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

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 pathological processes underlying the disease [125].

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 alterations of the mitochondrial proteome were observed in isolated cardiac mouse mitochondria 4 and 40 weeks after a 2 Gy local heart irradiation. Only a few alterations of the mitochondrial proteome and no effect on mitochondrial function were observed with 0.2 Gy [127, 128]. Finally, alterations of energy and lipid metabolism and perturbations of the insulin/insulin growth factor—phosphatidylinositol-4,5-bisphosphate 3-kinase—RAC-alpha serine/threonine-protein kinase (IGP-PI3K-Akt) signaling pathway were suggested in proteomic studies using cell lines or cells isolated from mice after irradiation with doses ranging from 3 to 16 Gy [129−131].

**3.5. Intercellular communication**

sure in mouse primary endothelial cells [161].

**4. Conclusion**

Traditionally, it was accepted that exposure to ionizing radiation only directly affects irradiated cells. However, in 1992, it was found that irradiation of 1% cells with α-particles leads to genetic damage in more than 30% of cells [148]. Exposure of cells to ionizing radiation results in significant biological effects occurring in both irradiated and non-irradiated cells through the radiation-induced bystander effect [149, 150]. Although the mechanisms of this effect are not fully elucidated yet, oxidative stress, different cytokines (e.g., TNF-α, IL-1, and

Selected Endothelial Responses after Ionizing Radiation Exposure

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Intercellular communication through gap junctions and paracrine signaling through hemichannels have been suggested to mediate bystander responses. Gap junctions and hemichannels are composed of multimeric transmembrane structures made of connexin (Cx) [150, 151]. In human, 21 Cx proteins have been identified, which are present in most organs, and display a tissue/cellular specificity [152, 153]. There are three different Cx isotypes expressed in endothelial cells of major arteries, namely, Cx37, Cx40, and Cx43 [154−156]. Cxs have important physiological roles (e.g., they support longitudinal and radial cell-cell communication in the vascular wall), and changes of their expression patterns have been observed during atherosclerosis. Although healthy endothelial cells mainly express Cx37 and Cx40, both Cxs are lost in the endothelium covering advanced atherosclerotic plaques. On the contrary, Cx43 is detectable at specific regions of advanced atherosclerotic plaques [157]. The mechanisms responsible for modification of the Cx expression pattern in atherosclerosis are not fully understood. However, it has been recently demonstrated that Cx37 is a regulator of endothelial NO synthase (eNOS) [158]. The altered Cx37 expression level could be responsible for decreased eNOS activity and decreased NO bioavailability, which may cause endothelial cell dysfunction and increased susceptibility to atherosclerosis. Therefore, Cx37 may play a protective role against atherosclerosis. In addition, Cx40 may play a similar role, as endothelial-specific deletion of Cx40 was reported to promote atherosclerosis by increasing CD73-dependent leukocyte adhesion to the endothelium [155]. In contrast to the atheroprotective effects of Cx37 and Cx40, Cx43 has been suggested to be pro-atherosclerotic. Indeed, downregulation of Cx43 expression inhibited monocyte-endothelial adhesion by decreasing the expression levels of cell adhesion proteins, whereas its upregulation enhanced the adhesion of monocytes to endothelial cells [159]. Besides their roles in atherosclerosis, Cxs were reported to be highly sensitive to ionizing radiation [156]. Indeed, it was observed that a low-dose irradiation exposure induced activation of Cx43 in a time- and dose-dependent manner in human neonatal foreskin fibroblasts [160]. Moreover, upregulation of Cx43 was noticed after 5 Gy of X-ray expo-

Research regarding CVD risk related to ionizing radiation is an important way forward to complement epidemiological data with the underlying biological and molecular mechanisms. This is especially important for doses <0.5 Gy, for which epidemiological data are suggestive rather than persuasive. Indeed, due to limited statistical power, the dose-risk relationship is undetermined below 0.5 Gy, but if this relationship proves to be without a threshold, it

IL-6), Ca2+, and kinases play a role in the damage to non-irradiated cells.

Water radiolysis instantly causes the formation of ROS (e.g., •OH<sup>−</sup> , •O2 − , H2 O2 ). However, cellular oxidative stress can also be observed long after irradiation, due to an increase in endogenous cellular ROS production [132]. Mitochondria are believed to be the major source of radiation-induced secondary ROS. For instance, Leach et al. have demonstrated that between 1 and 10 Gy, the amount of ROS-producing cells increased with the dose, which they suggested was dependent on radiation-induced propagation of mitochondrial permeability transition via a Ca2+-dependent mechanism [133, 134]. It has further been suggested that ROS can be transferred from cell to cell by gap junctions and paracrine communication pathways in order to propagate radiation-induced biological effects at the intercellular level. This phenomenon is commonly referred to as the radiation-induced "bystander effect" [135]. Multiple molecular signaling mechanisms involving oxidative stress, various kinases, inflammatory molecules, and Ca2+ are postulated to contribute to this effect [136].

#### **3.4. Premature senescence**

The culprit of radiation-induced premature senescence is most likely severe irreparable DSB [137], even if accelerated telomere shortening has also been suggested [138]. Furthermore, oxidative stress is seen as a major player in radiation-induced senescence and is involved in both radiation-induced DNA damage and accelerated telomere attrition [138−140].

In several in vitro studies, it has been demonstrated that ionizing radiation induces endothelial cell senescence, mainly with exposure to higher radiation doses [141–144]. An interesting study was carried out to examine the effect of chronic low-dose rate irradiation (1.4, 2.4, and 4.1 mGy/h) during 10 weeks [145, 146]. Exposure to 1.4 mGy/h did not accelerate the onset of senescence, whereas exposure to 2.4mGy/h and 4.1mGy/h did. Remarkably, a senescent profile was observed when the accumulated doses received by the cells reached 4 Gy. Proteomic analysis revealed a role for radiation-induced oxidative stress and DNA damage, resulting in induction of the p53/ p21 pathway. Also, a role for the PI3K/Akt/mechanistic target of rapamycin (mTOR) pathway was suggested. In a related transcriptomic study, authors suggested that premature senescence resulted from an early stress response with p53 signaling, cell cycle changes, DNA repair, and apoptosis observed after 1 week of exposure and an inflammation-related profile observed after 3 weeks. In addition, a possible role of insulin-like growth factor-binding protein 5 (IGFBP-5) signaling, known to be involved in the regulation of cellular senescence, was suggested for the induction of premature senescence after chronic low-dose rate irradiation [147].

Oxidative stress, inflammation, and cellular senescence are all consequences of a normal aging process but are observed early in irradiated tissues, including the heart, suggesting an intensification and acceleration of these molecular processes [71].
