Published date: Oct 25 2020

Journal Title: Journal of Ophthalmic and Vision Research

Issue title: October–December 2020, Volume 15, Issue 4

Pages: 470–480

DOI: 10.18502/jovr.v15i4.7781

Aneesh NeekhraGavin Herbert Eye Institute, University of California, Irvine, California

Julia TranGavin Herbert Eye Institute, University of California, Irvine, California

Khoa PhamGavin Herbert Eye Institute, University of California, Irvine, California

Ashish SharmaGavin Herbert Eye Institute, University of California, Irvine, California

Marilyn ChwaGavin Herbert Eye Institute, University of California, Irvine, California

Saurabh LuthraGavin Herbert Eye Institute, University of California, Irvine, California

Ana L. Grmajo aGavin Herbert Eye Institute, University of California, Irvine, California

Saffar MansoorGavin Herbert Eye Institute, University of California, Irvine, California

Baruch D. KuppermannGavin Herbert Eye Institute, University of California, Irvine, California

Cristina Kenneymkenney@hs.uci.eduDepartment of Pathology and Laboratory Medicine, University of California Irvine, Irvine, CA, USA

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.

Method: 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 7kChinduced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.

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