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Ping Yang, Nikolai P Skiba, Grace M Tewkesbury, Peter Baciu, Glenn J Jaffe; Complement-mediated Apolipoprotein E Accumulation in Cultured Human RPE Cell. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3772.
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Complement activation has been increasingly implicated in the pathogenesis of AMD. Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) have been detected in Bruch’s membrane and drusen from eyes with AMD. Herein, we investigate the effect of MAC formation on induction of ApoE accumulation in human RPE cells.<br />
Cultured human RPE cells from a 62 year-old donor (ApoE phenotype E3/E3) were primed with a complement-fixing antibody followed by treatment with 6% C1q-depleted human serum (C1q-Dep) to elicit complement activation and cell surface MAC formation. Controls included treatment of serum with an anti-C5 antibody (10 µg/ml) to block MAC formation, an endocytic pathway inhibitor (dynasore, 120 µM) to block MAC internalization, or heat inactivated C1q-Dep (HiC1q-Dep) to block complement activation. Mass spectrometry (LC-MS/MS) and western blot analysis were conducted on total protein extracts. mRNA expression of ApoE was evaluated by qPCR. Interleukin 6 (IL-6) expression was used as a positive control.
Complement attack up-regulated cell-associated ApoE protein but not apolipoprotein A1 (ApoA1), as determined by LC-MS/MS analysis (n=3 experiments), 6 hrs after C1q-Dep serum treatment. ApoE accumulation occurred as early as 1 hr. post-C1q-Dep treatment and peaked at 12 hrs (24h≥12h>6h>4.5h>2.5h>1h). Complement attack caused a 1.2-3.4 fold increase in ApoE accumulation vs C1q-Dep alone and a 2-5 fold increase vs Ab+HiC1q-Dep. ApoE accumulation was dependent on complement activation and MAC formation since the response was blocked by an anti-C5 antibody as well as heat inactived serum. Treatment with the endocytosis inhibitor dynasore enhanced ApoE accumulation. ApoE mRNA levels were not affected, while IL-6 mRNA levels were increased by complement activation (3h>2h>5h).<br />
Complement activation induces time-dependent ApoE accumulation in RPE cells. An understanding of the mechanisms by which complement affects RPE ApoE accumulation may help to better explain drusen formation and provide insights into potential therapeutic targets.<br />
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