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

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

Topical application of PPADS inhibits complement activation and choroidal neovascularization in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia

PPADS Inhibits Deposition of MAC at the site of laser induced CNV.

<p><b>A</b>) Representative images of C5b9 labeling at 7 days post laser in eyes treated with 0.9% NaCl or 4.17 mM PPADS topically for 3 days. <b>B</b>) Mean values ± SEM of C5b9 intensity/area of 0.9% NaCl treated eyes and 4.17 mM PPADS treated eyes 7 days post lasering. Studies were performed 3 times with 5 mice in each group, n = 15, NaCl = 91 laser spots, PPADS = 71 laser spots (** p<0.005).</p

PPADS Inhibits Complement Mediated Cell Lysis.

<p><b>A</b>) Representative flow cytometry plots indicating number of Hepa1c1c7 cells taking up propidium iodide (PI) when incubated in 1% normal human serum (NHS) and various concentrations of PPADS (blue lines) or 0.9% NaCl (red lines). <b>B</b>) Mean values ± SEM of PPADS mediated reduction of NHS mediated cell lysis from 5 independent studies. Significant differences are shown as capped lines between vehicle control (0.9% NaCl, 0 µM PPADS) and the various concentrations of PPADS (*** p<0.0005, ** p<0.005, * p<0.05).</p

PPADS inhibits formation of tubes by HUVECs.

<p><b>A</b>) Representative phase contrast images of tubes formed in the absence of supplements (negative control), in the presence of vehicle (0.9% NaCl) or vehicle plus 1.04 µM PPADS or 4.17 µM PPADS. <b>B</b>) Tabular summary of mean values ± SEM of master junctions, master segments and meshes in cells incubated with the indicated concentration of PPADS and calculated IC₅₀ for each marker. <b>C</b>) Dose dependent PPADS mediated reduction of master junctions, master segments and meshes. The punctuated line indicates the extrapolated inhibition trend. IC₅₀ values are indicated as intercept points in the trend line. Asterisks in each inset mark an example of a measured junction, segment or mesh, respectively. Studies were performed a total of 4 times in triplicate.</p

Hsp27 and Hsp90 expression analysis.

<p>(<b>A</b>) Quantification of realtime PCR expression analysis of Hsp27 and Hsp90 mRNAs in controls, ω-6 and ω-3 fatty acids pre-treated hTM normalized to controls. (<b>B</b>) Western blot detection of cellular Hsp27, Hsp90 and actin protein in controls, ω-6 and ω-3 fatty acids pre-treated hTM. (<b>C</b>) Plot of densitometric quantifications of Hsp27 and Hsp90 protein expression in controls, ω-6 and ω-3 fatty acids pre-treated hTM adjusted to actin expression and normalized to controls. Values represent m.a. ± sd of three independent experiments performed on cells of three different donors (n = 9); asterisks: p-values of statistical significances (*p≤0.05; **p≤0.01; ***p≤0.001).</p

Interleukin 1α, -6 and -8 expression analysis.

<p>Quantitative realtime PCR expression analysis of interleukins (<b>A</b>) -1α, (<b>B</b>) -6 and (<b>C</b>) -8 mRNAs in controls, hTM pre-treated with ω-6 and ω-3 before and after H₂O₂ exposition. ELISA quantification of (<b>D</b>) IL-6 and (<b>E</b>) IL-8 medium contents. Values are normalized to untreated controls and represent m.a. folds ± sd of three independent experiments performed on cells of three different donors (n = 9); asterisks: <i>p</i>-values of statistical significances (*<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001).</p

Primers used for realtime PCR.

<p>Primers used for realtime PCR.</p

BrdU incorporation analysis.

<p>Quantification of proliferation rate in hTM after 48 hours normalized to starting activity in controls. (<b>A</b>) Effects of ω-6 (16 µM) and ω-3 (50 µM) fatty acids compared to controls (Co). (<b>B</b>) Effects of H₂O₂ in controls, ω-6 and ω-3 fatty acids pre-treated hTM. (<b>C</b>) %-reduction of BrdU-incorporation after H₂O₂ exposition. Values represent m.a. ± sd of three independent experiments performed in triplicates of 5 different donors (n = 45); asterisks: p-values of statistical significances (*p≤0.05; **p≤0.01; ***p≤0.001).</p