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

  • Eftaxia-Konstantina Valanti
    Affiliations
    4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, ‘Attikon’ Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece

    Molecular Biology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
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  • Katerina Dalakoura-Karagkouni
    Affiliations
    Laboratory of Biochemistry, University of Crete Medical School, Heraklion, Greece

    Division of Gene Regulation and Genomics, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, Greece
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  • Author Footnotes
    1 Current address: Dren Bio, Inc., 400 Seaport Ct., Suite 102, Redwood City, CA 94063, San Carlos, California, USA.
    Panagiotis Fotakis
    Footnotes
    1 Current address: Dren Bio, Inc., 400 Seaport Ct., Suite 102, Redwood City, CA 94063, San Carlos, California, USA.
    Affiliations
    Molecular Genetics, Boston University Medical School, Boston, USA
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  • Elizabeth Vafiadaki
    Affiliations
    Molecular Biology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
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  • Christos S. Mantzoros
    Affiliations
    Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA
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  • Angeliki Chroni
    Affiliations
    Institute of Biosciences and Applications, National Center for Scientific Research “Demokritos”, Athens, Greece
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  • Vassilis Zannis
    Affiliations
    Molecular Genetics, Boston University Medical School, Boston, USA
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  • Dimitris Kardassis
    Affiliations
    Laboratory of Biochemistry, University of Crete Medical School, Heraklion, Greece

    Division of Gene Regulation and Genomics, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, Greece
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  • Despina Sanoudou
    Correspondence
    Corresponding author at: Clinical Genomics and Pharmacogenomics Unit, Center for New Biotechnologies and Precision Medicine, Building 15, Medical School, National and Kapodistrian University of Athens, Mikras Asias 75, Athens 11527, Greece.
    Affiliations
    4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, ‘Attikon’ Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece

    Molecular Biology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

    Center for New Biotechnologies and Precision Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece
    Search for articles by this author
  • Author Footnotes
    1 Current address: Dren Bio, Inc., 400 Seaport Ct., Suite 102, Redwood City, CA 94063, San Carlos, California, USA.
Published:December 03, 2021DOI:https://doi.org/10.1016/j.metabol.2021.154954

      Highlights

      • rHDL-apoE3 changes mRNA and protein levels of key molecules in pathways associated with human endothelial cell migration.
      • rHDL-apoE3 activates ERK1/2, AKT and p38 MAPK in human endothelial cells through MEK1/2 and PI3K, respectively.
      • rHDL-apoE3 increases migration of human endothelial cells, and themigration is attenuated by inhibition of MEK1/2 or PI3K.
      • ID1 silencing decreases human endothelial cell migration by inhibiting rHDLapoE3-mediated activation of ERK1/2 and AKT.
      • Administration of rHDL-apoE3 in apoE-deficient mice improves vascular permeability and ameliorates hypercholesterolemia.

      Abstract

      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.

      Abbreviations:

      AKT (AKT serine/threonine Kinase), ALP (Alkaline Phosphatase), ALT (Alanine Aminotransferase), ApoA-I (Apolipoprotein A-I), ApoE (Apolipoprotein E), apoE KO (apoE knockout), ApoER2 (Apolipoprotein E Receptor 2), ASCAD (Atherosclerotic Coronary Artery Disease), AST (Aspartate Aminotransferase), BAEC (Bovine Aortic Endothelial Cells), CAD (Coronary Artery Disease), CVD (Cardiovascular Disease), CYP1A1 (Cytochrome P450 family 1 subfamily A member 1), DPBS (Dulbecco's Phosphate Buffered Saline), EC (Endothelial Cell), EFNB2 (Ephrin B2), eNOS (Endothelial Nitric Oxide Synthase), EPC (Endothelial Progenitor Cells), ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), FAK (Focal Adhesion Kinase), FBS (Fetal Bovine Serum), FDR (False Discovery Rate), FLT1 (Fms-related Tyrosine kinase 1 (Vascular Endothelial Growth Factor Receptor 1)), FPLC (Fast protein liquid chromatography), GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase), HAEC (Human Aortic Endothelial Cells), HCAEC (Human Coronary Artery Endothelial Cells), HDL (High Density Lipoprotein), HDL-c (High Density Lipoprotein cholesterol), ID1 (Inhibitor of DNA binding 1, HLH protein), iNOS (Inducible Nitric Oxide Synthase), IPA (Ingenuity Pathway Analysis), LDL (Low Density Lipoprotein), LDL-c (Low Density Lipoprotein cholesterol), LPDS (Lipoprotein Deficient Serum), MEK1/2 (Mitogen-Activated Protein Kinase Kinase 1/2), MLCK (Myosin Light Chain Kinase), MMP2/9 (Matrix Metalloproteinase/Metallopeptidase 2/9), NO (Nitric Oxide), nNOS (Neuronal Nitric Oxide Synthase), p38 MAPK (p38 Mitogen-Activated Protein Kinase), PCA (Principal Components Analysis), PCC (Pearson's Correlation Coefficient), PCSK9i (Proprotein Convertase Subtilisin/Kexin type 9 inhibitors), PI3K (Phosphatidylinositol 3-Kinase), PIK3CG (Phosphatidylinositol-4,5-bisphosphate 3-Kinase Catalytic subunit Gamma), PTX3 (Pentraxin 3), PKA (Protein Kinase A), PKG (Protein Kinase G), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), RAC1 (Rac family Small GTPase 1), rHDL (reconstituted HDL), RHO-GTPases (Ras Homologous protein family GTPases), RHOA (Ras Homolog family member A), rhVEGF (recombinant human Vascular Endothelial Growth Factor), siRNA (Small Interfering RNA), SR-BI (Scavenger Receptor class B type I), Src (SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase), SEM (Standard Error of the Mean), TGFB2 (Transforming Growth Factor Beta 2), VLDL (Very Low Density Lipoprotein)

      Keywords

      1. Introduction

      Atherosclerosis is a chronic inflammatory disease triggered by pro-inflammatory factors that damage arterial endothelial integrity, and may lead to atherosclerotic plaque formation. Coronary artery occlusion caused by atherosclerotic plaque rupture is the predominant cause of Coronary Artery Disease (CAD), which represents the most common type of Cardiovascular Disease (CVD) [
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      ]. CAD is the leading cause of mortality worldwide with approximately 7.2 million deaths annually and an annual cost of > $10 billion, while by 2030, these numbers are anticipated to increase by ~18% and 43%, respectively [
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      ].
      Current treatments against atherosclerotic CΑD (ASCΑD) include low density lipoprotein cholesterol (LDL-c)-lowering drugs (e.g. statins), intestinal cholesterol absorption inhibitors (ezetimibe) and proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i). Although these drugs reduce the risk of cardiovascular events in a large percentage of patients, considerable patient-to-patient variability in efficacy and adverse-effects are reported, often leading to treatment discontinuation [
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      Lipid management for the prevention of atherosclerotic cardiovascular disease.
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      Pharmacogenomics in the development and characterization of atheroprotective drugs.
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      Advances in biological therapies for dyslipidemias and atherosclerosis.
      ]. Consequently, novel, safer and more effective therapeutic approaches are needed.
      Modulation of high density lipoprotein cholesterol (HDL-c) is an appealing alternative. Epidemiological studies have demonstrated an inverse association between plasma levels of HDL-c and the risk of early-onset ASCAD [
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      ]. However, genetic and clinical studies have suggested that enhancing HDL functionality is more important than increasing HDL-c levels alone, for achieving atheroprotection [
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      High-density lipoprotein: vascular protective effects, dysfunction, and potential as therapeutic target.
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      HDL dysfunction caused by mutations in apoA-I and other genes that are critical for HDL biogenesis and remodeling.
      ]. HDL functionality is dependent on its protein and lipid composition, and is associated with multiple atheroprotective features including cellular cholesterol efflux capacity, anti-inflammatory, anti-oxidative, anti-thrombotic, vasodilatory, anti-apoptotic, and endothelial repair properties [
      • Zannis V.I.
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      • et al.
      HDL biogenesis, remodeling, and catabolism.
      ]. Ongoing efforts are geared towards novel therapeutic approaches that could enhance HDL functionality including reconstituted forms of HDL containing human apolipoprotein A-I (apoA-I) and phospholipids (rHDL-apoA-I) [
      • van Capelleveen J.C.
      • Brewer H.B.
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      Novel therapies focused on the high-density lipoprotein particle.
      ,
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      Current and emerging reconstituted HDL-apoA-I and HDL-apoE approaches to treat atherosclerosis.
      ].
      As the therapeutic potential of rHDL is attracting increasing attention, rHDL containing apolipoprotein E (apoE) and phospholipids (rHDL-apoE) appears as an attractive therapeutic agent. The rationale for this strategy is based on existing data showing that deficiency of apoE in mice and humans leads to severe atherosclerosis, whereas deficiency of apoA-I does not [
      • Plump A.S.
      • Smith J.D.
      • Hayek T.
      • Aalto-Setala K.
      • Walsh A.
      • Verstuyft J.G.
      • et al.
      Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.
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      • et al.
      Familial apolipoprotein E deficiency.
      ,
      • Li H.
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      Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice.
      ]. The protection observed in apoA-I-deficient mice may be conferred by apoE-containing HDL particles (HDL-apoE) that are formed in the absence of apoA-I [
      • Kypreos K.E.
      • Zannis V.I.
      Pathway of biogenesis of apolipoprotein E-containing HDL in vivo with the participation of ABCA1 and LCAT.
      ]. In addition, apoE promotes hepatic clearance of circulating atherogenic triglyceride-rich lipoproteins [
      • Davignon J.
      Apolipoprotein E and atherosclerosis: beyond lipid effect.
      ]. The cholesterol-lowering properties of apoE, along with the increased anti-inflammatory effects of apoE as compared to apoA-I mimetics may also contribute to the greater protection against atherosclerosis in vivo [
      • Plump A.S.
      • Smith J.D.
      • Hayek T.
      • Aalto-Setala K.
      • Walsh A.
      • Verstuyft J.G.
      • et al.
      Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.
      ,
      • Nayyar G.
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      • Monroe C.E.
      • Keenum T.D.
      • Handattu S.P.
      • et al.
      Apolipoprotein E mimetic is more effective than apolipoprotein A-I mimetic in reducing lesion formation in older female apo E null mice.
      ,
      • Valanti E.K.
      • Chroni A.
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      The future of apolipoprotein E mimetic peptides in the prevention of cardiovascular disease.
      ].
      In this study, we assessed the atheroprotective potential of rHDL containing human apoE3, the most common variant of the polymorphic apoE [
      • Davignon J.
      Apolipoprotein E and atherosclerosis: beyond lipid effect.
      ], at the cellular, molecular and functional levels. Our findings demonstrated that rHDL-apoE3 stimulates endothelial cell (EC) migration, unveiled the molecular pathways mediating this effect, and identified ID1 as a key mediator of the rHDL-apoE3-induced EC migration. Through this mechanism rHDL-apoE3 could potentially promote re-endothelialization and preserve endothelial integrity. In support of these in vitro findings, we show that administration of a single dose of rHDL-apoE3 in mice lacking apoE markedly improved vascular permeability and ameliorated hypercholesterolemia suggesting that these reconstituted HDL particles containing apoE3 could be therapeutically exploited against atherosclerosis.

      2. Materials and methods

      Expanded materials and methods of expression and purification of apoE3 as well as preparation of rHDL-apoE3 particles can be found in Supplementary data (including Tables S1 and S2).

      2.1 Cell cultures

      Primary Human Aortic ECs (HAEC) were cultured in complete EC growth medium consisting of EBM-2 basal medium supplemented with growth factors, cytokines, 10% fetal bovine serum (FBS) and antibiotics. Primary Human Coronary Artery ECs (HCAEC) were cultured in complete MesoEndo Cell Growth medium consisting of MesoEndo basal medium and growth supplements. The human umbilical vein EC line EA.hy926 was cultured in DMEM supplemented with 10% FBS and antibiotics. All the experiments were performed on cells of passages 4–9 at 60–90% confluence. These three cell types were selected to confirm the consistency of our observations across different types of ECs, as established model systems for the study of EC biology and underlying mechanisms of CVD including atherosclerosis [
      • Bouis D.
      • Hospers G.A.
      • Meijer C.
      • Molema G.
      • Mulder N.H.
      Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research.
      ].

      2.2 Whole transcriptome microarray experiments

      HAEC were cultured in basal medium supplemented with 0.5% FBS and 5% bovine lipoprotein deficient serum (bLPDS) for 4 h. Thereafter, the cells were incubated with rHDL-apoE3 at 250 μg/ml or PBS 1× (baseline control) in basal medium containing 5% bLPDS for 12 h. Five biological replicates were used for the treated or control group. Total RNA was extracted using the Trizol reagent, as previously described [
      • Tsompanidis A.
      • Vafiadaki E.
      • Bluher S.
      • Kalozoumi G.
      • Sanoudou D.
      • Mantzoros C.S.
      Ciliary neurotrophic factor upregulates follistatin and Pak1, causes overexpression of muscle differentiation related genes and downregulation of established atrophy mediators in skeletal muscle.
      ]. All RNA samples had a 28S/18S rRNA ratio close to 2.0 on 1.5% agarose gels, and absorbance ratios 260/280 nm and 260/230 nm between 1.9 and 2.1. This high-quality (integrity and purity) RNA was used for target preparation and hybridization to GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's protocols. The 10 arrays were washed and stained on the Affymetrix 450 Fluidics station and scanned using the GeneChip Scanner-3000.

      2.3 Microarray data analysis

      The bioinformatical analysis of the 10 raw data datasets was performed using the Partek Genomics Suite software and involved Robust Multi-chip Analysis, Power Analysis and Principal Components Analysis (PCA). The statistically significant gene expression changes between treated and control groups (fold change ≥ |2.00| and false discovery rate (FDR) ≤ 0.05) were subjected to data mining using the Ingenuity Pathway Analysis (IPA) software and literature mining. Details are provided in Supplementary data.

      2.4 High-throughput quantitative Real-Time PCR (qRT-PCR)

      HAEC were treated as described in Section 2.2, cDNA was synthesized using Superscript II RNase H reverse transcriptase and oligo-dT primers, and it was applied to dynamic array chips (BioMark 96.96 Dynamic Array, Fluidigm Biomark, San Francisco, CA, USA), according to manufacturer's instructions. The qRT-PCR data were normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative CT method, and the ΔΔCT values were calculated relative to the PBS control group. A threshold of P ≤ 0.05 was applied for statistical significance.

      2.5 Protein expression and phosphorylation analysis

      HCAEC were cultured to 80% confluency and incubated in basal medium supplemented with 0.5% FBS and 5% human LPDS (hLPDS) for 4 h (for the study of AKT, ERK1/2, p38 MAPK, eNOS, ID1, PIK3CG, EFNB2) or 24 h (for FLT1 studies). The cells were then treated with 250 μg/ml rHDL-apoE3 or PBS 1× (negative control) in basal medium containing 5% hLPDS for 30 min (for AKT, ERK1/2, p38 MAPK, eNOS studies) or 12 h (for ID1, PIK3CG, EFNB2, FLT1 studies). Cells treated with 100 ng/ml recombinant human VEGF (rhVEGF) for the times indicated above were used as a positive control. In parallel, EA.hy926 cells were starved in 0.5% FBS containing DMEM for 4 h (for AKT, ERK1/2, p38 MAPK, eNOS studies) or 16 h (for ID1, PIK3CG, EFNB2, FLT1 studies) and then treated with 100 μg/ml rHDL-apoE3 or PBS 1× or 100 ng/ml rhVEGF for 30 min (for AKT, ERK1/2, p38 MAPK, eNOS studies) or 24 h (for ID1, PIK3CG, EFNB2, FLT1 studies). All protein extracts were analyzed by Western blotting to assess the expression of ID1, PIK3CG, EFNB2 and FLT1 and the phosphorylation of AKT, ERK1/2, p38 MAPK and eNOS. All experiments, for each treatment, were performed in biological triplicates. Details are provided in the Supplementary data.

      2.6 Wound healing assays

      HCAEC and EA.hy926 cells were cultured to 90–100% confluency and then incubated in basal medium containing 0.5% FBS and 1% hLPDS, or 0.5% FBS containing DMEM, respectively, for 16 h. The cell monolayers were scratched using a micropipette tip (200 μl). Thereafter, HCAEC and EA.hy926 cells were treated with rHDL-apoE3 at 100 μg/ml or PBS 1× (baseline control of cell migration) in basal medium containing 0.25% FBS and 1% hLPDS, or DMEM containing 0.5% FBS, respectively, for 24 h. These conditions were selected according to the literature [
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      High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I.
      ,
      • Gkolfinopoulou C.
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      • et al.
      Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.
      ,
      • Davis P.K.
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      Biological methods for cell-cycle synchronization of mammalian cells.
      ] and an extensive series of pilot experiments (data not shown). Cells treated with 100 ng/ml rhVEGF for 24 h served as a positive control of cell migration. Three biological replicates for each treatment were used. After 24 h, the cells were fixed with methanol, permeabilized in 0.2% Triton X-100/PBS and stained with hematoxylin. The cells were photographed on 10 high-power (10×) fields per replicate and treatment group on marked positions at 0 h and 24 h using a Leica DFC-500 inverted microscope. The number of migrated cells past the wound edge after 24 h was quantified using the ImageJ software.

      2.7 Transwell migration assay

      Quantitative cell migration assays on EA.hy926 cells were performed using a 24-well Transwell plate containing inserts with 8.0 μm pore polyester membrane filters. EA.hy926 cells were starved in 0.5% FBS containing DMEM for 16 h. The cells (1 × 105 cells/insert) were plated into the upper chamber in 300 μl DMEM containing 0.5% FBS. The lower chamber was filled with 700 μl DMEM supplemented with 10% FBS. rHDL-apoE3 at 100 μg/ml was added to the upper chamber of each insert and the cells were incubated for 20 h. Cells incubated with medium alone or medium plus rhVEGF at 100 ng/ml in the upper chamber for 20 h were used as a baseline and positive control of cell migration, respectively. Three biological replicates for each treatment were used. After 20 h, all non-migrated cells were removed from the upper side of the transwell membrane of each insert with a cotton swab. Cells migrated to the lower side of the transwell membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Migrated cells in each transwell insert were photographed on 25 random high-power (20×) fields using a Leica DFC-500 inverted microscope. The number of migrated cells was quantified using the ImageJ software.

      2.8 Analysis of the effects of kinase inhibitors on EC migration and protein phosphorylation

      Serum-starved HCAEC were pre-incubated for 1 h with the MEK1/2 inhibitor PD98059 (PD) (2 μM), or the PI3K inhibitor LY294002 (LY) (1 μM) in basal medium containing 0.25% FBS and 1% hLPDS. DMSO was used as control. The cells were then treated with rHDL-apoE3 (100 μg/ml) or PBS 1×, in the presence of PD or LY or DMSO, for 24 h in basal medium supplemented with 0.25% FBS and 1% hLPDS. After 24 h, the effect of each inhibitor on HCAEC migration was evaluated by wound healing assays, while the effect of LY on p38 MAPK and eNOS phosphorylation was assessed by Western blotting. ERK1/2 and AKT phosphorylation was studied in an independent set of experiments because of their different activation time window [
      • Kim A.L.
      • Labasi J.M.
      • Zhu Y.
      • Tang X.
      • McClure K.
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      Role of p38 MAPK in UVB-induced inflammatory responses in the skin of SKH-1 hairless mice.
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      ,
      • Theofilatos D.
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      • Kardassis D.
      HDL-apoA-I induces the expression of angiopoietin like 4 (ANGPTL4) in endothelial cells via a PI3K/AKT/FOXO1 signaling pathway.
      ]. In specific, serum-starved HCAEC were pre-incubated for 1 h with PD (5 μM) or LY (25 μM) or DMSO in basal medium containing 5% hLPDS and then treated with rHDL-apoE3 (100 μg/ml or 250 μg/ml) or PBS 1× for another 30 min. Three biological replicates for each treatment were performed in each experiment.

      2.9 siRNA silencing

      EA.hy926 cells were transfected with siRNA targeting ID1 (ID1 siRNA) or with control (scrambled) siRNA using Lipofectamine RNAiMAX [
      • Gkirtzimanaki K.
      • Gkouskou K.K.
      • Oleksiewicz U.
      • Nikolaidis G.
      • Vyrla D.
      • Liontos M.
      • et al.
      TPL2 kinase is a suppressor of lung carcinogenesis.
      ]. ID1 silencing was achieved following two rounds of 50 nM siRNA transfection. The cells were initially subjected to reverse transfection and 24 h later, to forward transfection. Twenty-four or forty-eight hours after the second round, the cells were treated with rHDL-apoE3 (100 μg/ml) or PBS 1× for 30 min or 24 h as described in 2.5, 2.6, respectively. Subsequently, protein phosphorylation studies or wound healing assays were performed, respectively. The silencing efficiency of ID1 was confirmed by Western blotting 48 h, 72 h and 96 h after the first transfection round. All experiments, for each treatment, were performed in biological triplicates.

      2.10 Animals

      C57BL/6 J apoE-deficient mice (apoE KO) [
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.
      ] were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained on a 12/12-h light/dark cycle and had ad libitum access to water and chow diet. All animal procedures were performed in accordance with the guidelines of the Institute of Molecular Biology and Biotechnology of Heraklion, Greece.

      2.11 Blood sampling and serum parameters

      Male mice 24 weeks-old received a single intravenous (tail vein) injection of 80 mg/kg rHDL-apoE3 or PBS. Blood was collected at different time points (1, 2, 4, 8, 12 h post-injection) and at the endpoint (24 h post-injection) by cardiac puncture. Serum samples were isolated rapidly following a 4 h fast and HDL was prepared by the dextran-Mg2+ method as previously described [
      • Warnick G.R.
      • Benderson J.
      • Albers J.J.
      Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol.
      ]. Concentration of total and HDL cholesterol, phospholipids and triglycerides in the serum was determined using Infinity™ Cholesterol Reagent, Phospholipids Reagent and Infinity™ Triglycerides Reagent, respectively, according to the manufacturers' instructions. Serum concentrations of ALT, ALP and AST were measured at the Laboratory of Clinical Chemistry of the University of Crete Medical School.

      2.12 Fast protein liquid chromatography (FPLC)

      Lipoprotein profile of 45 μl serum of rHDL-apoE3 treated and control mice 24 h post-injection was determined by FPLC gel filtration chromatography using a Superose 6 PC 3.2/30 column (GE Healthcare, Freiburg, Germany). A total of 40 fractions were collected and analyzed for total cholesterol and protein composition by SDS-PAGE and Coomassie Brilliant Blue staining. The FPLC fractions were also analyzed for the protein levels of human apoE and mouse apoA-I by Western blotting.

      2.13 Histological examination of the liver

      Histological analysis of lipid accumulation was performed in snap frozen liver specimens obtained from rHDL-apoE3 treated and control mice (24 h post-injection) using Oil Red O staining method. Sections of 10 μm were cut by Leica CM3050S cryostat and examined under an Axioskop 2 microscope (Zeiss, Germany).

      2.14 Vascular permeability assays

      Vascular permeability was evaluated using the Evans Blue Assay as previously described [
      • Radu M.
      • Chernoff J.
      An in vivo assay to test blood vessel permeability.
      ]. Essentially, 24 weeks-old male mice received a single intravenous injection (tail vein) of 40 mg/kg rHDL-apoE3 or PBS. Twenty-four hours later 0.5% sterile solution of Evans Blue dye was injected in lateral tail vein and 30 min later mice were sacrificed through cervical dislocation. Representative pictures to show differences in Evans Blue extravasation were taken and weight measurements of harvested tissues were performed. The concentration of Evans Blue in different organs was quantitated by measuring the absorbance at 610 nm.

      2.15 Statistical analysis

      Quantitative data are presented as mean ± SEM. Statistical significance for the in vitro experiments and Evans Blue assay was determined using the unpaired two-tailed Student's t-Test. Repeated measures ANOVA and the Bonferroni's post hoc tests were used to find significant differences between the treatments x time intervals in serum lipid measurements. For all results, P ≤ 0.05 was considered statistically significant. The Tukey's post hoc test was used to evaluate differences between three or more groups, and the Pearson's correlation coefficient (PCC) for inter-dataset comparisons. Analysis was performed using the GraphPad Prism software.

      3. Results

      3.1 rHDL-apoE3 induces distinct gene expression changes in HAEC

      To determine the effect of rHDL-apoE3 treatment on the global molecular pathways of HAEC, we performed whole genome expression analysis. The data were initially subjected to PCA which demonstrated a clear separation between rHDL-apoE3 treated and PBS control samples, indicating a distinct effect of the rHDL-apoE3 treatment on gene expression (Fig. 1A ). Consistent with this observation, hierarchical clustering resulted in two majorly distinct clusters of samples corresponding to the two sample groups (Fig. 1B). Therefore, rHDL-apoE3 appears to induce marked changes to the gene expression of HAEC.
      Fig. 1
      Fig. 1rHDL-apoE3 induces distinct gene expression signatures in HAEC. (A) Principal Component analysis of the gene expression profiles of rHDL-apoE3 treated and PBS control samples reveals distinct grouping. Each sphere represents a different sample. (B) Hierarchical clustering depicts two major distinct branches, representative of the markedly different expression patterns between the two sample groups, rHDL-apoE3 and PBS treated HAEC. Vertical and horizontal axes represent genes and individual samples, respectively. The heatmap color gradient from bright green to bright red represents levels of expression from high to low, respectively. (C) Significantly altered Biological Function Categories relevant to EC function, re-endothelialization and the atherosclerotic process detected between rHDL-apoE3 treated HAEC and PBS control by IPA (P-value ≤ 0.05 (orange line) threshold). (D) Venn diagram (Venny 2.1.0 - BioinfoGP – CSIC) of the 24 out of the 42 differentially expressed EC migration-related genes following rHDL-apoE3 treatment of HAEC that were validated with qRT-PCR and are involved in the four major EC migration pathways: i) MEK/ERK (17 genes), ii) PI3K/AKT/eNOS-MMP2/9 (16 genes), iii) small RHO-GTPases (RAC1 and RHOA) (17 genes) and iv) integrin (8 genes). In each section of the Venn diagram the significantly changed common or unique genes of these pathways are presented.

      3.2 rHDL-apoE3 affects multiple biological functions associated with atheroprotection

      To identify the specific differences between rHDL-apoE3 and PBS treated HAEC, we performed multilevel bioinformatical analysis with stringent thresholds (fold change ≥ |2.00| and FDR ≤ 0.05) and identified 198 significantly changed transcripts (89 upregulated/109 downregulated) (Table S3). The fold change ranged from +7.739 (CYP1A1) to −7.861 (PTX3).
      The role of these genes was investigated at the levels of Cellular and Molecular Biological Functions, using the IPA software along with extensive literature mining. The IPA Biological Function (Bio-Function) analysis revealed 23 significantly altered categories (P ≤ 0.05 calculated by the right-tailed Fisher's Exact Test) (Table S4). The top three were “Cellular Movement”, “Cellular Growth and Proliferation” and “Cell Death and Survival” (Table S4, Fig. 1C). These functions are crucial for the preservation of endothelial integrity and the endothelial repair process [
      • Werner N.
      • Junk S.
      • Laufs U.
      • Link A.
      • Walenta K.
      • Bohm M.
      • et al.
      Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury.
      ]. Other significantly changed Bio-Functions associated with atherosclerosis included: “Inflammatory Response”, “Lipid Metabolism”, “Cell-to-Cell Signaling and Interaction”, “Cell Cycle” and “Cell Signaling” (Table S5, Fig. 1C). The top three Biological Functions were analyzed further at the data mining level. Notably, over half of the significantly changed genes grouped under each of these Biological Functions belonged to a single sub-category. In specific, 42/64 “Cell Movement”-related genes are involved in EC migration pathways (Table S6), 36/75 “Cell Growth and Proliferation”-related genes are associated with EC proliferation and/or differentiation pathways, and 31/63 “Cell Death and Survival”-related genes participate in EC apoptosis and/or survival pathways. All of these functions are central steps of re-endothelialization following injury-induced endothelial dysfunction - the initial stimulus of atherogenesis [
      • Werner N.
      • Junk S.
      • Laufs U.
      • Link A.
      • Walenta K.
      • Bohm M.
      • et al.
      Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury.
      ,
      • Foteinos G.
      • Hu Y.
      • Xiao Q.
      • Metzler B.
      • Xu Q.
      Rapid endothelial turnover in atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein E-deficient mice.
      ].
      Among them, we specifically focused on the in-depth investigation of EC migration, because of the critical role of this biological function in the amelioration or even prevention of atherosclerosis, as well as the large and highly significant number of EC migration-related gene expression changes. Following extensive literature mining it emerged that the 42 EC migration-related genes were all involved in one or more of the following four main signaling pathways (Table S6) [
      • Munoz-Chapuli R.
      • Quesada A.R.
      • Angel Medina M.
      Angiogenesis and signal transduction in endothelial cells.
      ]: 1) MEK/ERK; 2) PI3K/AKT/eNOS-MMP2/9; 3) small RHO-GTPases (RHOA, RAC1); and 4) integrin. The common and unique genes of these pathways that were validated by qRT-PCR, as described in Section 3.3, are presented in a Venn diagram (Fig. 1D).

      3.3 qRT-PCR validation of microarray data

      To validate the microarray data, we performed high-throughput qRT-PCR for 25 significantly changed EC migration-associated genes following rHDL-apoE3 treatment of HAEC (Table S6). Τhe selection of these genes was based on their key roles in the four EC migration pathways affected by rHDL-apoE3. In addition, 12 of these genes were among the 30 most highly changed transcripts observed in this study. The qRT-PCR data were in full agreement with the microarray data (PCC, r = 0.818), albeit the latter fold changes were more modest, consistently with observations in the literature [
      • Morey J.S.
      • Ryan J.C.
      • Van Dolah F.M.
      Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR.
      ]. TGFB2 had a similar fold change but did not reach statistical significance in the qRT-PCR analysis and was therefore excluded from further consideration.
      To validate our results across different human EC types, the key transcriptomic observations from HAEC were confirmed in HCAEC and EA.hy926 cells at the protein, pathway activation and functional levels.

      3.4 rHDL-apoE3 induces the expression of key EC migration-associated proteins in HCAEC and EA.hy926 cells

      The expression of four key proteins identified in the four EC migration-related signaling pathways (MEK/ERK, PI3K/AKT/eNOS-MMP2/9, RHO-GTPases and integrin) - PIK3CG, EFNB2, FLT1 and ID1 - was assessed in rHDL-apoE3 treated HCAEC by Western blotting (Fig. 2A ). Treatment with PBS or rhVEGF was used as a negative and positive control, respectively [
      • Golan S.
      • Entin-Meer M.
      • Semo Y.
      • Maysel-Auslender S.
      • Mezad-Koursh D.
      • Keren G.
      • et al.
      Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment.
      ,
      • Yamanda S.
      • Ebihara S.
      • Asada M.
      • Okazaki T.
      • Niu K.
      • Ebihara T.
      • et al.
      Role of ephrinB2 in nonproductive angiogenesis induced by Delta-like 4 blockade.
      ,
      • Raikwar N.S.
      • Liu K.Z.
      • Thomas C.P.
      Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist.
      ,
      • Song X.
      • Liu S.
      • Qu X.
      • Hu Y.
      • Zhang X.
      • Wang T.
      • et al.
      BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1.
      ]. All four proteins were found to be significantly (P ≤ 0.0334) overexpressed following rHDL-apoE3 treatment (fold changes: PIK3CG = 2.321, EFNB2 = 1.479, FLT1 = 1.475, ID1 = 1.368) compared to the PBS control, in a fashion similar to rhVEGF treatment (Fig. 2B). These findings are consistent with the microarray and qRT-PCR data (Table S6).
      Fig. 2
      Fig. 2rHDL-apoE3 increases the protein expression of PIK3CG, EFNB2, FLT1 and ID1 that play key roles in the four EC migration pathways (MEK/ERK, PI3K/AKT/eNOS-MMP2/9, RHO-GTPases and integrin) in both HCAEC and EA.hy926 cells. HCAEC (A) and EA.hy926 (C) cells were treated with rHDL-apoE3, PBS (negative control) or rhVEGF (positive control) for 12 h and 24 h, respectively. Protein expression was measured by Western blotting using β-actin as a loading control. (B, D) The density of the bands corresponding to each protein was normalized to the β-actin bands and presented relative to the protein level of the PBS control. Values are expressed as mean ± SEM (n = 3 biological replicates per group). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
      To ensure these results were independent of the cell type used, the experiments were repeated in EA.hy926 cells (Fig. 2C). Similarly to HCAEC, rHDL-apoE3 treatment of EA.hy926 cells induced a significant (P < 0.045) increase in the protein levels of PIK3CG, FLT1 and ID1 (fold changes: PIK3CG = 1.708, FLT1 = 1.444, ID1 = 1.375) compared to PBS, which was similar to the changes induced by rhVEGF. Of note, rHDL-apoE3 caused a marked induction of EFNB2 protein expression by 5.736-fold (P < 0.0019) when compared with the PBS control, which was even greater to that induced by rhVEGF (Fig. 2D).

      3.5 rHDL-apoE3 activates EC migration-related molecules in HCAEC and EA.hy926 cells

      To assess whether the MEK/ERK, PI3K/AKT/eNOS-MMP2/9, RHO-GTPases, and integrin EC migration pathways are activated by rHDL-apoE3 in HCAEC, we measured the phosphorylation status of key downstream effectors - ERK1/2, AKT, eNOS and p38 MAPK - by Western blotting (Fig. 3A ). Treatment with PBS or rhVEGF was used as a negative and positive control, respectively [
      • Munoz-Chapuli R.
      • Quesada A.R.
      • Angel Medina M.
      Angiogenesis and signal transduction in endothelial cells.
      ]. Indeed, rHDL-apoE3 induced a significant (P < 0.0416) increase in the phosphorylation levels of all four EC migration-related effectors (fold changes: ERK1/2 = 1.487, AKT = 1.747, eNOS = 1.377, p38 MAPK = 2.621) compared to PBS, which was comparable to rhVEGF (Fig. 3B). Importantly, the total protein levels (phosphorylated and unphosphorylated forms) of all four molecules were unchanged (Fig. 3A), suggesting a shift from unphosphorylated to phosphorylated forms in the absence of gene/protein expression changes, indicating the activation of these pathways by rHDL-apoE3.
      Fig. 3
      Fig. 3rHDL-apoE3 induces the phosphorylation of AKT, ERK1/2, eNOS and p38 MAPK in both HCAEC and EA.hy926 cells. HCAEC (A) and EA.hy926 (C) cells were treated with rHDL-apoE3, PBS (negative control), or rhVEGF (positive control) for 30 min. The phosphorylated (p-) and total levels of AKT, ERK1/2, eNOS and p38 MAPK were measured by Western blotting using β-actin as a loading control. (B, D) The band density of both the phosphorylated (p-) and total (t-) protein forms was normalized to β-actin. The phosphorylated (p-) levels of each molecule were normalized to their respective total (t-) protein levels (ratio of phosphorylated/total protein levels) and presented relative to the phosphorylation of the PBS control. Values are expressed as mean ± SEM (n = 3 biological replicates per group). (A, C) The band corresponding to p-eNOS is indicated by an arrow (140 kDa). The upper band represents the phosphorylated levels of nNOS isoform (160 kDa) and the lower band the phosphorylated levels of iNOS isoform (130 kDa). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
      To ensure these results were independent of the cell type used, we repeated this entire analysis in EA.hy926 cells (Fig. 3C). Similarly to HCAEC, rHDL-apoE3 treatment of EA.hy926 cells induced a significant (P ≤ 0.0048) increase in the phosphorylation levels of ERK1/2, AKT, eNOS and p38 MAPK (fold changes: ERK1/2 = 1.375, AKT = 2.169, eNOS = 1.377, p38 MAPK = 2.576) compared to PBS, which was comparable to rhVEGF (Fig. 3D).

      3.6 rHDL-apoE3 induces migration of HCAEC and EA.hy926 cells through the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways

      Considering the significant molecular changes observed in the four EC migration-related pathways, we proceeded to assess the effect of rHDL-apoE3 on HCAEC migration through a series of wound healing assays (Fig. 4A). Treatment with PBS or rhVEGF was used as a baseline and positive control of cell migration, respectively [
      • Munoz-Chapuli R.
      • Quesada A.R.
      • Angel Medina M.
      Angiogenesis and signal transduction in endothelial cells.
      ]. As anticipated, rHDL-apoE3 significantly increased HCAEC migration after 24 h by 85.35% (P = 0.0002) compared to PBS, similarly to VEGF (81.33% increase, P = 0.0002) (Fig. 4B). The capacity of rHDL-apoE3 to stimulate EC migration was further confirmed in EA.hy926 cells (143.51% (P < 0.0001) increase compared to PBS, and similarly to rhVEGF (133.45% increase, P < 0.0001)) (Fig. 4C–D). To confirm these effects of rHDL-apoE3 on EC migration, transwell migration assays were carried out in EA.hy926 cells. The results were in agreement with the wound healing experiments. In specific, rHDL-apoE3 increased EA.hy926 cell migration after 20 h by 57.40% (P = 0.0052) compared to the baseline control, in a fashion similar to rhVEGF (73.99% increase, P = 0.0002) (Fig. S1A–B).
      We next sought to delineate the relative contribution of each of the different EC migration-related signaling cascades in the rHDL-apoE3-induced EC migration, with the use of selective inhibitors. In specific, we focused on the three signaling cascades with the largest number of significant gene/protein expression changes (MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases) (Fig. 1D, Table S6), using the MEK1/2 inhibitor PD98059 (PD) for the MEK/ERK pathway [
      • Santos E.
      • Crespo P.
      The RAS-ERK pathway: a route for couples.
      ], and the PI3K inhibitor LY294002 (LY) for the PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways [
      • Hemmings B.A.
      • Restuccia D.F.
      PI3K-PKB/Akt pathway.
      ,
      • Kimura T.
      • Sato K.
      • Malchinkhuu E.
      • Tomura H.
      • Tamama K.
      • Kuwabara A.
      • et al.
      High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors.
      ,
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.
      ].
      Fig. 4
      Fig. 4rHDL-apoE3 induces migration of both HCAEC and EA.hy926 cells. HCAEC (A) and EA.hy926 cells (C) were wounded and treated with rHDL-apoE3 or PBS (baseline control of cell migration) or rhVEGF (positive control of cell migration) for 24 h. Representative wound healing assay microscopic images of HCAEC (A) and EA.hy926 cell (C) migration at 0 h and 24 h are shown. The black lines indicated the scratch edge. The number of migrated HCAEC (B) and EA.hy926 cells (D) into the wound area after 24 h was quantified. The results are presented as % relative to the cell migration of the PBS control. Values are expressed as mean ± SEM (n = 3 biological replicates per group). ***P ≤ 0.001.
      Through a series of wound healing assays, we observed a dramatic inhibition (by 42.04%, P = 0.0028) of the rHDL-apoE3 effect on HCAEC migration in the presence of PD, which brought migration levels at baseline (PBS) (Fig. 5A–B ). Of note, PD did not affect basal migration (PBS) levels.
      Fig. 5
      Fig. 5rHDL-apoE3 induces HCAEC migration through activation of MEK1/2 and PI3K. HCAEC were wounded and treated with rHDL-apoE3 or PBS (baseline control of cell migration) for 24 h, in the absence (DMSO) or presence of PD98059 (PD) (MEK1/2 inhibitor) (A) or LY294002 (LY) (PI3K inhibitor) (C). (A, C) Representative wound healing assay microscopic images of HCAEC migration at 0 h and 24 h are shown. (B, D) The number of migrated cells into the wound area after 24 h was quantified, and the results are presented as % relative to the cell migration of the PBS + DMSO control. Values are expressed as mean ± SEM from one (PD) or two (LY) independent experiments performed in biological triplicates (n = 3 to 6 biological replicates per group). **P ≤ 0.01, ***P ≤ 0.001, ns = non-significant (P > 0.05).
      Remarkable was also the inhibition of the rHDL-apoE3 effect on HCAEC migration (by 43.28%, P < 0.0001) in the presence of LY, which also brought migration levels close to the PBS baseline (Fig. 5C–D). Of note, LY did not affect basal (PBS) migration.
      Collectively, these results indicate that the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways are directly involved in the stimulation of HCAEC migration by rHDL-apoE3.

      3.7 The rHDL-apoE3-triggered activation of ERK1/2, AKT and p38 MAPK in HCAEC is mediated by MEK1/2 and PI3K, respectively, while eNOS activation is PI3K-independent

      We next sought to determine whether the observed rHDL-apoE3-triggered activation of the EC migration-related effectors ERK1/2, AKT, eNOS and p38 MAPK (Fig. 3) was mediated through their respective pathways: MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases. PD was used for the inhibition of MEK1/2, the main activator of ERK1/2 [
      • Santos E.
      • Crespo P.
      The RAS-ERK pathway: a route for couples.
      ], and LY was used for the inhibition of PI3K, the main activator of AKT and an indirect activator of p38 MAPK and eNOS [
      • Hemmings B.A.
      • Restuccia D.F.
      PI3K-PKB/Akt pathway.
      ,
      • Kimura T.
      • Sato K.
      • Malchinkhuu E.
      • Tomura H.
      • Tamama K.
      • Kuwabara A.
      • et al.
      High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors.
      ,
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.
      ]. Because of their different activation time windows reported in the literature [
      • Kim A.L.
      • Labasi J.M.
      • Zhu Y.
      • Tang X.
      • McClure K.
      • Gabel C.A.
      • et al.
      Role of p38 MAPK in UVB-induced inflammatory responses in the skin of SKH-1 hairless mice.
      ,
      • Ghimire K.
      • Zaric J.
      • Alday-Parejo B.
      • Seebach J.
      • Bousquenaud M.
      • Stalin J.
      • et al.
      MAGI1 mediates eNOS activation and NO production in endothelial cells in response to fluid shear stress.
      ,
      • Theofilatos D.
      • Fotakis P.
      • Valanti E.
      • Sanoudou D.
      • Zannis V.
      • Kardassis D.
      HDL-apoA-I induces the expression of angiopoietin like 4 (ANGPTL4) in endothelial cells via a PI3K/AKT/FOXO1 signaling pathway.
      ], ERK1/2 and AKT were assessed at 30 min of rHDL-apoE3 treatment of HCAEC, whereas the AKT downstream targets p38 MAPK and eNOS were measured at 24 h, similarly to all cell migration experiments (Section 3.6). PD significantly decreased rHDL-apoE3-induced ERK1/2 phosphorylation by 39.25% (Ρ = 0.0058) compared to rHDL-apoE3 treatment alone, bringing ERK1/2 phosphorylation to the levels of the PBS control (Fig. 6A–B ). Strikingly, LY fully inhibited rHDL-apoE3-mediated AKT phosphorylation by 95.79% (P < 0.0001) compared to rHDL-apoE3 without LY, with rHDL-apoE3 treated cells displaying similar AKT phosphorylation levels to the PBS control (Fig. 6C–D). Similar, yet more modest observations were made for p38 MAPK phosphorylation where LY led to its marked (43.31%, P = 0.0294) inhibition in rHDL-apoE3 treated HCAEC, and brought its post rHDL-apoE3 treatment levels down to those of the PBS control (Fig. 6E–F). On the contrary, LY did not affect the rHDL-apoE3 mediated eNOS phosphorylation (Fig. 6G–H).
      Fig. 6
      Fig. 6rHDL-apoE3 activates ERK1/2, AKT and p38 MAPK in HCAEC through MEK1/2 and PI3K, respectively, while rHDL-apoE3-mediated activation of eNOS is PI3K-independent. HCAEC were treated with rHDL-apoE3 or PBS for 30 min (A, C) or 24 h (E, G), in the absence (DMSO) or presence of PD (A) or LY (C, E, G). The phosphorylated (p-) and total levels of ERK1/2 (A), AKT (C), p38 MAPK (E) and eNOS (G) were measured by Western blotting using β-actin as a loading control. The band density of both the phosphorylated (p-) and total (t-) protein forms of each molecule was normalized to β-actin. The phosphorylated (p-) levels of ERK1/2 (B), AKT (D), p38 MAPK (F) and eNOS (H) were normalized to their respective total (t-) protein levels (ratio of phosphorylated/total protein levels) and presented relative to the phosphorylation of the PBS + DMSO control. Values are expressed as mean ± SEM (n = 3 biological replicates per group). (G) The band corresponding to p-eNOS is indicated by an arrow (140 kDa). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = non-significant (P > 0.05).
      Overall, these results indicate that rHDL-apoE3 activates ERK1/2 through MEK1/2, and AKT along with p38 MAPK through PI3K in HCAEC, in a predominant, if not exclusive, fashion. Meanwhile, the rHDL-apoE3-mediated activation of eNOS appears to be taking place through a PI3K-independent mechanism.

      3.8 siRNA-mediated silencing of ID1

      We next investigated the potential upstream regulators mediating the rHDL-apoE3-induced activation of the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases EC migration pathways in human ECs. ID1 emerged as a promising candidate because it plays a key role in all three pathways, and it is significantly overexpressed following rHDL-apoE3 treatment of human ECs (Fig. 2, Table S6). To address this hypothesis we used siRNA to silence ID1 (ID1 siRNA), and compared its performance in HCAEC and EA.hy926 cells (Fig. S2A–D). EA.hy926 cells exhibited superior siRNA transfection efficiency compared to HCAEC (data not shown), consistently with observations in the literature showing that primary ECs are often poorly transfected [
      • Dennstedt E.
      • Bryan B.
      siRNA knockdown of gene expression in endothelial cells.
      ]. We therefore proceeded with the use of EA.hy926 cells. ID1 siRNA significantly decreased ID1 protein levels compared to control siRNA and non-transfected cells, at 48 h post-transfection (59.43% and 74.49%, respectively, P < 0.0001) (Fig. S2A–B), as well as 72 h post-transfection, when the ID1 silencing effect was found to be more pronounced (81.26% and 83.35%, respectively, P < 0.0001) (Fig. S2C–D).
      We next assessed the effect of the concomitant transfection with ID1 siRNA and treatment with rHDL-apoE3 on ID1 protein expression. In specific, at 48 h or 72 h post-transfection, rHDL-apoE3 or PBS was added to EA.hy926 cells for an additional 24 h (i.e. 72 h or 96 h of processing time in total). As anticipated, in the presence of ID1 siRNA, the effect of rHDL-apoE3 treatment on ID1 protein expression was drastically reduced compared to the control siRNA group, both at 72 h and 96 h (73.99% and 96.04%, respectively, P < 0.0001) (Fig. S2E–H). Furthermore, in the presence of ID1 siRNA, the effect of rHDL-apoE3 treatment on ID1 protein expression was reduced compared to the PBS control group, both at 72 h and 96 h (38.62% P = 0.0004 and 68.80% P = 0.0462, respectively). Meanwhile, in the presence of the control siRNA, rHDL-apoE3 significantly increased ID1 expression compared to PBS, both at 72 h and 96 h (38.16% P < 0.0001 and 23.72% P = 0.0414, respectively), as expected (Fig. S2E–H).
      Collectively, these findings indicated that ID1 silencing markedly attenuated rHDL-apoE3-mediated increase in ID1 protein levels at 72 h and 96 h post-transfection, with higher silencing efficiency being observed at 96 h.

      3.9 Induction of EA.hy926 cell migration by rHDL-apoE3 is mediated by ID1

      To determine whether endogenous ID1 is involved in rHDL-apoE3-induced EC migration, we performed a series of wound healing assays using ID1 siRNA (Fig. 7A ). In specific, 72 h post-transfection, EA.hy926 cells were treated with rHDL-apoE3 or PBS for 24 h. Strikingly, ID1 siRNA transfection led to markedly decreased cell migration (72.56%, P < 0.0001) upon rHDL-apoE3 treatment, compared to the control siRNA (Fig. 7B), indicating that ID1 plays an important role in mediating the rHDL-apoE3-induced effect on EA.hy926 cell migration.
      Fig. 7
      Fig. 7siRNA-mediated silencing of ID1 decreases rHDL-apoE3-induced migration of EA.hy926 cells. EA.hy926 cells were transfected with ID1 siRNA or control siRNA for 72 h, wounded and then treated with rHDL-apoE3 or with PBS (baseline control of cell migration) for 24 h. (A) Representative wound healing assay microscopic images of EA.hy926 cell migration at 0 h and 24 h are shown. (B) The number of migrated cells into the wound area after 24 h was quantified, and the results are presented as % relative to the cell migration of the Control siRNA + PBS group. Values are expressed as the mean ± SEM from three independent experiments performed in biological triplicates (n = 9 biological replicates per group). **P ≤ 0.01, ***P ≤ 0.001, and ###P ≤ 0.001 between ID1 siRNA and control siRNA transfected cells.

      3.10 ID1 mediates the rHDL-apoE3-induced activation of the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways in EA.hy926 cells

      ID1 overexpression has been shown to induce ERK1/2 and AKT activation through MEK1/2 and PI3K phosphorylation, respectively [
      • Su Y.
      • Gao L.
      • Teng L.
      • Wang Y.
      • Cui J.
      • Peng S.
      • et al.
      Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-kappaB/MMP-2 signaling pathways.
      ,
      • Ling M.T.
      • Wang X.
      • Ouyang X.S.
      • Lee T.K.
      • Fan T.Y.
      • Xu K.
      • et al.
      Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth.
      ]. To determine whether ID1 is responsible for the observed rHDL-apoE3-induced activation of ERK1/2 and AKT (Fig. 3), we measured their phosphorylation levels in EA.hy926 cells transfected with ID1 siRNA or control siRNA (76 h), followed by rHDL-apoE3 or PBS treatment (Fig. 8A–D ). Indeed, the presence of ID1 siRNA significantly reduced ERK1/2 phosphorylation by rHDL-apoE3 when compared to control siRNA (by 44.78%, P = 0.0011) or PBS (by 21.75%, P = 0.0179) (Fig. 8A–B). Similar findings were observed for AKT, where the presence of ID1 siRNA decreased AKT phosphorylation by rHDL-apoE3 when compared to control siRNA (by 33.26%, P = 0.0013), with rHDL-apoE3 treated cells displaying similar AKT phosphorylation levels to the PBS control (Fig. 8C–D).
      Fig. 8
      Fig. 8siRNA-mediated ID1 silencing attenuates rHDL-apoE3-induced activation of ERK1/2 and AKT in EA.hy926 cells. (A, C) EA.hy926 cells transfected with ID1 siRNA or control siRNA (76 h) were treated with rHDL-apoE3 or PBS for 30 min. The phosphorylated (p-) and total levels of ERK1/2 (A) and AKT (C) were measured using β-actin as a loading control. The band density of both the phosphorylated (p-) and total (t-) protein forms of each molecule was normalized to β-actin. The phosphorylated (p-) levels of ERK (B) and AKT (D) were normalized to their respective total (t-) protein levels (ratio of phosphorylated/total protein levels) and presented relative to the phosphorylation of the Control siRNA + PBS group. Values are expressed as mean ± SEM (n = 3 biological replicates per group). *P ≤ 0.05, **P ≤ 0.01, ns = non-significant (P > 0.05).
      Collectively, ID1 silencing attenuated rHDL-apoE3-induced activation of ERK1/2 and AKT in EA.hy926 cells. These results indicate that ID1 is required for the rHDL-apoE3-triggered activation of ERK1/2 and AKT, supporting the notion that rHDL-apoE3 activates the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways through the modulation of ID1 expression. Taken together, our overall data indicate that it is through these mechanisms that rHDL-apoE3 induces EC migration.
      Fig. 9
      Fig. 9rHDL-apoE ameliorates hypercholesterolemia and improves vascular permeability in apoE KO mice. (A) Experimental protocol for the rHDL-apoE3 or PBS administration in apoE KO mice. (B-E) Concentrations of total cholesterol, HDL cholesterol (HDL-c), triglycerides and phospholipids in the serum of rHDL-apoE3 treated and control (Ctr) mice at different time points post-injection. Data represent mean ± SEM (n = 5 per group) (F) FPLC analysis of plasma lipoproteins in pooled serum samples of rHDL-apoE3 treated and control mice 24 h post-injection (n = 3/group/pool). The fractions that correspond to different lipoprotein classes are indicated on top. (G) Histological examination of liver samples obtained from rHDL-apoE3 treated and control (CTR) mice (24 h post-injection) for lipids using Oil Red O staining. (H) Vascular permeability assays performed in tissues obtained from rHDL-apoE3 treated and control mice (24 h post-injection) using Evans Blue. The concentration of the Evans Blue dye in three different tissues (stomach, tongue and urinary bladder) as ng of dye/mg of tissue is shown in the histogram. Four mice per group were processed. Data expressed as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

      3.11 Administration of rHDL-apoE3 in apoE KO mice improves vascular permeability and ameliorates hypercholesterolemia

      In order to assess the atheroprotective properties of rHDL-apoE3 particles in vivo, we used a well-characterized model of atherosclerosis, the apoE KO mice [
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.
      ]. The experimental strategy is shown in Fig. 9A. More specifically, male 24-weeks old mice were split into two groups (n = 5 per group) and were injected either with rHDL-apoE3 (80 mg/kg) (rHDL-apoE3 group) or PBS (control group). Blood samples were obtained at multiple time intervals for lipid and lipoprotein analysis and the mice were sacrificed 24 h post-injection. Examination of liver enzymes revealed that mice that received the rHDL-apoE3 particles had slightly increased ALT and AST enzymes, but no difference in ALP levels (Table S7). As shown in Fig. S3A, apoE could be detected in the serum of mice that received the rHDL-apoE3 particles for 2 h post-injection and its concentration declined thereafter (lanes 1–6). In contrast, no apoE could be detected in the serum of apoE KO mice that received PBS (Fig. S3A lanes 7–12). Serum lipid analysis showed that rHDL-apoE3 treated mice had significantly reduced cholesterol levels even at 2 h after the injection of the rHDL-apoE3 particles and cholesterol levels remained low at 24 h post-injection (Fig. 9B). Triglyceride levels were increased at the early time points but were almost normalized at the end of the experiment (Fig. 9D), whereas a statistically significant increase in serum HDL-c was observed in rHDL-apoE3 treated mice at the late time points (Fig. 9C). No substantial changes in serum phospholipids could be detected between the two mouse groups (Fig. 9E). The data of Fig. 9B–E suggest that administration of rHDL-apoE3 in apoE KO mice caused a rapid but sustained clearance of LDL-c from the serum, and this was in agreement with the FPLC analysis at 24 h which showed that most of the reduced serum cholesterol was distributed in LDL and in slightly larger, compared to the control group, HDL particles suggesting remodeling of endogenous HDL containing apoA-I in the rHDL-apoE3 group (Fig. 9F and Fig. S3B–C). Histological examination of the liver with Oil Red O stain showed enhanced accumulation of lipids in rHDL-apoE3 treated mice compared with the control mice possibly reflecting the enhanced clearance of LDL particles in these mice (Fig. 9G). Importantly, administration of rHDL-apoE3 in apoE KO mice was associated with decreased vascular permeability as demonstrated by the reduced concentration of the Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder (Fig. 9H).

      4. Discussion

      Accumulating evidence indicates that HDL functionality is the appropriate therapeutic target against ASCAD compared to increasing HDL-c levels alone [
      • Luscher T.F.
      • Landmesser U.
      • von Eckardstein A.
      • Fogelman A.M.
      High-density lipoprotein: vascular protective effects, dysfunction, and potential as therapeutic target.
      ,
      • Chroni A.
      • Kardassis D.
      HDL dysfunction caused by mutations in apoA-I and other genes that are critical for HDL biogenesis and remodeling.
      ]. Towards this goal, rHDL-apoE may represent a highly promising yet largely unexplored therapeutic agent [
      • Valanti E.K.
      • Dalakoura-Karagkouni K.
      • Sanoudou D.
      Current and emerging reconstituted HDL-apoA-I and HDL-apoE approaches to treat atherosclerosis.
      ]. We studied the atheroprotective potential of rHDL-apoE3, and focused on the markedly enhanced EC migration pathways. A key upstream regulator (ID1) of the rHDL-apoE3 pro-migratory molecular effects was identified, along with multiple critical downstream effectors mediating this process. At the in vivo level, a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability and ameliorated hypercholesterolemia.
      Specifically, through the unbiased approach of whole genome expression profiling, we observed that rHDL-apoE3 induced significant changes in the expression of genes that affect important EC functions contributing to atheroprotection. The most statistically significant effects were on EC migration, proliferation/differentiation and survival-related genes. Each of these functions is crucial for the maintenance of endothelial integrity in vivo and promotes endothelial repair [
      • Werner N.
      • Junk S.
      • Laufs U.
      • Link A.
      • Walenta K.
      • Bohm M.
      • et al.
      Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury.
      ]. Significant changes were also evident in mechanisms regulating atherosclerosis progression, such as inflammatory response, lipid metabolism, as well as cell-to-cell signaling and interaction. The observed modulation of these functions by rHDL-apoE3 could contribute to the preservation of endothelial integrity, promote endothelial repair and retard progression of atherosclerosis.
      In support of this hypothesis, we obtained preliminary evidence in favor of an atheroprotective effect of rHDL-apoE3 in vivo. In an effort to evaluate rHDL-apoE3 under pathological conditions implicated in ASCAD, such as hypercholesterolemia which is a risk factor for ASCAD and atherosclerosis, we performed an in vivo study using a well-established mouse model of hypercholesterolemia, atherosclerosis and endothelial dysfunction, the apoE KO mice [
      • Meyrelles S.S.
      • Peotta V.A.
      • Pereira T.M.
      • Vasquez E.C.
      Endothelial dysfunction in the apolipoprotein E-deficient mouse: insights into the influence of diet, gender and aging.
      ]. We found that administration of a single dose of rHDL-apoE3 in apoE KO mice led to significantly reduced plasma LDL levels accompanied by an increased concentration of cholesterol in the liver suggesting a fast clearance of LDL particles through binding of apoE to liver receptors. Triglycerides were transiently increased in mice that received rHDL-apoE3. These data are in line with a previous study which had shown that administration of an apoE4-expressing adenovirus in apoE KO mice reduced slightly the cholesterol levels and resulted in severe hypertriglyceridemia, due to accumulation of cholesterol and triglyceride-rich very low density lipoprotein (VLDL) particles in plasma [
      • Kypreos K.E.
      • van Dijk K.W.
      • van Der Zee A.
      • Havekes L.M.
      • Zannis V.I.
      Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxyl-terminal region 203-299 promotes hepatic very low density lipoprotein-triglyceride secretion.
      ]. The transient increase of triglycerides in our study could be due to the fast (4–8 h) clearance of apoE from the circulation in contrast to the prolonged expression of apoE in mice that received the adenoviruses. A previous study showed that a single dose injection of rHDL containing human apoA-I and phospholipids (CSL-111) in wild type mice was able to elicit significant but transient changes in plasma lipids. Specifically, there was a rapid increase in total cholesterol and triglycerides at 1 h post-injection which returned to normal levels at 24 h, and these changes in plasma lipids paralleled the levels of exogenous human apoA-I [
      • Chen Z.
      • O’Neill E.A.
      • Meurer R.D.
      • Gagen K.
      • Luell S.
      • Wang S.P.
      • et al.
      Reconstituted HDL elicits marked changes in plasma lipids following single-dose injection in C57Bl/6 mice.
      ]. These findings combined with our observations provide insights into the pharmacodynamics of rHDL particles containing apoE or apoA-I which could be valuable for the future development of novel therapeutics based on HDL.
      Importantly, a single injection of rHDL-apoE3 in the tail vein of apoE KO mice was able to improve vascular permeability as evidenced by the reduced concentration of the dye in certain tissues such as the stomach, the tongue and the urinary bladder (Fig. 9H). Evans Blue binds albumin and, in physiologic conditions, the endothelium is impermeable to albumin but in pathologic conditions, such as in apoE KO mice with hypercholesterolemia, the endothelium becomes permeable to albumin allowing the transfer of Evans Blue to adjacent tissues [
      • Radu M.
      • Chernoff J.
      An in vivo assay to test blood vessel permeability.
      ,
      • Bommel H.
      • Kleefeldt F.
      • Zernecke A.
      • Ghavampour S.
      • Wagner N.
      • Kuerten S.
      • et al.
      Visualization of endothelial barrier damage prior to formation of atherosclerotic plaques.
      ]. This improvement of vascular integrity by rHDL-apoE3 in mice could be attributed to the activation of signaling cascades involved in cell migration such as those we observed in our in vitro study of human ECs (Fig. 10), since endothelial cell migration is critical for vascular repair following injury and regeneration [
      • Evans C.E.
      • Iruela-Arispe M.L.
      • Zhao Y.Y.
      Mechanisms of endothelial regeneration and vascular repair and their application to regenerative medicine.
      ].
      Fig. 10
      Fig. 10Molecular mechanisms mediating the enhancement of EC migration by rHDL-apoE3. Through these mechanisms, rHDL-apoE3 is anticipated to promote re-endothelialization, improve vascular endothelial permeability, preserve vascular endothelial integrity and promote atheroprotection.
      Indeed, EC migration was the most highly and significantly changed function at the gene expression level. When assessed at the functional level, rHDL-apoE3 was confirmed to significantly and highly increase both HCAEC and EA.hy926 cell migration. This effect of rHDL-apoE3 is of heightened importance, because EC migration is a crucial process of endothelial repair, involved in the early stages of re-endothelialization of an injured atherosclerotic vessel [
      • Foteinos G.
      • Hu Y.
      • Xiao Q.
      • Metzler B.
      • Xu Q.
      Rapid endothelial turnover in atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein E-deficient mice.
      ]. EC migration to the injury sites enables their direct incorporation into neovessels, differentiation into mature vascular ECs and promotion of endothelium revascularization [
      • Kong D.
      • Melo L.G.
      • Gnecchi M.
      • Zhang L.
      • Mostoslavsky G.
      • Liew C.C.
      • et al.
      Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries.
      ]. Indeed, migration of circulating endothelial progenitor cells (EPC) to the injured endothelium has been inversely correlated with atherosclerosis and future cardiovascular risk, while impairment of endothelial repair in itself can lead to atherosclerosis [
      • Ross R.
      Atherosclerosis--an inflammatory disease.
      ,
      • Werner N.
      • Kosiol S.
      • Schiegl T.
      • Ahlers P.
      • Walenta K.
      • Link A.
      • et al.
      Circulating endothelial progenitor cells and cardiovascular outcomes.
      ]. Therefore, activation of EC migration, as achieved by rHDL-apoE3, is a highly desirable attribute for a novel ASCAD treatment.
      We proceeded to determine the molecular pathways through which rHDL-apoE3 activates EC migration, and the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways emerged as significantly changed at the transcriptome level. Based on the known biological role of these genes and the observed fold changes, these pathways were expected to be activated. Indeed, these predictions were confirmed at the protein level that revealed significant overexpression of multiple key proteins - PIK3CG, EFNB2, ID1 and FLT1 -, as well as at the activation level manifested by significantly increased phosphorylation of the main downstream effectors of the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways - ERK1/2, AKT, eNOS and p38 MAPK. Overexpression of PIK3CG, EFNB2, ID1 and FLT1 has been shown to promote EC migration through multiple downstream mechanisms. In specific, PIK3CG, EFNB2, ID1 and FLT1 has each been shown to activate AKT [
      • Su Y.
      • Gao L.
      • Teng L.
      • Wang Y.
      • Cui J.
      • Peng S.
      • et al.
      Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-kappaB/MMP-2 signaling pathways.
      ,
      • Siragusa M.
      • Katare R.
      • Meloni M.
      • Damilano F.
      • Hirsch E.
      • Emanueli C.
      • et al.
      Involvement of phosphoinositide 3-kinase gamma in angiogenesis and healing of experimental myocardial infarction in mice.
      ,
      • Steinle J.J.
      • Meininger C.J.
      • Forough R.
      • Wu G.
      • Wu M.H.
      • Granger H.J.
      Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway.
      ,
      • Wang F.
      • Yamauchi M.
      • Muramatsu M.
      • Osawa T.
      • Tsuchida R.
      • Shibuya M.
      RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway.
      ], PIK3CG and EFNB2 activate ERK1/2, eNOS and RAC1-GTPase [
      • Siragusa M.
      • Katare R.
      • Meloni M.
      • Damilano F.
      • Hirsch E.
      • Emanueli C.
      • et al.
      Involvement of phosphoinositide 3-kinase gamma in angiogenesis and healing of experimental myocardial infarction in mice.
      ,
      • Madeddu P.
      • Kraenkel N.
      • Barcelos L.S.
      • Siragusa M.
      • Campagnolo P.
      • Oikawa A.
      • et al.
      Phosphoinositide 3-kinase gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions.
      ,
      • Heller R.
      • Chang Q.
      • Ehrlich G.
      • Hsieh S.N.
      • Schoenwaelder S.M.
      • Kuhlencordt P.J.
      • et al.
      Overlapping and distinct roles for PI3Kbeta and gamma isoforms in S1P-induced migration of human and mouse endothelial cells.
      ,
      • Steinle J.J.
      • Meininger C.J.
      • Chowdhury U.
      • Wu G.
      • Granger H.J.
      Role of ephrin B2 in human retinal endothelial cell proliferation and migration.
      ,
      • Wang M.
      • Collins M.J.
      • Foster T.R.
      • Bai H.
      • Hashimoto T.
      • Santana J.M.
      • et al.
      Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase.
      ,
      • Bochenek M.L.
      • Dickinson S.
      • Astin J.W.
      • Adams R.H.
      • Nobes C.D.
      Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding.
      ], EFNB2 and ID1 activate MMP2/9 [
      • Steinle J.J.
      • Meininger C.J.
      • Forough R.
      • Wu G.
      • Wu M.H.
      • Granger H.J.
      Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway.
      ] [
      • Sakurai D.
      • Tsuchiya N.
      • Yamaguchi A.
      • Okaji Y.
      • Tsuno N.H.
      • Kobata T.
      • et al.
      Crucial role of inhibitor of DNA binding/differentiation in the vascular endothelial growth factor-induced activation and angiogenic processes of human endothelial cells.
      ], and FLT1 activates RAC1-GTPase and p38 MAPK [
      • Wang F.
      • Yamauchi M.
      • Muramatsu M.
      • Osawa T.
      • Tsuchida R.
      • Shibuya M.
      RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway.
      ,
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • et al.
      Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.
      ]. In agreement with these data, we showed that rHDL-apoE3 leads to activation of ERK1/2, AKT, eNOS, and p38 MAPK in human ECs. Activated ERK1/2 phosphorylates MLCK, calpain or FAK, which regulate actin cytoskeleton re-organization, lamellipodia formation and myosin contraction, ultimately stimulating EC migration [
      • Huang C.
      • Jacobson K.
      • Schaller M.D.
      MAP kinases and cell migration.
      ]. Furthermore, ERK1/2 activation is involved in localized degradation of the endothelial extracellular matrix, a prerequisite for EC migration [
      • Munoz-Chapuli R.
      • Quesada A.R.
      • Angel Medina M.
      Angiogenesis and signal transduction in endothelial cells.
      ]. Similarly, phosphorylated AKT can activate eNOS leading to nitric oxide (NO) production and subsequent cytoskeletal re-organization through phosphorylation of FAK and Src kinases [
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.
      ,
      • Donnini S.
      • Ziche M.
      Constitutive and inducible nitric oxide synthase: role in angiogenesis.
      ], while phosphorylated RAC1-GTPase can activate p38 MAPK, which in turn stimulates lamellipodia formation [
      • Varon C.
      • Rottiers P.
      • Ezan J.
      • Reuzeau E.
      • Basoni C.
      • Kramer I.
      • et al.
      TGFbeta1 regulates endothelial cell spreading and hypertrophy through a Rac-p38-mediated pathway.
      ,
      • Rousseau S.
      • Houle F.
      • Landry J.
      • Huot J.
      p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells.
      ]. Therefore, the activation of the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways through the modulation of the described molecules - PIK3CG, EFNB2, ID1, FLT1, ERK1/2, AKT, eNOS, and p38 MAPK - may provide the mechanisms through which rHDL-apoE3 promotes EC migration.
      Since EC migration can be regulated by alternative pathways, while ERK1/2, AKT, eNOS and p38 MAPK are known to be implicated in a multitude of different EC functions besides migration [
      • Munoz-Chapuli R.
      • Quesada A.R.
      • Angel Medina M.
      Angiogenesis and signal transduction in endothelial cells.
      ], we proceeded to determine whether the specific molecules are the downstream effectors of the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways that mediate rHDL-apoE3-induced EC migration. Through direct inhibition of MEK1/2 which is the main ERK1/2 activator [
      • Santos E.
      • Crespo P.
      The RAS-ERK pathway: a route for couples.
      ], and PI3K which is the direct activator of AKT and an indirect activator of eNOS and p38 MAPK [
      • Hemmings B.A.
      • Restuccia D.F.
      PI3K-PKB/Akt pathway.
      ,
      • Kimura T.
      • Sato K.
      • Malchinkhuu E.
      • Tomura H.
      • Tamama K.
      • Kuwabara A.
      • et al.
      High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors.
      ], we showed a dramatic impact on the rHDL-apoE3-mediated phosphorylation of ERK1/2, AKT and p38 MAPK. Furthermore, MEK1/2 inhibition completely prevented rHDL-apoE3-induced EC migration, while PI3K inhibition led to a marked reduction. These findings indicate that MEK1/2 and PI3K phosphorylation is required for the rHDL-apoE3-mediated activation of ERK1/2, AKT and p38 MAPK, respectively, and that these mechanisms are necessary for the induction of EC migration by rHDL-apoE3 (Fig. 10).
      On the contrary, the inhibition of PI3K did not affect eNOS activation by rHDL-apoE3, suggesting that PI3K-independent mechanisms are involved. This is in agreement with previous studies showing that eNOS can be phosphorylated by several other kinases, such as PKA and PKG [
      • Boo Y.C.
      • Sorescu G.
      • Boyd N.
      • Shiojima I.
      • Walsh K.
      • Du J.
      • et al.
      Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.
      ]. Taking into consideration that PI3K inhibition markedly attenuated rHDL-apoE3-induced EC migration, independently of eNOS phosphorylation, it is interesting to speculate that eNOS activation may not be necessary for the induction of EC migration by rHDL-apoE3. Nevertheless, the detected increase of eNOS activation by rHDL-apoE3 may contribute to other atheroprotective functions, since eNOS-derived NO promotes endothelial monolayer integrity and attenuates EC–leukocyte adhesion [
      • Shaul P.W.
      Regulation of endothelial nitric oxide synthase: location, location, location.
      ].
      In search of the upstream regulator(s) mediating the rHDL-apoE3-triggered activation of EC migration, we focused on the transcription factor ID1, as it was significantly overexpressed by rHDL-apoE3 treatment of human ECs, and had been previously associated with PI3K-mediated AKT phosphorylation and increased EC migration [
      • Su Y.
      • Gao L.
      • Teng L.
      • Wang Y.
      • Cui J.
      • Peng S.
      • et al.
      Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-kappaB/MMP-2 signaling pathways.
      ,
      • Sakurai D.
      • Tsuchiya N.
      • Yamaguchi A.
      • Okaji Y.
      • Tsuno N.H.
      • Kobata T.
      • et al.
      Crucial role of inhibitor of DNA binding/differentiation in the vascular endothelial growth factor-induced activation and angiogenic processes of human endothelial cells.
      ]. Indeed, upon ID1 silencing the effect of rHDL-apoE3 on the activation of MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways was significantly hampered, as evidenced by the inhibition of rHDL-apoE3-induced ERK1/2 and AKT phosphorylation. Importantly, silencing of ID1 significantly reduced the pro-migratory effect of rHDL-apoE3, without completely abolishing it. This suggests that ID1 is a major, but not an exclusive upstream regulator of rHDL-apoE3-induced EC migration (Fig. 10). The effect of rHDL-apoE3 on ID1 is of great significance because ID1 has been shown to play a direct role in the regulation of EC migration, proliferation, differentiation and survival, processes that are crucial for re-endothelialization and vascular repair [
      • Su Y.
      • Gao L.
      • Teng L.
      • Wang Y.
      • Cui J.
      • Peng S.
      • et al.
      Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-kappaB/MMP-2 signaling pathways.
      ,
      • Yu Y.
      • Liang Y.
      • Liu X.
      • Yang H.
      • Su Y.
      • Xia X.
      • et al.
      Id1 modulates endothelial progenitor cells function through relieving the E2-2-mediated repression of FGFR1 and VEGFR2 in vitro.
      ,
      • Li W.
      • Du D.
      • Li Y.
      Id-1 promotes reendothelialization in the early phase after vascular injury through activation of NFkB/survivin signaling pathway.
      ]. Based on these findings, it has been proposed that ID1 may represent a promising therapeutic target for endothelium repair [
      • Yu Y.
      • Liang Y.
      • Liu X.
      • Yang H.
      • Su Y.
      • Xia X.
      • et al.
      Id1 modulates endothelial progenitor cells function through relieving the E2-2-mediated repression of FGFR1 and VEGFR2 in vitro.
      ,
      • Li W.
      • Du D.
      • Li Y.
      Id-1 promotes reendothelialization in the early phase after vascular injury through activation of NFkB/survivin signaling pathway.
      ]. Additional studies indicated that ID1 overexpression promoted rapid re-endothelialization and vascular repair in a rat model of carotid artery injury [
      • Li W.
      • Du D.
      • Li Y.
      Id-1 promotes reendothelialization in the early phase after vascular injury through activation of NFkB/survivin signaling pathway.
      ]. Furthermore, Id1-deficient (Id1 −/−) mice displayed increased vascular permeability and endothelial apoptosis in their lungs following injury [
      • Zhang H.
      • Lawson W.E.
      • Polosukhin V.V.
      • Pozzi A.
      • Blackwell T.S.
      • Litingtung Y.
      • et al.
      Inhibitor of differentiation 1 promotes endothelial survival in a bleomycin model of lung injury in mice.
      ]. Collectively these findings indicate that ID1 may represent a major mediator of the rHDL-apoE3 effects on EC migration, and support the notion that rHDL-apoE3 could promote re-endothelialization and vascular endothelial repair. However, since ID1 has also been associated with tumorigenesis [
      • Zhao Z.
      • Bo Z.
      • Gong W.
      • Guo Y.
      Inhibitor of differentiation 1 (Id1) in cancer and cancer therapy.
      ], this parameter should be taken into consideration in future preclinical and clinical studies evaluating rHDL-apoE3.
      It is interesting to speculate on the mechanism through which rHDL-apoE3 activates the observed molecular EC migration-related cascades. A previous study showed that rHDL-apoA-I (apoA-I/POPC/cholesterol complexes) interacts with the scavenger receptor SR-BI in bovine aortic ECs (BAEC) leading to migration through activation of the Src/PI3K/AKT/RAC-GTPase and Src/PI3K/MEK/ERK/RAC-GTPase pathways. Importantly, rHDL-apoA-I signaling through SR-BI enhanced re-endothelialization in a mouse model of carotid artery injury [
      • Seetharam D.
      • Mineo C.
      • Gormley A.K.
      • Gibson L.L.
      • Vongpatanasin W.
      • Chambliss K.L.
      • et al.
      High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I.
      ]. rHDL-apoE3 (apoE3/POPC/cholesterol complexes) was also shown to interact with SR-BI in vitro [
      • Chroni A.
      • Nieland T.J.
      • Kypreos K.E.
      • Krieger M.
      • Zannis V.I.
      SR-BI mediates cholesterol efflux via its interactions with lipid-bound ApoE. Structural mutations in SR-BI diminish cholesterol efflux.
      ]. Furthermore, apoE3 was demonstrated to activate the PI3K/AKT/eNOS pathway through binding to the ApoER2 receptor, leading to BAEC migration. The same study showed that adenovirus-mediated administration of human apoE3 in a mouse model of carotid artery injury accelerated re-endothelialization [
      • Ulrich V.
      • Konaniah E.S.
      • Herz J.
      • Gerard R.D.
      • Jung E.
      • Yuhanna I.S.
      • et al.
      Genetic variants of ApoE and ApoER2 differentially modulate endothelial function.
      ]. These findings combined with our in vitro and in vivo observations support the hypothesis that rHDL-apoE3 could promote activation of EC migration-related signaling pathways, at least in part, through SR-BI and/or ApoER2.
      The value of the observed pro-migratory effect of rHDL-apoE3 for therapeutic purposes is supported by preclinical and clinical studies involving atheroprotective drugs or therapeutic approaches under clinical trials including statins, PCSK9i, ezetimibe and rHDL-apoA-I. Specifically, in vitro as well as in vivo studies have demonstrated that these agents promote EC migration through signaling pathways similar to the pathways activated by rHDL-apoE3, and enhance re-endothelialization in mammalian models of arterial injury or atherosclerosis, as well as endothelial repair in CAD patients [
      • Seetharam D.
      • Mineo C.
      • Gormley A.K.
      • Gibson L.L.
      • Vongpatanasin W.
      • Chambliss K.L.
      • et al.
      High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I.
      ,
      • Meyer N.
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      • von Versen-Hoynck F.
      Pravastatin promotes endothelial colony-forming cell function, angiogenic signaling and protein expression in vitro.
      ,
      • Lee C.H.
      • Chang S.H.
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      • Liu S.J.
      • Wang C.J.
      • Hsu M.Y.
      • et al.
      Acceleration of re-endothelialization and inhibition of neointimal formation using hybrid biodegradable nanofibrous rosuvastatin-loaded stents.
      ,
      • Honda K.
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      • et al.
      Lipid-lowering therapy with ezetimibe decreases spontaneous atherothrombotic occlusions in a rabbit model of plaque erosion: a role of serum oxysterols.
      ,
      • Schmidt-Lucke C.
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      Improvement of endothelial damage and regeneration indexes in patients with coronary artery disease after 4 weeks of statin therapy.
      ,
      • Itzhaki Ben Zadok O.
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      ]. These processes are essential for prevention and treatment of atherosclerosis.
      It should be noted that our focus was on EC migration, nevertheless, a number of other atheroprotective molecular mechanisms were also significantly changed by rHDL-apoE3 and should be further investigated to unveil the full spectrum of rHDL-apoE3 effects. Furthermore, we obtained preliminary evidence in favor of the atheroprotective properties of rHDL-apoE3 in vivo using a well-characterized model of hypercholesterolemia, endothelial dysfunction and atherosclerosis, the apoE KO mice. Importantly, administration of a single dose of rHDL-apoE3 in apoE KO mice was able to ameliorate hypercholesterolemia and to improve vascular permeability. The capacity of rHDL-apoE3 to stimulate human EC migration in vitro and to improve vascular permeability along with its cholesterol-lowering properties in vivo suggests its heightened potential to promote vascular endothelial repair and to improve hypercholesterolemia in ASCAD patients, respectively. However, in depth studies will be needed to evaluate the full effects of rHDL-apoE3 in vivo, including its long-term effects on hypercholesterolemia, endothelial repair and atherosclerotic plaque development. The present findings also merit further investigation in additional animal models of hypercholesterolemia, endothelial dysfunction and atherosclerosis, as well as extended characterization of the molecular mechanisms mediating the rHDL-apoE3 effects.
      In conclusion, rHDL-apoE3 has a significant pro-migratory effect on human ECs, which predominantly involves the MEK/ERK, PI3K/AKT/eNOS-MMP2/9 and RHO-GTPases pathways, requires the activation of ERK1/2 and AKT, and is further facilitated by p38 MAPK. The central mediator of the rHDL-apoE3 effect on these pathways is ID1, a molecule that may play a significant role in atheroprotection (Fig. 10). Importantly, rHDL-apoE3 markedly improved vascular permeability and ameliorated hypercholesterolemia in apoE KO mice. The current novel insights on the role of rHDL-apoE3 functions suggest its important potential to promote re-endothelialization and preservation of endothelial integrity, as well as to improve hypercholesterolemia that may find clinical applications in ASCAD in the near future.
      Fig. S1
      Fig. S1rHDL-apoE3 promotes migration of EA.hy926 cells. (A) EA.hy926 cells were either left untreated (baseline control of cell migration) or treated with rHDL-apoE3 or rhVEGF (positive control of cell migration) in the upper chamber of the transwell insert for 20 h. Representative transwell migration assay microscopic images of EA.hy926 cell migration at 20 h are shown. (B) The number of migrated cells to the lower side of the transwell membrane after 20 h was quantified. The results are presented as % relative to the cell migration of the baseline control. Values are expressed as mean ± SEM (n = 3 biological replicates per group). **P ≤ 0.01, ***P ≤ 0.001.
      Fig. S2
      Fig. S2siRNA-mediated ID1 silencing decreases ID1 protein levels in EA.hy926 cells. EA.hy926 cells were transfected with ID1 siRNA or control siRNA for 48 h (A) and 72 h (C) or were mock-transfected or non-transfected, and ID1 protein expression was assessed by Western blotting using β-actin as a loading control. (E, G) EA.hy926 cells were transfected with ID1 siRNA or control siRNA for 48 h or 72 h and then treated with rHDL-apoE3 or PBS for 24 h (i.e. 72 h or 96 h of processing time in total). 72 h (E) and 96 h (G) post-transfection, ID1 protein levels were measured by Western blotting using β-actin as a loading control. The band density of ID1 was normalized to the β-actin bands and presented relative to the protein levels of the non-transfected control (B, D) or the Control siRNA + PBS group (F, H). Values are expressed as mean ± SEM of one or two independent experiments performed in biological triplicates (n = 3 to 6 biological replicates per group). *P ≤ 0.05, ***P ≤ 0.001, and ###P ≤ 0.001 between ID1 siRNA and control siRNA transfected cells.
      Fig. S3
      Fig. S3Serum protein analysis in rHDL-apoE3 treated and control (CTR) apoE KO mice. (A) Equal volumes of mouse sera at various time intervals post-injection (n = 3/group/pooled serum) were analyzed by SDS-PAGE and Coomassie Brilliant blue staining for protein visualization. The position of human apoE3 and mouse apoA-I protein in serum of rHDLapoE3 treated mice is indicated with an arrow and an asterisk, respectively. (B–C) Equal volumes of the FPLC fractions 10–37 of rHDL-apoE3 treated and control mice corresponding to LDL and HDL lipoproteins were analyzed by SDS-PAGE. Gels were stained with Coomassie dye and blotted against human apoE and mouse apoA-I proteins in control (B) and rHDL-apoE3 treated (C) groups. FPLC analysis of plasma lipoproteins was performed in pooled serum samples (n = 3/group/pool) of rHDL-apoE3 treated and control mice 24 h post-injection.

      Funding sources

      The authors were supported by: (a) a grant from the Ministry of Education, Lifelong Learning and Religious Affairs of Greece (THALIS MIS 377286 ) (DS, EKV, AC, DK); (b) the grant “In vitro and in vivo evaluation of the atheroprotective role of reconstituted HDL containing apolipoprotein E3” ( MIS 5006782 ) (DS, EKV, KDK), which was co-financed by Greece and the European Union (European Social Fund - ESF) through the “ Operational Programme Human Resources Development, Education and Lifelong Learning 2014–2020 ”; (c) the CURE-PLaN grant from the Leducq Foundation for Cardiovascular Research (DS, EV, EKV); (d) the Alexander S. Onassis Public Benefit Foundation (EKV).

      CRediT authorship contribution statement

      Eftaxia-Konstantina Valanti: Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Visualization. Katerina Dalakoura-Karagkouni: Methodology, Validation, Formal analysis, Investigation, Visualization. Panagiotis Fotakis: Investigation. Elizabeth Vafiadaki: Methodology. Christos S. Mantzoros: Writing – review & editing. Angeliki Chroni: Methodology, Resources, Writing – review & editing. Vassilis Zannis: Conceptualization, Writing – review & editing, Supervision, Funding acquisition. Dimitris Kardassis: Conceptualization, Methodology, Resources, Writing – original draft, Supervision, Funding acquisition. Despina Sanoudou: Conceptualization, Writing – review & editing, Supervision, Project administration, Funding acquisition.

      Declaration of competing interest

      The authors declare no conflict of interest.

      Acknowledgements

      We would like to thank Drs Ioannis Dafnis and Christina Gkolfinopoulou and Christina Mountaki for their valuable assistance with apoE3 production/purification and rHDL-apoE3 particle formation protocols, and Dr. Dimitris Theofilatos for advice on the wound healing assays. We also thank Professor Aristides Eliopoulos for expert guidance with siRNA protocols.

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