Cellular Uptake and Clearance of Oxidatively-modified Apolipoprotein E3 by Cerebral Cortex Endothelial Cells.

Siobanth Cruz,Vasanthy Narayanaswami

doi:10.3390/ijms20184582

Abstract

Apolipoprotein E3 (apoE3) plays a critical role in the metabolism of lipoproteins and lowers plasma lipid levels by serving as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins and by promoting macrophage cholesterol efflux. The current study examines the effect of acrolein (an endogenously generated metabolite and an environmental pollutant) modification on the structure and function of apoE3. Acrolein modification was confirmed in Western blots by reactivity with acrolein–lysine-specific antibody and by the presence of oligomeric species due to cross-linking. LC-MS/MS analysis revealed modification of 10 out of 12 lysines in apoE3, with Nε-(3-methylpyridinium)-lysine being the predominant form of modification, and Lys75 being a ‘hot spot’ in terms of susceptibility to oxidation. Circular dichroism spectroscopy showed no major change in overall secondary structure compared to unmodified apoE3. Reconstituted high density lipoprotein (HDL) bearing acrolein modified apoE3 showed loss of binding to soluble LDLr; however, incubation with mouse endothelioma bEnd.3 cells showed that it was internalized. Incubation with excess LDL did not abolish cellular uptake of acrolein modified apoE3, suggesting alternative mechanism(s) not involving LDLr. Incubation with anti-CD36 antibody did not show a decrease in internalization while incubation with anti- lectin-like oxidized LDL receptor 1 (LOX1) showed partial internalization. However, incubation with anti-scavenger receptor class B type I (SRB1) antibody abolished internalization of acrolein modified apoE3. Taken together, our studies suggest that acrolein modification of apoE3 at lysine residues leads to increase in net negative charge, and as a consequence, results in clearance by LOX1 and SRB1 on endothelial cells. Overall, oxidative modification of apoE3 likely impairs its role in regulating plasma cholesterol homeostasis, eventually leading to lipid disorders.

Highlights

Aging and several disease states such as neurodegenerative diseases, cardio- and cerebrovascular diseases, and inflammation are characterized by heightened cellular and plasma oxidative stress accompanied by increased levels of reactive oxygen and nitrogen species
Whereas the role of ox-LDL in the etiology of atherosclerosis and ischemic stroke [6] has been recognized for decades, that of ox-high density lipoprotein (HDL) has only gained attention over the last decade, and very little is known about its role at the neurovascular junction
The objective of the study is to investigate the fate of oxidatively modified Apolipoprotein E3 (apoE3) when exposed to endothelial cells lining the cerebral vasculature; apoE3 is one of the critical anti-atherogenic protein components on VLDL, VLDL remnants and a sub-fraction of HDL

Summary

Introduction

Aging and several disease states such as neurodegenerative diseases, cardio- and cerebrovascular diseases, and inflammation are characterized by heightened cellular and plasma oxidative stress accompanied by increased levels of reactive oxygen and nitrogen species Whereas the role of oxidative stress and ox-LDL in the etiology of atherosclerosis [5] and ischemic stroke is well-established [4,6], that of oxidized high density lipoprotein (ox-HDL), and, associated proteins and lipids have gained attention only in recent years (reviewed in [7]) This is relevant as lipoprotein researchers have made a paradigm shift in focus from identifying approaches to increasing HDL cholesterol levels (for improved cardiovascular outcomes) to improving the quality and functionality of HDL [8]. It plays a crucial role cholesterol efflux and reverse cholesterol transport [15], and bears anti-oxidant and anti-inflammatory properties [16] It contributes towards maintaining the integrity of the blood–brain barrier (BBB) [17], and endothelial function at the neurovascular junction [18]. These studies report that apoE-null mice display increased endothelial cell permeability, with aging exacerbating the process

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