**3. Endothelial cell responses after ionizing radiation exposure**

The endothelium could be a critical target in ionizing radiation-related CVD [74]. The endothelium is a single layer of cells that lines the interior of the vascular system and of the heart and has thus a strategic position between the blood and the surrounding tissues. It is a highly active organ system that is constantly sensing and responding to changes of the extracellular environment to maintain a normal function of the vascular system [75]. Endothelial cells are involved in a wide range of physiological processes, such as regulation of vascular tone, vascular permeability, blood coagulation/fibrinolysis, and inflammation, which are needed to maintain proper vascular functioning (**Figure 4**) [76]. Endothelial dysfunction has been observed in patients with atherosclerosis and in patients that exhibit CVD risk factors such as smoking, dyslipidemia, obesity, and diabetes mellitus [77] and is considered to be one of the first predictive indicators of cardiovascular morbidity and mortality [78].

of the atherosclerotic plaque. Radiation has been shown to upregulate several of such adhesion molecules. For instance, exposure of endothelial cells to 5 Gy increases the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin 6 h after irradiation [111]. Platelet endothelial cell adhesion molecule-1 (PECAM-1), ICAM-1 and ICAM-2, and vascular cellular adhesion molecule-1 (VCAM-1) were also observed to increase in mouse heart cells 10 weeks after local thorax irradiation with 8 Gy [112]. Interestingly, ICAM-1 and VCAM-1 remained upregulated 20 weeks after irradiation. Besides induction of adhesion molecules, the expression of cytokines, such as interleukin (IL)-6 and IL-8, and other inflammatory molecules such as transforming growth factor-β (TGF-β) was shown to increase after high and moderate irradiation doses in human endothelial cell cultures [113, 114]. In this context, the Japanese atomic bomb survivors' cohort also showed signs of a generally increased inflammation state, with

Selected Endothelial Responses after Ionizing Radiation Exposure

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Ionizing radiation is known to induce a wide range of DNA lesions, of which double-strand breaks (DSBs) are most severe in a direct manner but also indirectly through the formation of reactive oxygen species (ROS) [116, 117]. Upon DNA damage, a response is initiated, and cells activate cell cycle checkpoints that slow down or stop cell cycle progression [118]. This gives them time to repair damaged DNA or to prevent division when chromosomes are damaged or incompletely replicated. If cells fail to repair their DNA, they undergo programmed cell death, apoptosis, or premature senescence (described below) [119]. Consequently, DSB leads

Whereas high doses are known to induce apoptosis in endothelial cells [120], less is known about the effect of low radiation doses. A subtle but significant increase in DSBs was observed in human umbilical vein endothelial cells (HUVEC) and EA.hy926 endothelial cells 30 min after exposure to 0.05 Gy. In addition, irradiation with 0.05 Gy and 0.1 Gy induced relatively more DSB/Gy in comparison to 0.5 Gy and 2 Gy [121]. This observation could be caused either by an underestimation due to DNA damage spot merging [122] or by the induction of a global chromatin reorganization at low doses of ionizing radiation [123]. Furthermore, a dose-dependent increase in the number of apoptotic cells was observed, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells [121]. Another study showed no increase in the number of apoptotic endothelial cells after exposure to 0.2 Gy, whereas apoptosis was observed after exposure to 5 Gy [124].

Mitochondria are often regarded as the powerhouse of the cell by generating the ultimate energy transfer molecule, adenosine triphosphate or ATP. Mitochondrial dysfunction is part of both normal and premature agings, but it can also contribute to inflammation, cell senescence, oxidative stress, and apoptosis. Increasing evidence indicates that mitochondrial damage and dysfunction occur in atherosclerosis and may contribute to the multiple patho-

An increased accumulation of mitochondrial DNA damage was observed in several human fibroblast cell lines after exposure to doses as low as 0.1 Gy [126]. Furthermore, functional impairment of mitochondria (reduced mitochondrial respiration and electron transport chain activity) and

**3.3. Oxidative stress, mitochondrial dysfunction, and metabolic changes**

increased levels of IL-6 and C-reactive protein (CRP) [115].

**3.2. DNA damage and apoptosis**

to a high lethality of the affected cells.

logical processes underlying the disease [125].

A dysfunctional endothelium is characterized by inflammation, DNA damage, oxidative stress, alterations of coagulation and platelet pathways, senescence, and cell death, all of which are observed after radiation doses above 1–2 Gy, as shown in many in vitro and in vivo studies [6, 28, 79–81]. Comparatively, both protective and detrimental effects have been reported for low-dose exposure, suggesting that multiple mechanisms may influence radiation-induced atherosclerosis [6, 62]. Increasing evidence also suggests a role of intercellular communication in the endothelial cell response to ionizing radiation [82]. All of these endpoints are briefly discussed in the following paragraphs. In addition, other pathological effects of ionizing radiation on the endothelium are observed like impaired endothelial regulation of vascular tone [83−87], loss of the endothelial monolayer integrity [88–92], and procoagulant and prothrombic conditions [28, 93−108].

#### **3.1. Inflammation**

Endothelial expression of adhesion molecules plays an important role in recruiting inflammatory cells from the bloodstream into the vessel intima where they transform into foam cells, elements

**Figure 4.** Overview of the major physiological functions of the arterial endothelium. (A) The endothelium (ECs, endothelial cells) forms a selective barrier regulating solute flux and fluid permeability between the blood and surrounding tissues [78]. (B) Formation of a thrombus or blood clot is referred to as intravascular coagulation, and the breakdown of a thrombus is referred to as fibrinolysis. Normal endothelium has antithrombotic and profibrinolytic properties and actively represses platelet adhesion and aggregation. Vessel damage or exposure to pro-inflammatory molecules shifts the balance toward more prothrombic/antifibrinolytic activities [75, 109]. (C) To regulate the vascular tone, the endothelium releases various vasodilatory agents, such as nitric oxide (NO) and endothelial-derived hyperpolarizing factor (EDHF), or vasoconstrictive agents, such as endothelin-1 (ET-1), which modify vascular smooth muscle cell (VSMC) contractility [110]. (D) In the case of inflammation, endothelial permeability is increased. Endothelial cells recruit immune cells via the expression of adhesion molecules and mediate their transmigration toward the inner vascular wall [75, 76] .

of the atherosclerotic plaque. Radiation has been shown to upregulate several of such adhesion molecules. For instance, exposure of endothelial cells to 5 Gy increases the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin 6 h after irradiation [111]. Platelet endothelial cell adhesion molecule-1 (PECAM-1), ICAM-1 and ICAM-2, and vascular cellular adhesion molecule-1 (VCAM-1) were also observed to increase in mouse heart cells 10 weeks after local thorax irradiation with 8 Gy [112]. Interestingly, ICAM-1 and VCAM-1 remained upregulated 20 weeks after irradiation. Besides induction of adhesion molecules, the expression of cytokines, such as interleukin (IL)-6 and IL-8, and other inflammatory molecules such as transforming growth factor-β (TGF-β) was shown to increase after high and moderate irradiation doses in human endothelial cell cultures [113, 114]. In this context, the Japanese atomic bomb survivors' cohort also showed signs of a generally increased inflammation state, with increased levels of IL-6 and C-reactive protein (CRP) [115].
