Diminazene enhances stability of atherosclerotic plaques in ApoE-deficient mice

Abstract

Angiotensin (Ang) II contributes to the development of atherosclerosis, while Ang-(1–7) has atheroprotective actions. Accordingly, angiotensin-converting enzyme 2 (ACE2), which breaks-down Ang II and forms Ang-(1–7), has been suggested as a target against atherosclerosis. Here we investigated the actions of diminazene, a recently developed ACE2 activator compound, in a model of vulnerable atherosclerotic plaque. Atherosclerotic plaque formation was induced in the carotid artery of ApoE-deficient mice by a shear stress (SS) modifier device. The animals were treated with diminazene (15 mg/kg/day) or vehicle. ACE2 was strongly expressed in the aortic root and low SS-induced carotid plaques, but poorly expressed in the oscillatory SS-induced carotid plaques. Diminazene treatment did not change the lesion size, but ameliorated the composition of aortic root and low SS-induced carotid plaques by increasing collagen content and decreasing both MMP-9 expression and macrophage infiltration. Interestingly, these beneficial effects were not observed in the oscillatory SS-induced plaque. Additionally, diminazene treatment decreased intraplaque ICAM-1 and VCAM-1 expression, circulating cytokine and chemokine levels and serum triglycerides. In summary, ACE2 was distinctively expressed in atherosclerotic plaques, which depends on the local pattern of shear stress. Moreover, diminazene treatment enhances the stability of atherosclerotic plaques.

Introduction

Vulnerable plaque, characterized by the presence of intense inflammation and a lipid-rich necrotic core covered by a thin fibrous cap, have a higher propensity to rupture and lead to subsequent thrombotic occlusion. Indeed, it is well recognized that the risk of thrombosis in atherosclerosis largely depends on plaque composition [1], [2].

Local shear stress forces have been documented as a biomechanical factor which modulates atherogenesis and plaque composition [3], [4], [5]. For instance, it has been shown that local low shear stress (LSS) may induce plaque formation and a vulnerable composition [5]. Several molecular pathways are involved in the shear-stress inducing plaque vulnerability process; however, such mechanisms are poorly known.

The renin–angiotensin system (RAS), a major regulator of cardiovascular function, is highly involved in the genesis and progression of atherosclerosis [6], [7], [8]. Moreover, this system seems to actively influence the composition of atherosclerotic plaques. Angiotensin (Ang) II, the main effector of RAS, contributes to atherosclerosis development by increasing vascular permeability, inflammatory cell infiltration and LDL oxidation and uptake [9], [10], [11]. Moreover, Ang II may increase plaque vulnerability by modulating macrophage trapping, increasing reactive oxygen species production and weakening the fibrous cap by activation of matrix metalloproteinase (MMP) [10], [12], [13].

Contrarily to Ang II, emerging data indicates that Ang-(1–7), another effector of RAS, has atheroprotective actions [14], [15], [16]. It has been shown that Ang-(1–7) infusion promotes a reduction in plaque size and improves vascular endothelial function in hypercholesterolemic mice [17]. Moreover, long-term treatment with Ang-(1–7) enhances atherosclerotic plaque stability by increasing intraplaque collagen content, decreasing MMP-9 expression and reducing neutrophil and macrophage infiltration [18].

Angiotensin-converting enzyme 2 (ACE2) is a key regulator of Ang II and Ang-(1–7) levels. This enzyme breaks-down Ang II, by cleaving the C-terminal phenylalanine and, consequently, forms Ang-(1–7); therefore, ACE2 reduces Ang II and favors Ang-(1–7) actions [19]. ACE2 enzyme is expressed in animal models [20] and human [21] atherosclerotic plaques, playing a protective role in atherosclerosis [14]. Indeed, ACE2 overexpression attenuates the progression of atherosclerotic lesions and increases plaque stability [22]. Contrarily, ACE2 deficiency in low-density lipoprotein receptor (Ldlr^−/−) or apolipoprotein E (ApoE^−/−)-deficient mice worsens atherogenesis [23], [24], [25]. Based on these observations, ACE2 has being suggested as a potential target for the treatment of atherosclerosis.

In the present study, an investigation on the effects of diminazene (a recently developed pharmacological ACE2 activator) [26], [27], in a mouse model of vulnerable atherosclerotic plaque was performed.