INTRODUCTION: Atherosclerotic Coronary Artery Disease (ASCAD) is the leading 
cause of mortality worldwide. Novel therapeutic approaches aiming to improve the 
atheroprotective functions of High Density Lipoprotein (HDL) include the use of 
reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given 
the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 
may represent an attractive yet largely unexplored therapeutic agent.
OBJECTIVE: To evaluate the atheroprotective potential of rHDL-apoE3 starting 
with the unbiased assessment of global transcriptome effects and focusing on 
endothelial cell (EC) migration as a critical process in re-endothelialization 
and atherosclerosis prevention. The cellular, molecular and functional effects 
of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the 
potential translatability of these findings in vivo.
METHODS: Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was 
analyzed by whole genome microarrays. Expression and phosphorylation changes of 
key EC migration-associated molecules were validated by qRT-PCR and Western blot 
analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human 
EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was 
assessed by wound healing and transwell migration assays. The contribution of 
MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration 
and activation of EC migration-related effectors was assessed using specific 
inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, 
respectively. The capacity of rHDL-apoE3 to improve vascular permeability and 
hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia 
(apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the 
serum, respectively.
RESULTS: rHDL-apoE3 induced significant expression changes in 198 genes of HAEC 
mainly involved in re-endothelialization and atherosclerosis-associated 
functions. The most pronounced effect was observed for EC migration, with 42/198 
genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 
2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes 
in 24 representative transcripts of these pathways in HAEC, increasing the 
expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and 
EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and 
EA.hy926 cells, and the migration was markedly attenuated in the presence of 
PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, 
AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited 
rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY 
had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly 
decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and 
AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in 
apoE KO mice markedly improved vascular permeability as demonstrated by the 
reduced concentration of Evans Blue dye in tissues such as the stomach, the 
tongue and the urinary bladder and ameliorated hypercholesterolemia.
CONCLUSIONS: rHDL-apoE3 significantly enhanced EC migration in vitro, 
predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and 
PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and 
improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 
functions suggest a potential clinical use to promote re-endothelialization and 
retard development of atherosclerosis.