Antihypertensive Drugs Accelerate the Accumulation of Lipofuscin in the RPE by Dysregulating Autophagy Flux

Abstract

Purpose: : Hypertension has been identified as a risk factor for age-related macular degeneration. We have previously reported that antihypertensive drugs accelerate lipofuscin deposition and now wished to determine if these drugs accelerate lipofuscin accumulation in vivo and if this is due to a change in autophagy flux.

Methods: : Human RPE cultures were exposed over 14 days to a) calcium antagonists - Verapamil, and Diltiazem; b) the adrenergic antagonists - Propranolol and Phentolamine at concentrations of 0.5µM to 50µM. Autofluorescence was assessed by FACs analysis. To evaluate autophagy flux, cells were transfected with an RFP-GFP-LC3 construct, and exposed to medium with or without drug for 3h or 6h and autophagic vacuoles counted. Cell lysates were prepared for Western blotting to determine changes in the autophagy proteins, LC3, Beclin, Atg5, Atg7 and Atg9. For in vivo studies, rats received Propanolol, Phentolamine or saline for 21 days using osmotic pumps (1mg/day). Eyes were enucleated and examined for lipofuscin accumulation using confocal microscopy and autophagy proteins by immunohistochemistry.

Results: : All four antihypertensive drugs caused a significant increase in lipofuscin accumulation in RPE cells. Propranolol, Phentolamine, Verapamil and Diltiazem induced a 125, 77, 60 and 82% increase in autofluorescence respectively compared to untreated controls. Lipofuscin accumulation was blocked by the autophagy inhibitor, 3-methyladenine. Autophagy flux was enhanced by antihypertensive drugs and the number of autophagic vacuoles was increased by up to 90% compared to untreated controls after 3 hours. The increase in autophagic vacuoles was substantiated by an elevation in the LC3-II to I ratio in drug-treated cells compared to control. There was also elevated expression of Beclin, Atg7, Atg5 and Atg9 compared to control. Animals receiving Propanolol or Phentolamine demonstrated a 6-fold and 3-fold increase in lipofuscin respectively in the RPE compared to untreated controls. Immunohistochemistry showed elevated expression of autophagy proteins in the RPE of drug treated animals compared to controls.

Conclusions: : We conclude that antihypertensive drugs can rapidly induce lipofuscin accumulation and that the reported link between AMD and hypertension may actually be a function of treatment rather than disease.

Keywords: ipofuscin • retinal pigment epithelium • drug toxicity/drug effects