Abstract
Purpose :
Accumulation of oxidized forms of cholesterol, including 7-ketocholesterol (7-KC), in Bruch’s membrane under the pigment epithelium (RPE) occurs in aging and in patients at risk of early age-related macular degeneration (AMD). However, the mechanisms whereby 7-KC leads to vision threatening late AMD are incompletely understood. We addressed the hypothesis that 7-KC is sufficient to induce cellular senescence and promote senescence-associated secretory phenotype (SASP) in AMD. We tested our hypothesis in cultured primary RPE.
Methods :
In cultured primary RPE (Lonza, Walkersville, MD ) incubated with 7-KC (10 µM) or its solvent, HPBCD (control), for 3 days, a series of experiments were performed: 1) western blots of cellular senescence markers, p21 and p16, 2) β-Galactosidase staining of cellular senescence; 3) western blots of IL-1 β and VEGF, 4) assessment of RPE barrier integrity by immunoprecipitation of junction protein complexes, cadherin and β-catenin, and 5) western blots of RPE65, a specific RPE cell marker. Experiments were repeated three times (n=4-6). Statistics were performed using ANOVA.
Results :
Compared to control (HPBCD), 7-KC treatment significantly induced protein expression of the cellular senescence markers, p21 (p=0.022) and p16 (p=0.016), and increased the number of β-Galactosidase stained RPE (p=0.033). Senescence associated secretory phenotype was observed in 7-KC-treated RPE as increased protein concentrations of IL-1β (p= 0.0008 vs. control), and VEGF (p=0.000433 vs. control). In 7-KC-treated RPE compared to control, the adherens junction protein complex, cadherin/β-catenin, was significantly reduced (p=0.004). However, RPE65 protein was not affected by 7-KC treatment, suggesting that 7-KC does not induce RPE dedifferentiation.
Conclusions :
Accumulation of 7-KC in RPE is sufficient to promote RPE senescence and senescence-associated secretory phenotype. Senescent RPE have compromised barrier integrity shown by reduced cadherin/β-catenin complexes even though they maintain RPE phenotype.
This is a 2020 ARVO Annual Meeting abstract.