Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE) induces cell loss, cellular senescence, and extracellular matrix (ECM) synthesis in primary human retinal pigment epithelial (RPE) cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA) fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J), connective tissue growth factor (CTGF), fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration

Assessing the adherence behavior of glaucoma patients to topical eye drops

Purpose: The goal of this study was to determine the adherence of glaucoma patients to their topical glaucoma medication. Furthermore, the relationships between the adherence behavior and the patients' demographic data, clinical characteristics, and their knowledge about glaucoma were evaluated. Methods: This was a prospective study of 123 patients with primary open-angle glaucoma who were given two standardized questionnaires. The first questionnaire at time point T1 comprised a knowledge assessment and the self-reported adherence measures Adherence to Refills and Medication Scale 2 (ARMS2),visual analogue scale for adherence (VAS-AD),and missed doses in the past 14 days. Two months later at time point T2, a second questionnaire reevaluated the adherence measures ARMS2, VAS-AD, and missed doses in the past 14 days. Results: There was a good correlation among all the three adherence measures at T1 and T2. The mean values of ARMS2 were in the lower range, with 3.38 at T1 and 2.8 at T2. The VAS-AD detected that 18.5% of patients always took their eye drops correctly, and 77.9% of patients reported not to have missed a single dose in the past 14 days. There was no significant correlation between the patients' demographic data or knowledge about glaucoma and the adherence measures ARMS2 or VAS-AD. Among the clinical characteristics, only single-eye blindness showed a significant correlation with VAS-AD. Conclusion: In this study, no general relationships were found between medication adherence and the patients' demographic data, clinical characteristics, or knowledge about glaucoma. It may be assumed that more individualized strategies are required to optimize adherence behavior

Assessing the adherence behavior of glaucoma patients to topical eye drops

Purpose: The goal of this study was to determine the adherence of glaucoma patients to their topical glaucoma medication. Furthermore, the relationships between the adherence behavior and the patients' demographic data, clinical characteristics, and their knowledge about glaucoma were evaluated. Methods: This was a prospective study of 123 patients with primary open-angle glaucoma who were given two standardized questionnaires. The first questionnaire at time point T1 comprised a knowledge assessment and the self-reported adherence measures Adherence to Refills and Medication Scale 2 (ARMS2),visual analogue scale for adherence (VAS-AD),and missed doses in the past 14 days. Two months later at time point T2, a second questionnaire reevaluated the adherence measures ARMS2, VAS-AD, and missed doses in the past 14 days. Results: There was a good correlation among all the three adherence measures at T1 and T2. The mean values of ARMS2 were in the lower range, with 3.38 at T1 and 2.8 at T2. The VAS-AD detected that 18.5% of patients always took their eye drops correctly, and 77.9% of patients reported not to have missed a single dose in the past 14 days. There was no significant correlation between the patients' demographic data or knowledge about glaucoma and the adherence measures ARMS2 or VAS-AD. Among the clinical characteristics, only single-eye blindness showed a significant correlation with VAS-AD. Conclusion: In this study, no general relationships were found between medication adherence and the patients' demographic data, clinical characteristics, or knowledge about glaucoma. It may be assumed that more individualized strategies are required to optimize adherence behavior

Retinal pigment epithelium is protected against apoptosis by

PURPOSE. The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein ␣B-crystallin in the RPE in response to oxidative stress and to explore whether ␣B-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS. Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of ␣B-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42°C for 20 minutes) or oxidant-mediated injury (50 -300 M H 2 O 2 for 1 hour). Induction of ␣B-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of ␣B-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing ␣B-crystallin cDNA or a control vector without ␣B-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS. ␣B-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in ␣B-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of ␣B-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with ␣B-crystallin were more resistant to H 2 O 2 -induced cellular injury. CONCLUSIONS. ␣B-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of ␣B-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress. (Invest Ophthalmol Vis Sci. 2002;43:3575-3582) A ge-related macular degeneration (AMD) is the leading cause of severe visual impairment in elderly individuals

TGF-beta(2)- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-beta(2) (TGF-beta(2))- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant alpha-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-beta(2) for 24 h or 200 mu M hydrogen peroxide (H2O2) for 1 h. Lipid peroxidation was measured by a decrease in cis-pari-naric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-beta 2 or H2O2 exposure. Expressions of the heat shock protein (Hsp) alpha B-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-beta(2) and H2O2 increased lipid peroxidation. Treatment of astrocytes with TGF-beta(2) and H2O2 upregulated the expression of alpha B-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-beta(2)- and H2O2-stimulated gene expressions. Conclusion: We showed that TGF-beta(2)- and H2O2-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy. Copyright (C) 2012 S. Karger AG, Base

Preventive Effects of Omega-3 and Omega-6 Fatty Acids on Peroxide Mediated Oxidative Stress Responses in Primary Human Trabecular Meshwork Cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease

CSE induced SA-ß-Gal activity in cultured human RPE cells.

<p>(<b>A</b>) Morphology and SA-ß-Gal activity of untreated human RPE cells. Only single cells were stained blue indicating SA-ß-Gal activity. Scale bar: 100 µm. (<b>B</b>) In contrast, RPE cells of the same passage exposed to 8% of CSE showed a marked increase of SA-ß-Gal activity. Scale bar: 100 µm. (<b>C</b>) Quantification of the number of SA-ß-Gal positive cells. The percentage of SA- ß-Gal activity was analyzed after exposure to 2, 4, and 8% of CSE and scored by counting at least 300 cells in phase contrast photomicrographs of representative fields. Data (mean ± s.d.) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P<0.05). Co, control.</p

CSE increased fibronectin, laminin protein secretion.

<p>Protein secretion of (<b>A</b>) fibronectin (FN) and (<b>B</b>) laminin into culture media. Error bars: ± s.d. of results from three experiments with three different cell cultures (*P<0.05). Co, control.</p