Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E.

Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signalling phenomena. ApoE-deficient (apoE(-/-)) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE(-/-) mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trial for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (epsilon3) and a defective receptor-binding mutant (epsilon2) human apoE transgene in apoE(-/-) mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (ie <10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.

Cell-derived apolipoprotein E (ApoE) particles inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells.

Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.

Localization of apolipoprotein E receptor 2 to caveolae in the plasma membrane.

The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.

Inability of plasma high-density lipoproteins to inhibit cell adhesion molecule expression in human coronary artery endothelial cells.

High-density lipoproteins (HDL) have several antiatherogenic actions, including the ability to sequester cellular cholesterol, to protect low-density lipoproteins from oxidation and to inhibit platelet aggregation. An early event in atherogenesis is the adhesion and recruitment of blood monocytes, a process mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly synthesized by endothelial cells in response to cytokines. It has been reported that HDL limits CAM expression in cultured human umbilical vein endothelial cells (HUVECs), implying that HDL also protects at an early stage in lesion development. Here, we have studied HDL suppression of CAM induction in human coronary artery endothelial cells (HCAECs), a model directly relevant to blood vessels susceptible to atherosclerosis. Arterial endothelial cells were preincubated with increasing amounts of total HDL, or different subfractions, and then activated with the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Flow cytometric analysis failed to detect any downregulation of VCAM-1 or E-selectin expression by HDL in this model of vascular endothelium. Moreover, we were unable to confirm that HDL could suppress CAM induction in well-characterized, low-passage HUVECs, even though positive controls, 17beta-estradiol or a nitric oxide donor, did cause downregulation and factors such as variability in donors and HDL preparation, or culture conditions, were excluded. We tentatively conclude that, as isolated HDL did not downregulate CAM expression in cultured HCAECs or HUVECs, attenuation of CAM induction in arterial endothelium is unlikely to contribute to HDL antiatherogenic actions in vivo.

Identification and characterization of LRP8 (apoER2) in human blood platelets.

Recently, we reported that apoE inhibits platelet reactivity by stimulating NO release and postulated apoE-receptor activation of intracellular NO synthase (eNOS). Here, we implicate a low density lipoprotein receptor (LDL-R) family member by studying ligand requirements using purified apoE isoforms, synthetic peptides, and the receptor antagonist, receptor-associated protein (RAP). Then, using a homology cloning approach and degenerate PCR primers to amplify the conserved Cys-rich binding domain of the LDL-R family, this receptor was identified as LRP8 (formerly termed, apoER2), a newly described brain protein with several splice variants. Immunoprecipitation of platelet membranes with anti-peptide antisera confirmed protein expression, while analysis of RNA from platelets and two megakaryocytic cell lines (Meg-01 and HEL) disclosed that the major LRP8 transcript lacked binding repeats 4-6 (LRP8delta4-6) but contained the full-length cytoplasmic tail. Sequence analysis of cytoplasmic LRP8 revealed several peptide motifs with potential for cellular signaling and we propose this as a rational mechanism through which apoE inhibits platelet aggregation.

Apolipoprotein E and regulation of cytokine-induced cell adhesion molecule expression in endothelial cells.

Atherosclerotic plaques develop in the arterial wall from complex multicellular processes following the early recruitment of circulating monocytes. Infiltration of monocytes is mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly induced in endothelial cells in response to cytokines. Apolipoprotein E (apo E), a 34-kDa polypeptide, helps protect against atherosclerosis, in part, because apo E phospholipid particles secreted by macrophages may have local protective effects within lesions. Here we have investigated whether purified plasma apo E, complexed with dimyristoyl phosphatidylcholine (DMPC) vesicles, can inhibit cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs). Expression of VCAM-1 in endothelial cells after exposure to tumour necrosis factor-alpha (TNF-alpha) or interleukin 1beta (IL-1beta) was quantified by ELISA and shown to be partially inhibited by 17beta-estradiol (40-60% inhibition) or by S-nitroso-L-glutathione, a nitric oxide donor (20-25%). However, preincubations with physiological concentrations (10-100 microg protein/ml) of apo E DMPC did not downregulate VCAM-1 expression, even with extended preincubation times. These findings were confirmed using a fluorescence-activated cell sorter (FACS) for analysis which indicated additionally that apo E-DMPC had no effect on sub-populations within the HUVEC cultures. Finally, apo E-DMPC vesicles were also unable to suppress TNF-alpha-induced upregulation of E-selectin or intercellular adhesion molecule-1 (ICAM-1). We conclude that plasma apo E is unlikely to be important in limiting endothelial activation.