Regulation of LDLR in hRPE and loss of retinal function in Ldlr -/- mice exposed to NaIO3

Abstract

Purpose : Lipid peroxidation induces oxidative stress (OS) which is one of the major underlying causes of age-related diseases including AMD. In early AMD, extracellular deposits, cholesterol, lipids, and proteins accumulate between the RPE and the Bruch membrane. Cholesterol content is abundant in the retina and circulating low density lipoprotein (LDL) is taken up by low density lipoprotein receptor (LDLR) in the RPE and Müller cells. Lldr-/- mice develop thickened Bruch membrane and show increased VEGF with high fat diet; however, the role of LDLR in dry AMD has not been systematically investigated. The aim of this study is to determine the role of LDLR in an OS-induced model of dry AMD.

Methods : Confluent RPE cells were treated with150 µM tBH and expression of LDLR was studied using immunofluorescence. Kinetic activation of caspase-3/7 in Ldlr silenced RPE cells co-treated with 300 µM tBH and 10 or 40 µg/ml of an HDL- mimetic HM 10/10 peptide was quantified by live-cell imaging using the Incucyte system. In vivo studies were conducted with male C57BL6/J (7-8 weeks old) and Ldlr ^-/- mice. OS was imposed in mice with intravenous NaIO3 (20mg/kg BW). On day 7, fundus imaging, OCT and ERG were performed, and retinal thickness was measured from OCT images. Posterior eye cups were processed for histology, TUNEL staining and immunostaining for cleaved caspase 3.

Results : In primary human RPE cells, LDLR is predominantly found in the cytosol and tBH-induced OS altered its expression pattern. tBH markedly increased caspase activation in a time-dependent manner for 24 h in Ldlr silenced RPE cells. Co-treatment with HM-10/10 (10 µg/ml) significantly blocked caspase activation. Severe pathophysiological changes and functional impairment were observed in the retinas of NaIO3-treated Ldlr^-/- mice. LDLR deficiency resulted in significant thinning of retina and compromised rod and cone functions with a total abolition of the rod function. Furthermore, RPE/BM was discontinuous with patchy RPE loss and irregular degenerated cell aggregates were more prominent in the NaIO3-treated retina of Ldlr^-/- mice. Moreover, the percentage of apoptotic cells and cleaved caspase 3 was significantly elevated in stressed Ldlr^-/- mice vs WT mice.

Conclusions : Our data suggest that loss of LDLR impairs retinal function which could play a significant role in the development of AMD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.