Abstract
Purpose:
Age-related macular degeneration is a complex disease that is caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure has been shown individually to lead to oxidative stress, complement activation, endoplasmatic reticulum (ER) stress and lipid dysregulation, which have all been proposed to be associated with the pathogenesis of AMD. Here we examine the effects of smoke exposure on retinal pigment epithelium (RPE) of mice, and cigarette smoke extract (CSE) on immortalized human RPE cells (ARPE-19).
Methods:
Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% smoke extract. Barrier function of monolayers was determined by transepithelial resistance (TER) measurements; and effects of smoke were determined by biochemical, molecular and histological measures. Effects of the alternative pathway (AP) of complement as well as anaphylatoxin receptor signaling were analyzed using knockout mice or specific inhibitors.
Results:
ER stress markers were elevated in the RPE of smoke exposed mice, which could be prevented by eliminating the AP. To further examine this mechanism, RPE monolayers were exposed to 5% CSE, a concentration which did not alter TER. Short-term smoke exposure resulted in epithelial C3 release, generation of the complement anaphylatoxins, C3a and C5a, oxidative stress, complement activation on the cell membrane, and ER stress. In addition, long-term exposure resulted in lipid accumulation and secretion. All measured outcomes were significantly reduced by blocking anaphylatoxin receptor function, and AP signaling. Lipid deposition induced by tunicamycin-mediated ER stress, however was not susceptible to AP inhibition.
Conclusions:
Our results provide clear evidence that smoke exposure results in oxidative stress and complement activation involving the AP, resulting in ER stress-mediated lipid accumulation. Taken together our data suggests that smoking and complement activation act synergistically in the disease process.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
557 inflammation