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
reendothelial ization 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 miaoarrays. 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 reendothelialization 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 P13K, 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. (C) 2021 Elsevier Inc. All rights reserved