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Yujuan Wang, Xiaoguang Cao, Defen Shen, Jingsheng Tuo, Rafael Villasmil, Chi-Chao Chan; Pro-inflammatory Mediator And Oxidative Stress Enhance Apoptosis In Cultured Retinal Pigment Epithelial Cells Of Ccl2-/-/Cx3cr1-/- Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4441.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate and compare the in vitro effects of pro-inflammatory mediator and oxidative stress on apoptosis of retinal pigment epithelium (RPE) from C57/B6 (wild type, WT) and Ccl2-/-/Cx3cr1-/- (DKO) mouse, a murine model of age-related macular degeneration (AMD).
RPE cells of 4-8 week old DKO and WT mice were isolated with dispase II digestion and grown in DMEM/F12 culture medium with 15% FBS. After 2-3 weeks, the confluent primary RPE cells were incubated with serum-free medium in addition with 1 µg/ml lipopolysaccharide (LPS, a major component of Gram negative bacterial cell wall that promotes pro-inflammatory cytokines), 10 nM 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, a byproduct of incomplete combustion that induces formation of reactive oxygen species and DNA damage), or 100 µM H2O2 (an oxidant that is a reactive oxygen species) for 24 hours. RPE cell survival rate was assessed by MTT assay and apoptosis was detected by Annexin V-FITC staining followed by flow cytometry and immunofluorescence assay; Fas and FasL mRNA expressions were detected by RQ-PCR; and cleaved caspase-3 and caspase-9 staining were detected by indirect immunofluorescence using confocal microscopy.
MTT assay showed that RPE survival rate was the lowest under H2O2 (p<0.05) compared to LPS and TCDD. DKO RPE was more affected compared to WT RPE, co-cultured with LPS (p=0.19), TCDD (p=0.04), and H2O2 (p=0.17). Flow cytometry confirmed a higher rate of apoptosis in DKO RPE compared to WT RPE. RQ-PCR also showed increased Fas and FasL expressions in DKO RPE compared to WT RPE stimulated with LPS (p<0.01), TCDD (p=0.10), and H2O2 (p<0.01). Immunofluorescence illustrated positive cleaved caspase-3, caspase-9 and Annexin V-FITC in both DKO and WT RPE under stress.
The data suggest that DKO RPE is more apoptosis-prone to low grade inflammation and oxidative stress that may play a pathogenic role in AMD.
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