Mechanism of Cholesterol-Mediated Retinal Pigment Epithelium Dysfunction as a Model for Dry Age-Related Macular Degeneration
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Choi, Eun Young
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CitationChoi, Eun Young. 2019. Mechanism of Cholesterol-Mediated Retinal Pigment Epithelium Dysfunction as a Model for Dry Age-Related Macular Degeneration. Doctoral dissertation, Harvard Medical School.
AbstractPurpose: There is currently no established treatment for dry age-related macular degeneration (AMD), which is characterized by a progressive degeneration and death of the retinal pigment epithelium (RPE). While the etiology of AMD is not completely understood, lipid accumulation and inflammation are strongly associated with disease progression. Studies suggest that lipoproteins which accumulate in sub-retinal space and become oxidized are a major contributor to RPE dysfunction and death. Peroxisome proliferator-activated receptors (PPARs) are a family of lipid-activated transcription factors that are involved in lipid metabolism and inflammatory processes. We examined a variety of PPARg agonists and found that troglitazone is uniquely effective in protecting the RPE against oxLDL-induced cell death. The purpose of this study is to elucidate the mechanism of troglitazone in its protection of RPE against oxLDL-induced cell death.
Methods: SiRNA was used to knock down PPARg in ARPE-19 cells, and the expression of PPARg was determined by RT-PCR. After treatment with oxLDL ± troglitazone, rosiglitazone, or trolox, cell death was measured by lactate dehydrogenase (LDH) release into the conditioned media. Reactive oxygen species (ROS) formation was detected using a fluorescent ROS staining kit. Lysosomal integrity was assessed by LysoTracker staining. NF-kB activation was determined by immunostaining for nuclear translocation of NF-kB.
Results: Transfection with PPARg siRNA achieved a significant reduction in the expression of PPARg in ARPE-19 cells (P < 0.001). Treatment with troglitazone resulted in a significant decrease of oxLDL-induced LDH release, regardless of transfection with PPARg siRNA or scrambled siRNA (P < 0.001 for both conditions), whereas rosiglitazone did not. Treatment with oxLDL induced formation of reactive oxygen species (ROS) in ARPE-19 cells at 28 hours. Troglitazone and trolox, but not rosiglitazone, inhibited oxLDL-induced ROS formation. Furthermore, oxLDL treatment significantly decreased the number of intact lysosomes at 36 hours (P < 0.05). Treatment with troglitazone and trolox, but not rosiglitazone, significantly restored lysosomal integrity (P < 0.05 for troglitazone, P < 0.001 for trolox) in a dose-dependent manner. Finally, immunostaining revealed that oxLDL treatment induced NF-kB nuclear translocation at 36 hours. Troglitazone and trolox, but not rosiglitazone, inhibited NF-kB nuclear translocation in a dose-dependent manner.
Conclusions: These data suggest that troglitazone prevents oxLDL-induced ARPE-19 cytotoxicity, not through the PPARg pathway, but rather through its antioxidant phenolic component, trolox. Troglitazone and trolox suppressed oxLDL-induced ROS formation, lysosomal destabilization, and NF-kB activation in ARPE-19 cells. Future direction of this research will investigate the link between oxidative stress and lysosomal integrity, investigate novel analogs of trolox their efficacy in protecting the RPE against oxLDL-induced cell death, and explore the efficacy of these drugs in a mouse model of dry AMD.
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