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KATAYOON B. EBRAHIMI, MARISOL CANO, NATALIA FIJALKOWSKI, NAOSHI KONDO, JAMES T. HANDA; Oxidized Lipoproteins Induce Complement Mediated Inflammation in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2293.
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© ARVO (1962-2015); The Authors (2016-present)
Genetic studies have identified the alternative complement pathway as an important component of AMD. Our lab has shown that oxidized lipoproteins accumulate in drusen in early AMD, presumably due to the high oxidative stress environment of the macula, and induce a pathologic phenotype to the RPE, a target during early disease. This study was conducted to determine if oxidized lipoproteins promote complement pathway activation in the RPE cells.
The ARPE-19 cells were cultured to confluence in DMEM/Ham’s F12 with 10% FBS, and then serum-deprived for 24 h. Oxidized LDL (ox-LDL) 100 µg/ml, a viable dose, or vehicle was added to the medium for 24 h. RNA and proteins were extracted for RT-qPCR, end point RT-PCR, and Western Blot analysis. RNA interference experiments using siRNA against IRE1-α, a highly conserved ER-localized type I transmembrane ribonuclease, which plays a central role in the unfolded protein response, or control siRNA were also performed.Each condition was examined in triplicate, and the experiments were repeated at least three times. Significance was determined using the unpaired Student’s t-test.
Taqman RT-qPCR analysis showed thatCD36 was increased 2.5 fold (p<0.05), and C3 expression was increased 1.6 fold (p<0.05), but C5 showed no change after ox-LDL treatment. Complement regulators CD55, CFB, and CFH showed increased expression of 1.7, 2, 1.5 fold respectively (all P<0.01 except CFH P<0.05). CD46 and CD59 showed no significant change in mRNA expression. However, western blot analysis showed that CD59 was decreased 7 fold (p<0.01) and CD46 was decreased 2.5 fold by ox-LDL treatment. IRE1α is known to splice XBP1during ER stress, and to cleave CD59. Ox-LDL treatment increased its mRNA expression 1.9 fold (P<0.05). IRE1-α knockdown increased the CD59 protein level 1.7 fold compared to control siRNA.
Our results suggest that ox-LDL promotes complement mediated inflammation by suppressing the MAC complex inhibitory regulatory factor CD59, through activation of IRE1 α. Further evaluation is required to evaluate the functional consequences on RPE cells, the role of the XBP1 transcription factor, and ultimately their roles in AMD.
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