Immunization with the Gly¹¹²⁷-Cys¹¹⁴⁰ amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies

Figures

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  Figure 1. P3 immunization raises serum levels of specific anti-P3 antibodies. (A) Graphs showing the results of standardized enzyme-linked immunosorbent assay (ELISA) in pre-serum and serum at the check points (Check1 and Check2) from control (A), IrP- (B) and P3-treated rabbits (C) against P3-BSA used as antigenic source. Results are shown as mean±SD. Control (N=3); IrP (N=12); P3 (N=15) (D) Confocal images of rabbit SMCs exposed to serum from P3-treated rabbits (middle panel) or to commercial anti-LRP1 (right panel).

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  Figure 2. P3 immunization reduces HFD-induced atherosclerotic burden. (A) Representative images of longitudinally open sections of aorta stained with Herxheimer in control, IrP and P3-immunized rabbits fed either chow or HFD. (B) Graph showing quantification of atherosclerotic burden in HFD-fed rabbits from control (N=3), IrP (N=6) and P3 (N=9). Results are shown as mean±SD of 8 aortic sections/rabbit. #P<0.005, P3 versus control and IrP groups. (C) Representative immunohistochemical images of lipid and macrophage staining in cross-sections of aortas from control and P3-immunized rabbits, both on HFD m: media; i: intima.

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  Figure 3. P3 immunization reduces HFD-induced SMC accumulation in the arterial intima. Representative confocal microscopy images of SMCs detected with anti-a-actin antibodies in cross-sections of aortas from control and P3- immunized rabbits (A) and of SMC detected with falloidin and macrophages detected with anti-RAM antibodies (B). Fluorescent images were acquired in a scan format of 1024 x 1024 pixels and were processed with the Leica Standard Software TCS-AOBS. m: media; i: intima.

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  Figure 4. P3 immunization did not induce alterations in lipoprotein mass subclasses or lipoprotein profile of HFD fed rabbits. VLDL, LDL and HDL were isolated by sequential ultracentrifugation and their composition in terms of total, free cholesterol, triglycerides, phospholipids and protein were determined as explained in methods. Lipoprotein composition was used to calculate the total mass of each lipoprotein. Results are shown as mean±SD of control (N=3), IrP (N=6) and P3-immunized rabbits (N=9).

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  Figure 5. P3 immunization reduces HFD-induced cholesterol accumulation in the aorta but not in the liver. Representative thin-layer chromatography of aorta (A) and liver (B). Bar graphs showing the quantification of cholesteryl ester (CE), triglycerides and free cholesterol content of aorta and liver. Results are expressed as µg cholesterol per mg protein and shown as mean ± SD in chow IrP (N=6), chow P3 (N=6), HFD Ctr (N=3), HFD IrP (N=6) and HFD P3 (N=9). *P<0.005 versus chow diet; #P<0.005 versus control or IrP-rabbits.

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  Figure 6. P3 immunization reduces aortic pro-inflammatory mediators in the aorta. Representative Western blot analysis of LRP1, TNFR1, pNF-kB (p65), total NF-kB (p65), and β-actin bands (A) and bar graphs showing band quantification of LRP1 and TNFR1 normalized by a-actin (B,C) and pNF-kB (p65)/total NF-kB (p65) ratio (E). Results are shown as mean ± SD in chow IrP (N=6), chow P3 (N=6), HFD Ctr (N=3), HFD IrP (N=6) and HFD P3 (N=9). *P<0.005 versus chow diet; #P<0.005 versus control or IrP-rabbits. Representative confocal microscopy images of TNFR1 (D) and NF-kB levels (F) in aortic samples from the different groups. Scale bar = 50 µm.

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  Figure 7. HFD serum from control and IrP serum, but not P3 serum, dramatically increased intracellular CE and pro-inflammatory mediators in human macrophages. Quiescent human macrophages (hMΦ) were exposed to serum from the different groups (0.5%, 2 hours). Cells were then exhaustively washed and collected in NaoH 0.1N for lipid extraction or in lysis buffer for Western blot analysis. A) Representative TLC and bar graphs showing the cholesteryl ester (CE) /free cholesterol ratio. Representative Western blot analysis of LRP1, SREBP-2, TNFR1, pNF-kB (p65), total NF-kB (p65), and a-actin bands (B) and bar graphs showing band quantification of LRP1, SREBP-2 and TNFR1 normalized by a-actin (C,D,E) and pNF-kB (p65)/total NF-kB (p65) ratio (F). Results are shown as mean ± SD of three experiments performed in duplicate. *P<0.005 versus chow serum; #P<0.005 versus control or IrP-serums.

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  Figure 8. HFD serum from control and IrP serum, but not P3 serum, dramatically increased intracellular CE and pro-inflammatory mediators in human coronary vascular smooth muscle cells. Quiescent human coronary vascular smooth muscle cells (hcVSMC) were exposed to serum from the different groups (0.5%, 2 hours). Cells were then exhaustively washed and collected in NaoH 0.1N for lipid extraction or in lysis buffer for Western blot analysis. A) Representative TLC and bar graphs showing the cholesteryl ester (CE) /free cholesterol ratio. Representative Western blot analysis of LRP1, SREBP-2, TNFR1, pNF-kB (p65), total NF-kB (p65), and a-actin bands (B) and bar graphs showing band quantification of LRP1, SREBP-2 and TNFR1 normalized by a-actin (C,D,E) and pNF-kB (p65)/total NF-kB (p65) ratio (F). Results are shown as mean ± SD of three experiments performed in duplicate. *P<0.005 versus chow serum; #P<0.005 versus control or IrP-serums.

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  Figure 9. P3 immunization counteracts aortic 18F-FDG uptake induced by HFD in rabbits. (A) Representative PET/CT longitudinal images of the aorta in control and P3 immunized rabbits fed HFD. (B) Graphs showing the SUVmean progression from pre-diet to post-diet time points in the upper and middle parts of the aorta in HFD-fed rabbits (IrP and P3 groups) (N=6/group). *P<0.005 versus pred-diet; #P<0.005 versus IrP-rabbits.

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  Figure 10. P3 immunization reduces the HFD-induced arterial resistance index (RI) and cholesteryl ester content in the carotids of rabbits. (A) Representative image of Eco Pulsed-Wave Doppler in the external carotid of rabbits. The systolic (1, 3 and 5), diastolic (2, 4 and 6), and pulse frequency measurement points (vertical dotted lines) are marked. (B) Arterial resistance index (ARI) was measured at baseline (pre-diet) and after 1 month of HFD feeding (post-diet) in external and internal carotids by Doppler ultrasonography in IrP and P3 groups (N=4/group). (C) Representative thin-layer chromatography of external and internal carotids and bar graphs showing the cholesteryl ester (CE)/free cholesterol (FC) ratio in carotids. Results are shown as mean ± SD. ***P<0.001, P3 versus control. *P<0.005 versus pred-diet; #P<0.005 versus IrP-rabbits.

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  Fig 11. Graphical Abstract