Secretion of matrix metalloproteinase-3 by co-cultured pigmented and non-pigmented human trabecular meshwork cells following selective laser trabeculoplasty.

BACKGROUND: Matrix metalloproteinases (MMPs) are reported to contribute to the mechanism of argon laser trabeculoplasty, but it is unknown whether they are also secreted after selective laser trabeculoplasty (SLT). The aim of this study was to investigate whether human primary trabecular meshwork (HTM) cells secrete MMP-3 after stimulation by SLT. The relationship between SLT and the two principal forms of cell death, apoptosis and necrosis, was also examined. METHODS: Non-pigmented primary HTM cells were challenged with melanin granules to artificially introduce pigmentation. Isolated non-pigmented HTM cells and 1:1 co-cultures of pigmented and non-pigmented cells were treated with 0.5-1.5 mJ SLT. Cellular metabolic activity (CMA) was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and necrosis were measured using a cell death ELISA and a lactate dehydrogenase assay, respectively. MMP-3 secretion was measured by ELISA. RESULTS: Co-cultures exhibited a dose-dependent decline in CMA and a dose-dependent increase in necrosis 4 and 24 h after SLT. Non-pigmented cells did not undergo necrosis and displayed a trend towards increased CMA. Apoptosis was reduced in non-pigmented cells but elevated in co-cultures. Increased MMP-3 secretion was observed from co-cultures but not isolated non-pigmented cells. CONCLUSIONS: Pigmentation is necessary for both post-SLT cell death and MMP-3 secretion. SLT appears to have a hormetic effect on non-pigmented HTM cells.

Mechanisms of benzalkonium chloride toxicity in a human trabecular meshwork cell line and the protective role of preservative-free tafluprost.

BACKGROUND: Benzalkonium chloride (BAK) is a controversial ophthalmic preservative because of its prominent side-effect profile. In this study, we examined the mechanism of BAK toxicity in human trabecular meshwork cells (HTMC) and compared the effects of BAK with tafluprost free acid, which is an active form of tafluprost commercially available in a preservative-free formulation. METHODS: Primary HTMC were treated with different BAK concentrations over various exposure times. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide assay, and apoptosis was measured by enzyme-linked immunosorbent assay. The cell viability of primary HTMC exposed to various concentrations and times of tafluprost free acid was also determined. Cells were treated with BAK and tafluprost free acid for 30 min at 37°C, and cell viability was again assessed. The effect of BAK on the gap junction protein Connexin-43 (Cx43) expression was subsequently examined. RESULTS: BAK treatment resulted in a dose- and time-dependent decline in cell viability. Apoptosis increased following BAK treatment. Tafluprost-free acid treatment did not significantly affect cell viability. Tafluprost co-treatment with BAK resulted in an increase in cell viability as compared with BAK treatment alone. BAK treatment upregulated Cx43 expression in HTMC. CONCLUSIONS: These results demonstrate that BAK is harmful to the health of cultured HTMC. Tafluprost is both safe and cytoprotective against BAK for these HTMC. The effect of tafluprost on the gap junctions of the HTM should be further investigated.

Mechanisms of selective laser trabeculoplasty: a review.

Selective laser trabeculoplasty is a safe and effective treatment for glaucoma, with greater cost effectiveness than its pharmacological and surgical alternatives. Nevertheless, although the basic science literature on selective laser trabeculoplasty continues to grow, there remains uncertainty over the mechanism by which laser trabeculoplasty reduces intraocular pressure. To address this uncertainty, the evidence behind several potential biological and mechanical mechanisms of selective laser trabeculoplasty were reviewed. In particular, cytokine secretion, matrix metalloproteinase induction, increased cell division, repopulation of burn sites and macrophage recruitment were discussed. Refining our understanding of these mechanisms is essential both to understanding the pathophysiology of ocular hypertension and developing improved therapies to treat the condition.

Ciliary body lymphangiogenesis in uveal melanoma with and without extraocular extension.

PURPOSE: To determine whether peritumoral ciliary body lymphatics are found in uveal melanoma in the absence of extraocular extension. DESIGN: Consecutive case series from 1999 to 2005. PARTICIPANTS: Thirty-two uveal melanoma cases involving the ciliary body from the Ophthalmic Pathology Laboratory, University of Toronto, of which 23 showed no extraocular extension. METHODS: All immunofluorescence studies and quantitative analyses were performed in a masked fashion. Sections were immunostained for the presence of lymphatic endothelium using podoplanin (D2-40 antibody) and blood vessel endothelium using CD34. MAIN OUTCOME MEASURES: Identification and quantification of D2-40-positive lymphatic vessels in the ciliary body. RESULTS: In every case (n = 32), D2-40-positive lymphatics were detected in the peritumoral ciliary body. Lymphatic signal was significantly increased in the peritumoral ciliary body compared with the nonperitumoral ciliary body (P < 0.0001). There was no difference in lymphatic signal between cases with and without extraocular extension (P > 0.05). Lymphatics were not detected within the tumors. CONCLUSIONS: Peritumoral lymphangiogenesis was present in the ciliary body in uveal melanomas with and without extraocular extension, and as such, the presence of peritumoral lymphatics is not recommended as a prognostic marker in uveal melanoma.

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.

Efficacy of various antioxidants in the protection of the retinal pigment epithelium from oxidative stress.

BACKGROUND: Oxidative stress induced retinal pigment epithelium (RPE) dysfunction is hypothesized to be fundamental in the pathogenesis of age-related macular degeneration (AMD). This study investigated whether vitamin C, vitamin C phosphate, vitamin E, propofol, betaxolol, and N-acetyl cysteine (NAC) protect human RPE cells from oxidative stress. METHODS: ARPE-19 cells were pretreated with the compounds under investigation. The chemical oxidant tert-butyl hydroperoxide (t-BOOH) was used to induce oxidative stress. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Exposure to t-BOOH resulted in a dose- and time-dependent reduction in ARPE-19 cell viability. Compared with cells given t-BOOH alone, vitamin E and NAC pretreated cells had significantly improved viability, propofol and betaxolol pretreated cells had no significant difference in viability, and vitamin C and vitamin C phosphate pretreated cells had significantly reduced viability. CONCLUSION: Of the compounds studied, only vitamin E and NAC significantly mitigated the effects of oxidative stress on RPE cells. Because of their potential therapeutic value for AMD patients, these and other RPE protective compounds continue to merit further investigation.