Cigarette Smoke-Derived Toxic Chemicals Disrupt the Balance Between VEGF /PEDF and the Activity of MMP-2 in Human Retinal Pigment Epithelium Cells

Abstract

Purpose:: Cigarette smoking has been shown to be the most important environmental risk factor for both dry and wet AMD. Several studies have suggested that the RPE may modulate CNV through pro- and anti-angiogenesis factors such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, as well as through matrix metalloproteinases (MMPs). We postulate that several tar components of cigarette smoke, might enhance CNV by deregulating the balance between VEGF/PEDF and expression of MMP-2 in RPE cells. We have shown that RPE oxidant injury with a quinone tar compound, hydroquinone (HQ), diminishes MMP-2 activity. In this study, we sought to determine the impact of cigarette smoke-derived toxic chemicals in the balance between PEDF/VEGF and MMP-2 activity in RPE cells.

Methods:: Confluent cultures of human ARPE-19 cells were incubated with quinones (HQ and chatecol) and nicotine at different concentrations, alone or in combination, for 24 hrs. In additional experiments, RPE cells were incubated with different concentrations of cigarette smoke extracts (CSE). Cell count and MTS assay were used to detect cell viability. Based on these results, the cells were treated with a nonlethal concentration of 50 µM quinones, (10^-6 and 10^-8M) nicotine, and a final concentration of 10% CSE for 24 hrs. Supernatants and cell homogenates were collected. PEDF and VEGF mRNA levels were assessed by real-time PCR, protein secretion by ELISA, and MMP-2 activity by zymography.

Results:: Dose of HQ or chatecol ranging from100 to 80µM killed a significant fraction of cells. However, concentrations of 50 µM of both quinones and lower alone or in combination were nonlethal within 24 hours. In addition, no toxic effect of 10% CSE was found since viability was consistently established to be >80%. PEDF was suppressed by HQ alone or in combination with chatecol and/or nicotine. However, chatecol and nicotine alone did not change PEDF mRNA or protein secretion. Nicotine and chatecol up-regulated VEGF. This effect was potentated by the combination of both compounds. Exposure to nicotine was associated with increased MMP-2 activity.

Conclusions:: These data suggest that HQ, chatecol, and nicotine may promote CNV by disrupting the balance between VEGF/PEDF and MMP-2 activity, partially explaining the association between smoking and neovascular AMD.

Keywords: choroid: neovascularization • growth factors/growth factor receptors • extracellular matrix