Bone Marrow Transplantation Shows Superior Atheroprotective Effects of Gene Therapy With Apolipoprotein A-I Milano Compared With Wild-Type Apolipoprotein A-I in Hyperlipidemic Mice
Lai Wang, MD,
Behrooz G. Sharifi, PhD,
Theresa Pan, MS,
Lei Song, MD,
Ada Yukht, MS and
Prediman K. Shah, MD, FACC*
Atherosclerosis Research Center and the Division of Cardiology, Burns and Allen Research Institute and Department of Medicine, Cedars Sinai Medical Center and David Geffen School of Medicine at UCLA, Los Angeles, California.
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Figure 1 Expression of apolipoprotein A-I (apoA-I) or apolipoprotein A-I Milano (apoA-IM) in the serum of recipient double-knockout mice. Serum levels of wild-type apoA-I or apoA-IM at 4, 8, 12, and 24 weeks after bone marrow transplantation are shown. Serum from a group of mice transplanted with the empty vector is used as a control. The data represent mean ± SD from 5 mice per group.
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Figure 2 Comparative serum lipoprotein profile of mice on a high-fat diet for 8 and 20 weeks. Distribution of cholesterol was determined in serum from the recipient mice transplanted with wild-type apolipoprotein A-I (A), apolipoprotein A-I Milano (M) or vector control (C) using standard superose-6 chromatography.
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Figure 3 Representative aortic sinus lesions showing apolipoprotein A-I (apoA-I) or apolipoprotein A-I Milano (apoA-IMilano) expression in mice samples reconstituted with cells expressing apoA-I (B) or apoA-IMilano (C) compared with no expression in vector controls (A). Data were consistent in all 5 mice per group examined.
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Figure 4 Average percent lesion area (mean ± SD) in en fasse aortas after oil red O staining in vector control group (VC; n = 12), wild-type apolipoprotein A-I (apoA-I) gene recipients (A-I; n = 11), and apolipoprotein A-I Milano (apoA-IMilano) gene recipients (A-IM; n = 15) are shown.
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Figure 5 Average percent lesion area (mean ± SD) in innominate artery (A) and aortic sinus (B) in vector control group (VC; n = 5) , wild-type apolipoprotein A-I (apoA-I) gene recipients (A-I; n = 5), and apolipoprotein A-I Milano (apoA-IMilano) gene recipients (A-IM; n = 5) are shown. Mice expressing apoA-IMilano had significantly smaller lesion areas compared with vector control mice and recipients of wild-type apoA-I.
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Figure 6 Representative macrophage immunoreactivity in the innominate artery lesions of mice are shown (A to C) and are presented as quantitative data (D). The macrophage immunoreactive area was significantly lower in recipients of apolipoprotein A-I Milano (A-IM) compared with vector controls (VC) or recipients of wild-type apolipoprotein A-I (A-I).
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Figure 7 (A) Representative frozen sections of aortic sinus plaques before and after laser microdissection. The serial sections from control, apolipoprotein A-I (apoA-I), and apoA-IM recipient mice were selected (5 mice per group) and the macrophage-rich areas were dissected. (B) Total RNA was extracted from the dissected segments from recipient mice transplanted with bone marrow transduced empty vector (VC), wild-type apoA-I (A-I), or apolipoprotein A-I Milano (A-IM) genes. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using primers specific for beta-actin (lanes 1 to 3, 410 bp) or apoA-I (lanes 6 to 8, 301 bp). Lane 4 is a positive control (PC) for apoA-I derived from packaging cell lines; lane 5 is negative control (NC) from PCR in the absence of primers.
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Figure 8 Cholesterol efflux from cholesterol-loaded macrophages. Transduced RAW cells expressing genes for green fluorescent protein (GFP), apolipoprotein A-I (A-I), or apolipoprotein A-I Milano (A-IM) were used for cholesterol efflux in the absence of exogenous extracellular acceptors. In addition, nontransduced RAW cells were loaded with 3H-cholesterol, and then cholesterol efflux was measured 4 h after loading in the presence of dipalmitoyl phosphatidylcholine (DPPC) alone or DPPC with 200 µg/ml of either apoA-I (A-I) or apoA-IMilano (A-IM). A similar experiment was performed with the peritoneal macrophages that were isolated from apoA-I–/–/apoE–/– double-knockout mice. Each bar shows the mean ± SD of a triplicate determination. The experiments were repeated twice with similar results.
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