Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits

Abstract

Objective: Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions.

Approach and results: Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I-expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%-32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)-stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%-39% less; P=0.06-0.1).

Conclusions: In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I-expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

Keywords: apolipoprotein; atherosclerosis; carotid artery; gene therapy; rabbits.

Figure 1

Vector genome persistence and apo A-I mRNA expression. Rabbit carotid arteries were transduced with HDAdNull or HDAdApoAI and harvested 24 weeks later. (A) HDAd vector genomes were measured in carotid artery extracts and normalized to the number of vascular wall cells as determined by measurement of total DNA in extracts. P value from rank-sum test. (B) Apo A-I mRNA was measured in carotid artery extracts, normalized to GAPDH mRNA, and expressed as arbitrary units (AU, with the median value in the HDAdNull group defined as 1). Dotted line is assay background (see Materials and Methods). Data points represent individual arteries; bars are group medians. P value from rank-sum test.

Figure 2

Carotid atherosclerotic lesions from rabbits fed a high-fat diet for 24 weeks after gene therapy. Sections are from arteries treated either with HDAdNull (A–D, I–L) or HDAdApoAI (E–H, M–P). (A, E) hematoxylin and eosin (H&E) stain; (B, F) Oil Red O (ORO) stain; (C, G) immunostaining with RAM-11 antibody to detect macrophages; (D, H) immunostaining with HHF-35 antibody to detect muscle actin; (I, M) immunostaining to detect VCAM-1 or (J, N) ICAM-1; (K, L, O, P) immunostaining with KEN-5 antibody to detect T-cells. Sections A–D, and I–K are from the same artery, as are sections E–H and M–O. Sections L and P are higher magnification images of different arteries showing characteristic KEN-5 staining of T-cells. All panels except B and F: hematoxylin counterstain. (A) Arrows indicate medial thickening below large intimal lesions. Scale bars = 200 μm.

Figure 3

Impact of apo A-I expression on arterial morphology. Arteries were removed 24 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (ApoAI), sectioned, stained, and analyzed with computer-assisted planimetry. (A) Intimal areas measured on HHF-35-stained sections. (B) Medial areas measured on H&E-stained sections. (C) Medial thickness calculated from measuring the medial area and the internal elastic lamina (IEL) perimeter, both on H&E-stained sections. (D) Ratio of intimal area to medial area. (E) Area within the IEL, calculated from the IEL perimeter, measured on H&E-stained sections. (F) Luminal stenosis calculated as intimal area divided by area within the IEL. Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which the HDAdApoAI-treated artery had a lower value than the contralateral HDAdNull-treated artery are indicated in black; rabbits in which the HDAdApoAI-treated artery had a higher value than the contralateral HDAdNull-treated artery are indicated in grey. P values are from Wilcoxon signed-rank test comparing HDAdApoAI- and HDAdNull-treated carotids in the same rabbit.

Figure 4

Intimal lesion composition. Arteries were removed 24 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (ApoAI), sectioned, stained, and analyzed with computer-assisted color thresholding and planimetry. Intimal areas staining with: (A) Oil Red O (ORO); (B) RAM-11 antibody (detects macrophages); (C) HHF-35 antibody (detects muscle actin); or (D) the KEN-5 antibody (detects T-cells). Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which the HDAdApoAI-treated artery had a lower value than the contralateral HDAdNull-treated artery are indicated in black; rabbits in which the HDAdApoAI-treated artery had a higher value than the contralateral HDAdNull-treated artery are indicated in grey. P values are from Wilcoxon signed-rank test comparing HDAdApoAI- and HDAdNull-treated carotids in the same rabbit.

Figure 5

Intimal adhesion molecule expression. Arteries were removed 24 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (ApoAI) sectioned, stained, and analyzed with computer-assisted color thresholding and planimetry. Intimal areas staining with: (A) antibody to VCAM-1; or (B) antibody to ICAM-1. Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which the HDAdApoAI-treated artery had a lower value than the contralateral HDAdNull-treated artery are indicated in black; rabbits in which the HDAdApoAI-treated artery had a higher value than the contralateral HDAdNull-treated artery are indicated in grey. P values are from Wilcoxon signed-rank test comparing HDAdApoAI- and HDAdNull-treated carotids in the same rabbit.

Figure 6

Expression of inflammation-related markers in carotid arteries. Rabbit carotid arteries were transduced with HDAdNull (Null) or HDAdApoAI (ApoAI) and harvested 24 weeks later. mRNA was measured, normalized to GAPDH mRNA measured in the same extract, and expressed as arbitrary units (AU). (A) TNF‐α; (B) MCP-1; (C) MRC-1 (a M2 macrophage marker); (D) ARG-1 (a M2 macrophage marker); and (E) IL-1β (a M1 macrophage marker). Data points are values for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which the HDAdApoAI-treated artery had a lower value than the contralateral HDAdNull-treated artery are indicated in black; rabbits in which the HDAdApoAI-treated artery had a higher value than the contralateral HDAdNull-treated artery are indicated in grey. P values are from Wilcoxon signed-rank test comparing HDAdApoAI- and HDAdNull-treated carotids in the same rabbit.