Apolipoprotein A-I vascular gene therapy reduces vein-graft atherosclerosis

Figure 1. Experimental timeline and rabbit plasma cholesterol levels (Top) Rabbits were fed 0.3% cholesterol for 2 weeks, then fed with diets containing 0.3%–0% cholesterol, with diets adjusted every 2 weeks as needed to maintain plasma cholesterol in the range indicated. (Bottom) Plasma cholesterol levels in all rabbits (n = 22) for the duration of the experiment. Vein grafting was performed 4 weeks after beginning the high-cholesterol diet, followed by vector infusion and vein graft harvest at the indicated time points. Data points are individual rabbits; bars are group means.

Figure 2. Vector genomes and apoAI transgene expression Vector genomes (A and B), total APOAI mRNA (C), and transgenic APOAI mRNA (D) were measured in vein-graft extracts (n = 22 graft pairs) harvested 12 weeks after vector infusion. (A–D) Data points are from individual grafts. (A and B) Values from grafts in the same rabbit (infused with either HDAdNull or HDAdApoAI) are connected by lines. (C) Median value for the HDAdNull group was arbitrarily assigned a value of 1; all data points are expressed as a ratio of this value. Bar for HDAdApoAI is median. (D) Mean value for the HDAdNull group was arbitrarily assigned a value of 1; all data points are expressed as a ratio of this value. Bar for HDAdApoAI is mean. (A and B) p values are from paired t test. (C and D) Dotted line is the lower limit of assay detection. p values are from signed rank tests.

Figure 3. Vein graft hemodynamics Blood flow (A and B) was measured and pulsatility/shear stress (C–F) were calculated from measurements made during the vector infusion (A, C, and E) and during the harvest surgeries (B, D, and F). Data points are from individual grafts. Results from grafts in the same rabbit (infused with either HDAdNull or HDAdApoAI; n = 22 for both) are connected by lines. p values are from comparisons of the HDAdNull and HDAdApoAI groups with t tests or rank-sum tests.

Figure 4. Vein graft cholesterol content, lipid accumulation, and macrophage markers (A–D) Cholesterol (esterified and unesterified) was measured by mass spectrometry performed on vein-graft extracts; total cholesterol and percent cholesterol ester were calculated. (E) Total intimal ORO-stained area was measured on histologic sections, then calculated (F) as a percentage of total intimal area. CD68 mRNA (G) was measured in vein-graft extracts using quantitative RT-PCR. (H) Total intimal area stained for the macrophage marker RbM2 was measured on histologic sections, then calculated (I) as a percentage of total intimal area. Data points are from individual grafts (cholesterol measurements: n = 20–21 pairs; ORO area and percent ORO-stained area: n = 17 pairs; RbM2 area and percent RbM2-stained area: n = 18 pairs; CD68 mRNA: n = 22 pairs). Values from grafts in the same rabbit (infused with either HDAdNull or HDAdApoAI) are connected by lines. p values are from paired t tests or signed-rank tests.

Figure 5. Histology and immunohistochemistry of vein grafts Sections of vein grafts transduced in vivo with either HDAdNull or HDAdApoAI were stained with (A–D) ORO, (E–H) antibodies to the macrophage marker RbM2, (I–L) VVG stain, (M–P) H&E, or (Q–T) antibodies to VCAM-1. Portions of low-magnification images indicated by rectangles are portrayed in adjacent high-magnification images. The stains illustrated here are quantified in Table 2 and Figure 4. Dashed lines delineate the intimal-medial border. ∗Lumen. scale bars, 100 μm in high-magnification images and 500 μm in low-magnification images.

Figure 6. Expression of cytokines and macrophage subset markers (A–I) The indicated mRNA were measured in vein graft extracts, using quantitative RT-PCR. Data points are from individual grafts (n = 22 pairs). Results from grafts in the same rabbit (infused with either HDAdNull or HDAdApoAI) are connected by lines. Data points for samples with undetectable levels are superimposed at 0 AU. p values are from signed-rank tests.