**1. Introduction**

The (in)stability of atherosclerotic plaques determines the incidence of major cardiovascular events such as myocardial infarction and stroke [1]. Lack of oxygen within the plaque, or intraplaque hypoxia, has been identified as one of the major contributors to plaque instability [2,3]. It has been detected in advanced human atherosclerotic lesions [4] as well as in murine atherosclerotic lesions [5,6].

The intraplaque lack of oxygen is provoked by progressive thickening of the neointimal layer [7] and overconsumption of O2 by plaque inflammatory cells [4]. The key regulator of hypoxia is the transcription factor Hif1a [8]. Low oxygen levels, or hypoxia, prevents degradation of Hif1a, promoting its dimerization with the Hif1b subunit. This complex activates the transcription of multiple genes, the most important being Vegfa, that triggers the formation of neovessels in the plaque. Intraplaque neovessels are often immature and therefore leaky, leading to intraplaque hemorrhage, a phenomenon characterized by extravasation of inflammatory cells and red blood cells inside the plaque. Hypoxia also upregulates the expression of transcription factors that cause vascular calcification in vascular smooth muscle cells [9], a characteristic feature of atherosclerosis. Both intraplaque neovessels and vascular calcification are regulated by hypoxia [9] and contribute to plaque instability. The combination of those processes results in a larger plaque which is more unstable and prone to rupture [10,11]. Hif1a was shown to be present in macrophage-rich and foam cell-rich areas and its expression in macrophages was correlated with accelerated atherosclerosis development in LDLR-/- mice [12]. Moreover, it has been shown that hypoxia can influence gene expression in macrophages, leading to an inflammatory response with increased production of pro-inflammatory cytokines [13,14].

Thus, the reoxygenation of the atherosclerotic plaque would be expected to prevent intraplaque hypoxia and atherosclerotic plaque progression. Previously, Marsch et al. showed that plaque reoxygenation in LDLR-/- mice via breathing of the hyperoxic gas carbogen, composed of 95% O2 and 5% CO2, prevented necrotic core expansion by enhancing e fferocytosis [15]. The response of intraplaque angiogenesis to reoxygenation could not be studied in this model, as intraplaque angiogenesis is virtually nonexistent in plaques of LDLR-/- mice. To examine the e ffect of carbogen treatment on intraplaque angiogenesis we used vein grafts in hypercholesterolemic ApoE3\*Leiden mice that do harbor extensive intraplaque angiogenesis, and have been shown to be morphologically similar to rupture-prone plaques in humans. The lesions in this model have the typical characteristics of late stage atherosclerosis, including the presence of foam cells, intraplaque neovascularization, calcification and cholesterol clefts [16] and eventually also occlusion of the graft.

We hypothesized that carbogen gas exposure would reduce hypoxia in vein grafts in the ApoE3\* Leiden mice and consequently would reduce intraplaque angiogenesis and increase lesion stability. Thus, we used this model to study plaque reoxygenation and its e ffect on vein graft remodeling, intraplaque neovascularization, inflammation and vein graft patency. Moreover, since prolonged hyperoxia has the risk of introducing reactive oxygen species [17], we investigated the e ffect of reactive oxygen species in vitro on bone marrow derived macrophages and in vivo in the atherosclerotic lesions and their e ffect on the plaque environment.

#### **2. Materials and Methods**
