesthesia, 20 eyes from 20 patients with persistent DME after pars plana vitrectomy were treated with trans-Tenon's retrobulbar infusion of 40 mg TA through an inferotemporal approach. The mean duration (+/-SD) between vitrectomy and trans-Tenon's retrobulbar TA infusion was 11.4+/-7.9 months. The mean follow-up period (+/-SD) after trans-Tenon's retrobulbar TA infusion was 13.3+/-2.8 months. RESULTS: At 1 week after trans-Tenon's retrobulbar TA infusion, the mean central retinal thickness (+/-SD) measured by optical coherence tomography was 381+/-99 mum, which was a statistically significant decrease in comparison with the preoperative thickness (555+/-112 mum) (P<0.001). Additional trans-Tenon's retrobulbar TA infusions were performed in ten eyes (50%), due to the recurrence of DME at 6.6+/-3.0 months after the first TA infusion. At the final examination, macular edema resolved in 13 (65%), improved in four (20%), and remained unchanged in three (15%) of the 20 eyes. At 1 month after trans-Tenon's retrobulbar TA infusion, the mean laser flare value (+/-SD) was 9.6+/-3.0 photon/ms, which was a statistically significant decrease in comparison with the preoperative value (15.5+/-5.9 photon/ms) (P<0.01). Furthermore, in ten eyes (50%) with recurrent DME, re-elevated laser flare values were observed prior to the recurrence of DME. The final best-corrected Snellen visual acuity improved by two or more lines in nine eyes (45%), and remained unchanged in 11 eyes (55.0%). IOP elevation equal to or higher than 21 mmHg was observed in three (15%) of the 20 eyes with TA infusion, and was controlled by topical medications. No other injection-related complications were observed. CONCLUSION: Trans-Tenon's retrobulbar TA infusion is an effective and safe method for the treatment of refractory DME, which is present even after vitrectomy.