Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation

Abstract

Passage of apolipoprotein B-containing lipoproteins (apoB-LPs), i.e., triglyceride-rich lipoproteins (TRLs), intermediate-density lipoproteins (IDLs), and low-density lipoproteins (LDLs), through the endothelial monolayer occurs in normal and atherosclerotic arteries. Among these lipoproteins, TRLs and IDLs are apoE-rich apoB-LPs (E/B-LPs). Recycling of TRL-associated apoE has been shown to form apoE-carrying high-density lipoprotein (HDL)-like (HDL_E) particles in many types of cells. The current report studied the formation of HDL_E particles by transcytosis of apoB-LPs through mouse aortic endothelial cells (MAECs). Our data indicated that passage of radiolabeled apoB-LPs, rich or poor in apoE, through the MAEC monolayer is inhibited by filipin and unlabeled competitor lipoproteins, suggesting that MAECs transport apoB-LPs via a caveolae-mediated pathway. The cholesterol and apoE in the cell-untreated E/B-LPs, TRLs, IDLs, and LDLs distributed primarily in the low-density (LD) fractions (d ≤ 1.063). A substantial portion of the cholesterol and apoE that passed through the MAEC monolayer was allotted into the high-density (HD) (d > 1.063) fractions. In contrast, apoB was detectable only in the LD fractions before or after apoB-LPs were incubated with the MAEC monolayer, suggesting that apoB-LPs pass through the MAEC monolayer in the forms of apoB-containing LD particles and apoE-containing HD particles.

Keywords: apolipoprotein B-containing lipoprotein; apolipoprotein E; high-density lipoprotein formation; mouse aortic endothelial cells; transendothelial transport.

Figure 1

Transendothelial transport of radiolabeled apoB-LPs is inhibited by filipin and unlabeled competitor lipoproteins. (A): The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of ³H-Ch-labeled E/B-LPs (³H-Ch E/B) or ³H-Ch-labeled LDLs (³H-Ch LDL) in the presence of 800 µg/mL of unlabeled E/B-LPs (uE/B) or unlabeled LDLs (uLDLs) or in the absence of unlabeled lipoproteins as a control (Ctrl) for 24 h. (B): The AP side of the MAEC monolayer was pre-incubated with 20 µg/mL chlorpromazine, 5 µg/mL filipin, or 1 µL/mL dimethyl sulfoxide as a Ctrl for 1 h, followed by incubation with 20 µg/mL (167,000 DPM) of ¹²⁵I-labeled TRLs, IDLs, or LDLs for 24 h. The radioactivity in the BL medium were counted. Values represent the mean ± SE of 3–5 experiments; * p < 0.05 vs. Ctrl.

Figure 2

The free/total cholesterol ratio of transported apoB-LPs remains unchanged. The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of ³H-Ch-labeld TRLs, IDLs, or LDLs for 24 h. The free ³H-Ch and esterified ³H-Ch in the AP and BL media and cell lysates, as well as in the cell-untreated TRLs, IDLs, and LDLs (control), were separated by digitonin precipitation and assessed by scintillation counting. The percentage of free to total ³H-Ch was calculated as follows: (free ³H-Ch/(esterified ³H-Ch + free ³H-Ch)) × 100. Values represent the mean ± SE of three experiments; * p < 0.05 vs. control.

Figure 3

Transendothelial transport of apoB-LPs resulted in cholesterol redistribution among lipoprotein fractions. The AP side of the MAEC monolayer was incubated with 20 µg/mL of ³H-Ch-labeld E/B-LPs (E/B) (A), TRLs (D), IDLs (E), or LDLs (F); the BL side was incubated with medium alone for 24 h. The AP and BL media (Mdm), and 20 µg of cell-untreated control ³H-Ch-labeled lipoproteins (i.e., Ctrl E/B, TRL, IDL, or LDL), were subjected to DG ultracentrifugation. Twenty fractions were collected for determination of density and radioactivity. The level of ³H-Ch was expressed by radioactivity counts: 1/100, 1/50, 1/10 (radioactivity counts in Ctrl lipoproteins and AP medium were divided by 100, 50, or 10). A representative density curve was presented in panel A. The arrow pointing to the density curve indicated d = 1.063 g/mL. (B): The level of ³H-Ch in the HD fractions of the AP and BL media and control lipoproteins (Ctrl) was expressed as the % of the HD ³H-Ch in the total ³H-Ch (HD/total ³H-Ch), which was calculated as follows: (The radioactivity in fractions 10–20/the radioactivity in fractions 1–20) × 100. (C): The average density of the lipoproteins in low density fractions (LDF), i.e., fractions 1–9, was calculated as follows: (${{\displaystyle \sum }}_{i=1}^{9}DiRi/{{\displaystyle \sum }}_{i=1}^{9}Ri$), where D and R are the hydrated density and radioactivity count of each fraction, respectively. Values represent the mean ± SE of four experiments; * p < 0.05 vs. the Ctrl EB-LPs, TRLs, IDLs, or LDLs; ^† p < 0.05 vs. BL medium containing ³H-Ch EB-LPs, TRLs, or IDLs.

Figure 4

Transendothelial transport of apoB-LPs induced redistribution of apoE, but not apoB, among lipoprotein fractions. The AP side of the MAEC monolayer was incubated with 20 µg/mL of E/B-LPs (E/B) (A,C) or LDLs (B,D); the BL side was incubated with medium alone for 24 h. The AP and BL media, and 20 µg of cell-untreated Ctrl E/B or LDLs, were subjected to DG ultracentrifugation. Fractions 1–9 and 10–20 were pooled separately and designated as LD and HD fractions, respectively. Proteins in the LD and HD fractions and the cell lysate were precipitated with 75% methanol for Western blot analysis of apoB and apoE. The protein in the BL medium was pooled from six BL chambers. The level of apoE was expressed by the immunoblot band intensity (C,D). (E): The % of apoE in the HD fraction to the total apoE (HD/total apoE) in the AP and BL media, and the Ctrl E/B and LDLs, was calculated as follows: (apoE in the HD fraction/(apoE in the HD fraction + apoE in the LD fraction)) × 100. Values represent the mean ± SE of three independent experiments; * p < 0.05 vs. the Ctrl EB-LPs or LDLs, and ^† p < 0.05 vs. AP medium.

Figure 5

Transendothelial transport of apoB-LPs resulted in apoE redistribution among lipoprotein fractions. The AP side of the MAEC monolayer was incubated with ¹²⁵I-apoE E/B-LPs (E/B) (A–C) or TRLs (D–F); the BL side was treated with medium alone for 24 h (1st 24 h). (B,E): The MAEC monolayer, treated as above, was washed with heparin solution (B), or low ionic strength solution (E), and followed by incubation with fresh medium for another 24 h (2nd 24 h). The AP and BL media collected from the 1st 24 h and the 2nd 24 h incubations, as well as the cell-untreated Ctrl ¹²⁵I-apoE E/B-LPs and TRLs, were subjected to DG ultracentrifugation. Twenty fractions were collected for determination of radioactivity. (C,F): The % of the HD fraction ¹²⁵I-apoE in the total ¹²⁵I-apoE of the AP and BL media, and the Ctrl E/B-LPs and TRLs (HD/total ¹²⁵I-apoE) was calculated using the following equation: (The radioactivity in fractions 10–20/the radioactivity in fractions 1–20) × 100. Values represent the mean ± SE of three experiments; * p < 0.05 vs. the cell-untreated Ctrl E/B-LPs or TRLs, and ^† p < 0.05 vs. AP medium.

Scheme 1

Schematic of the association of transendothelial transport of apoB-LPs with HDL_E formation.