- •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.
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)
2. Materials and methods
2.1 Cell cultures
2.2 Whole transcriptome microarray experiments
- Tsompanidis A.
- Vafiadaki E.
- Bluher S.
- Kalozoumi G.
- Sanoudou D.
- Mantzoros C.S.
2.3 Microarray data analysis
2.4 High-throughput quantitative Real-Time PCR (qRT-PCR)
2.5 Protein expression and phosphorylation analysis
2.6 Wound healing assays
- Gkolfinopoulou C.
- Soukou F.
- Dafnis I.
- Kellici T.F.
- Sanoudou D.
- Mavromoustakos T.
- et al.
2.7 Transwell migration assay
2.8 Analysis of the effects of kinase inhibitors on EC migration and protein phosphorylation
2.9 siRNA silencing
2.11 Blood sampling and serum parameters
2.12 Fast protein liquid chromatography (FPLC)
2.13 Histological examination of the liver
2.14 Vascular permeability assays
2.15 Statistical analysis
3.1 rHDL-apoE3 induces distinct gene expression changes in HAEC
3.2 rHDL-apoE3 affects multiple biological functions associated with atheroprotection
3.3 qRT-PCR validation of microarray data
3.4 rHDL-apoE3 induces the expression of key EC migration-associated proteins in HCAEC and EA.hy926 cells
3.5 rHDL-apoE3 activates EC migration-related molecules in HCAEC and EA.hy926 cells
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
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
3.8 siRNA-mediated silencing of ID1
3.9 Induction of EA.hy926 cell migration by rHDL-apoE3 is mediated by ID1
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
3.11 Administration of rHDL-apoE3 in apoE KO mice improves vascular permeability and ameliorates hypercholesterolemia
Supplementary Materials and Methods
CRediT authorship contribution statement
Declaration of competing interest
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