Introduction

For many years after its discovery in the 1800s, the vascular endothelium was believed to be a mere inert, semipermeable barrier between circulating blood and underlying subendothelial tissues. Numerous subsequent studies have led to the current view of the endothelium as a dynamic heterogeneous and distributed organ with essential secretory, synthetic, metabolic, and immunologic functions [1]. In the presence of irritant stimuli, such as dyslipidemia [2, 3], hypertension [4,5,6,7], and pro-inflammatory agents [8,9,10,11], the normal physiological functions of the arterial endothelium are adversely affected [12, 13], starting a chain of molecular changes that leads to atherosclerosis and cardiovascular diseases (CVDs), including coronary artery disease, carotid artery disease, peripheral artery disease, and ischemic stroke [14,15,16].

When cells are exposed to ionizing radiation, they undergo a stress response within less than a microsecond after the hit [17]. This response is initiated by the interaction of ionizing radiation with biological matter, causing damage by interacting directly or indirectly through the formation of reactive oxygen species (ROS) with cellular biomolecules such as DNA, proteins, and lipids. This reaction interferes with all cellular organelles and has the ability to affect their molecular mechanisms. As a result, endothelial activation occurs, causing the quiescent phenotype to switch towards a pro-inflammatory one [18,19,20]. When exposure is prolonged and/or repeated, it can exhaust the protective physiological effect of the endothelium, leading to endothelial dysfunction [21]. This pathological state can thus be seen as a maladaptive response to pathological stimuli and refers to a failure of the endothelium to perform its normal, physiologic functions [22]. As a result, deterioration of the vascular tone, blood hemostasis problems, inflammation, and edema occurs at the site of the affected endothelium [23]. Because the endothelium is a key integrator of vascular risk, pathogenic signals, including ionizing radiation, may converge to produce several pathological conditions [22], atherosclerosis as typified example [24]. Atherosclerosis itself perpetrates vascular damage, resulting in radiation-induced heart disease [25, 26]. According to the current consensus, the term “low dose” is defined as a dose of 0.1 Gy or less throughout this review [27, 28]. The terms “moderate dose” and “high dose” are, respectively, defined in this review as doses between 0.1 Gy and 2 Gy, and equal or higher than 2 Gy. It has been shown that high doses of ionizing radiation-induced cardiovascular diseases in atomic bomb survivors [29] and cancer therapy patients [30].

In the sections below, we summarize current knowledge on the effects of ionizing radiation exposure on the different aspects of endothelial activation and dysfunction. Progress in the knowledge of endothelial pathophysiology has mainly been a consequence of investigations performed with endothelial cells in culture (Table 1) [31], human umbilical vein endothelial cells (HUVECs) being the most commonly used [32]. To standardize experimental conditions, immortalized, well-characterized endothelial cell lines were developed, of which EA.hy926 is the most frequently used [33]. Additional models of endothelial pathophysiology encompass in vitro co-culture and 3D models that mimic in vivo complexity [34,35,36,37,38]. Ex vivo explanted blood vessel segments from animal (commonly used are canine, bovine, porcine, rat and mouse) or human origin are also used, mainly in the field of vascular tone research [39]. Finally, a number of animal models have been used for understanding the mechanisms involved in cardiovascular disease development as they can replicate complex cell–cell and cell–matrix interactions. The most common animal model being the ApoE−/− mice [40, 41]. However, it is difficult to draw general conclusions, because the current literature often describes different endothelial cell models, timepoints, and radiation doses.

Table 1 Non-exhaustive list of the most commonly used endothelial cell models in endothelial pathophysiological research
Full size table

Endothelial activation: a pro-inflammatory state

Endothelial cell activation can be defined by the manifestation of a pro-inflammatory phenotype characterized by the expression of chemokines, cytokines, and adhesion molecules that facilitate the recruitment and attachment of circulating leukocytes on the vascular wall [21]. Endothelial cells are typically activated by pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6, released by immune cells upon contact with pathogens [42]. After ionizing radiation exposure, however, endothelial cell activation occurs in a sterile environment without the presence of pathogens, i.e., sterile inflammation (Fig. 1). The prime cause of sterile inflammation following ionizing radiation exposure is activation of the genotoxic stress-induced nuclear factor (NF)-κB pathway, recently reviewed by Hellweg [43]. NF-κB is a heterodimeric transcription factor that is normally sequestered in the cytoplasm as an inactive complex with inhibitor of κB (IκB) [44]. DNA double-strand breaks (DSBs), produced by direct or indirect radiation damage to DNA, act as an initial trigger that results in activation of ataxia telangiectasia mutated protein (ATM). Activated ATM promotes nuclear export of IKK-γ/NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase complex that is able to activate NF-κB in the cytoplasm. During this process, a nucleoplasmic signalosome is required for NEMO posttranslational modification and NEMO shuttling to the cytoplasm. While the composition of the nucleoplasmic signalosome is not fully elucidated, p53-induced protein with a death domain (PIDD), receptor interacting protein 1 (RIP1), and poly(ADP-ribose)-polymerase-1 (PARP-1) are known to play supporting roles [43]. In this context, a dose of 8–10 Gy of either γ-rays or X-rays was found to activate the genotoxic stress-induced NF-κB pathway in HUVECs [45, 46].

Fig. 1
figure 1

Radiation-induced sterile inflammation in endothelial cells. Ionizing radiation exposure activates redox-sensitive transcription factor NF-κB via DSB and ATM signaling, induces oxidative stress, and triggers the release of DAMPs. The resulting inflammation leads to the production and secretion of pro-inflammatory cytokines as well as to the expression of a modified repertoire of adhesion molecules by irradiated endothelial cells

Full size image

Another possible cause of sterile inflammation is oxidative stress, a recognized consequence of endothelial cell exposure to radiation (Fig. 1) [47,48,49,50]. Besides reacting with cellular biomolecules, ROS directly activate redox-sensitive transcription factors nuclear factor (erythroid-derived 2)-like 2 (NRF2), activator protein 1 (AP-1), and NF-κB [44]. AP-1 is a heterodimeric transcription factor composed of members of the Jun, Jun dimerization protein (JDP), FOS, and related activating transcription partner families [51, 52]. Depending on its composition, it plays a role in the expression of several genes involved in cellular differentiation, proliferation, and apoptosis. Examples of AP-1-target genes are transforming growth factor (TGF)α, TGFβ, and IL-2 [51]. Activation of AP-1 during oxidative and inflammatory stimuli is predominantly mediated by mitogen-activated protein kinase (MAPK) signaling [44]. NF-κB is also a redox-regulated transcription factor: inflammatory and/or oxidative stimuli activate a series of upstream kinases, such as MAPKs, IκB kinase, protein kinase C (PKC), and phosphatidylinositide 3-kinases (PI3 K), which then activate NF-κB by phosphorylation-mediated degradation of IκB. Activated NF-κB translocates to the nucleus and induces the expression of a wide array of genes regulating pro-inflammatory mediators TNF-α, IL-8, IL-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 [44]. In endothelial cells, NF-κB is involved in the transcriptional regulation of most cytokines and adhesion molecules [53,54,55,56,57].

Another possible cause of endothelial activation is the release of damage-associated molecular patterns (DAMPs) by stressed and dying cells (Fig. 1). Tissue injury emits DAMPs that serve as danger signals to activate danger control (i.e., inflammation for host defense). DAMPs can either be intracellular molecules that signal cell stress and necrosis [high-mobility group box 1 (HMGB1), histones, purine metabolites, uric acid, S100 proteins, heat-shock proteins, and DNA/RNA outside nucleus or mitochondria], matrix constituents that signal extensive matrix remodeling (hyaluronan fragments and glycosaminoglycan fragments) and luminal factors that signal barrier destruction (uromodulin, oxidized low-density lipoprotein). DAMPs activate toll-like receptors, purinergic receptors, and inflammasomes in parenchymal cells and leukocytes. DAMP binding on endothelial cells upregulates pro-inflammatory signaling pathways that lead to NF-κB, MAPK, and interferon regulatory factor 3 (IRF3) signaling [58, 59], resulting in expression of adhesion molecules [intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and E-selectin] and the release of cytokines [IL-6, IL-8, chemokine C–C motif ligand (CCL) 2, and interferon (IFN) γ] [42, 60,61,62,63]. In this respect, exposure to doses ≥ 2 Gy of X-rays was found to induce a dose-dependent in vitro and in vivo release of HMGB1 [64], known to induce endothelial expression of IL-6, CCL2, ICAM-1, and VCAM-1 [65]. In the murine microvascular endothelial cell line, bEnd.3, irradiation with 10 Gy has been shown to promote HMGB1gene expression [66]. Moreover, NF-κB signaling was found to be upregulated in irradiated arteries of patients treated with radiotherapy, even months or years after radiation exposure [67].

In general, high doses (> 2 Gy) of ionizing radiation induce endothelial activation. Endothelial adhesion molecules ICAM-1 and E-selectin are upregulated in a time- and dose-dependent manner [68,69,70], in part due to NF-κβ activation [71]. Furthermore, the expression of cytokines IL-6 and IL-8 as well as TGF-β was shown to increase after exposure to high doses of ionizing radiation [18, 72, 73] and was further differentially affected by dose quality [74]. For example, in obese ApoE−/− mice, a 14 Gy exposure induced an inflammatory phenotype, accelerating atherosclerotic plaque formation and rupture [75]. In addition, atomic bomb survivors exposed to high doses are more prone to the development of atherosclerosis [29] and demonstrated signs of general inflammation, with increased levels of IL-6 and C-reactive protein (CRP) [76]. Comparatively, the effects of low doses (≤ 2 Gy) of ionizing radiation on endothelial activation are still under debate (also discussed in [77]). On one hand, increased ICAM-1 expression and concomitant leukocyte attachment were detected in in vitro endothelial cell cultures after 0.125–0.5 Gy [78]. In addition, we detected elevated IL-6 and CCL2 levels in human endothelial cells exposed to 0.5 Gy [79]. On the other hand, a decrease in endothelial ICAM-1 and E-selectin expression has been observed after exposure of mice to 0.3 Gy and 1 Gy [69], which caused decreased endothelial adhesiveness to monocytes [69, 80]. This anti-inflammatory effect of low-dose radiation, which was confirmed by others [50, 81,82,83,84,85,86], requires a pre-activation of endothelial cells with pro-inflammatory stimuli TNF-α, IL-1β, or lipopolysaccharide. When these mice were exposed to low amounts of 137Cs delivered in the drinking water, the pro-inflammatory plaque phenotype was diminished [87]. The dampening effect of radiation exposure on endothelial activation has been used for decades for the treatment of benign inflammatory diseases [88, 89]. Today, the use of low-dose radiotherapy for the treatment of chronic inflammatory diseases is rare, due to the debate on possible cancer and non-cancer risks [85].

It must be emphasized that endothelial cell activation is a normal part of bodily defense mechanisms. In physiological circumstances, it draws immune cells to sites of infection or tissue injury. The difference between normal physiological and detrimental pathological activation of the endothelium lies in the nature, extent, duration, and combination of pro-inflammatory stimuli. As a consequence of prolonged and/or repeated exposure to a combination of cardiovascular risk factors, the protective effect of endogenous anti-inflammatory systems of endothelial cells can ultimately be depleted, resulting in endothelial dysfunction [21]. An overview of findings supporting endothelial inflammation in different endothelial cell cultures and organs by different radiation qualities and doses is given in Table 2.

Table 2 Experimental findings to support the induction of an endothelial pro-inflammatory state by ionizing radiation
Full size table

Deterioration of the vascular tone

One of the key consequences of endothelial dysfunction is impairment of endothelium-dependent vasodilation due to reduced bioavailability of vasodilators, particularly nitric oxide (NO), and/or to elevated levels of endothelium-derived contracting factors, i.e., endothelins, prostaglandin, and thromboxane [23, 90,91,92,93]. The role of NO and its reactive intermediates in the endothelial radiation response largely remains an open question [94]. What is known is that, after exposure of endothelial cells to ionizing radiation, NO is rapidly deactivated by superoxide radicals, resulting in the formation of vasotoxic peroxynitrites [95, 96] (Fig. 2). Irradiation-induced oxidative stress also causes endothelial NO synthase (eNOS) uncoupling due to inadequate availability of its redox-sensitive cofactor tetrahydrobiopterin, resulting in eNOS-dependent production of superoxide and diminished release of NO [97]. From 1 to 4 days after irradiation, doses of 6 Gy and higher were found to promote eNOS expression and activity, leading to NO production and NO-induced angiogenesis with a concomitant increase in tumor blood flow [98, 99]. eNOS activation after endothelial irradiation depends on components of the DNA damage response pathway, namely, ATM and heat-shock protein 90, which phosphorylate Ser1179 of eNOS, leading to enhanced eNOS activity [100]. However, most of endothelial DNA damage signaling ceases within 24 h after irradiation [79], explaining why irradiation acutely but not chronically enhances NO availability. At later timepoints, endothelium-dependent vasodilation is compromised. Timing also depends on the dose and on the nature of the irradiated endothelial bed. For example, reduced endothelium-dependent vasodilation was found in rabbit carotid arterial rings 20 h after irradiation with 8 Gy and 16 Gy [95], in rabbit ear arteries 1 week after irradiation with 10 Gy, 20 Gy, and 45 Gy [101, 102], in rabbit aorta 9 days after whole-body irradiation with 1 Gy, 2 Gy, and 4 Gy [103] and in rat aorta 6 months after irradiation with 15 Gy [104]. In humans, endothelium-dependent vasodilation was found to be impaired both in vitro and in vivo in carotid arteries 4–6 weeks after neck irradiation (total pre-operative dose of radiation averaged 47.9 Gy ± 2.8 Gy) [105]. In addition, impaired endothelium-dependent vasodilation of axillary arteries was reported in breast cancer radiotherapy patients more than 3 year after radiotherapy (no average dose assigned) [106].

Fig. 2
figure 2

Irradiation-induced deterioration of the vascular tone. Ionizing radiation exposure induces oxidative stress and DNA damage in endothelial cells (left), leading to decreased NO levels and altered production and/or secretion of vasoactive compounds resulting in an initial vasodilation followed by vasoconstriction. In addition, VSMC irradiation induces oxidative stress and DNA damage, resulting in an initial reduction of cellular viability and proliferation as well as vasodilation (right). In the long run, oxidative stress results in Ca2+ release from intracellular stores and increased VSMC proliferation, supporting vasoconstriction

Full size image

NO is not the sole vasoactive substance produced and released by the endothelium. The production of prostacyclin, a potent endothelium-derived vasodilator, is also affected by radiation exposure. Basal prostacyclin release was found to be unaffected in irradiated HUVECs at doses up to 25 Gy [107]. However, when endothelial cells were stimulated with exogenous arachidonic acid, a precursor of endothelial prostacyclin, prostacyclin levels decreased 15 min after irradiation [108], increased within 1 day after irradiation [109,110,111,112,113] and then decreased again thereafter in a radiation dose-dependent way [107, 111, 114, 115]. The short-term stimulatory effect of radiation on prostacyclin production is believed to be caused by oxidative stress [116, 117] and cell damage [111]. Endothelium-dependent hyperpolarization-related signaling was unaffected after endothelial irradiation, thereby serving as a reserve defense mechanism for vasorelaxation [103, 118]. Conversely, levels of vasoconstrictor endothelin-1 were increased after in vitro [119, 120] and in vivo [121, 122] radiation exposure with doses ranging from 0.2 to 20 Gy. In addition, the endothelial production and release of vasoconstrictor angiotensin II by endothelial cells, in bovine pulmonary arterial endothelial cells and HUVECs [123, 124] and in pulmonary endothelial cells collected from irradiated rats [125, 126] increased dose- and time-dependently starting 24 h after exposure to 5–30 Gy. Overall, one can conclude that endothelial irradiation induces initial vasodilation during the first couple of days after irradiation, followed by chronic vasoconstriction with compromised endothelium-dependent vasodilation.

Besides affecting the endothelial layer of blood vessels, ionizing radiation can also directly affect vascular smooth muscle cells (VSMCs; Fig. 2). In culture in the absence of endothelial cells, VSMCs underwent decreased proliferation after a 1.25–20 Gy exposure [127,128,129], with a reduction of viable cells only 15 days after exposure [128, 129]. Surviving VSMCs demonstrated reduced contractibility [129], but maintained a contractile phenotype after exposure to 10–20 Gy [130]. In contrast, when VSMCs were co-cultured with endothelial cells and both were irradiated together with 2–10 Gy, VSMCs changed from a normal contractile to a fibrogenic phenotype [73] associated with the pathogenesis of atherosclerosis [131]. Fibrosis was induced by TGFβ released by irradiated endothelial cells, resulting in small mothers against decapentaplegic (SMAD) signaling in VSMCs [73]. Exposure to 6 Gy also mediated increased myofilament Ca2+ sensitivity in isolated rat thoracic aortic VSMCs 9 and 30 days after exposure [132, 133]. Furthermore, oxidative stress has been shown to induce vasoconstriction by promoting Ca2+ release from VSMC intracellular stores [134] and by upregulating VSMC proliferation by either their secretion of cyclophilin A [135] or by the binding of oxidative stress products hydroperoxyoctadecadienoic acids and 4-hydroxy-2-nonenal to VSMCs [136, 137]. An overview of findings supporting deterioration of vascular tone by different radiation qualities and doses is given in Table 3.

Table 3 Experimental findings to support the deterioration of the vascular tone by ionizing radiation
Full size table

Procoagulatory and prothrombotic phenotype

In addition to altered vascular tone, vascular damage shifts the homeostatic balance towards a procoagulant and prothrombotic endothelial cell phenotype [138]. Because prostacyclin and NO are the main anticoagulatory agents secreted by endothelial cells [139], their decreased production after radiation exposure results in platelet aggregation and blood clot formation (Fig. 3). However, molecular mechanisms responsible for loss of endothelial thromboresistance are more complex. An irradiated endothelium indeed increases the synthesis of von Willebrand factor (vWF) [140,141,142,143,144] and platelet-activating factor [145] while reducing thrombomodulin [68, 146, 147] and prostacyclin production [108, 117, 148], as well as its fibrinolytic activity [149,150,151]. These changes promote platelet adhesion and aggregation and the development of platelet–fibrin thrombi [152,153,154,155]. Cytokines produced during endothelial activation (e.g., IL-6 and CCL2) further affect hemostasis by inducing the expression of tissue factor, tissue plasminogen activator, and vWF [156,157,158]. In this context, irradiation with 14 Gy was shown to induce atherosclerotic plaques with an inflammatory phenotype prone to hemorrhage in ApoE−/− obese mice [75], which may accelerate atherosclerosis [159]. An overview of findings supporting the procoagulatory and prothrombotic effect on endothelial cells by different radiation qualities and doses is given in Table 4.

Fig. 3
figure 3

Irradiation-induced procoagulatory and prothrombotic state in endothelial cells. Endothelial irradiation results in a decreased production of anticoagulants prostacyclin and NO, resulting in a procoagulatory state. In addition, endothelial cell activation and general vascular damage result in elevated secretion of prothrombotic proteins (e.g., vWF) and a reduced fibrinolytic activity producing a prothrombotic state

Full size image
Table 4 Experimental findings to support the induction of a procoagulatory and prothrombotic phenotype in endothelial cells by ionizing radiation
Full size table

Endothelial cell retraction and death

Besides edema formation in surrounding tissues caused by endothelial inflammation and tissue injury [160, 161], exposure to radiation doses as low as 2 Gy can induce a transient and rapid decrease in the integrity of in vitro human endothelial barriers through cell detachment and loss of platelet endothelial cell adhesion molecule (PECAM)-1 [162, 163] (Fig. 4). Rapid loss of endothelial monolayer integrity depends on cytoskeletal reorganization due to actin stress fiber formation and redistribution of vascular endothelial (VE)-cadherin junctions, resulting in endothelial retraction [164,165,166,167]. At higher doses, a more direct cause of increased vascular permeability is of course endothelial cell death [168, 169]. Sensitivity of endothelial cells to cell-reproductive death after ionizing radiation can be assessed by clonogenic assays, the method of choice in such situation [170]. Radiosensitivity varies between endothelial cells from different vascular beds, with HUVECs being the most sensitive and HHSEC being the most radioresistant among the tested ones [171]. In addition, sensitivity to cell-reproductive death depends on radiation quality, with the relative biological effectiveness of α-particles estimated at 5.5 and 4.6 for 10% survival of A549 cells and EA.hy926 cells, respectively [172]. Doses as low as 0.1 Gy can reduce the surviving fraction of EA.hy926 cells [172, 173]. Doses higher than 5 Gy induce endothelial cell apoptosis by the production of ceramide [174, 175]: irradiation activates stress-activated c-Jun N-terminal kinases (JNKs), resulting in the conversion of sphingomyelin to ceramide by neutral sphingomyelinase and the subsequent activation of caspase-3 [176, 177]. In addition, endothelial apoptosis at doses higher than 5 Gy can also be induced by persistent DNA damage, resulting in p53 accumulation and activation of the caspase pathway [178, 179]. Mechanisms behind endothelial cytotoxicity of lower doses are less known. For example, apoptotic EA.hy926 cell death was not increased after exposure to 0.2 Gy, but well after exposure to 5 Gy [180]. In another study, TNF-α-activated endothelial cells were shown to have a discontinuous induction of apoptosis, with a relative maximum at 0.3 Gy and 3 Gy and a relative minimum at 0.5 Gy [82]. In addition, our group observed a dose-dependent increase in endothelial cell apoptosis from 0.5 Gy in HUVECs and from 0.1 Gy in EA.hy926 cells [173]. In vivo, compromised barrier function is involved in the pathogenesis of vascular failure, including atherosclerosis [23, 181, 182]. An overview of findings supporting the induction of endothelial cell retraction and cell death by different radiation qualities and doses is given in Table 5.

Fig. 4
figure 4

Irradiation-induced retraction and death of endothelial cells. Ionizing radiation exposure is able to decrease PECAM-1 expression, redistribute VE-cadherin, and produce actin stress fibers leading to endothelial retraction. Depending on the radiation dose, radiation quality, and inherent radiation sensitivity, ionizing radiation can activate the caspase pathway by ceramide formation and persistent p53 signaling, causing endothelial cell death. As a consequence of endothelial retraction and cell death, the physiological endothelial barrier is compromised

Full size image
Table 5 Experimental findings to support the induction of endothelial cell retraction and death by ionizing radiation
Full size table

Mitochondrial dysfunction

Recent years have seen increasing interest for radiation-induced mitochondrial dysfunction as a cause of endothelial dysfunction in the context of cardiovascular disease [183,184,185,186,187]. In most mammalian cells, mitochondria are primarily considered as the major suppliers of cellular energy in the form of ATP produced by oxidative phosphorylation (OXPHOS) [186]. However, mitochondria are only present in modest number in endothelial cells [185, 188] and produce a low proportion of the total amount of cellular energy [189,190,191]. Thus, endothelial mitochondria are more likely to primarily serve as important signaling organelles [192]. While mitochondria are linked to endothelial function (reviewed in [185]) and endothelial mitochondria are known to play a role in vascular diseases (reviewed in [186]), data on the effect of ionizing radiation on endothelial mitochondria in general are scarce. It was shown that in vitro endothelial cells exposed to 5–20 Gy of γ-rays lose their mitochondrial membrane potential and that mitochondrial ROS production increased 24–72 h after exposure [193]. Furthermore, murine cardiac microvascular endothelial cells irradiated with 8 and 16 Gy X-rays acquired protein expression profiles associated with mitochondrial dysfunction [194]. In light of the caveats in current knowledge, sections below will focus on three main mitochondrial functions hypothesized to be disturbed in endothelial cells after exposure to ionizing radiation: Ca2+ regulation, control of cell death, and oxidative stress signaling.

Normal cytosolic Ca2+ concentrations are maintained approximately 10,000 times lower than extracellular Ca2+ concentrations by plasma membrane and endoplasmic reticulum Ca2+ ATPases. Because these transport proteins require ATP for Ca2+ transport, mitochondria are indirectly involved in this form of Ca2+ regulation [195, 196]. In addition, mitochondria can also directly sequester Ca2+ and, thereby, regulate intracellular concentrations by their inner membrane uniporter rapid mode of Ca2+ uptake into heart mitochondria (RaM), which is driven by the proton electrochemical potential. Conversely, mitochondria release Ca2+ via the 2Na+/Ca2+- and 2H+/Ca2+-exchanger. Increased mitochondrial Ca2+ activates dehydrogenase enzymes in mitochondria and increases ATP synthase activity, leading to increased NADH and ATP production [197]. Sparse evidence exists that altered mitochondrial calcium contributes to endothelial dysfunction in cardiovascular diseases. For example, in diabetes, high glucose levels were shown to elevate mitochondrial Ca2+ levels in human endothelial cells, thereby increasing mitochondrial free radical production [198]. Furthermore, mitochondrial Ca2+ regulates the intensity of TNF-α-induced inflammation in mouse lung microvascular endothelium [199]. In addition, flow-induced dilation of human coronary arterioles was found to be mediated by Ca2+ influx via the transient receptor potential vanilloid type 4 (TRPV4) channel that is in closely apposition with endothelial mitochondria, resulting in mitochondrial ROS release in coronary artery endothelial cells [200]. Finally, the mitochondrial Ca2+ uniporter can potentiate endothelial cell migration [201], and its levels are markedly decreased in endothelial cells derived from CVD patients [202]. While it is known that mitochondrial Ca2+ signaling is affected by ionizing radiation (reviewed in [203, 204]), there is a lack of experimental studies on the role of mitochondrial Ca2+ in the irradiated endothelium.

Importantly, mitochondria are also central executioners of apoptosis. In normal state, anti-apoptotic proteins of the B-cell lymphoma (Bcl)-2 family located on the outer mitochondrial membrane inhibit pro-apoptotic effector proteins Bcl-2-associated protein X (BAX) and Bcl-2 homologous antagonist killer (BAK) [205]. In response to cytotoxic stress, Bcl-2 homology 3 (BH3)-only proteins inhibit Bcl-2 proteins, resulting in BAX and BAK activation. BAX and BAK form oligomers that permeabilize the mitochondrial outer membrane, mediating the release of cytochrome c into the cytosol [206, 207]. Cytosolic cytochrome c promotes the activation of caspase 9 by apoptotic protease activating factor 1 (APAF1), which in turn activates effector caspases that induce cell death [208]. Dysregulation of these vital functions can promote endothelial inflammation, apoptosis, and senescence, which are all linked to the development and progression of atherosclerosis [169, 185,186,187, 209].

As a byproduct of OXPHOS, a small amount of O2 undergoes monoelectronic reduction mainly at complexes I and III of the mitochondrial electron transport chain, resulting in the generation of O ·−2 [210]. As a result, mitochondria are a main generation site of ROS within cells [210]. Formed O ·−2 is converted to H2O2 by SOD2 inside mitochondria, which is able to activate the redox-sensitive transcription factors NRF2, AP-1, and NF-κB [44]. Because mitochondria are an important cellular source of ROS [169, 211, 212], they are closely related to oxidative stress signaling. At relatively low levels, mitochondria-derived ROS are signaling molecules that support normal or compensatory cellular functions involved in hypoxic adaptation, immunity, cellular differentiation, and longevity [213]. However, excessive mitochondrial ROS levels can cause oxidative stress [186]. This is problematic, because mitochondria themselves are critical targets of ROS [214], leading to irreversible damage to mitochondrial DNA, membrane lipids, and proteins [215].

In an effort to combat oxidative challenge, ROS and RNS activate cellular transcription factors, NRF2, NF-κB, and AP-1, resulting in increased expression of ROS-detoxifying enzymes catalase, SOD, glutathione peroxidase (GPx), glutathione S-transferases (GST), and heme oxygenase-1 (HO-1) [216,217,218]. NRF2 is believed to be the main regulator of cellular resistance to pro-oxidants. Because NRF2 controls basal and induced expression of an array of antioxidant response element-dependent genes, including HO-1, SOD2, and GPx [219, 220], it is not surprising that this factor is induced after radiation exposure in both normal and cancerous cells (0.05–8 Gy) [217, 221, 222]. NRF2 also confers cellular radioresistance [223,224,225,226] by mediation of DNA repair, by regulating genes from the homologous recombination DNA repair pathway, and oxidative defense in both normal and cancerous cells [222, 227]. In addition, NRF2 upregulation has been implicated in oxidative stress-induced endothelial dysfunction [228]. Because NRF2 mediates gene expression resulting in both high NADPH production and the production, regeneration and utilization of GSH, thioredoxin, and peroxiredoxin, upregulation of NRF2 leads to increased levels of these antioxidants after irradiation (0.25–20 Gy) in lymphocytes and glioma cells [229, 230]. Of note, elevated levels of several mammalian peroxiredoxin isoforms have been evidenced after a 10 Gy radiation exposure of mouse testis and liver, further enhancing cellular defense mechanisms [231,232,233,234]. Both cumulative and acute radiation exposure can disrupt the cellular redox balance. However, oxidative stress only prevails when pro-oxidant levels eventually overwhelm cellular antioxidant systems, an event marked by enzyme inactivation, a low GSH/glutathione disulfide ratio, and a decreased pool of low molecular weight antioxidants. The consequence of such redox imbalance is manifested by modifications of nucleic acids, lipids, proteins, and other biomolecules [216, 235].

If radiation doses are high enough to overwhelm cellular antioxidant responses, oxidative stress can induce mitochondrial dysfunction by ROS-induced ROS release [236]. As a consequence, radiation-induced oxidative stress that normally disappears within seconds after exposure [214] can lead to the initiation of a self-amplifying cycle, giving rise to long-term ROS production [237] and concomitant mitochondrial dysfunction [169]. During this process, mitochondrial DNA seems to be particularly sensitive to oxidative damage because of its limited DNA repair capacity, lack of protective histones, a high exon to intron ratio and its close proximity to the electron transport chain [238]. In agreement, a range of studies demonstrated changes in mitochondrial function and number after exposure of cells or tissues to high doses of ionizing radiation [239,240,241,242]. Doses of 5–20 Gy of γ-radiation were found to induce a dose-dependent increase in ROS levels with a decrease in mitochondrial activity [193].

Furthermore, 15 Gy of X-rays induced persistent oxidative stress in endothelial cells, linked to mitochondrial dysfunction and premature senescence [243]. Effects of low radiation doses have been less studied on mitochondrial dysfunction in endothelial cells. Doses of 1.5 Gy, 4 Gy, and 10 Gy were found to influence mitochondrial membrane potential in HUVECs 2 days after exposure. While the mitochondrial potential reverted back to control level by days 5 and 6 in 1.5 and 4 Gy irradiated cells, respectively, 10 Gy resulted in persistently decreased mitochondrial activity [244]. In another example, the respiratory capacity of cardiac mitochondria was significantly reduced 40 weeks after local heart irradiation in ApoE−/− mice with a single X-ray dose of 2 Gy [245]. In addition, 0.1 Gy and 0.5 Gy were found to reduce mitochondrial signaling in murine hippocampus and cortex [246]. An overview of findings supporting the induction of mitochondrial dysfunction in endothelial cells by different radiation qualities and doses is given in Table 6.

Table 6 Experimental findings to support the induction of mitochondrial dysfunction in endothelial cells by ionizing radiation
Full size table

Premature endothelial senescence

Aging of the vascular system predisposes the cardiovascular system to the development of diseases, even in the absence of other risk factors [247]. On a cellular level, vascular aging corresponds to endothelial cell senescence [248, 249], a phenomenon that refers to irreversible arrest of endothelial cell renewal. At a molecular level, senescence is induced and maintained by p53 and p16-Rb pathways that inhibit cell-cycle progression [250]. Both pathways are activated either during attrition of telomeres, referred to as replicative senescence [251, 252], or during stress situations independently of telomere shortening, referred to as stress-induced premature senescence [253]. For instance, limited availability of nutrients and growth factors, chromatin perturbations, improper cell contacts, and oxidative stress prematurely induces senescence via cell stress [254]. Oxidative stress is of special importance, because it induces and accelerates senescence at multiple molecular levels: accelerated telomere shortening [254], induction of DNA damage leading to p53 activation [255], NO scavenging decreasing its bioavailability [256], and mitochondrial dysfunction [257].

Endothelial senescence is currently emerging as a contributor to the pathogenesis of atherosclerosis by increasing ROS production, decreasing NO availability, and increasing the production of pro-inflammatory molecules IL-6, IL-1, IL-8, CCL2, and ICAM-1 [257,258,259]. All these molecules indeed contribute to the development and progression of atherosclerosis [209, 257], as indirectly evidenced by the presence of senescent endothelial cells in human atherosclerotic plaques [258]. These observations have to be taken with caution: identification of senescent endothelial cells suggests an association with atherosclerosis, but cannot be used to evidence a causal relationship between endothelial senescence and development and/or progression of atherosclerosis. Considering that ionizing radiation induces oxidative stress [254, 260], DSBs [261], and telomere shortening [260], it is not surprising that it constitutes a stressor that can evoke premature senescence in cells. Several in vitro studies demonstrated that high dose (4–50 Gy) [45, 262,263,264,265,266,267] and medium dose (0.5 Gy) [79] radiation exposure as well as chronic radiation exposure to low doses [268,269,270] induces premature endothelial cell senescence. An overview of findings supporting the induction of premature senescence in endothelial cells by different radiation qualities and doses is given in Table 7.

Table 7 Experimental findings to support the induction of premature senescence in endothelial cells by ionizing radiation
Full size table

Conclusions

In current times, it has become a common practice to use ionizing radiation during justified diagnostic and therapeutic medical procedures. High doses of ionizing radiation lead to cardiac dysfunction over time [29, 30]. However, precise biological and molecular mechanisms are still elusive. In this review, we substantiated the hypothesis that ionizing radiation exposure can induce endothelial activation and dysfunction (Fig. 5). Mechanisms of action predominantly involve induction of a pro-inflammatory state by genotoxic stress, oxidative stress and DAMP release, deterioration of the vascular tone by compromising endothelium-dependent vasodilatation, induction of a procoagulatory and prothrombotic endothelial phenotype by perturbing hemostasis, inducing endothelial cell retraction and death leading to loss of vessel integrity, and induction of mitochondrial dysfunction and premature senescence. Together with other vascular risk factors (e.g., dyslipidemia and hypertension), ionizing radiation can initiate and/or trigger the progression of several pathological conditions, such as atherosclerosis and subsequent radiation-induced heart disease. While these effects are well documented at high doses, there is to date little knowledge on the effect at low doses of ionizing radiation. Furthermore, little is known about the effects of chronic radiation exposure, the exposure to different kinds of radiation qualities (in particular particle radiation), the evolution of endothelial dysfunction in time, and the role of endothelial heterogeneity. Future studies are required to advance the understanding of the mechanisms that lead to endothelial dysfunction, as they may relate to the induction and/or progression of cardiovascular diseases following exposure to ionizing radiation. Further understanding of radiation-induced endothelial dysfunction could lead to advances in the development of countermeasures, such as antioxidant therapy [271, 272], for cardiovascular disease in radiation-exposed individuals.

Fig. 5
figure 5

Ionizing radiation can induce both endothelial cell activation and dysfunction. The resulting vasoconstrictive, pro-inflammatory, procoagulatory, prothrombotic, and prohypertrophic environment can initiate and/or trigger the progression of several pathological cardiovascular conditions, together with other vascular risk factors (e.g., dyslipidemia and hypertension)

Full size image