Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro

Abstract

Purpose: 7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro.

Methods: Two populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28) were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts.

Results: Exposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P $<$ 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P $<$ 0.01), memantine (decreased 47.8% at 1 µM, P = 0.0039 and 81.9% at 1 mM, P $<$ 0.001), simvastatin (decreased 85.3% at 0.01 µM, P $<$ 0.001 and 84.8% at 0.05 µM, P $<$ 0.001) or epicatechin (83.6% decrease, P $<$ 0.05), Z-IETD-FMK (68.1% decrease, P $<$ 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase-3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P $>$ 0.05) regardless of the pretreatment.

Conclusion: Several current drugs protect ARPE-19 cells but not R28 cells from 7kCh-induced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.

Keywords: 7-Ketocholesterol; Memantine; Epicatechin.

Figure 1

Caspase-3/7 activity in 7kCh-treated ARPE-19 and R28 cells in response to 7kCh alone or pretreatment with Z-VAD-FMK. Cells exposed to 7kCh alone increased caspase-3/7 activity*** (A–B). ARPE-19 cells pretreated with Z-VAD-FMK decreased caspase-3/7 activity*** (A). In contrast, R28 cells pretreated with Z-VAD-FMK did not have decreased caspase-3/7 activity (B). *P $<$ 0.05, **P $<$ 0.01, ***P $<$ 0.001. The error bars represent the standard error of the mean (SEM) of measurements for the three conditions in three separate runs (n = 9, A – B ).

Figure 2

Caspase-3/7 activity in 7kCh-treated ARPE-19 and R28 cells after pretreatment with memantine, simvastatin, or epicatechin. All cells exposed to 7kCh alone increased caspase-3/7 activity (A–F). Pretreatment of ARPE-19 cells with 1 µM** and 1 mM*** memantine (A) 0.01 µM*** and 0.05 µM*** simvastatin (C), or 5 µM epicatechin* (E) showed decreased caspase-3/7 activity. In contrast, R28 cells pretreated with 1 µM and 1 mM memantine (B) 0.01 µM and 0.05 µM simvastatin (D), or 5 µM epicatechin (F) did not have significantly decreased caspase-3/7 activity. Representative IncuCyte live-cell images of DMSO-Control, 7kCh, and 7kCh with epicatechin-treated ARPE-19 cells at 24 hours (G). There are no major differences among treatments detected with brightfield microscopy (G, first column). ARPE-19 cells stressed with 7kCh demonstrate increased nuclear staining number, increased caspase-3/7 signal number, and an increased overlap signal number (G, second row). These counts decrease when ARPE-19 cells are pretreated with epicatechin before 7kCh (G, third row). *P $<$ 0.05, **P $<$ 0.01, ***P $<$ 0.001. The error bars represent the standard error of the mean (SEM) of measurements for the four conditions in three separate runs (n = 12, A – D ) and three conditions in three separate runs (n = 9, E – F ). Scale bar = 400 μM ( G ).

Figure 3

Caspase-8 and -12 activity in 7kCh-treated ARPE-19 and R28 cells in response to 7kCh alone or pretreatment with Z-IETD-FMK or Z-ATAD-FMK. All cells exposed to 7kCh alone showed increased caspase-3/7 activity (A–F). ARPE-19 cells pretreated with Z-IETD-FMK** (A) or Z-ATAD-FMK** (C) showed decreased caspase-8 and -12 activity, respectively. In contrast, R28 cells pretreated with Z-IETD-FMK (B) or Z-ATAD-FMK (D) did not show decreased caspase-8 nor -12 activities, respectively. *p $<$ 0.05, **p $<$ 0.01, ***p $<$ 0.001. The error bars represent the standard error of the mean (SEM) of measurements for the three conditions in three separate runs (n = 9, A – D ).