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M. E. Marin Castano, O. Alcazar, M. Pons; Regulation of Monocyte Chemoattractant Protein-1 (MCP-1), Growth Factors, and Pigment Epithelium-Derived Factor in Response to Transient and Repetitive Non Lethal Oxidative Injury in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1444.
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Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and tobacco smoking has been shown to be a major risk factor. Cumulative oxidative injury caused by cigarette smoke to the RPE and inflammation have been implicated in the pathogenesis of choroidal neovascularization (CNV), the advanced form of AMD. Although the exact contribution of macrophages remains unknown, they may play crucial roles both in early AMD scavenging accumulated debris, and wet AMD stimulating CNV. The study was undertaken to investigate the expression of cytokines involved in inflammation and angiogenesis (monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and pigment epithelium-derived factor (PEDF)) by ARPE-19 cells when exposed to transient and repetitive cigarette smoke-related hydroquinone (HQ)-induced oxidative stress. Moreover, since nuclear factor-ΚB (NF-ΚB) is known to induce MCP-1 gene, we also investigated whether HQ-induced MCP-1 expression was mediated by NF-ΚB.
Confluent ARPE-19 cells were treated with HQ 100µM for either 24 hours (transient injury) or 5 weeks (repetitive injury). In some experiments, cells were pretreated with either proteasome inhibitor MG-132, or IΚBα phosphorylation inhibitor Bay 11-7082, or NF-ΚB inhibitor pyrrolidine dithiocarbamate (PDTC). After treatment, cells were harvested for RNA extraction and samples were assayed by real-time PCR. Nuclear translocation of NF-ΚB p65 was visualized by immunofluorescence staining.
Transient exposure of ARPE-19 cells to HQ increased MCP-1, VEGF and TGF-β mRNA expression, and decreased PEDF mRNA expression. Induction of MCP-1 by HQ was mediated by NF-ΚB because inhibition of this pathway by MG-132, PDTC and Bay11-7082 resulted in the impairment of MCP-1 transcription activation. Also, exposure to HQ resulted in nuclear translocation of p65/RelA as well as an increase in p65/RelA and IΚBα mRNA expression. Sustained oxidative injury with HQ decreased MCP-1 expression and increased VEGF/PEDF ratio.
Taken together, our findings suggest a differential regulation of MCP-1 expression in RPE cells in response to cigarette-smoke related HQ-induced oxidative injury. HQ might be a key contributor to the progression of late AMD by stimulating inflammation and angiogenesis leading to CNV.
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