Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis

Abstract

Objective: Gene therapy, delivered directly to the blood vessel wall, could potentially prevent atherosclerotic lesion growth and promote atherosclerosis regression. Previously, we reported that a helper-dependent adenoviral (HDAd) vector expressing apolipoprotein A-I (apoA-I) in carotid endothelium of fat-fed rabbits reduced early (4 weeks) atherosclerotic lesion growth. Here, we tested whether the same HDAd-delivered to the existing carotid atherosclerotic lesions-could promote regression.

Approach and results: Rabbits (n=26) were fed a high-fat diet for 7 months, then treated with bilateral carotid gene transfer. One carotid was infused with an HDAd expressing apoA-I (HDAdApoAI) and the other with a control nonexpressing HDAd (HDAdNull). The side with HDAdApoAI was randomized. Rabbits were then switched to regular chow, lowering their plasma cholesterols by over 70%. ApoA-I mRNA and protein were detected in HDAdApoAI-transduced arteries. After 7 weeks of gene therapy, compared with HDAdNull-treated arteries in the same rabbits, HDAdApoAI-treated arteries had significantly less vascular cell adhesion molecule-1 expression (28%; P=0.04) along with modest but statistically insignificant trends toward decreased intimal lesion volume, lipid and macrophage content, and intercellular adhesion molecule-1 expression (9%-21%; P=0.1-0.4). Post hoc subgroup analysis of rabbits with small-to-moderate-sized lesions (n=20) showed that HDAdApoAI caused large reductions in lesion volume, lipid content, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression (30%-50%; P≤0.04 for all). Macrophage content was reduced by 30% (P=0.06). There was a significant interaction (P=0.02) between lesion size and treatment efficacy.

Conclusions: Even when administered on a background of aggressive lowering of plasma cholesterol, local HDAdApoAI vascular gene therapy may promote rapid regression of small-to-moderate-sized atherosclerotic lesions.

Keywords: apolipoprotein A-I; atherosclerosis; carotid arteries; genetic therapy; macrophages; rabbits.

Figure 1

Experimental protocol and plasma cholesterol levels. Rabbits were fed a high-fat diet for 4 weeks, containing the indicated percentages of cholesterol. Rabbits then underwent neck surgery with bilateral common carotid artery infusions of DMEM to help initiate atherosclerosis development. Plasma cholesterol was measured at this time and every 2 weeks thereafter. From the end of week 4 through the time of gene transfer (GT), dietary cholesterol was adjusted according to a sliding scale with a goal of maintaining plasma cholesterol in the range of 200–800 mg/dL (gray horizontal bar). Most of the rabbits (~ 80%) underwent gene transfer during week 28. Due to scheduling issues, ~ 20% were treated during week 29. Gene transfer in one rabbit was delayed until week 31 due to an anesthetic complication. Gene transfer included infusion of HDAdNull and HDAdApoAI in contralateral carotids, with the side for each vector randomized. After gene transfer, rabbits were switched to normal chow and carotids removed 3 days or 7 weeks later.

Figure 2

Vector genome persistence and apo A–I expression. Rabbit carotid arteries were transduced with HDAdNull or HDAdApoAI and harvested 3 days or 7 weeks later. (A) Vector genomes in carotid artery extracts were measured by qPCR and normalized to the number of vascular wall cells represented by the total amount of DNA in the extract. (B) Apo A–I mRNA in carotid artery extracts was measured by qRT-PCR, with normalization to GAPDH mRNA in the same extracts. Data points are individual arteries; bars are group medians. Dotted line indicates assay background. (C) Representative western blot of medium conditioned by explanted HDAdNull- or HDAdApoAI-transduced carotid arteries. Paired samples (1 and 5; 2 and 6; 3 and 7; 4 and 8) are from the same rabbits. Size markers are in kilodaltons.

Figure 3

Carotid atherosclerotic lesions 35 weeks after beginning high-fat diet and 7 weeks after infusion of HDAdNull or HDAdApoAI. Arteries were removed, embedded in OCT, and sectioned at multiple steps. Sections of representative lesions treated either with HDAdNull (Null) or HDAdApoAI (Apo A–I) are shown, stained with: hematoxylin and eosin (H & E) (A, E); Oil Red O (ORO) (B, F); RAM-11 antibody to detect macrophages (C, G); HHF-35 antibody to detect smooth muscle actin (D, H); anti-VCAM-1 (I, K); or anti-ICAM-1 (J, L). Sections A, B, C, and D are from the same artery, as are: sections E, F, G, and H; sections I and J; and sections K and L. All panels except B and F: hematoxylin counterstain. Scale bars = 200 μm.

Figure 4

Intimal lesion size and composition. Arteries were removed 7 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (Apo A–I), sectioned, stained, and analyzed with computer-assisted planimetry (n=26 rabbits; 52 arteries). (A) Intimal areas measured on H&E-stained sections. (B) Intimal areas staining with Oil Red O. (C) Intimal areas staining with the RAM-11 antibody (detects macrophages). (D) Intimal areas staining with the HHF-35 antibody (detects smooth muscle actin). Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which HDAdApoAI-treated arteries had lower values than HDAdNull-treated arteries are indicated in black; rabbits in which HDAdNull-treated arteries had lower values than HDAdApoAI-treated arteries are indicated in grey.

Figure 5

Intimal lesion size and composition of small-to-moderate lesions. Data are from a subgroup of 20 rabbits (40 arteries), none of which had an intimal lesion area more than one standard deviation above the mean of all lesions. Arteries were removed 7 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (Apo A–I), sectioned, stained, and analyzed with computer-assisted planimetry. Data for these 20 rabbits are the same as in Figure 4, with the y-axis re-scaled to improve readability. (A) Intimal areas measured on H&E-stained sections. (B) Intimal areas staining with Oil Red O (ORO). (C) Intimal areas staining with the RAM-11 antibody (detects macrophages). (D) Intimal areas staining with the HHF-35 antibody (detects smooth muscle actin). Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which HDAdApoAI-treated arteries had lower values than HDAdNull-treated arteries are indicated in black; rabbits in which HDAdNull-treated arteries had lower values than HDAdApoAI-treated arteries are indicated in grey.

Figure 6

Intimal adhesion molecule expression. Arteries were removed 35 weeks after beginning high-fat diet and 7 weeks after treatment with either HDAdNull (Null) or HDAdApoAI (Apo A–I), sectioned, stained, and analyzed with computer-assisted planimetry. (A) and (B) Data for all rabbits (n=26 rabbits; 52 arteries). (A) Intimal areas staining for vascular cell adhesion molecule-1 (VCAM-1). (B) Intimal areas staining for intercellular adhesion molecule 1 (ICAM-1). (C) and (D): data are from a subgroup of 20 rabbits (40 arteries), none of which had an intimal lesion area more than one standard deviation above the mean of all lesions. Data for these 20 rabbits are the same as in (A) and (B), respectively, with the y-axis rescaled in (C) to improve readability. Data points are means for each artery; points from arteries in the same rabbit are connected by bars. Rabbits in which HDAdApoAI-treated arteries had lower values than HDAdNull-treated arteries are indicated in black; rabbits in which HDAdNull-treated arteries had lower values than HDAdApoAI-treated arteries are indicated in grey.