%0 Thesis
%9 Doctoral
%A Stannard, Anita Katharine
%D 1999
%F discovery:10111204
%I UCL (University College London)
%P 237
%T Apolipoprotein E and expression of endothelial cell adhesion molecules
%U https://discovery-pp.ucl.ac.uk/id/eprint/10111204/
%X I have hypothesized that: 'apolipoprotein E (apoE) has an anti- inflammatory/anti-atherogenic action by inhibiting cytokine-mediated upregulation of endothelial cell adhesion molecules (CAMs).' After rejecting ECV304 cells as a model of human vascular endothelium, I showed that primary cultures of human umbilical vein endothelial cells (HUVECs) were activated by cytokines and quantified CAM expression by ELISA and flow cytometry. Although HUVECs responded to known down-regulators of CAMs, a nitric oxide (NO) donor and 17?-estradiol, no inhibition of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) or E-selectin was observed with apoE complexed to phospholipid vesicles (apoE:DMPC). Additionally, apoE:DMPC did not affect HUVEC sub-populations by flow cytometry, nor did they suppress monocyte adhesion to activated HUVEC monolayers. ApoE-rich high density lipoprotein (HDL-E) was also benign. I concluded that plasma apoE is unlikely to limit endothelial activation in vivo. By contrast, when HUVECs were transfected with an apoE expression plasmid, VCAM-1 induction was inversely correlated with the apoE secreted, while co-culturing HUVECs and recombinant CHQ cells synthesizing apoE also suppressed VCAM-1. Incubating cell-conditioned media containing apoE with HUVECs reduced VCAM-1 in a dose-dependent manner. Characterization of this highly active apoE, revealed similarities to the minimally-lipidated, spherical particles secreted by macrophages. ApoE most likely inhibits endothelial activation by stimulating NO synthase (NOS): apoE increased cGMP, as expected for NO release; a NOS inhibitor blocked its effect; and lipoprotein receptor-related protein 8 (LRP8), implicated in coupling apoE-NOS in platelets, was detected in HUVECs by RT-PCR and immunoprecipitation. Moreover, consistent with protein-protein module formation and cell signalling to activate NOS, a synthetic cytoplasmic motif within LRP8 bound [35S]proteins in HUVEC cytosol. I propose, therefore, that apoE, secreted locally at lesion sites by macrophages, suppresses VCAM-1 expression on endothelium and that this anti-inflammatory action is mediated by activation of a LRP8-linked signalling cascade to generate intracellular NO.
%Z Thesis digitised by ProQuest.