Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK.

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.