Atherosclerosis (ATS) is a chronic inflammatory disease characterized by formation of a plaque or lesion (also called atheroma) in the intimal layer of the arterial wall. Atherosclerotic plaque is composed of oxidized lipids and lipoproteins, inflammatory cell infiltrates, areas of cell death and fibrosis [1]. ATS is the leading cause of morbidity and mortality in developed nations. ATS is a dynamic disease evolving in time often-taking decades to develop advanced lesions responsible for clinical symptoms. Plaques may remain asymptomatic (subclinical disease), become occlusive (intermittent claudication or stable angina pectoris), or become thrombosis-prone (vulnerable) leading to athero-thrombotic events, such as myocardial infarction or stroke [2]. Pathogenesis of ATS is not yet fully elucidated; however clear evidence indicate that the Renin-Angiotensin System (RAS), and in particular its final product Angiotensin (Ang) II, play a pivotal role in atherogenesis.

The RAS is intimely involved in the maintenance of cardiovascular homeostasis, including regulation of blood pressure, blood flow, fluid volume and electrolyte balance. Secretion of renin is the first step in the RAS cascade. Renin is secreted in a regulated manner by the juxtaglomerular cells of the kidneys. Renin cleaves angiotensinogen, mainly synthesized in the liver, to form the inactive peptide Ang I. This is in turn converted by the Angiotensin Converting Enzyme (ACE) to form the biologically active peptide Ang II, the primary effector of the RAS. Ang II exerts its physiological actions through membrane-bound receptors of the G-protein coupled receptor family. The two main Ang II receptors are the Ang II receptor subtypes 1 (AT1) and 2 (AT2) which induce different signaling pathways and cellular responses. Proatherosclerotic actions of Ang II are mediated by the AT1 receptor whereas the role of AT2 in ATS is still controversial. AT1 receptor is expressed in a variety of organs, blood vessel, and in bone marrowderived cells, such as macrophages and T cells [3,4].

The importance of Ang II in atherosclerotic cardiovascular disease has been widely demonstrated both indirectly and directly in humans and mouse models. Indeed, pharmacological inhibition of RAS system using ACE inhibitors or Angiotensin Receptor Blocker (ARBs) prevents or delays progression of ATS and reduces cardiovascular events in patients [5,6]. Along the same line, pharmacological inhibition or genetic inactivation of RAS prevents ATS development and stabilizes plaques in animal models of ATS [7-10]. Observational studies in humans have demonstrated that patients with high levels of circulating renin are at increased risk for myocardial infarction and other atherosclerotic cardiovascular events, suggesting a direct role for Ang II in promoting ATS [11,12]. During the past decades, specific mouse models with increased plasma concentrations of Ang II have been generated in hypercholesterolemic apolipoprotein E-deficient (ApoE^-/-) and low-density lipoprotein-deficient (LDLr^-/-) mice, allowing study of mechanism through which Ang II promotes ATS [13,14].

This review emphasizes the pro-atherosclerotic effects and underlying mechanisms of Ang II derived from mouse studies with Ang II-mediated ATS.

To define the direct role of Ang II in atherogenesis, nonphysiological and physiological models have been generated in ApoE^-/- or LDLr^-/- mice.

Non-physiological model: subcutaneous infusion of Ang II

Daugherty et al. initiated studies in which Ang II was subcutaneously and chronically infused into ApoE^-/- mice for 28 days by using osmotic pumps [13]. Results showed that Ang II promoted a significant increase in the extent of atherosclerotic lesion in the thoracic aorta, and that lesions were predominantly rich in lipid-laden macrophages and lymphocytes. Of note, authors reported no significant differences between mice infused with increasing doses of Ang II (500 compared to 1000 ng/min/Kg). Unexpectedly, mice also developed large abdominal aortic aneurysm. These vascular effects of Ang II occurred independent of blood pressure elevations or changes in plasma lipid concentration [13]. Weiss et al. also showed that subcutaneous Ang II infusion induced-hypertension (8 weeks) specifically increased plaque size, in thoracic and abdominal aorta, as well as macrophage infiltration in ApoE^-/- mice receiving standard chow or atherogenic diet [15]. Ni et al. have first demonstrated evidence of plaque vulnerability in response to Ang II infusion in ApoE^-/- mice [16]. Authors reported that Ang II infusion into ApoE^-/- mice not only increased plaque size confirming previous findings, but also promoted atherosclerotic plaque transformation to a more destabilized phenotype (increased macrophages and lipids, decreased collagen and smooth muscle cells content) [16]. Ang II continued to accelerate ATS in ApoE^-/- mice even after discontinuation of Ang II infusion [17].

Physiological model: 2-Kidneys, 1-Clip renovascular hypertension

To study the contribution of Ang II in ATS in physiological conditions, Mazzolai et al. generated hypertensive ApoE^-/- mice with endogenously increased (two-kidney, one clip, 2K1C) Ang II production (renovascular hypertension model) [14]. The 2K1C ApoE^-/- mice are characterized by a pathophysiologically and continuously stimulated RAS with an endogenous increase in renin and consequently Ang II production secondary to unilateral renal artery clipping producing artery stenosis [18,19]. Contrary to models were high doses of Ang II are exogenously administered via minipumps as described above, the 2K1C ApoE^-/- mouse model allows studying the effect of Ang II and hypertension on ATS development/progression in a context that is very much similar to that found in humans. In fact, in the 2K1C ApoE^-/- mice, increased endogenous Ang II production is achieved through stimulation of renal renin secretion, as it occurs in humans, thus taking into account all the physiological regulatory mechanisms. Results revealed that 2K1C ApoE^-/- mice presented increased lesion surface compared to normotensive ApoE^-/- mice. However, extent of increased lesion surface was comparable to that observed in similarly hypertensive ApoE^-/- mice but with normal circulating Ang II levels (1-kidney, 1-clip [1K1C] model) [14]. Nevertheless, 2K1C mice developed more advanced and vulnerable plaques than 1K1C mice (thinner fibrous cap, larger lipid core, increased macrophage content and decreased smooth muscle cells), confirming the hypothesis that Ang II mediates ATS independently of its hemodynamic effects but via a direct vascular effect.

Ang II modulates pro-atherosclerotic genes expression in vascular wall

Several studies have shown that Ang II increases expression of pro-atherosclerotic and/or decreases expression of anti-atherosclerotic genes in the aorta (Table 1). For example, Tham et al. reported a downregulation of the anti-inflammatory genes PPAR-α and PPAR-γ, and an upregulation of pro-inflammatory genes such as the MCP-1, the M-CSF, the E-selectin, the ICAM-1, the VCAM-1, the iNOS, and the COX-2 through a nuclear factor-kappaB-dependent pathway in aorta from ApoE^-/- mice infused with Ang II [20]. Increased expression of other pro-inflammatory cytokines in response to Ang II has also been shown in aorta and culture of explanted aorta from mice with Ang IIinduced ATS [16,21]. More recently, it has been shown that Ang II upregulated extracellular matrix metalloproteinase inducer (EMMPRIN) expression in aortic plaques from ApoE^-/- mice, which could play a role in plaque destabilization [22].

Table 1: Effects of Angiotensin II on pro-atherosclerotic and anti-atherosclerotic genes expression in mouse aorta [16,20,21].

Ang II induces Th1/Th2 imbalance

Inflammatory cells, including CD4 T lymphocytes, are commonly found in ATS plaque during all stages of the disease [1]. CD4⁺ T cell subtype Th1 (pro-inflammatory cells) plays a major role in ATS while the role of CD4⁺ T cell subtype Th2 (anti-inflammatory cells) is still unclear [1]. Mazzolai et al. investigated for the first time the specific role of Th1/Th2 balance in Ang II-induced ATS [14]. Results showed that IFN-γ production (the Th1 signature cytokine) from spleen lymphocytes was stimulated in ApoE^-/- 2K1C one week after renal artery clipping whereas IL-4 production (the Th2 signature cytokine) remained unchanged [14]. These findings indicate that Ang II skews the Th1/Th2 balance towards a pro-inflammatory Th1 profile in the early phase of ATS process, which may induce plaque vulnerability.

Other mechanisms

Recent studies using pharmacological or genetic approaches in Ang II-infused hypercholesterolemic mice, showed that numerous factors/proteins are involved in Ang II-induced ATS, including myeloid differentiation factor 88 (a mediator of signaling cascade that directly influences leukocytes involved in innate immunity and has indirect effects on adaptive immunity) [23], endothelin-1 [24], or receptor-associated protein (a regulator of low density lipoprotein receptor-related protein 1) [25]. Endothelial dysfunction is a critical element in the pathogenesis of atherosclerotic disease. Impaired aortic endothelium-dependent relaxation as well as decreased eNOS protein phosphorylation has been recently demonstrated in ApoE^-/- miceinfused Ang II [26].

Mouse models with high circulating Ang II levels provide direct evidence that Ang II is a local mediator of plaque development and vulnerability. Modulation of Th1/Th2 phenotype appears to be a major downstream atherosclerotic mechanism of Ang II. Further research is needed to determine the time course of Th1/Th2 modulation in ATS process.

Th17 has been recently identified as the third CD4 T cell subset, and has been suggested to play an important role in Ang II-induced ATS [27]. However, no experimental data are available yet to substantiate this hypothesis.

Similarly, to CD4 T cells, macrophages can polarize into two different subsets: classically pro-inflammatory M1 macrophages, driven by Th1 cytokines, or alternatively anti-inflammatory M2 macrophages, driven by Th2 cytokines. Although recent evidence indicates that the macrophage polarization balance affects pathogenesis, evolution, and complications of ATS [28], its specific implication in Ang II-induced ATS is unknown.

In conclusion, although there are good experimental proofs demonstrating a pro-atherosclerotic role of Ang II, the precise mechanisms whereby Ang II exerts its actions need further clarification. Ang II-dependent signaling pathways may refer to a complex and multi factorial process including, at least in part, the involvement of various immuno-inflammatory cell types, and a pro-and anti-inflammatory cytokines network. More work is needed to define the specific networks of pathways activated by Ang II.

A better understanding of cellular and molecular mechanisms underlying how Ang II mediates atherosclerosis and plaque vulnerability may help develop new and more selective therapeutic strategies aiming at preventing Ang II-related atherosclerotic cardiovascular disease.

Results reported in mouse studies, if confirmed in clinical studies, should have clinical implications. In fact, hypertensive or nonhypertensive patients presenting with clinical or subclinical atherosclerosis would benefit from pharmacological blockade of key pro-atherosclerotic Angiotensin II-dependent signaling pathways.