Carotid inflammation is unaltered by exercise in hypercholesterolemic Swine

Abstract

Introduction: Reduction of vascular inflammation might contribute to the beneficial effects of exercise. We hypothesized that 1) exercise would reduce carotid endothelial vascular cell adhesion molecule-1 (VCAM-1) and that 2) in vivo detection of carotid inflammation can be achieved in a large animal model using contrast-enhanced ultrasound (CEU) with VCAM-1-targeted microbubbles (MBs).

Methods: Familial hypercholesterolemic (FH) swine were divided into sedentary (Sed) and exercise-trained (Ex) groups. Ex pigs underwent 16-20 wk of treadmill aerobic exercise. At the end of the study, in vivo CEU with VCAM-1-targeted MBs and assessment of endothelial-dependent dilation (EDD) were performed in carotid arteries. VCAM-1 mRNA and protein expression were compared with markers of atherosclerotic disease and health, and in vitro EDD was assessed in carotid arteries.

Results: Exercise training neither reduced inflammation nor improved EDD in carotid arteries of FH swine. Markers of atherosclerosis including VCAM-1 were prominent in the bifurcation compared with the proximal or distal common carotid artery and inversely associated with phosphorylated and total endothelial nitric oxide synthase. Signal intensity from VCAM-1-to-control MBs positively correlated with carotid VCAM-1 protein expression, validating our technique.

Conclusion: These results first demonstrate that aerobic exercise has no effect on carotid endothelial inflammatory markers and EDD in FH swine. Second, our findings indicate that CEU using VCAM-1-targeted MBs can detect inflammation in vivo, providing strong foundations for longitudinal studies examining the effect of therapeutic interventions on the inflammatory status of the endothelium.

Figure 1. Representative contrast-enhanced ultrasound images of the left distal common carotid artery (CCA)

A, Longitudinal image of the distal CCA using 2D-brightness mode. The distal CCA was first located and depth optimized. The large white arrows delimit the external border of the artery. The lumen and near wall of the artery are indicated. B–C: Image of the same arterial segment before (B) and after injection of VCAM-1-targeted microbubbles (C), using ultrasound contrast mode. The presence of microbubbles adhered to the vessel wall resulted in increased signal intensity (small white arrows). Gain settings and depth were kept constant throughout the procedure. The scale located at the right of each sonographic image indicates centimeters of depth. MBs, microbubbles.

Figure 2. Effect of training on contrast-enhanced ultrasound signal in the proximal and distal common carotid arteries (CCA)

Signal intensity of VCAM-1 and control microbubbles measured at 3 and 6 min after injection were normalized to background and averaged. A–B, No difference in signal intensity was found between Ex and Sed in the proximal (A) or distal (B) CCA. The signal intensity of VCAM-1-to-control microbubbles was greater than 1.0 for Seds in the proximal CCA. Mean VCAM-1-to-control microbubbles signal intensity ± SE are represented. C–D, Endothelial VCAM-1 protein expression for Ex and Sed in the proximal and distal CCA. Mean VCAM-1 protein expression normalized to beta-actin in each group ± SE are represented. Similar levels of endothelial VCAM-1 protein were found in Ex and Sed. E–F, Relation between in vivo and in vitro detection of endothelial VCAM-1 protein expression. For each animal (n = 14), in vivo detection of VCAM-1-targeted microbubbles using contrast-enhanced ultrasound was compared to Western Blot measure of VCAM-1 protein expression. The signal intensity (SI) of VCAM-1-to-control microbubbles ratio was positively correlated with endothelial VCAM-1 protein expression in both proximal (E) and distal (F) CCA. *P < 0.05; CCA, common carotid arteries; Bckg, background; SI, signal intensity; MBs, microbubbles; EX, exercised-trained; SED: Sedentary animals.

Figure 3. Effect of training on endothelial VCAM-1, macrophage infiltration and oxidative stress staining in the bifurcation, proximal and distal common carotid arteries (CCA)

Training did not result in a significant difference in endothelial VCAM-1 (A), percent intimal macrophage infiltration (B) and oxidative stress (C) protein expression. A–B, A 3 to 5-fold increase in endothelial VCAM-1 expression and macrophage infiltration was seen in the bifurcation compared to the proximal and distal CCA. C, Oxidative stress as measured by nitrotyrosine staining was approximately 1.5-fold greater in the distal CCA compared to proximal CCA, whereas nitrotyrosine staining was similar in the bifurcation vs. proximal or distal CCA. Values are means ± SE. CCA, common carotid artery. *P < 0.05 vs. Bifurcation, †P < 0.05 vs. Proximal CCA.

Figure 4. Effect of training on protein levels of phosphorylated- and total-eNOS and phosphorylated-to-total eNOS ratio in proximal and distal common carotid arteries (CCA)

Phosphorylated- (A, B), total-eNOS (C, D) and as phosphorylated-to-total eNOS ratio (E, F) protein levels assessed by immunoblot were not significantly different between Ex vs. Sed; however, a trend for increase in phosphorylated- and total-eNOS protein expression was seen in the proximal CCA. Values are means ± SE (n = 7 in each subgroup).

Figure 5. Effect of training on endothelial agonist-induced dilation in proximal and distal common carotid arteries (CCA)

A–B, Proximal and distal carotid luminal diameter was measured in vivo during 5 min of acetylcholine (10⁻⁶ and 10⁻⁵M) infusion. Ex and Sed demonstrated similar acetylcholine induced dilation in proximal (A) and distal B) CCA. For each group (Ex vs. Sed) and carotid segment (Proximal and Distal CCA), mean percent changes in diameter after each dose of acetylcholine compared to saline ± SE are represented (n = 7 in each group for ACH 10⁻⁶M, and n = 6 in each group for ACH 10⁻⁵M). C–F, Concentration-dependent acetylcholine (ACH) - and bradykinin (BK)-induced relaxation in mid CCA in vitro. Relaxation was expressed as percent reduction in force from PGF2-α(30 μM)-induced tension. Values are means ± SE (n = 5 in each subgroup). C, Relaxation to acetylcholine was similar between Ex and Sed. D, N^G-nitro-L-arginine methyl ester (L-NAME) significantly inhibited acetylcholine- (E) and BK-induced (F) relaxation to a similar extent in both groups.