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G. Malek, P. Hu, S. Cousins; Oxidant Injury and Pro-Inflammatory Poly-Unsaturated Fatty Acids Stimulate Extracellular Matrix Synthesis in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5166.
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The pathophysiology of ‘early’ age-related macular degeneration (AMD), characterized by accumulation of lipid and protein-rich sub-retinal pigment epithelium (RPE) deposits, remains controversial. Dysregulated turnover and accumulation of extracellular matrix (ECM) molecules in sub-RPE deposits play a role in AMD. Oxidant injury is a stimulator of ECM production and turnover. Epidemiological studies have shown an increased risk for AMD associated with higher intake of omega-6 poly-unsaturated fatty acids (PUFA) vs. omega-3. These PUFAs are vulnerable to oxidant modification and can activate members of the MAPK cascade. We examined the effects of PUFAs and oxidant injury on production of extracellular matrix molecules in RPE cells, and activation of members in the MAPK-PPARγ pathway.
ARPE19 cells were fed 20 uM PUFAs (arachidonic acid, linoleic acid and docosahexanoic acid) chronically (at least four serial passages) or acutely (24hours). Oxidant injury was induced using a combination of myeloperoxide and hydrogen peroxide or hydroquinone for 24 hours. Cell viability was determined via MTT assay. Expression of ECM molecules (vitronectin, fibronectin, collagen-IV and laminin) and members of the MAPK pathway (p38, JNK and PPARγ) were determined by qRT-PCR, ELISA and Western blot analysis. PPARγ activity was measured in the presence and absence of PPARγ antagonist GW9962 and pre-treatment with antioxidants.
Treatment of post-confluent RPE cells with PUFAs did not affect cell viability. Fibronectin and laminin expression was significantly higher in cells treated with omega-6 PUFAs compared to omega-3, and increased by approximately 1.5 fold in all cells following oxidant treatment regardless of PUFA. Increased expression of vitronectin and MAPK, JNK and PPARγ was seen only in linoleic acid treated cells following injury. Studies on PPARγ activity are ongoing.
PUFAs are membrane targets of lipid peroxidation and natural ligands for PPARγ. This study demonstrates that PUFAs and especially the pro-inflammatory omega -6, linoleic acid, can induce the production of extracellular matrix molecules found in sub-RPE deposits. Members of the MAPK signaling pathway appear to be differentially expressed and may prove to be a potential therapeutic target in preventing ECM production by RPE cells.
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