Up-regulation of apoptosis of Ccl2-/-/Cx3cr1-/- mouse and human retinal pigment epithelium under inflammatory and oxidative stress

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NEI	CANCER-27

Authors

• Y Wang
• X Cao
• D Shen
• J Tuo
• R Villasmil
• C Chan

Abstract

Purpose: To compare apoptosis of retinal pigment epithelium (RPE) of C57/B6 (WT) and Ccl2-/-/Cx3cr1-/- (DKO) mice, an age-related macular degeneration (AMD) model, and explore anti-apoptotic effects of decoy receptor 3 (DcR3) on RPE under inflammatory and oxidative stress. Methods: Human ARPE-19 and primary mouse RPE were incubated with lipopolysaccharide (LPS), 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), or H2O2. ARPE-19 was pre-incubated with DcR3 or DcR3 siRNA. RPE viability was assessed by MTT and apoptosis was detected by Annexin-V via flow cytometry. Fas and FasL mRNA expression were detected by qRT-PCR and cleaved caspase-3/-9 was detected by immunofluorescence. Results: MTT showed less cell viability in DKO than WT RPE under physiological conditions or stimulated with TCDD (p<0.05). Under LPS, TCDD, and H2O2 stimulation, increased apoptosis and Fas/FasL expression were detected in DKO RPE compared with WT RPE (p<0.05). The addition of DcR3 resulted in less ARPE-19 apoptosis (p<0.05) and lower Fas/FasL mRNA expression (p<0.05). Immunofluorescence showed positive staining for cleaved caspase-3/-9 in both human and mouse RPE. Conclusions: Under inflammatory and oxidative stress, DKO RPE is more prone to Fas-mediated apoptosis than WT RPE, suggesting RPE apoptosis may play a role in AMD pathogenesis. DcR3 showed anti-apoptotic effects in ARPE-19 under stress.

back to top