Cigarette Smoke Increases Severity of Experimental Choroidal Neovascularization (CNV): Role of Inflammation

Abstract

Abstract: : Purpose: Cigarette smoking is the greatest environmental risk factor for all forms of age–related macular degeneration (AMD). Previously we showed that oral administration of nicotine increased the severity of experimental choroidal neovascularization (CNV). In addition, we and others have shown that inflammation, especially macrophage infiltration, contributes to CNV severity. Smoking is known to cause macrophage activation in the lung, but the effect of cigarette smoke on the systemic and ocular inflammatory response is unknown. We investigated the effects of nicotine and cigarette smoking as an immune stimulant for CNV severity in a mouse model of neovascular AMD and its effects on macrophages in culture. Methods: C57BL/6 mice were exposed to whole cigarette smoke (2 hrs per day), or nicotine (orally in the drinking water), to achieve serum levels of nicotine in both groups consistent with chronic smokers. After 2 weeks of exposure, laser–induced CNV was induced and flat mounts were made to measure CNV severity 28 days later. Splenic macrophages were recovered at 3 days and 28 days after smoke exposure and placed in culture for production of various factors or the mRNA was extracted for real time RT PCR. In addition normal macrophages were incubated with nicotine or "smoke conditioned" media, and their capacity to produce various factors spontaneously or after activation with bacterial lipopolysaccharide (LPS) was determined. Results: Both cigarette smoke and nicotine administration resulted in increased size and vascularity of CNV compared to untreated controls. Freshly isolated splenic macrophages recovered from nicotine–treated or smoke–exposed mice demonstrated similar changes, including decreased PGE2 synthesis, decreased mRNA for iNOS and increased mRNA for MMP–9, but no difference in mRNA for TNF–alpha or VEGF compared to controls. However, nicotine or smoke–exposed macrophages in culture demonstrated markedly increased production of TNF–alpha after stimulation with LPS. Conclusions: Both cigarette smoke and nicotine increase size and severity of experimental CNV. The effect of cigarette smoke and nicotine on macrophages in vitro and in vivo is pro–inflammatory and pro–angiogenic. In particular, the data suggest smoke and nicotine decrease NO, increase MMP–9 and increase the stimulated production of TNF–alpha. The magnitude of the changes in production of these three factors by infiltrating macrophages would be predicted to increase severity of CNV.

Keywords: age-related macular degeneration • choroid: neovascularization • pathobiology