Genetic variants of ApoE and ApoER2 differentially modulate endothelial function

It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE–ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte–endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2^−/− mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2^−/− mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.Cardiovascular disease risk is modified by common genetic variants of apolipoprotein E (apoE) and its receptor apolipoprotein E receptor 2 (ApoER2), which is a member of the LDL receptor family. Compared with the most common allele apoE3, individuals with the apoE4 allele have an increased risk of atherosclerosis and coronary heart disease (1, 2). The LRP8 gene, which encodes ApoER2, is a major gene locus for premature atherosclerosis and acute myocardial infarction identified in four independent human populations. In particular, homozygous carriers of the ApoER2-R952Q variant have a twofold increased risk of these conditions (3–5). ApoER2-R952Q also has an additive effect with apoE4, with the combined genotype QQ/E4 showing a 3.9-fold greater susceptibility to cardiovascular disease (5). ApoER2 polymorphism-associated risk is independent of cholesterol levels (3–5), and although apoE4 may impact LDL abundance (2), there is also evidence that apoE4-associated risk goes well beyond changes in lipoprotein status (6–9). Whereas there is considerable understanding of the biology of the apoE–ApoER2 tandem in the central nervous system and in Alzheimer’s disease (10), the basis for the cardiovascular impact of the receptor and apoE variants remains unclear.Our prior work demonstrated that ApoER2 is expressed in endothelial cells, where it plays a critical role in the pathogenesis of the antiphospholipid syndrome (APS) (11). The receptor is enriched in caveolae/lipid rafts in which signaling molecules regulating endothelial NOS (eNOS) are compartmentalized (12, 13). We now know that in APS, antiphospholipid antibody recognition of the cell surface protein β2-GPI on endothelial cells promotes β2-GPI dimerization and interaction with the extracellular domain of ApoER2, causing the activation of PP2A and eNOS antagonism. The resulting decrease in bioavailable NO underlies APS-related thrombosis (11). However, the normal function of the receptor in endothelium, and whether and how it modulates apoE actions on endothelium, are unknown.In addition to regulating thrombogenesis, eNOS-derived NO plays a major role in cardiovascular protection via promotion of the integrity of the endothelial cell monolayer and attenuation of endothelial cell–leukocyte adhesion (13). Recognizing that eNOS enzymatic activity is both positively and negatively modulated by signaling molecules in endothelial caveolae/lipid rafts (14), to better understand the biology of ApoER2 in endothelium we hypothesized that apoE3 binding to the receptor activates eNOS. Experiments were performed in cell culture and in mice to test this hypothesis and to determine whether genetic variants in apoE or ApoER2 disrupt this process and thereby adversely impact endothelial function.