Noninvasive ultrasound molecular imaging of the effect of statins on endothelial inflammatory phenotype in early atherosclerosis

Abstract

Background/objectives: Inflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.

Methods: Mice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MB(VCAM)) and control microbubbles (MB(Ctr)). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.

Results: Plaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MB(VCAM) in non-treated animals (MB(VCAM) 2±0.3 vs MB(Ctr) 0.7±0.2, p<0.01), but not in statin-treated animals (MB(VCAM) 0.8±0.2 vs MB(Ctr) 1.0±0.2, p = ns; p<0.01 for the effect of statin on MB(VCAM) signal).

Conclusions: Non-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.

Figure 1. Effect of statin treatment on aortic atherosclerotic plaque burden.

Percent of total luminal plaque area at the base of aorta (A) and in the ascending aorta (B) in non-treated animals versus statin treated animals (A, n = 11 non-treated animals, n = 7 statin treated animals; B, n = 7 non-treated and n = 7 statin treated animals), *p<0.05 vs non-treated animals. Examples of Movat’s pentachrom stains at the base of aorta in a non-treated mouse (C, E) and a statin treated mouse (D, F). The arrows denote a large plaque in a magnified view from a non-treated animal (E) and a small plaque in a magnified view from a statin treated animal (F).

Figure 2. Effect of statin treatment on aortic expression of VCAM-1.

(A) VCAM-1 protein expression in the ascending aorta assessed by Western blot in non-treated (lanes 1–5) versus statin treated (lanes 6–10) animals, n = 5 per group, *p<0.01 vs non-treated animals. Representative examples of fluorescent immunohistochemistry images of the base of the aorta demonstrating endothelial VCAM-1 expression (red fluorescence) in a non-treated animal (B) and a statin treated animal (C), the green fluorescence is autofluorescence delineating vessel anatomy.

Figure 3. Effect of statin treatment on vascular inflammation.

Fluorescent immunohistochemistry for the expression of macrophage Mac-2 at the base of the aorta (A) and in the ascending aorta (B) of non-treated versus statin treated mice (A, n = 12 non-treated animals, n = 9 statin treated animals; B, n = 8 non-treated and n = 8 statin treated animals), *p<0.05 versus non-treated animals. Examples of Mac-2 staining in a non-treated mouse (C) and a statin treated mouse (D), arrows denote stained Mac-2 positive macrophages.

Figure 4. High frequency ultrasound imaging (40 MHZ) of the ascending aorta.

(A) Wall thickness of the ascending aorta measured at the sinus of valsalva (SV), the greater curvature (GC), the lesser curvature (LC) and at the origin of brachoicephalic artery (BC) in non-treated and statin treated animals (n = 10 per group). (B) Example of high frequency ultrasound imaging of the ascending aorta illustrating the measurements obtained.

Figure 5. Molecular imaging of the ascending aorta.

(A) Mean ± SEM background-subtracted signal intensity for microbubbles targeted to VCAM-1 (MB_VCAM) and control microbubbles (MB_Ctr) in non-treated (n = 10) and statin treated animals (n = 12). *p<0.01 vs MB_ctr in non-treated animals, ¥ p<0.01 vs MB_VCAM in statin treated animals. Examples of color coded CEU images from a non-treated animal after injection of MB_VCAM (B), and of MB_Ctr (C). Images from a statin treated animal after injection of MB_VCAM (D), and of MB_Ctr (E). The color scale for the CEU images is shown at the bottom of each frame. (F) and (G) illustrate the outline of the ascending aorta on B-mode ultrasound images which was used as a region of interest for acoustic intensity measurements.

Figure 6. CEU molecular imaging of the regional endothelial expression of VCAM-1.

(A) Mean ± SEM background-subtracted signal intensity for microbubbles targeted to VCAM-1 at the base of the aorta and in the distal ascending aorta in non-treated (n = 9) and statin treated animals (n = 10) *p<0.05 vs non-treated animals. Two dimensional ultrasound imaging illustrating outline of ( B) base of the aorta and (C) ascending aorta.