Introduction

Endothelial activation is a key step both in the initiation of atherosclerotic lesions and in their progression to a late stage, where inflammatory cell burden and susceptibility to acute atherothrombotic complications are high. Oxidative stress plays a major role in supporting and amplifying the endothelial activation in atherosclerosis.¹ The family of NOX nicotinamide adenine dinucleotide phosphate (NADPH) oxidase present in plaque macrophages and in native endothelial and smooth muscle cells is a major source of reactive oxygen species (ROS) and, therefore, represents a potential therapeutic target.²

Apocynin is a polyphenolic drug that has been isolated from plant extracts and inhibits assembly of the NOX-2 isoform of the NADPH oxidase enzyme complex.³ In mice with advanced atherosclerosis, long-term therapy with apocynin has been shown to reduce endothelial adhesion molecule expression, platelet adhesion, and plaque growth, whereas in hypercholesterolemic rabbits, apocynin started at a much earlier stage of disease has been shown to prevent development of atherosclerotic lesions.⁴ It is unknown whether the beneficial effects of apocynin occur early after initiation of therapy. With regards to mechanism, it is unknown whether apocynin’s effects are entirely caused by a reduction in oxidative stress because polyphenolic drugs, such as apocynin, have anti-inflammatory effects independent of their antioxidant properties.^5,6 Direct anti-inflammatory action independent of antioxidant properties has been substantiated by the reduced adhesion molecule expression in cultured endothelial cells exposed to apocynin.^7,8

In this study, we addressed many of these knowledge gaps. We performed in vivo ultrasound molecular imaging to test the hypothesis that short-term administration of apocynin in a model of early atherosclerosis reduces endothelial activation and platelet adhesion, 2 factors that are recognized to play an important role in plaque progression. Ex vivo techniques were used to evaluate whether these effects were associated with a reduction in vessel oxidative stress.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Effect of Apocynin on Vascular Cell Adhesion Molecule 1 Expression and Endothelial Platelet Adhesion

Apocynin treatment for 1 week reduced total aortic wall expression of vascular cell adhesion molecule 1 (VCAM-1) on Western blot by 50% (Figure 1A). On immunohistochemistry, small regions of neointimal formation were observed in the aortic root and proximal ascending portion. VCAM-1 staining was present on the endothelial lining and on macrophages in fibrofatty lesions in nontreated animals. In apocynin-treated animals, VCAM-1 expression was reduced both on endothelial cells and on macrophages within lesions, whereas the total amount of macrophages present in plaques remained unchanged (Figure 2). The reduction in endothelial VCAM-1 expression was evident both in regions overlying plaques and on endothelium in regions without plaques (Figure 1B and 1E).

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In vivo labeling of platelets with rhodamine-6G allowed visualization of platelet/leukocyte aggregates on the vascular endothelial surface. Platelet/leukocyte aggregates were present not only in regions with early plaques but also in regions of the aortic endothelial surface with a normal appearance. Both the number of platelet/leukocyte aggregates per square millimeter (5.0±0.44 in apocynin-treated animals versus 9.6±0.52 in nontreated animals; P<0.01) and the percentage of endothelial surface covered by platelet/leukocyte aggregates were significantly reduced in animals that were treated with apocynin (Figure 3).

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Effect of Apocynin on Vascular Oxidative Stress

Lucigenin assays showed robust NADPH oxidase activity in whole aortic rings of nontreated mice. In mice treated with apocynin, NADPH-dependent lucigenin chemiluminescence was not different compared with nontreated mice. Given the potential of lucigenin to undergo redox cycling and generate artifactual signals, high pressure liquid chromatography of tissue extracts after exposure of aortic rings to hydroethidine was performed to directly assess tissue superoxide content. In accordance with the results of the lucigenin assays, hydroethidine oxidation to 2-hydroxyethidium was not different between the 2 animal groups (Figure 4).

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Molecular Imaging of VCAM-1 Expression and Platelet Adhesion

High-frequency ultrasound imaging was not of sufficient quality for evaluation in 1 animal in each group. In the remaining animals, there were no differences in left ventricular ejection fraction, peak aortic flow velocity, or aortic diameter, indicating that apocynin treatment did not lead to hemodynamic differences that could potentially influence targeted microbubble adhesion (Table).

Contrast-enhanced ultrasound molecular imaging in the ascending aorta at baseline showed greater signal enhancement for VCAM-1–targeted and platelet-targeted microbubbles compared with control microbubbles (Figure 5). After 7 days of treatment with apocynin, signal for VCAM-1–targeted and platelet-targeted microbubbles was not different from control microbubble signal (Figure 6). In contrast, in animals treated with saline injections, the signal for VCAM-1 and platelets was elevated significantly over control signal to a degree that was similar to baseline. In the subgroup of animals that were imaged before and after treatment, apocynin leads to a significant decrease in VCAM-1–targeted signal (from 1.80±0.51 to 0.30±0.10; P=0.046) and a strong trend for decrease in platelet-targeted signal (2.21±0.80 to 0.35±0.07; P=0.078). In animals imaged before and after treatment with saline injections, signal for VCAM-1 (1.44±0.55 versus 1.00±0.23,; P=1.00) and platelet-targeted signal (1.84±0.88 versus 1.26±0.40; P=0.69) did not decrease significantly.

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Discussion

Endothelial activation plays a crucial role in the initiation and progression of atherosclerotic plaque formation. In this study, short-term treatment with the polyphenol apocynin in a murine model of early mild atherosclerosis leads to a reduction in endothelial inflammatory phenotype and platelet adhesion. These relatively acute changes were not associated with a measurable reduction in vascular NADPH oxidase activity or superoxide content.

Endothelial adhesion molecule expression is an early and important step in the pathogenesis of atherosclerosis. Deposition of oxidized low-density lipoprotein in the vascular wall leads to endothelial expression of proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α. Locally increased cytokine levels result in an upregulation of cell adhesion molecules, such as VCAM-1, mediated by the transcriptional factor–activated protein-1 and nuclear factor κB, which in turn promotes the recruitment of leukocytes to the vessel wall.

ROS generated by NADPH oxidases both in endothelial cells and in leukocytes in nascent plaques are thought to amplify vascular inflammation throughout the pathogenesis of atherosclerosis. Accordingly, mouse models with knockouts of the NADPH oxidase isoforms, NOX-1 and NOX-2, or the cytosolic NADPH oxidase subunit, p47phox, have shown a reduction in atherosclerotic plaque formation.^9–11 In humans, functional deficiency of the GP91phox subunit is associated with smaller carotid intima-media thickness.¹² These findings have generated interest in using inhibitors of NADPH oxidases for the treatment of atherosclerosis. Apocynin inhibits NADPH oxidase activity in leukocytes allegedly by impeding the assembly of the cytosolic subunits at the cell membrane.¹³ In cell culture experiments it has been noted that the inhibitory action of apocynin occurs with a delay, suggesting that it has to undergo activation before inhibiting ROS generation. In the presence of H₂O₂ and myeloperoxidase, apocynin is converted to an apocynin radical and subsequently forms apocynin dimers, which are thought to be the active compounds that result in inhibition of NADPH oxidase activity.¹⁴ Thus, the effect of apocynin on NADPH oxidase inhibition during the later stages of atherosclerosis may depend on its activation in vascular tissue and require a sufficient inflammatory cell and oxidative stress burden. Notably, however, apocynin also exerts anti-inflammatory effects that are independent of NADPH oxidase inhibition.⁶ Such effects may be of importance during the early stages of atherosclerosis, when inflammatory cell load and oxidative burden are low.

We examined the effects of treatment with apocynin in a murine model of early atherosclerosis in mice with fibrofatty lesions. Assessing treatment effects of potential drug candidates during the early stages of atherosclerosis is of clinical significance because interventions that are started early during the pathogenesis of atherosclerosis are thought to afford a larger risk reduction for cardiovascular events than interventions that are initiated when clinical atherosclerotic disease is established.¹⁵ Our data indicate that treatment with apocynin results in a rapid decrease in endothelial expression of VCAM-1. These results are in line with observations in cell culture showing a decrease in VCAM-1 expression in response to apocynin⁸ and extend observations made in advanced atherosclerosis to earlier stages of plaque development.⁴ The decrease in signal from VCAM-1–targeted microbubbles was more pronounced than differences in VCAM-1 expression determined with Western blot. We believe this to be because of the capability of ultrasound molecular imaging to specifically assess endothelial inflammatory phenotype¹⁶ and thus to detect a more pronounced decrease in endothelial VCAM-1 compared with Western blot which assesses expression in the whole vascular wall. In addition, the observation that the decrease in vascular inflammation was not associated with a reduction of vascular NADPH oxidase activity or tissue superoxide content indicates that the anti-inflammatory effect of apocynin observed in our study was probably mediated through a ROS-independent mechanism in the very early stages of atherosclerotic plaque development, possibly mediated instead by its effects on cytochrome P450 pathways.¹⁷

In addition to endothelial cell inflammatory activation, platelet–endothelial interactions play a role in vascular inflammation and the pathogenesis of atherosclerosis. Platelet–endothelial interactions mediated by P-Selectin and von Willebrand factor-glycoprotein Ibα ligation in the absence of plaque rupture accelerate plaque formation in murine atherosclerosis.^18–20 The interaction of activated platelets with the endothelial surface results in the secretion of proinflammatory cytokines CD40L and interleukin-1β, as well as of chemokines such as RANTES (regulated on activation, normal T cell expressed and secreted) and platelet factor 4 to the vascular wall, all of which facilitate increased monocyte recruitment.^21–23 Platelets from patients with functional deficiency of GP91phox subunit of NADPH oxidase or control subject platelets treated with apocynin have reduced in vitro platelet recruitment and aggregation, indicating a direct role of NADPH oxidase in platelet reactivity.²⁴ Our molecular imaging results indicate a decrease in platelet–endothelial interactions after treatment with apocynin. Although we did not specifically investigate the pathways responsible for the action of apocynin in platelets, previous data indicate that apocynin influences platelet aggregation by mechanisms that are dependent not only on NADPH oxidase activity²⁵ but also on changes in arachidonic acid metabolism with a decrease in thromboxane A2 formation.⁵

Several limitations of our study deserve attention. First, as the aim of our study was to assess the acute effects of apocynin treatment on the endothelial inflammatory phenotype, we did not expect an influence of treatment on plaque size and thus did not perform histological analysis. However, long-term treatment with apocynin has been shown to reduce plaque formation in our mouse model.⁵ Also, the dose used in our study was in the low range of doses used in published animal studies; however, there is no established optimal dose, and our data demonstrate an effect of the treatment on both endothelial inflammatory phenotype and platelet adhesion. Furthermore, although we applied well-established techniques to measure NADPH oxidase activity and superoxide content without finding an effect of apocynin therapy, we cannot exclude that locally restricted (eg, endothelial) and changes in other ROS species contributed to the observed effect. Finally, our methods for evaluating platelet adhesion did not allow differentiation of direct endothelial attachment and platelet–monocyte complexes.

In summary, we show that in a murine model of early atherosclerosis, treatment with apocynin leads to a rapid decrease in endothelial inflammation and platelet adhesion, which is detectable using ultrasound molecular imaging. Our data indicate that these effects of apocynin are not associated with a measurable decrease in ROS generation.

Footnotes