Regulation of HDL dysfunctionality by phosphatidylethanolamine links poly-unsaturated fatty acids with atherosclerotic cardiovascular diseases

Figures

• 

  Figure 1. Total PE and PE (36:5) content of HDL2 was associated with atherosclerosis in women with Metabolic Syndrome. Human HDL2 particles were isolated from plasma of 86 women with Metabolic Syndrome by sequential ultracentrifugation for 48 h. Total PE and PE (36:5) contents in HDL2 was quantified by LC-ESI/MS/MS. Carotid intima-media thickness (cIMT) was evaluated by ultrasonography and correlated with total PE and PE (36:5) content in HDL2. A. Unadjusted and adjusted linear regression analysis for the mean cIMT (in mm) according to total PE content of HDL2 B. Unadjusted and adjusted linear regression analysis for the mean cIMT (mm) according to PE (36:5) content of HDL2. The analysis was adjusted for traditional risk factors, including age, diabetes mellitus, hypertension, smoking, plasma total cholesterol and triglycerides levels. HDL: high density lipoprotein; PE: Phosphatidylethanolamine.

• 

  Figure 2. Injection of ARA-PE rHDL did not reduce the size of aortic atherosclerotic plaques as compared to Soy-PC rHDL in CETP transgenic-LDLr KO female mice. A. CETP transgenic/LDL receptor KO female mice fed a high cholesterol diet for 8 weeks were injected with 15 mg/kg of Soy-PC rHDL (n = 5) or ARA-PE rHDL (n = 6), retro-orbitally, every second day. The mice received 10 injections of either treatment, and were maintained on a chow diet during rHDL treatment. B. Sections of aortic root at the level of the three valves were labeled with hematoxylin-eosin to quantify the atherosclerotic plaque size. The extent of the atherosclerotic plaque was quantified using ImageJ software. Values are shown as mean (±SEM), n ≥ 5, ∗p < 0.001 vs. Soy-PC rHDL. CETP: cholesteryl ester transfer protein; HC: high cholesterol; rHDL: reconstituted high density lipoprotein; PC: phosphatidylcholine; ARE-PE: arachidonic acid-phosphatidylethanolamine.

• 

  Figure 3. The presence of Soy-PE, ARA-PE or EPA-PE alters the biological activities of Soy-PC rHDL. A. The phospholipidome composition of the rHDLs determined by LC/MS–MS. B. The CEC of rHDLs was determined using THP-1 macrophages loaded with radiolabeled [3H] cholesterol and incubated with 5, 10, 20, or 50 μg ApoA-1/mL of Soy-PC, Soy-PE, ARA-PE, and EPA-PE rHDLs for 4 h. C. DCFH fluorescent probe was used to assess the oxidation kinetics of LDL induced by copper sulfate (CuSO4) in the presence of Soy-PC, Soy-PE, ARA-PE, or EPA-PE rHDLs (50 μg ApoA-I/ml), or none for 24 h at 37C° using Spectromax Gemini fluorometer. D. Maximum DCF (oxidized DCFH) formed after 24 h. E. Oxidation rate during the propagation phase. F. Lag phase duration, G. Oxidability of rHDL expressed as relative fluorescence units (RFU) for different rHDLs (50 μg ApoA-I/ml), incubated with CuSO4, and DCFH (0.2 mg/mL) in the absence of LDL. ∗vs. LDL + Cu+2, #vs. Soy-PC rHDL, ϕvs. Soy-PE rHDL δvs. ARA-PE rHDL with p values < 0.05. Values are shown as mean ± SEM from at least 3 independent experiments that were performed in triplicate for each condition.

• 

  Figure 4. Soy-PE diminished the capacity of rHDL to accept phospholipids from LDL, while ARA-PE and EPA-PE reversed this effect, potentially by enhancing the fluidity of HDL lipids. A. The percentage of Dil PL transferred from DiI labelled LDL to rHDLs. To evaluate the capacity of rHDLs to acquire phospholipids (PL), Dil-Labelled LDL was mixed with rHDL (4 mg ApoA-I/dL), or reference ApoB deficient plasma (1:30 v/v), and incubated for 1 h at 37 °C to achieve PL transfer. Then, mixtures were ApoB-depleted and the fluorescence intensity of Dil was measured in rHDL. For the fluidity assay, DPH or TMA-DPH were incorporated in rHDL particles, and the anisotropy of the probe fluorescence was recorded. B. Fluorescence anisotropy of TMA-DPH probe, which is inversely related to surface lipid fluidity of rHDL. C. Fluorescence anisotropy of DPH probe, which is inversely related to core lipid fluidity of rHDL. D. Scheme illustrates the influence of Soy-PE, ARA-PE and EPA-PE on the arrangement of the phospholipid monolayer of rHDLs, according to their head group and the unsaturation level in their fatty acid moieties. Our data indicate that Soy-PE reduced the capacity of rHDL to accept phospholipids…

• 

  Figure 5. Soy-PE reduced the AIA of rHDL, while ARA-PE endowed rHDL with pro-inflammatory activity with EPA-PE reversing it and improving the AIA of rHDL. AIA of rHDLs was evaluated in LPS-stimulated THP-1 macrophages A. Relative mRNA and protein levels of IL-1β after short-term (4 h), and B. long-term (16 h) treatment of THP-1 cells with 20 μg ApoA-I/mL of different rHDL followed by 4 h stimulation with 100 ng/mL of LPS. C-E. Western blot analysis for the phosphorylated and non-phosphorylated ERK, p38, and JNK MAPKs in THP-1 cells treated with rHDLs (20 μg ApoA-I/mL) of for 4 h, followed by 0.5 h stimulation with 100 ng/mL of LPS. ∗vs. LPS, #vs. Soy-PC rHDL, ϕvs. Soy-PE rHDL, δvs. ARA-PE rHDL, with p values < 0.05. Values shown are from at least 3 independent experiments that were performed in triplicate for each condition. IL-1β: Interleukin-1 beta; LPS: lipopolysaccharide; JNK: Jun kinase; ERK: Extracellular signal-regulated kinase; MAPK: mitogen-activated protein kinase.

• 

  Figure 6. ARA-PE exacerbated Soy-PE rHDL pro-inflammatory effects while EPA-PE reversed it and even improved the basal AIA of rHDL. A. Pro-inflammatory eicosanoid production after 16 h treatment of THP-1 cells with 20 μg/mL of rHDLs (20 μg APOA-1/mL). The pro-inflammatory effects of rHDLs were evaluated in THP-1 macrophages in the absence of LPS stimulation. B. Relative mRNA and protein levels of IL-1β after long-term (16 h) treatment of THP-1 cells with 20 μg ApoA-I/mL of rHDL.∗vs. None, #vs. Soy-PC rHDL, ϕvs. Soy-PE rHDL, δvs. ARA-PE rHDL, with p values less than 0.05. Values are from at least 3 independent experiments that were performed in triplicate for each condition. HETE: hydroxyeicosatetraenoic acids; PGE2: prostaglandin E2, TXB2: thromboxane B2, LOX: lipoxygenase, and COX: cyclooxygenase enzymes.