Molecular imaging of inflammation and platelet adhesion in advanced atherosclerosis effects of antioxidant therapy with NADPH oxidase inhibition

Abstract

Background: In atherosclerosis, local generation of reactive oxygen species amplifies the inflammatory response and contributes to plaque vulnerability. We used molecular imaging to test whether inhibition of NADPH oxidase with apocynin would reduce endothelial inflammatory activation and endothelial-platelet interactions, thereby interrupting progression to high-risk plaque phenotype.

Methods and results: Mice deficient for both the low-density lipoprotein receptor and Apobec-1 were studied at 30 weeks of age and again after 10 weeks with or without apocynin treatment (10 or 50 mg/kg per day orally). In vivo molecular imaging of vascular cell adhesion molecule-1 (VCAM 1) P-selectin, and platelet glycoprotein-1bα (GPIbα) in the thoracic aorta was performed with targeted contrast-enhanced ultrasound molecular imaging. Arterial elastic modulus and pulse wave transit time were assessed using ultrahigh frequency ultrasound and invasive hemodynamic measurements. Plaque size and composition were assessed by histology. Molecular imaging in nontreated mice detected a 2-fold increase in P-selectin expression, VCAM-1 expression, and platelet adhesion between 30 and 40 weeks of age. Apocynin reduced all of these endothelial events in a dose-dependent fashion (25% and 50% reduction in signal at 40 weeks for low- and high-dose apocynin). Apocynin also decreased aortic elastic modulus and increased the pulse transit time. On histology, apocynin reduced total monocyte accumulation in a dose-dependent manner as well as platelet adhesion, although total plaque area was reduced in only the high-dose apocynin treatment group.

Conclusions: Inhibition of NADPH oxidase in advanced atherosclerosis reduces endothelial activation and platelet adhesion, which are likely responsible for the arrest of plaque growth and improvement of vascular mechanical properties.

Figure 1

(A) Elastic modulus (mean ±SEM) of the thoracic aorta (E_Ao) in wild type mice and DKO mice at baseline (30 wks) and at 40 wks after 10 wks of either no treatment (control) or apocynin given at low- and high-dose. (B) Examples of source data for from a non-treated animal illustrating age-dependent worsening of E_Ao as a result of both an increase in the aortic pulse pressure (ΔP) and a reduction in the systolic aortic diameter change (ΔD) from M-mode echocardiography. (C) Pulse transit time (mean ±SEM) in wild type mice and DKO mice at baseline and at 40 wks. (D) Examples of pulse-wave spectral Doppler data from an untreated and high-dose apocynin-treated DKO mouse at 40 wks illustrating differences in pulse transit time measured as the time delay (ΔT) for the onset of systolic flow from the ascending aorta (top) to the femoral artery (bottom). *p<0.05 vs. 30 wk wild type; †p<0.05 vs.30 wk DKO; ‡p<0.05 versus untreated (control) 40 wk DKO.

Figure 2

Mean (±SEM) number of microbubbles attached to platelet complexes per optical field (OF) for flow chamber studies when comparing microbubbles targeted to GPIbα (MB_GPIb) to either (A) control microbubbles not bearing a ligand (MB_C) suspended in PBS; or (B) control microbubbles bearing a mutated non-binding ligand (MB_Ib-mut) suspended in whole blood. Examples show fluorescent MB_GPIb attaching to platelet complexes at shear rates of (C) 300 s⁻¹ and (D) 1,000 s⁻¹.

Figure 3

(A to D) Molecular imaging signal (mean ±SEM) for P-selectin, VCAM-1, or GPIbα, and for control non-targeted microbubbles. *p<0.05 vs. 30 wk wild type; †p<0.05 vs. 30 wk DKO; ‡p<0.05 versus untreated (control) 40 wk DKO. (E) Example from an untreated 40 wk DKO mouse of a 2-D ultrasound image of the aortic arch used to define regions-of-interest; and CEU molecular imaging from the same mouse after injection of either (F) GP_Ibα-targeted or (G) control microbubbles (color scale at bottom). As previously described, an ultrasound beam elevation (out-of-plane) dimension larger than the aortic diameter produced volume averaging and extension of molecular imaging signal enhancement into the apparent “lumen”. Illustration of method for background-subtraction are provided in Supplemental Figure A.

Figure 4

Vessel thickness (mean ±SEM) measured by high-frequency 2-D ultrasound imaging of the aortic arch measured at (A) the lesser curvature of the aortic arch, and (B) at the origin of the brachiocephalic artery. Images illustrate examples obtained from DKO mice (C) at 30 wks, (D) at 40 wks without treatment, or (E) at 40 wks with high-dose apocynin. Arrows illustrate regions of thickening at the lesser curvature of the arch and at the origin of the brachiocephalic artery. *p<0.05 vs. 30 wk wild type; †p<0.05 vs.30 wk DKO; ‡p<0.05 versus untreated (control) 40 wk DKO.

Figure 5

(A and B) Mean (±SEM) plaque area in absolute terms and expressed as a ratio to the total vessel area for DKO mice at 40 wks of age. (C and D) Mean (±SEM) area staining positive for Mac-2 in absolute terms and expressed as a ratio to total plaque area. Histology illustrates plaque formation on Masson’s trichrome staining from: (D and E) two separate untreated 40 wk DKO mice, and (F) a high-dose apocynin-treated mouse. (G) Examples of Mac-2 staining illustrating differences in monocyte/macrophage accumulation at 40 wks in the different cohorts. Control staining staining conditions are provided in Supplemental Figure B. *p<0.05 vs. untreated control; and †p<0.05 vs. untreated controls and low-dose.

Figure 6

(A and B) Histology with Masson’s trichrome illustrating inflammatory cells or platelets (arrows) adherent to the endothelial surface of plaques from two separate untreated 40 wk DKO mice. (C and D) Examples of immunofluorescent histology for GPIIb from two separate mice confirming the presence of platelets (arrows) on the endothelial surface and also within the plaque milieu. Composite images of VCAM-1 immunofluorescent staining from (E) an untreated 40 wk DKO mouse, and (F) a high-dose apocynin treated mouse.