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

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

F-actin labelling.

<p>Phalloidin labeling of the F-actin cytoskeleton in controls, ω-6 and ω-3 supplemented hTM (<b>A</b>) before and (<b>B</b>) after H₂O₂ exposition; scale bar: 100 µm.</p

Analysis of nuclear NFκB.

<p>(<b>A</b>) Quantitative realtime PCR expression analysis of NFκB mRNA in controls, hTM pre-treated with ω-6 and ω-3 before and after H₂O₂ exposition. (<b>B</b>) Quantification of nuclear NFκB protein 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).</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

Antibodies used for Western blots (WB) and Immunofluorescence (IF) labeling.

<p>Antibodies used for Western blots (WB) and Immunofluorescence (IF) labeling.</p

FN, PAI-1 and CTGF expression analysis.

<p>Quantitative realtime PCR expression analysis of FN, PAI-1 and CTGF mRNAs in controls, ω-6 and ω-3 fatty acids pre-treated hTM normalized to controls (<b>A</b>) before and (<b>B</b>) after H₂O₂ exposition. (<b>C</b>) Western blot detection of cellular FN, PAI-1, CTGF, and actin in controls, ω-6 and ω-3 fatty acids pre-treated hTM. Plots of densitometric quantifications to deduce fold expressions of intracellular (<b>D</b>) FN, (<b>E</b>) PAI-1 and (<b>F</b>) CTGF, before and after H₂O₂ exposition. (<b>G</b>) ELISA quantification of FN medium contents normalized to controls. Values 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

CCK-8 assay.

<p>Quantification of mitochondrial activity 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 mitochondrial activity after H₂O₂ exposition. Values represent mean averages (m.a.) ± standard deviations (sd) of three independent experiments performed in triplicates of 5 different donors (n = 45); asterisks: p-values of statistical significances (**p≤0.01; ***p≤0.001).</p