Abstract
Purpose: :
Smoking is a primary risk factor associated with the prevalence and the incidence of AMD. To better understand the cellular and molecular bases for the epidemiologic association between smoke and AMD, we determined the effects of Benzo(a)Pyrene (B(a)P), a toxic element in cigarette smoke, on ARPE-19 cells and examined retinas from mice exposed to chronic cigarette smoke. We have previously demonstrated increased mtDNA damage, autophagy and increased exosome activity by the aged RPE and suggested that these age-related changes may contribute to the formation of drusen.
Methods: :
We exposed ARPE-19 cells to B(a)P and measured: (1) mtDNA damage; (2) phagocytosis activity; (3) lysosomal enzymes: cathepsin D and B-glucuronidase; (4) exosome markers: CD63, CD81 and LAMP2, (5) complement C3a, C5, C5b-9, CFH, and (6) chemokines. Similarly, lysosomal enzymes, exosome markers, and complement activation were determined in young (4mos) and old (24mos) mouse RPE/choroids, eyes from mice exposed to cigarette smoke and human donor eyes (AMD eyes and age-matched controls). Realtime RT-PCR, western blots, and immunohistochemistry were used to identify lysosomal enzymes, exosome markers and complement pathway compenents, Phagocytosis was measured using fluorescence-based assays. Release of chemoattractants was measured using ELISA.
Results: :
We have found that drusen in AMD donor eyes and eyes from mice exposed to smoke contain markers for complement and exosomes. Furthermore, these markers are also found in the region of Bruch’s membrane in old mice. We found that mtDNA damage occurs in RPE cells treated with BaP at a concentration that was not cytotoxic (10 uM). In the present of B(a)P, RPE cell phagocytic activity was not altered but there was: (1) increased lysosomal activity; (2) increased complement activity; (3) increased exocytotic activity and (4) the release of chemoattractants MCP-1 and MIF.
Conclusions: :
Our findings link smoking with oxidative stress, mtDNA damage, lysosomal activity, exocytotic activity and chemoattractants as altered processes of RPE cell biology that have changed in the aged eye and may be risk factors for AMD. These altered cell biological processes in old eyes and eyes exposed to toxic components of cigarette smoke may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.
Keywords: age-related macular degeneration • mitochondria • retinal pigment epithelium