Pathway of biogenesis of apolipoprotein E-containing HDL in vivo with the participation of ABCA1 and LCAT

Abstract

We have investigated the ability of apoE (apolipoprotein E) to participate in the biogenesis of HDL (high-density lipoprotein) particles in vivo using adenovirus-mediated gene transfer in apoA-I-/- (apolipoprotein A-I) or ABCA1-/- (ATP-binding cassette A1) mice. Infection of apoA-I-/- mice with 2x10(9) pfu (plaque-forming units) of an apoE4-expressing adenovirus increased both HDL and the triacylglycerol-rich VLDL (very-low-density lipoprotein)/IDL (intermediate-density lipoprotein)/LDL (low-density lipoprotein) fraction and generated discoidal HDL particles. ABCA1-/- mice treated similarly failed to form HDL particles, suggesting that ABCA1 is essential for the generation of apoE-containing HDL. Combined infection of apoA-I-/- mice with a mixture of adenoviruses expressing both apoE4 (2x10(9) pfu) and human LCAT (lecithin:cholesterol acyltransferase) (5x10(8) pfu) cleared the triacylglycerol-rich lipoproteins, increased HDL and converted the discoidal HDL into spherical HDL. Similarly, co-infection of apoE-/- mice with apoE4 and human LCAT corrected the hypercholesterolaemia and generated spherical particles, suggesting that LCAT is essential for the maturation of apoE-containing HDL. Overall, the findings indicate that apoE has a dual functionality. In addition to its documented functions in the clearance of triacylglycerol-rich lipoproteins, it participates in the biogenesis of HDL-sized apoE-containing particles. HDL particles generated by this pathway may account at least for some of the atheroprotective functions of apoE.

Figure 1. Cholesterol FPLC profiles, apoE distribution and electron microscopy analyses of HDL fractions of control apoA-I^−/− mice and apoA-I^−/− mice infected with recombinant adenoviruses expressing GFP or apoE4 alone or a combination of apoE4 and human LCAT

(A–C) FPLC profiles. (D–F) ApoE distribution among different densities (d). (G–I) Electron micrographs of fractions 6–8 of HDL. Particle diameters (d) are given. (A, D, G) Analyses of samples obtained from apoA-I^−/− mice infected with 2×10⁹ pfu of control adenoviruses expressing GFP (AdGFP). (B, E, H) Analyses of samples obtained from apoA-I^−/− mice infected with 2×10⁹ pfu of adenoviruses expressing apoE4 (AdGFP-E4). (C, F, I) Analyses of samples obtained from apoA-I^−/− mice infected with a mixture of adenoviruses expressing apoE4 (AdGFP-E4) (2×10⁹ pfu) and human LCAT (Ad-LCAT) (5×10⁸ pfu). CE/TC, cholesteryl ester/total cholesterol ratio.

Figure 2. Cholesterol FPLC profiles, apoE distribution and electron microscopy analyses of HDL fractions of control ABCA1^−/− mice and ABCA1^−/− mice infected with recombinant adenoviruses expressing apoE4 or LCAT

(A–C) Cholesterol FPLC profiles. (D–F) ApoE distribution among different densities (d). Lanes M, molecular-mass markers. (G–I) Electron micrographs of fractions 6–8 of HDL. (A, D, G) Analyses of samples obtained from ABCA1^−/− mice infected with 2×10⁹ pfu of the control adenovirus expressing GFP (AdGFP). (B, E, H) Analyses of samples obtained from ABCA1^−/− infected with 2×10⁹ pfu of adenoviruses expressing apoE4 (AdGFP-E4). (C, F, I) Analyses of samples obtained from ABCA1^−/− mice infected with 5×10⁸ pfu of adenoviruses expressing LCAT (Ad-LCAT). CE/TC, cholesteryl ester/total cholesterol ratio.

Figure 3. Cholesterol FPLC profiles, apoE distribution and electron microscopy analyses of HDL of apoE^−/− mice and apoE^−/− mice infected with recombinant adenoviruses expressing human LCAT or apoE4 or a combination of apoE4 and human LCAT

(A–D) Cholesterol FPLC profiles of (A) apoE^−/− mice treated with 2×10⁹ pfu of control adenovirus expressing GFP (AdGFP), (B) apoE^−/− mice treated with 5×10⁸ pfu of adenoviruses expressing LCAT (Ad-LCAT), (C) apoE^−/− mice treated with 2×10⁹ pfu of adenoviruses expressing apoE4 (AdGFP-E4), and (D) apoE^−/− mice treated with a mixture of 2×10⁹ pfu of adenoviruses expressing apoE4 (AdGFP-E4) and 5×10⁸ pfu of adenoviruses expressing human LCAT (Ad-LCAT). (E and F) ApoE distribution among different densities (d) of human apoE in apoE^−/− mice infected with the adenoviruses expressing apoE4 alone (E), or a combination of apoE4 and human LCAT (F). (G and H) Electron micrographs of HDL obtained from mice infected with adenoviruses expressing apoE4 alone (G), or apoE4 and human LCAT (H). CE/TC, cholesteryl ester/total cholesterol ratio.

Figure 4. Cholesterol FPLC profile, apoE distribution and electron microscopy analysis of HDL fraction of apoE^−/− mice infected with recombinant adenoviruses expressing apoE4mut1

(A) FPLC profiles of total, free and esterified cholesterol of apoE^−/− mice infected with 2×10⁹ pfu of adenoviruses expressing apoE4mut1 (AdGFRP-E4[L261A,T264A,F265A,L268A,V269A]). (B) ApoE distribution among different densities (d). (C) Electron micrographs of fractions 6–8 of HDL obtained from mice infected with adenoviruses expressing apoE4mut1. CE/TC, cholesteryl ester/total cholesterol ratio.

Figure 5. Participation of apoE in the biogenesis of apoE-containing HDL and the clearance of triacylglycerol-rich lipoproteins

Schematic representation of the participation of apoE in the biogenesis of apoE-containing HDL (branches III–V) and the clearance of triacylglycerol-rich lipoproteins (branches I and II). LDLr, LDL receptor. The Figure is based on the data of Figures 1–3 and [3,4].