draft - university of toronto t-space · lazou, antigone; aristotle ... draft 1 potencial markers...

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Potencial markers and metabolic processes involved in

mechanism of radiation-induced heart injury

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2017-0121.R2

Manuscript Type: Review

Date Submitted by the Author: 12-May-2017

Complete List of Authors: Slezak, Jan; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia,, Kura, Branislav; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia,, Babal, Pavel; Faculty of Medicine, Comenius University and University Hospital, Institute of Pathological Anatomy

Barancik, Miroslav; Institute for Heart Research, Slovak Academy of Sciences, Ferko, Miroslav; Institute for Heart Research, Slovak Academy of Sciences, Frimmel, Karel; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia, Kalocayova, Barbora; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia, Kukreja, Rakesh; Virginia Commonwealth University School of Medicine, Internal Medicine Lazou, Antigone; Aristotle University of Thessaloniki, Mezesova, Lucia; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia,

Okruhlicova, Ludmila; Institute for Heart Research SAS Ravingerova, Tatiana; Slovak Academy of Sciences Singal, Pawan; Institute of Cardiovascular Sciences, Szeiffova Bacova, Barbara; Institute fr Heart Research Bratislava, Slovakia, Viczenczova, Csilla; Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia, Vrbjar, Norbert; Institute for Heart Research Slovak Academy of Sciences Tribulova, Narcis; Institute fr Heart Research

Is the invited manuscript for consideration in a Special

Issue?: IACS Sherbrooke 2016 special issue Part 2

Keyword: radiation induced heart disease, ischemia, apoptosis, adaptation, markers

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Potencial markers and metabolic processes involved in

mechanism of radiation-induced heart injury

Jan Slezak1*, Branislav Kura

1, Pavel Babal

2, Miroslav Barancik

1, Miroslav Ferko

1,

Karel Frimmel1, Barbora Kalocayova

1, Rakesh C. Kukreja

3, Antigone Lazou

4, Lucia

Mezesova1, Ludmila Okruhlicova

1, Tanya Ravingerova

1, Pawan K. Singal

5, Barbara

Szeiffova Bacova1, Csilla Viczenczova

1, Norbert Vrbjar

1, Narcis Tribulova

1

1Institute for Heart Research, SAS, Bratislava, Slovakia

2Institute of Pathology, Medical Faculty of Comenius University, Bratislava, Slovakia

3Division of Cardiology, Medical College of Virginia, Virginia Commonwealth University,

Richmond, Virginia, USA

4School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece

5University of Manitoba, St Boniface Research Ctr., Winnipeg, Canada

*Corresponding author. Present address: Institute for Heart Research, Slovak Academy of

Sciences, Dúbravská cesta 9, 840 05 Bratislava, Slovak Republic. Tel.: +421 903 620 181, e-

mail address: [email protected]

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Abstract

Irradiation of normal tissues leads to acute increase in reactive oxygen/nitrogen species that

serve as intra and intercellular signaling to alter cell and tissue function. In the case of chest

irradiation it can affect the heart, blood vessels and lungs, with consequent tissue

remodelation and adverse side effects and symptoms. This complex process is orchestrated by

a large number of interacting molecular signals, including cytokines, chemokines and growth

factors. Inflammation, endothelial cell dysfunction, thrombogenesis, organ dysfunction and

ultimate failing of the heart occur as a pathological entity - "radiation-induced heart disease"

(RIHD) that is major source of morbidity and mortality. The purpose of the review is to bring

insights into the basic mechanisms of RIHD that may lead to the identification of targets for

intervention in the radiotherapy side effect. Studies of authors also provide knowledge how to

select targeted drugs or biological molecules to modify the progression of radiation damage in

the heart.

New prospective studies are needed to validate that assessed factors and changes are useful as

early markers of cardiac damage.

Key words: radiation induced heart disease, ischemia, apoptosis, adaptation, markers

Introduction

Treatment of oncological patients often requires radiation therapy (RT). Despite of modern

radiotherapy techniques in some cases irradiation of tumor inevitably involves also exposure

of normal tissues that causes undesired side effects. Relative risk of fatal heart disease in

patients treated with mediastinal radiotherapy increases from 1.5 to more than 3.0 times that

of unirradiated patients and it is especially frequent in young patients undergoing mediastinal

irradiation (Heidenreich et al. 2007).

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There are just few studies using biomarkers of radiation injury in patients and they are

showing conflicting results. Some authors demonstrate no change in serum troponin (Hughes-

Davies et al. 1995) and no significant elevation in creatine kinase-myocardial band (CK-MB),

troponin, or NT-proBNP (Kozak et al. 2008). However, in another study, both, troponin and

BNP increased significantly after radiation therapy (Nellessen et al. 2010). Therefore cardiac

biomarkers are not fully accepted for evaluation of radiation-induced cardiotoxicity in clinical

settings however, they may remain a useful research tool (Yusuf et al. 2011).

Undesired side effects of radiation and combination of symptoms are manifested as pathology

unit referred as “radiation-induced heart disease (RIHD)”. Ionizing radiation passing through

living tissues generates reactive oxygen species and highly reactive hydroxyl and nitrosyl

radicals and hydrogen peroxide, all of which can damage critical macromolecules such as

DNA, proteins or membranes and induce cell damage (Zhao et al. 2007). Endothelial and

microvascular injury seems to be one of the keys to the unique nature of radiation injury

(Wang et al. 2002a). Learning more detailed mechanisms of radiation injury would help to

develop interventions that could attenuate the severity of normal tissue injury without

compromising tumor control.

The mechanisms whereby these cardiac effects occur are not fully understood. After high

therapeutic doses different factors leading to radiation damage are involved. These various

mechanisms probably result in different cardiac pathologies, e.g. microvascular damage and

fibrosis leading to congestive heart failure versus coronary artery atherosclerosis leading to

myocardial ischemia (Stewart et al. 2013). The cellular response to injury initiates a chronic

active process that ultimately leads to progressive damage, impaired function and failure of

the heart. Most importantly, however, knowing detailed mechanisms of radiation injury,

evidence is emerging to suggest that this process can be modulated by therapies directed at

mitigating the cascade of events resulting from normal tissue injury (Moulder 2004).

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Effect of tissue radiation.

There are several crucial factors determining the intensity of radiation tissue damage. These

can be shortly summarized as dose- (the higher dose the greater injury), speed of dose

delivered- (the faster delivery results in more injurious effect), size of exposed body- (the

bigger part of body the more severe injury), sensitivity of tissue to radiation, age, health status

and genetic abnormalities.

Dependence of damage from radiation dose targeted on thoracic region is very prominent.

Results showed that irradiation with 5 Gy resulted only in a modest increase in right

ventricular weight and a reduction in lung angiotensin converting enzyme activity. Rats

receiving 10 Gy exhibited pulmonary vascular dropout, right ventricular hypertrophy,

increased pulmonary vascular resistance, increased dry lung weight, and decreases in total

lung angiotensin converting enzyme (ACE) activity, as well as pulmonary artery distensibility

after 4-6 weeks (Ghosh et al. 2009; Slezak et al. 2011; Slezak et al. 2012; Slezak et al. 2013a).

Activation of protein kinase C was involved in radiation-induced adaptive responses, and the

intracellular signal transduction pathway induced by protein phosphorylation with protein

kinase C was a key step in the signal transduction pathways induced by low-dose irradiation

(Matsumoto et al. 2004). Radiation at doses of 14 and 25 Gy increased cGMP, increased

iNOS activity and nitrite content. Both doses of radiation significantly decreased the L-

arginine transport and increased iNOS gene expression. It was proposed that radiation induces

the NO generation by up-regulating the iNOS activity (Zhong et al. 2004). Radiation damage

to vasculature can be demonstrated by the fact that breast cancer patients exposed to post-

operative radiotherapy showed in later stages a significant increase in mortality from ischemic

heart disease (Rutqvist et al. 1992). About 50% of the patients had new scintigraphic defects

which could be related to radiation damage to the micro-circulation (Gyenes et al. 1996)

resulting in reduced myocardial capillary density (Baker et al. 2009), and increased

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expression of von Willebrand factor (Boerma et al. 2004b). It was estimated that 1 Gy added

to the mean dose would increase the cardiotoxic risk by 4% (Mège et al. 2011).

Radiation is affecting tissues directly and indirectly. DNA is the principle target for biological

effects of radiation. Radiation may damage the DNA directly, causing ionization of the atoms

in the DNA molecule. Radiation-induced DNA double strand breaks play an important role in

the induction of apoptosis and cell cycle arrest (Han and Yu 2009). Radiation produces a

variety of DNA and other cellular lesions that elicit a stress response. Altered gene profiles

are one characteristic feature of this response. Increased expression of pro-inflammatory and

other genes has been demonstrated within hours following irradiation (Hong et al. 1995;

Kyrkanides et al. 2002). These include genes of transcription factors such as nuclear factor–

kappa B (NF-κB), cytokines such as tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β),

and basic fibroblast growth factor (bFGF) involved in inflammatory processes.

From the early 1990s, development in single-cell irradiation has led to an immense interest in

the bystander effects. Generally, radiation induced bystander effect (RIBE) can be defined as

the phenomenon whereby the irradiated cells can release some signaling molecule, which is

transferred via the medium or intercellular gap-junctions, so that the same cytotoxicity or

genotoxicity can be observed in the nonirradiated cells (Han and Yu 2009).

Indirect action of radiation is executed via production of oxygen free radicals. Radiation

interacts with non-critical target atoms or molecules, usually water. This results in the

production of free radicals. Free radicals can then attack critical targets such as the DNA,

because they are able to diffuse some distance in the cell. The initial ionization event does not

have to occur so close to the DNA in order to cause damage. Moreover, radiation treatment

may cause direct damage to blood vessels by the generation of reactive oxygen species (ROS)

that disrupt DNA strands leading to an inflammatory cascade (Hatoum et al. 2006).

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Mechanisms of free radicals action. Free radiacals are molecules containing one or more

unpaired electrons in atomic or molecular orbitals (Gutteridge and Halliwell 2000). Unpaired

electrons, result in high chemical reactivity. Most of the energy deposited in cells is absorbed

initially in water, which is the main component of cells leading to a rapid production of

oxidizing and reducing reactive hydroxyl radicals. Reactive free radicals play a crucial part in

different physiological processes ranging from cell signaling, inflammation and the immune

defense (Elahi and Matata 2006). Some defensive systems or responses in cells exists that can

protect the cells from the damage (Han and Yu 2009). Formation of ROS is originating from a

variety of sources such as nitric oxide synthase (NOS), xanthine oxidases (XO), the

cyclooxygenases (COX), nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase

isoforms and metal-catalyzed reactions (Elahi et al. 2009). Abnormal production of free

radicals leads to changes in molecular pathways resulting in pathogenesis of several important

pathological states including inflammation, heart disease, neurological disease and cancer,

and is involved in the process of physiological aging.

Reduction and oxidation can render the reduced molecule unstable and make it free to react

with other molecules to cause damage to cellular and sub-cellular components. This includes

free radicals such as superoxide anion (O2·−), hydroxyl radical (HO·), lipid radicals (ROO−)

and nitric oxide (NO). Although other reactive oxygen species, hydrogen peroxide (H2O2),

peroxynitrite (ONOO−) and hypochlorous acid (HOCl), are not free radicals, they have

oxidizing effects that contribute to oxidative stress. ROS have been implicated in cell damage,

necrosis and cell apoptosis due to their direct oxidizing effects on macromolecules such as

lipids, proteins and DNA (Valko et al. 2005; Valko et al. 2006). Reaction between radicals

and polyunsaturated fatty acids within the cell membrane can result in fatty acid peroxyl

radicals, which accumulate in the cell membrane and alter protein function and signal

transduction.

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Lipid peroxidation can be described generally as a process under which oxidants such as free

radicals attack lipids containing carbon double bond(s), especially polyunsaturated fatty acids

(PUFAs). Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-

HNE), reveal physiological and protective function as signaling molecule stimulating gene

expression and cell survival, but also its cytotoxic role inhibiting gene expression and

promoting cell death. MDA appears to be the most mutagenic product of lipid peroxidation,

whereas 4-HNE is the most toxic (Esterbauer et al. 1990; Ayala et al. 2014).

Malondialdehyde (MDA): The degree of lipid peroxidation can be estimated by the amount of

malondialdehyde (MDA) in tissues. MDA has been widely used for many years as a

convenient biomarker for lipid peroxidation and form facile reaction with thiobarbituric acid

(TBA). MDA is reliable marker that determine oxidative stress (Fig. 1A) (Giera et al. 2012;

Esterbauer et al. 1991).

Radicals that attack biomolecules located just a few nanometres from its site of generation,

the lipid peroxidation-derived aldehydes can easily diffuse across membranes and covalently

modify proteins in the cytoplasm and nucleus, far from their site of origin (Negre-Salvayre et

al. 2008).

4-HNE is the most intensively studied lipid peroxidation end-product, in relation not only to

its physiological and protective function as signaling molecule stimulating gene expression,

but also to its cytotoxic role inhibiting gene expression and promoting the development and

progression of different pathological states (Esterbauer et al. 1991).

MDA and 4-HNE depending of their cellular level and the pathway activated by them may

enhance survival or promote cell death (Negre-Salvayre et al. 2008).

ROS can also induce the opening of the mitochondrial membrane permeability transition pore

and cause a release of cytochrome C and other factors that can lead to apoptosis-mediated cell

death (Tatton et al. 2003; Tsutsui et al. 2009). O2.- radicals can further interact with the

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signaling molecule nitric oxide (NO) resulting in the formation of reactive nitrogen species

(RNS), which reduce NO bioavailability and at the same time cause NO toxicity known as

“nitrosative stress” (Elahi et al. 2007). Excessive production of RNS results in nitrosylation

reactions that change the structure of proteins (Ridnour et al. 2004) leading to the loss or

change of protein function. The oxidation and nitration of cellular proteins, lipids and nucleic

acids, and formation of aggregates of oxidized molecules underlie the loss of cellular

function, cellular aging and/or the inability of cells to withstand various stresses.

Under the physiological situation, defences such as specialized enzymes and antioxidants can

cope with the situation and maintain reduction-oxidation (redox) balance. However, during

excessive production of ROS, enzymes and antioxidants can get exhausted resulting in

oxidative/nitrosative disbalance, a process that is an important mediator of cell damage

(Pacher and Szabo 2008; Vassalle et al. 2008; Elahi et al. 2009).

Inflamatory effect of ionizing radiation

Ionizing radiation is associated with induction of inflammatory markers including cytokine

expression. An increase in cyclooxygenase-2 (COX-2) expression and COX-2-mediated

prostanoid production was observed in the irradiated mouse brain. COX-2 is one of two

isoforms of the obligate enzyme in prostanoid synthesis and a principal target of non-steroidal

anti-inflammatory drugs (NSAIDs). Inhibition of COX-2 attenuates prostanoid induction and

cerebral edema in mice after radiation therapy (Moore et al. 2004). This suggests that

radiation causes an oxidative stress and activation of nuclear factor-kappa B induced

inflammatory response and in later phase, oxidative damage in large vessels that in

combination with high cholesterol, may increase oxidation of low density lipoproteins and

allows them to be ingested by macrophages, thus triggering the atherosclerotic process. Once

the atherosclerotic process is initiated, the lipid cells secrete further inflammatory cytokines

and growth factors, which stimulate proliferation and migration of the smooth muscle cells

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(Stewart et al. 2010). Certain cytokines and growth factors, such as TGF-beta1 and IL-1 beta,

may stimulate radiation-induced endothelial proliferation, fibroblast proliferation, collagen

deposition, and fibrosis leading to advanced lesions of atherosclerosis (Weintraub et al. 2010).

Indirect association of inflammation with radiation induced vascular damage comes from

studies showing elevated levels of proinflammatory cytokines IL-6, CRP, TNF-α, and INFγ

(Hayashi et al. 2005).

Sources of ROS, physiological and/or pathophysiological conditions, and cellular oxidant

targets determine the characteristic feature of a disease process and resultant outcomes (Elahi

et al. 2009). In this context, cytokines and growth factors probably play a central role in this

process and in particular, TGF-β1, TGF-β2, and TGF-β3 are highly pleiotropic cytokines

secreted by all cell types. TGF-β molecules are proposed to act as cellular switches that

regulate processes such as immune function, proliferation, and epithelial-mesenchymal

transition. TGF-β1 is the isoform most frequently implicated in the fibro-proliferative process,

and it appears to be a key-molecule and a master switch for the general fibrotic program

(Lawrence 1996; Hendry et al. 2008).

As mentioned earlier, ROS is an important intermediate second messenger of nuclear kappa B

(NF-κB) activation. NF-κB belongs to a family of inducible transcription factors (Baeuerle

and Henkel 1994), and is one of the most commonly studied transcriptional factors influenced

by cellular redox state (Imbert et al. 1996). NF-κB regulates diverse biological processes,

including immune responses, inflammation, cell proliferation, and apoptosis. The NF-κB

protein complex is retained in an inactive state in the cytoplasm by binding to inhibitory

proteins IκBs family (inhibitor of κB (IκB), regulator of NF-κB). Various cellular stimuli,

such as oxidative stress results in nuclear translocation of NF-κB complex where it can bind

to various promoter areas of its target genes and induce gene transcription of the

corresponding genes, most of which are implicated in the regulation of inflammation (Morgan

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and Liu 2011; Siomek 2012). NF-κB targets multiple genes involved in inflammation

including Intercellular Adhesion Molecule (ICAM), vascular cell adhesion molecule

(VCAM), and IL-1, the production of cytokines, upregulation of prothrombotic markers, and

pathogenesis of atherosclerosis (Wilson et al. 2000; Kim et al. 2001). Postirradiation

activation of NF-κB was prevented by NO, and thus a reduction in the bioavailability of NO

may result in epigenetic changes that promote vascular inflammation and atherosclerosis

(Peng et al. 1995). NF-κB is found to be upregulated in atherosclerotic vessels and its nuclear

translocation has been detected in the intima and media of atherosclerotic lesions and in

smooth muscle cells, endothelial cells, macrophages and T cells of atherosclerotic plaques. It

has also been reported that NF-κB plays a role in mediating of T-cell signaling in

atheromatous plaques (Brand et al. 1997; Landry et al. 1997; Mach et al. 1998; Kawano et al.

2006; Barlic et al. 2007).

C-reactive protein (CRP). Damage caused by free oxygen and nitrogen radicals to DNA

alongside with inflammation, belongs to the main features of cardiovascular system radiation

injury. The radiation elicit acute phase response that develops in a wide range of

inflammatory conditions. C-reactive protein (CRP) has been proposed as an independent risk

factor for cardiovascular disease (Tchernof et al. 2002).

CRP is an acute-phase protein found in the blood, the levels of which rise in response to

inflammation. Its physiological role is to bind to phosphocholine expressed on the surface of

dead or dying cells (and some types of bacteria) in order to activate the complement system

via the C1Q complex (Thompson et al. 1999). CRP is synthesized by the liver (Pepys and

Hirschfield 2003) in response to factors released by macrophages and adipocytes (Lau et al.

2005). During the acute phase response, levels of CRP rapidly increase. On the other hand

elevated CRP level can provide support for the presence of an inflammation. Thus, CRP

indirectly participates in the clearance of necrotic and apoptotic cells (Volanakis and Kaplan

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1971; Du Clos 1989; Lagrand et al. 1997; Nijmeijer et al. 2003, Pepys and Hirschfield 2003;

Lau et al. 2005).

After 6 weeks of irradiation, plasma assessement of C-reactive protein (CRP) showed that

irradiated rats in our experiments had only half the CRP values compared with controls

(Slezak et al. 2015). TNF-α in these experiments was after 6 weeks also down regulated to

about ½. This finding could be explained by a compensatory and or defense mechanisms,

which in the subacute phase (after 6 weeks) in irradiated rats predominate. This phenomenon

is consistent with our other results (Fig. 1B).

Tumor necrosis factor alpha (TNF-α) is a cell signaling protein involved in systemic

inflammation and is one of the cytokines that make up the acute phase reaction. It is produced

chiefly by activated macrophages, although it can be produced by many other cell types such

as neutrophils, mast cells, eosinophils, e.t.c. The primary role of TNF-α is in the regulation of

immune cells. TNF is able to induce apoptotic cell death. TNF and IL-1 are involved in the

regulation of stress responses, expression of cytokines and cell adhesion molecules

(Olszewski et al. 2007).

Six weeks after irradiation the acute phase of inflammation fades, the tissue adapts and

inflammatory markers (CRP and TNF) in experimental rats decrease (Fig. 1B,C) (Slezak et al.

2015).

Effect of radiation on endothelial cells

The vascular endothelium plays a pivotal role in vascular tone and remodeling as well as

regulating thrombosis and inflammation. Early changes in endothelial function are indicators

of cardiovascular pathologies (Fig. 2) (Okruhlicova et al. 2012; Triggle et al. 2012). The

thrombotic and inflammatory pathways are regulated by nitric oxide (NO) produced from

endothelial nitric oxide synthase (NOS), thrombin, and the thrombin receptor (PAR1), and

fibrinogen among other factors. Nitric oxide, through its anti-inflammatory and

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antithrombotic effects, is able to diminish leukocyte adhesion and arterial thrombosis. Nitric

oxide decreases thrombosis by inhibiting the expression of the prothrombotic protein

plasminogen activator inhibitor-1 and decreasing platelet aggregation (Loscalzo 2001), but on

the other side, thrombin enhances nitric oxide production through the thrombin receptor

(Hirano et al. 2007; Baker et al. 2011). Endothelial cell injury markers secreted after

irradiation includes thrombomodulin (Zhou et al. 1992).

Radiation damages the vascular endothelium. Damage to the capillary vessels manifests as

teleangiectasia, whereas thrombotic, inflammatory, and fibrogenic complications in larger

vessels can result in peripheral, coronary and carotid artery disease. Following a single dose

of radiation to the heart, from 3 months onwards changes in coronary arteries of the irradiated

hearts included endothelial cell loss, a loss of smooth muscle cells, and fibrosis in media and

adventitia (Boerma et al. 2004a).

Following radiation, the endothelial cell neutrophil chemotactic activity is increased, with

greater adherence of polymorphonuclear leucocytes to irradiated endothelial cells (Dunn et al.

1986).

Changes in endothelial cells and endothelial dysfunction may contribute to profibrotic and

proinflammatory environments, which are common aspects of normal tissue radiation.

Endothelial cells (at EM level) seems to be the most radiation sensitive part of vasculature.

Indeed, the earliest morphological changes described by Schultz-Hector (1992) in the

irradiated rodent heart were reversible changes in the function of capillary endothelial cells,

lymphocyte adhesion and extravasation followed by thrombus formation, obstruction of the

microvessels and decreases in capillary density, accompanied by loss of the endothelial cell

marker alkaline phosphatase (Schultz-Hector 1992; Boerma et al. 2004b). Our experimental

findings of early postirradiation phase support findings of other authors and suggest that

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radiation injury to the myocardial capillary network may represent myocardial degeneration

after heart irradiation (Hendry et al. 2008).

Later postirradiation phase (after 3 months and later) described by Stewart et al. (2010) has

been characterized by radiation damage to the myocardium caused primarily by inflammatory

changes in the microvasculature, leading to microthrombi and occlusion of vessels, reduced

vascular density, perfusion defects and focal ischemia. This is followed by progressive

myocardial cell death and fibrosis (Schultz-Hector et al. 1992; Stewart et al. 2010).

Reduction in the total number of blood vessels in a vascular bed in later stage increase

vascular resistance by reducing the number of paralleled pathways through that circulation.

Rarefaction of vessels in the pulmonary circulation has been reported in human subjects with

pulmonary hypertension (Ryland and Reid 1975; Rabinovitch et al. 1979) and in animal

models (Jones and Reid 1995).

It is well known from previous experiments that a single exposure to 15–60 Gy exerts an

adverse long-term effect on cardiovascular function in the rat, resulting in morphological

changes of different severity (Kruse et al. 2001) including damage to the endothelium

(Boerma et al. 2004b), micro-vascular injury caused by inflammation and oxidative stress

(Schultz-Hector and Trott 2007). Following radiation, the endothelial cell neutrophil

chemotactic activity is increased, with greater adherence of polymorphonuclear leucocytes to

irradiated endothelial cells. Leukocyte adhesion to endothelial cells and thrombi can block the

vascular lumen (Fajardo 1992; Dunn et al. 1986). Vascular injury is indeed a prominent

feature of normal tissue radiation injury in animals and humans (Hopewell and Young 1978;

Fajardo 1989; Slezak et al. 2015).

Radiation exposure causes excessive production of eicosanoids (prostaglandins, prostacyclin,

thromboxane and leukotrienes), which are endogenous mediators of inflammatory reactions

like vasodilation, vasoconstriction, vascular permeability, microthrombi formation and

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extravasation of leukocytes. In larger arteries, monocytes adhere to the irradiated endothelium

and transmigrate into the intima. Monocytes transformed into activated macrophages (foam

cells), may later initiate the process of atherosclerosis (Basavaraju and Easterly 2002; Dent et

al. 2003; Stewart et al. 2010).

Our ultrastructural studies showed that the early changes might be associated with endothelial

cell degeneration and at the same time with activation, proliferation and angiogenesis (Fig.

2A). Capillary endothelial cells respond to damage by increased proliferation (Maeda 1980;

Lauk and Trott 1990; Hopkins and McLoughlin 2002). 6 weeks after irradiation we have

found also increase in capilarization of the left ventricle that can be explained by

compensatory capilarogenesis and opening of capillary reserves to meet the demands for

increased blood flow that was found in our physiological studies (Fig. 2A,C,F). This could be

explained by compensatory mechanisms operating in vivo and masking the extent of

functional damage (Slezák et al. 2016). Other experimental studies point to radiation injury to

the capillary network as the underlying cause of later myocardial degeneration and heart

failure after irradiation (Stewart et al. 2010).

As shown in our experiments early radiation damage to the myocardium was represented

primarily by inflammatory cells infiltration and increased amount of mast cells in the left

ventricular myocardium 6 weeks after 25 Gy (Slezak et al. 2015) which is in consent with

some other authors. Mast cells contain proteases that can activate matrix metalloproteinases

(Maurer et al. 2004; Janicki et al. 2006). As demonstrated by us, microvascular density was

not decreased (Fig. 2A, F). at this time sequence (after 6 weeks and 25 Gy). In this stage, the

inflammation may cause angiogenesis serving to prevent or attenuate increased vascular

resistance (Hopkins and McLoughlin 2002).

Myocardial and microvascular inflammatory changes were leading to extravasation of blood

cells, creation of microthrombi and signes of fibrosis (Slezák et al. 2016). A number of acute

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effects, including endothelial damage (seen in EM), inflammatory cell infiltration, and

lysosomal activation, have been described also by other authors (Konings et al. 1975).

Endothelial cell derived NO plays an important anti-inflammatory role that, in part, is

mediated by the inhibition of adhesion molecule expression (Kubes et al. 1991; Niu et al.

1994). Reduced bioavailability of NO promotes endothelial and vascular dysfunction not only

via profound effects on vascular tone and blood flow, but also via promotion of cell

proliferation and enhanced expression of adhesion molecules. In addition, a reduced

bioavailability of NO and (or) PGI2 will also enhance the potential for platelet aggregation

(Bates 2010; Triggle et al. 2012).

The activation of mastocytes, macrophages and monocytes during the inflammatory process

results in the continuous secretion of cytokines and growth factors, including tumor necrosis

factor (TNF), Interleukins (IL)-1, IL-6, IL-18 and monocytes chemotactic factor. Besides

induction of adhesion molecules, up-regulation of some cytokines (namely IL-6 and IL-8) has

been observed after endothelial cell irradiation in a time- and dose-related fashion manner

(Burger et al. 1998; Van der Meeren et al. 1999).

Although microvascular injury is a major underlying cause of radiation-induced myocardial

damage, radiation could also damage the major arteries leading to an accelerated development

of age-related atherosclerosis. The initial event in radiation-induced atherosclerosis is

endothelial cell damage and transmigration of monocytes into the intima, with subsequent

ingestion of low-density lipoproteins and formation of fatty streaks (Konings et al. 1978; Vos

et al. 1983; Lauk and Trott 1988).

Radiation may cause micro-vascular disease characterised by a decrease in capillary density

causing chronic ischemic heart disease and focal myocardial degeneration, and macrovascular

disease through the faster development of age-related atherosclerosis in the coronary arteries

(Schultz-Hector and Trott 2007). In early period after irradiation (compensatory phase)

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temporary about 20% increase in number of capillaries was observed 6 weeks after 25 Gy

irradiation (Fig. 2 E,F) (Slezak et al. 2015).

Even if acute myocardial damage is moderate, the process of myocardial remodeling can lead

to progressive myocardial dysfunction over time and eventually induce myocardial

dysfunction and heart failure (Yusuf et al. 2011). Treatment with ACE inhibitors, angiotensin

receptor blockers, aldosterone antagonists, and beta blockers is usually recommended (Adams

et al. 2003).

Progressive decrease of capillary density occurrs later (after two-three months onwards) both

as a random rarefication by disappearance of individual capillaries and as a focal loss of

groups of capillaries which gradually lead to ischemic necrosis. Before the focal loss of

capillaries, focal disappearance of alkaline phosphatase activity was observed (Lauk 1987;

Schultz-Hector and Balz 1994; Seddon et al. 2002). Focal loss of capillaries is preceded by

increased endothelial proliferation but in the enzyme-negative areas only (Schultz-Hector et

al. 1993).

Radiation-induced vascular injury and endothelial dysfunction are mediated also in part by

Transforming Growth Factor-β (TGF-β) a pluripotent growth factor (Kruse et al. 2009).

Also, there is evidence of prothrombotic effects of radiation (Verheij et al. 1994; van Kleef et

al. 1998; Boerma et al. 2004a) which may be the cause of the increased platelet adherence and

thrombus formation observed in irradiated capillaries and arteries (Schultz-Hector et al. 1992;

Darby et al. 2005; Ivanov et al. 2006; Hussein et al. 2008). Experimental evidence suggests

that RIHD is the result of indirect myocytes secondary effect caused by microvascular and

macrovascular damage (Corn et al. 1990; Gagliardi et al. 2001; Jaworski et al. 2013).

Endothelial dysfunction is believed to be a precipitating factor in the development of cardiac

sequelae (Paris et al. 2001) and is most likely a combination of impaired endothelial function,

stimulation of growth factors, and eventual fibrosis (Darby et al. 2010).

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Interestingly, according to animal studies, the pathophysiology of RIHD seems to be

fundamentally different from non-radiation-related chronic heart failure. In the latter, the

reduction of cardiac output induces a sustained activation of the sympathetic nervous system

and, subsequently, a down-regulation of cardiac β-receptors. In RIHD, the adrenal

catecholamine synthesis is unchanged and cardiac catecholamine content is reduced, leading

to an increase of β-receptor density (Schultz-Hector et al. 1992; Gyenes 1998).

EAs mentioned, endothelial damage leads to an acute inflammatory reaction (due to acute

swelling of the endothelial cells). The activation of the coagulation mechanisms leads to fibrin

deposition. These early effects are followed by organized fibrin formation, endothelial

proliferation and collagen deposition (Slezak et al. 2013a; Slezak et al. 2014), and, in the late

phase, fibroblastic proliferation and enhanced atherosclerosis. Microscopy revealed an

increased amount of collagen and a higher proportion of type I collagen (relative to type III)

amount measured by morphometry of collagen 6 weeks after irradiation. Significant increase

of collagen I, enhances the rigidity of myocardium (Schultz-Hector et al. 1992; Chello et al.

1996; Gyenes 1998; Slezak et al. 2014.).

Early and late side-effects of radiation limit dose escalation and affect the patient's quality of

life. Irradiated endothelial cells acquire a proinflammatory, procoagulant and prothrombotic

phenotype. Reduced myocardial capillary density in later stages (Baker et al. 2009), focal loss

of endothelial alkaline phosphatase (Schultz-Hector and Balz 1994), and increased expression

of von Willebrand factor (Boerma et al. 2004a) are hallmarks of irradiation damage.

Von Willebrand factor (vWF) is a blood glycoprotein involved in hemostasis. vWF is

produced constitutively in endothelial Wiebel-Palade bodies, megakaryocytes (alfa-granules

of platelets) and subendotelial connective tissue (Schick et al. 1997).

Radiation induced changes of left ventricular tissues may lead to a cascade of changes in

blood vessels, resulting in an impaired blood supply (Hallahan et al. 1995). Upon stimulation,

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endothelial cells produce increased amounts of vWF (Smith et al. 1989; Hallahan et al. 1995;

Jahroudi et al. 1996) and elevated expression of vWF in blood plasma or tissue samples was

suggested to be a marker of endothelial cell damage (te Poele et al. 2001; Wang et al. 2002b;

Gabriels et al. 2012).

vWF mediates the adherence of platelets to one another and to the sites of vascular damage. It

is important in modulation of platelets and leukocyte recruitment and formation of blood clots

(Gabriels et al. 2012). In our experiments six weeks after irradiation vWF expression was not

increased significantly (Fig. 3) (Slezak et al. 2013b). The results might indicate that six weeks

after irradiation overall damage to the endothelial cells may need not to be statistically

significant. Some authors report endothelial changes in later period after irradiation (Schultz-

Hector et al. 1992).

Effect of irradiation on heart function and myocardial response to ischaemia.

Shortly after 25 Gy dose, cardiac function was slightly reduced, then maintained in a steady

state for several weeks, probably due to a compensatory up-regulation of cardiac β-adrenergic

receptors (Slezak et al. 2015).

In our experiments, the state of rats six weeks after 25 Gy irradiation of mediastinal area

could be characterized by general alteration of the animals, e.g. body and heart weight

retardation, presence of exudate in the chest and abdominal cavity. On the other hand, in

isolated Langendorff-perfused hearts, the effect of irradiation on the heart function was

manifested by mild bradycardia and surprisingly enhanced coronary flow, but no

deteriorations in heart contractile function were observed. After exposure of hearts to

ischaemia/reperfusion (I/R), occurrence of reperfusion arrhythmias measured in the most

vulnerable early phase of reperfusion (10 min after its onset) was higher in the hearts of

irradiated rats than in the hearts of control ones (Fig. 4A). Interestingly, postischemic

functional recovery at the end of reperfusion (left ventricular developed pressure, LVDP) was

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not altered but rather improved in the irradiated group (Fig. 4B). Moreover, the size of

infarction was smaller in the hearts of irradiated animals than in the non-irradiated hearts

(Slezak et al. 2011; Slezak et al. 2012; Carnicka et al. 2013; Slezak et al. 2014). These

differential effects of irradiation on the manifestations of ischaemia/reperfusion injury can be

attributed to the differences between the pathophysiological mechanisms of lethal injury and

early reperfusion-induced events (Ravingerová et al. 2007), and a major dependence of

reperfusion arrhythmias on the generation of free radicals (Ravingerová et al. 1999). Hence,

myocardium of the irradiated animals may be more susceptible to arrhythmias at the onset of

reperfusion associated with a burst of ROS production.

In addition, investigation of the involvement of transcription factors peroxisome proliferator-

activated receptors (PPARs) in the effects of irradiation revealed (real-time RT-PCR)

significantly lower mRNA levels of PPARα in the left ventricular tissue of rats six weeks

after irradiation compared to non-irradiated controls (Fig. 5).

PPARs are key transcriptional regulators of lipid metabolism and energy production (Huss

and Kelly 2004; Lopaschuk et al. 2010; Ravingerova et al. 2011). Decreased gene expression

of PPARα in the hearts of irradiated animals indicates their higher reliance on glucose as a

source of ATP production than on the oxidation of fatty acids, which is characteristic for the

normal myocardium (Lopaschuk et al. 2010).

Activation of PPAR by exogenous PPARα ligands (fibrates) and PPARγ ligands

(thiazolidinediones or glitazones) has been shown to exert various protective non-metabolic

vasculoprotective (Touyz and Schiffrin 2006), anti-inflammatory, antioxidant and

antiapoptotic actions (Smeets et al. 2007; Barrera et al. 2008; Barlaka et al. 2016). These

effects are often related to activation of „survival“ PI3K/Akt-NOS pathways both in normal

(Ravingerová et al. 2012) and diseased (diabetic) animals (Bulhak et al. 2009), as well as in

hypertrophy and heart failure (Barlaka et al. 2016).

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While up-regulation of PPARα exerts beneficial effects under conditions of acute myocardial

injury (Yue et al. 2003; Ravingerová et al. 2012), in the longer-lasting processes associated

with increased ROS production and tissue hypoxia, it is considered as unfavourable

(Sambandam et al. 2006). Thus, chronic downregulation of PPARα and a subsequent shift in

substrate selection (from fatty acids to glucose) may be considered as an adaptive response

(Razeghi et al. 2001). This finding is in line with the unchanged degree of myocardial

stunning (improved LVDP recovery), and, even more importantly, with the reduced extent of

lethal injury (smaller size of infarction) in the hearts of irradiated rats. The latter indicates that

irradiation may induce not only deleterious effects but also some adaptive mechanisms may

be activated as well, to counteract unwanted consequences of irradiation and to preserve heart

function.

Connexin-43 (Cx43) cardiac gap junction channels play the crucial role in synchronizing

myocardium allowing impulse propagation from pacemaker cells along the conduction system

and throughout the atria and ventricles. The channels, in addition, are permeable to ions and

small molecules (up to 1 kD) that is important for direct cell-to-cell communication. Cx43

channels are opened and closed (gated) by various treatments. Likewise Cx43 expression and

distribution can be modulated by various physiological and pathophysiological stimuli

(Salameh and Dhein 2005). Impaired intercellular communication due to disease-related

alterations in myocardial Cx43 distribution and/or expression promotes development of life-

threatening arrhythmias and contractile dysfunction (Severs et al. 2004; Tribulova et al. 2008;

2009).

In our experiments Cx43 immunoblotting revealed the expression of three obvious forms of

myocardial Cx43, i.e. two functional phosphorylated (P1+P2-Cx43) and one un-

phosphorylated form (P0-Cx43) in all examined rats. Comparing to non-treated rats the

expression of total Cx43 as well as its functional phosphorylated forms was significantly

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increased 6 weeks after irradiation. These findings point out to up-regulation of myocardial

Cx43 that is most likely associated with enhanced cardiomyocyte communication at the early

compensatory phase of irradiation (Tribulova et al. 2009; Viczenczova et al. 2016).

In the field of oncology, it has been demonstrated that intercellular communication is

enhanced by ionizing radiation in lung or skin via up-regulation of Cx43 at mRNA and

protein levels (Azzam et al. 1998; Glover et al. 2003; Viczenczova et al. 2016). The increase

of intercellular communication is believed to play an important role for the enhancement of

radiation-induced effects such as modulation of gene expression, mutagenesis and cell

survival (bystander effect) (Azzam et al. 2001). Moreover, it has been reported that external

heavy ion beam irradiation increases Cx43 mRNA and protein levels in the left ventricle of

control as well as postischemic rabbit heart (Amino et al. 2006). However, the molecular

mechanisms responsible for the radiation-induced Cx43 up-regulation are not elucidated yet.

Mitochondrial Mg2+

-ATPase represents, in fact, the enzyme responsible for oxidative

production of ATP from ADP in the mitochondria. Data about its activity may provide

representative information about participation of mitochondria in energy metabolism of cells

(Ferko et al. 2006). Mitochondria not only appear susceptible to damage mediated by

increased oxidative and nitrosative stress, but also play significant roles in the regulation of

cardiovascular cell function. In addition, accumulating evidence suggests that a common

theme among cardiovascular disease development and cardiovascular disease risk factors is

increased mitochondrial damage and dysfunction. For technical reasons ATP-synthase is

usually estimated in opposite direction of its catalytic activity i.e., splitting ATP to ADP.

However, the latter procedure requires to make the membranes of all mitochondria in the

preparation permeable for Mg2+

ions. The resulting enzyme activity is than indicated as Mg2+

-

dependent 2,4-dinitrophenol stimulated ATPase, also termed as total mitochondrial Mg2+

-

ATPase activity (Ziegelhöffer et al. 2012).

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Results presented in Fig. 6 indicate that ionizing radiation decreases significantly the total

Mg2+

-ATPase activity of mitochondria isolated from left ventricles of the rat hearts. This

finding testifies for decreased energy generation by the mitochondria. Nevertheless, the

resulting energy deficit may be, but it need not to be unconditionally crucial for myocardial

bioenergetics. In an our earlier study (Muráriková et al. 2013) we observed in hearts of

healthy rats an approximately 12 % regeneration and in hearts of diabetic animals even a 47

% regeneration of mitochondrial Mg2+

-ATPase activity after ischaemia/reperfusion injury.

The Na, K-ATPase is an integral membrane protein complex responsible for establishing the

electrochemical gradients of Na+ and K

+ ions across the plasma membranes. Na

+-pump

extrudes 3 Na+ ions from a cell and recovers 2 K

+ per each hydrolyzed ATP molecule (for

review see Ziegelhöffer et al. 2000). Decrease of the Na, K-ATPase activity leads to higher

levels of intracellular sodium in cardiomyocytes, what is subsequently accompanied with

development of pathophysiological complications (Jelicks and Gupta 1994).

Our experiments revealed that irradiation reduced the cardiac Na, K-ATPase activity by 38 %.

Another experiments demonstrated that radiation is altering functional properties of cardiac

sarcolemmal Na, K-ATPase (Mezesova et al. 2014).

Concerning the severity of cardiac injury caused by irradiation various conflicting

contradictory data showing the importance of dose and time after the application were

published. Local thorax irradiation with 10 Gy was not followed by increase of risk factor of

cardiovascular disease in time interval of 60-240 days after application (Baker et al. 2009). In

contrast, local irradiation of the heart/thorax using higher doses of 15-30 Gy resulted in

delayed injury to the heart in the same time frame (Wondergem et al. 1991). Thus, the

decreased functionality of the Na, K-ATPase shown in our study suggests that this enzyme may

be one of the the most rapidly reacting systems to irradiation-induced changes observable on

molecular level in the cardiac tissue. The Na, K-ATPase was sensitive to irradiation in similar

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time interval also in intestine (Lebrun et al. 1998), kidney (Balabanli et al. 2006) and

erythrocytes (Moreira et al. 2008).

MicroRNAs (miRNAs, miRs) have recently emerged as one of the central players in

regulating gene expression. Numerous studies have documented the implications of miRNAs

in nearly every pathological process of the cardiovascular system, including cardiac

arrhythmia, cardiac hypertrophy, heart failure, cardiac fibrosis, cardiac ischemia and vascular

atherosclerosis. More surprisingly, forced expression or suppression of a single miRNA is

enough to cause or alleviate the pathological alteration, underscoring the therapeutic potential

of miRNAs in cardiovascular diseases (Chua et al. 2009; Li et al. 2009; Pan et al. 2010).

MicroRNAs (miRNAs) have been shown to be essential for normal heart development and

cardiac function. Recent data suggest that miRNAs are involved in the etiology of cardiac

disease and the remodeling of hearts, including cardiac hypertrophy, myocardial infarction,

and cardiac arrhythmias (Wang et al. 2009; Kura et al. 2016; Slezak et al. 2016).

Our experiments demonstrated that 6 weeks after irradiation miR-15b was down regulated

almost 42 % which is indicating that these hearts are probably protected or there is an

adaptive protective mechanism triggered upon irradiation (Fig. 7). Interestingly, we also saw

decrease in the pro-apoptotic protein Bax - which is also pointing towards protection (Slezak

et al. 2015).

MicroRNA have been shown to be essential for normal heart development and cardiac

function. Recent data suggest that miRNAs are involved in the etiology of cardiac disease and

remodeling of hearts, including cardiac hypertrophy, myocardial infarction and cardiac

arrhythmias. Evidence suggests that miRNAs are differentially expressed in the failing

myocardium and play an important role in progression of heart failure by targeting genes that

govern diverse functions in cardiac remodeling process. MiRNA-based therapeutics may

allow for modulation of cardiac and/or systemic levels of specific miRNAs in situatons with

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heart failure (Latronico and Condorelli 2009; Topkara and Mann 2011; Kura et al. 2016; 2017

in press), remodeling and disease (Wang et al. 2009).

Matrix metalloproteinases (MMPs) are enzymes that play an important role in degradation

and remodeling of extracellular matrix. These proteins are also suggested to play an important

role in pathogenesis of several diseases and their functions are associated with several

physiological and pathological processes (vasculogenesis, morphogenesis, angiogenesis,

tissue healing, chronic inflammation, tumour growth etc.). Recent studies implicate the MMPs

also in the effects induced by tissue irradiation. MMPs act on various intracellular and

extracellular targets to mediate tissue damage (Wang et al. 2002b; Mukherjee et al. 2006).

Increased levels of circulating MMPs in patients with acute coronary syndrome or with post-

acute coronary syndrome have consistently been found to be markers of left ventricular (LV)

dysfunction, remodelling, future cardiovascular events and poor prognosis (Squire et al. 2004;

Webb et al. 2006).

We studied the effect of irradiation on protein levels and activities of matrix

metalloproteinases in plasma. The effects of irradiation on MMPs activities were determined

by gelatin zymography in gels containing 0.2 % gelatin as a substrate. In our samples of

plasma were by gelatin zymography detected activities of several metalloproteinases. In

plasma of rats exposed to mediastinal irradiation we observed significantly increased

activities of 72 kDa MMP-2. In plasma samples were detected also activities of MMP-9 but

these were not significantly changed in consequence of irradiation (Fig. 8A). Investigation of

protein levels of MMPs in plasma revealed that the changes in plasma MMP-2 activities were

not connected with significant changes in protein levels of this enzyme (Fig. 8C). The protein

levels of plasma MMP-9 were also not influenced after irradiation (Fig. 8B).

Our study brings new informations about the effects of irradiation of mediastinal area on

matrix metalloproteinases in rats. We have shown that activation of circulating MMP-2 is

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closely associated with progression of radiation effects. The increase in MMP-2 activities was

not connected with changes in protein content of this enzyme. The latter finding suggests that

regulation of MMP-2 activities observed in rats exposed to irradiation is not realized at the

transcriptional level. The observed activity of 72 kDa MMP-2 corresponds to form of MMP-

2, which is activated through conformational changes induced by oxidative stress. Radiation-

induced injury can primarily be attributed to radiation generated free radicals. Thus, the

observed increase in activities of 72 kDa form of MMP-2 can also be interpreted in the light

of increased radical production after irradaiation. It has been described a direct correlation

between MMP-1 activity and ROS generation (Brown et al. 2000). ROS secreted by tumour-

associated macrophages have also been related to MMP-2 activation, possibly through a

reaction between ROS and thiol groups of MMP-2 (Rajagopalan et al. 1996). We have shown

that the activation of myocardial and circulating MMPs, especially MMP-2, is closely

associated with the progression of toxic effects of DOX (Ivanova et al. 2012).

It has been found that early elevations of MMP-2 in plasma correlated strongly with infarct

size and left ventricular dysfunction in a STEMI population, indicating that MMP-2 might

play an important role in injury induced by ischaemia/reperfusion (Nilsson et al. 2012). The

study showed a strong positive correlation between plasma levels of MMP-2 and infarct size

and LV dysfunction.

Conclusion

The review provides an overview of current knowledge on molecular mechanisms in early

and delayed cardiovascular response to heart irradiation caused by sequence of overlaping

events orchestrated by a large number of interacting molecular signals, including cytokines,

chemokines, and growth factors. This complex process is refelected by changes in cardiac

functional parameters, morphology, inflamatory markers, endothelial dysfunction, expression

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of PPAR, von Willebrand factor, Mg2+

-ATPase and Na, K-ATPases, miRNAs, connexins and

matrix metalloprotenases in rats.

Better understanding of molecular pathways of cardiac radiation injury will help to unravel

basic mechanisms of RIHD, with the ultimate goal to identify potential targets for

intervention and mitigation of pathological processes caused by ionizing radiation.

Acknowledgements

This work was supported by the grants APVV-0241-11, APVV-0102-11, APVV-15-0376, AP

VV-15-0119 and VEGA SR 2/0021/15, 2/0201/15 and 2/0133/15.

References

Adams, M.J., Hardenbergh, P.H., Constine, L.S., and Lipshultz, S.E. 2003. Radiation-associated

cardiovascular disease. Crit. Rev. Oncol. Hematol. 45(1): 55-75.

Amino, M., Yoshioka, K., Tanabe, T., Tanaka, E., Mori, H., Furusawa, Y., et al. 2006. Heavy ion

radiation up-regulates Cx43 and ameliorates arrhythmogenic substrates in hearts after myocardial

infarction. Cardiovasc. Res. 72(3): 412-421.

Ayala, A., Muñoz, M.F., and Argüelles, S. 2014. Lipid peroxidation: production, metabolism, and

signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid. Med. Cell Longev.

2014(2014): 31.

Azzam, E.I., de Toledo, S.M., and Little, J.B. 2001. Direct evidence for the participation of gap

junction-mediated intercellular communication in the transmission of damage signals from alpha-

particle irradiated to nonirradiated cells. Proc. Natl. Acad. Sci. U.S.A. 98(2): 473-478.

Azzam, E.I., de Toledo, S.M., Gooding, T., and Little, J.B. 1998. Intercellular communication is

involved in the bystander regulation of gene expression in human cells exposed to very low fluences

of alpha particles. Radiat. Res. 150(5): 497-504.

Baeuerle, P.A., and Henkel, T. 1994. Function and activation of NF-kappa B in the immune system.

Annu. Rev. Immunol. 12: 141-179.

Baker, E., Fish, B.L., Su, J., Haworth, S.T., Strande, J.L., Komorowski, R.A., et al. 2009. 10 Gy total

body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a

rat model. Int. J. Radiat. Biol. 85(12): 1089-1100.

Page 27 of 48



Draft

27

Baker, J.E., Moulder, J.E., and Hopewell, J.W. 2011. Radiation as a Risk Factor for Cardiovascular

Disease. Antioxid. Redox. Signal. 15(7): 1945-1956.

Balabanli, B., Türközkan, N., Akmansu, M., and Polat, M. 2006. Role of free radicals on mechanism

of radiation nephropathy. Mol. Cell. Biochem. 293(1-2): 183-186.

Barlaka, E., Mellidis, K., Ravingerová, T., and Lazou, A. 2016. Role of pleiotropic properties of

peroxisome proliferator-activated receptors in the heart: focus on the non-metabolic effects in cardiac

protection. Cardiovascular Therapeutics 34(1): 37–48.

Barlic, J., Zhang, Y., and Murphy, P.M. 2007. Atherogenic lipids induce adhesion of human coronary

artery smooth muscle cells to macrophages by upregulating chemokine CX3CL1 on smooth muscle

cells in a TNFalpha-NFkappaB-dependent manner. J. Biol. Chem. 282(26): 19167-19176.

Barrera, G., Toaldo, C., Pizzimenti, S., Cerbone, A., Pettazzoni, P., and Dianzani, M.U. 2008. The

Role of PPAR Ligands in Controlling Growth-Related Gene Expression and their Interaction with

Lipoperoxidation Products. PPAR Res. 2008(2008): 524671.

Basavaraju, S.R., and Easterly, C.E. 2002. Pathophysiological effects of radiation on atherosclerosis

development and progression, and the incidence of cardiovascular complications. Med. Phys. 29(10):

2391-2403.

Bates, D.O. 2010. Vascular endothelial growth factors and vascular permeability. Cardiovasc. Res.

87(2): 262-271.

Boerma, M., Kruse, J.J.C.M., van Loenen, M.M., Klein, H.R., Bart, C.I., Zurcher, C., et al. 2004a.

Increased deposition of von Willebrand factor in the rat heart after local ionizing irradiation.

Strahlenther. Onkol. 180(2): 109-116.

Boerma, M., Zurcher, C., Esveldt, I., Schutte-Bart, C.I., and Wondergem, J. 2004b. Histopathology of

ventricles, coronary arteries and mast cell accumulation in transverse and longitudinal sections of the

rat heart after irradiation. Oncol. Rep. 12(2): 213-219.

Brand, K., Page, S., Walli, A.K., Neumeier, D., and Baeuerle, P.A. 1997. Role of nuclear factor-

kappaB in atherogenesis. Exp. Physiol. 82(2): 297-304.

Brown, N.S., Jones, A., Fujiyama, C., Harris, A.L., and Bicknell, R. 2000. Thymidine phosphorylase

induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors. Cancer. Res.

60(22): 6298-6302.

Bulhak, A.A., Jung, C., Ostenson, C.G., Lundberg, J.O., Sjöquist, P.O., and Pernow, J. 2009. PPAR-

alpha activation protects the type 2 diabetic myocardium against ischemia-reperfusion injury:

involvement of the PI3-Kinase/Akt and NO pathway. Am. J. Physiol. Heart. Circ. Physiol. 296(3):

H719-H727.

Burger, A., Loffler, H., Bamberg, M., and Rodermann, H.P. 1998. Molecular and cellular basis of

radiation fibrosis. Int. J. Radiat. Biol. 73(4): 401-408.

Carnicka, S., Murarikova, M., Ferko, M., Lazou, A., Kukreja, R.C., Ziegelhöffer, A., et al. 2013. The

effects of chest irradiation on myocardial function and response to ischemia in isolated rat hearts.

Cardiologa Hungarica 43(Suppl. G): G11.

Page 28 of 48



Draft

28

Chello, M., Mastroroberto, P., Romano, R., Zofrea, S., Bevacqua, I., and Marchese, A.R. 1996.

Changes in the proportion of types I and III collagen in the left ventricular wall of patients with post-

irradiative pericarditis. Cardiovasc. Surg. 4(2): 222-226.

Chua, J.H., Armugam, A., and Jeyaseelan, K. 2009. MicroRNAs: biogenesis, function and

applications. Curr. Opin. Mol. Ther. 11(2): 189-199.

Corn, B.W., Trock, B.J., and Goodman, R.L. 1990. Irradiation – related ischemic heart disease. J. Clin.

Oncol. 8(4): 741-750.

Darby, S.C., Cutter, D.J., Boerma, M., Constine, L.S., Fajardo, L.F., Kodama, K., et al. 2010.

Radiation-related heart disease: current knowledge and future prospects. Int. J. Radiat. Oncol. Biol.

Phys. 76(3): 656-665.

Darby, S.C., McGale, P., Taylor, C.W., and Peto, R. 2005. Long-term mortality from heart disease and

lung cancer after radiotherapy for early breast cancer: prospective cohort study of about 300,000

women in US SEER cancer registries. Lancet Oncol. 6(8): 557-565.

Dent, P., Yacoub, A., Contessa, J., Caron, R., Amorino, G., Valerie, K., et al. 2003. Stress and

radiation-induced activation of multiple intracellular signaling pathways. Radiat. Res. 159(3): 283-

300.

Du Clos, T.W. 1989. C-reactive protein reacts with the U1 small nuclear ribonucleoprotein. J.

Immunol. 143(8): 2553−2559.

Dunn, M.M., Drab, E.A., and Rubin, D.B. 1986. Effects of irradiation on endothelial cell-

polymorphonuclear leukocyte interactions. J. Appl. Physiol. 60(6): 1932-1937.

Elahi, M.M., and Matata, B.M. 2006. Free radicals in blood: evolving concepts in the mechanism of

ischemic heart disease. Arch. Biochem. Biophys. 450(1): 78-88.

Elahi, M.M., Kong, Y.X., and Matata, B.M. 2009. Oxidative stress as a mediator of cardiovascular

disease. Oxid. Med. Cell. Longev. 2(5): 259-269.

Elahi, M.M., Naseem, K.M., and Matata, B.M. 2007. Nitric oxide in blood. The nitrosativeoxidative

disequilibrium hypothesis on the pathogenesis of cardiovascular disease. Febs. J. 274(4): 906-923.

Esterbauer, H., Eckl, P., and Ortner, A. 1990. Possible mutagens derived from lipids and lipid

precursors. Mutat. Res. 238(3): 223-233.

Esterbauer, H., Schaur, R.J., and Zollner, H. 1991. Chemistry and biochemistry of 4-hydroxynonenal,

malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11(1): 81-128.

Fajardo, L.F. 1989. The unique physiology of endothelial cells and its implications in radiobiology.

Front. Radiat. Ther. Oncol. 23: 96-112.

Fajardo, L.F. 1992. Morphology of radiation effects on normal tissues. In: Principles and practice of

radiation oncology (2nd edn.). Edited by: Perez, C.A., and Brady, L.W., Philadelphia: JB Lippincott

Company, 114-123.

Page 29 of 48



Draft

29

Ferko, M., Gvozdjaková, A., Kucharská, J., Mujkosová, J., Waczulíková, I., Styk, J., et al. 2006.

Functional remodeling of heart mitochondria in acute diabetes: interrelationships between damage,

endogenous protection and adaptation. Gen Physiol Biophys. 25(4): 397-413

Gabriels, K., Hoving, S., Seemann, I., Visser, N.L., Gijbels, M.J., Pol, J.F., et al. 2012. Local heart

irradiation of ApoE(-/-) mice induces microvascular and endocardial damage and accelerates coronary

atherosclerosis. Radiother. Oncol. 105(3): 358-364.

Gagliardi, G., Lax, I., and Rutqvist, L.E. 2001. Partial irradiation of the heart. Semin. Radiat. Oncol.

11(3): 224-233.

Ghosh, S., Wu, Q., Mäder, M., Fish, B.L., Moulder, J.E., Jacobs, E.R., et al. 2009. Vascular injury

following whole thoracic X-ray irradiation in the rat. Int. J. Radiat. Oncol. Biol. Phys. 74(1): 192-199.

Giera, M., Lingeman, H., and Niessen, W.M. 2012. Recent Advancements in the LC- and GC-Based

Analysis of Malondialdehyde (MDA): A Brief Overview. Chromatographia 75(9-10): 433-440.

Glover, D., Little, J.B., Lavin, M.F., and Gueven, N. 2003. Low dose ionizing radiation-induced

activation of connexin43 expression. Int. J. Radiat. Biol. 79(12): 955-64.

Gutteridge, J.M., and Halliwell, B. 2000. Free radicals and antioxidants in the year 2000. A historical

look to the future. Ann. N. Y. Acad. Sci. 899: 136-147.

Gyenes, G. 1998. Radiation-induced ischemic heart disease in breast cancer – a review. Acta. Oncol.

37(3): 241-246.

Gyenes, G., Fornander, T., Carlens, P., Glas, U., and Rutqvist, L.E. 1996. Myocardial damage in

breast cancer patients treated with adjuvant radiotherapy: a prospective study. Int. J. Radiat. Oncol.

Biol. Phys. 36(4): 899-905.

Hallahan, D., Clark, E.T., Kuchibhotla, J., Gewertz, B.L., and Collins, T. 1995. E-selectin gene

induction by ionizing radiation is independent of cytokine induction. Biochem. Biophys. Res.

Commun. 217(3): 784-795.

Han, W., and Yu, K.N. 2009. Response of cells to ionizing radiation. In: Advances in biomedical

sciences and engineering. Edited by: S. C. Tjong. Bentham Science Publishers Ltd., Hong Kong, pp.

204-262.

Hatoum, O.A., Otterson, M.F., Kopelman, D., Miura, H., Sukhotnik, I., Larsen, B.T., et al. 2006.

Radiation induces endothelial dysfunction in murine intestinal arterioles via enhanced production of

reactive oxygen species. Arterioscler. Thromb. Vasc. Biol. 26(2): 287-294.

Hayashi, T., Morishita, Y., Kubo, Y., Kusunoki, Y., Hayashi, I., Kasagi, F., et al. 2005. Long-term

effects of radiation dose on inflammatory markers in atomic bomb survivors. Am. J. Med. 118(1): 83-

86.

Heidenreich, P.A., Schnittger, I., Strauss, H.W., Vagelos, R.H., Lee, B.K., Mariscal, C.S., et al. 2007.

Screening for coronary artery disease after mediastinal irradiation for Hodgkin's disease. J. Clin.

Oncol. 25(1): 43-49.

Hendry, J.H., Akahoshi, M., Wang, L.S., Lipshultz, S.E., Stewart, F.A., and Trott, K.R. 2008.

Radiation-induced cardiovascular injury. Radiat. Environ. Biophys. 47(2): 189-193.

Page 30 of 48



Draft

30

Hirano, K., Nomoto, N., Hirano, M., Momota, F., Hanada, A., and Kanaide, H. 2007. Distinct Ca2+

Requirement for NO production between proteinase-activated receptor 1 and 4 (PAR1 and PAR4) in

vascular endothelial cells. J. Pharmacol. Exp. Ther. 322(2): 668-677.

Hong, J.H., Chiang, C.S., Campbell, I.L., Sun, J.R., Withers, H.R., and McBride, W.H. 1995.

Induction of acute phase gene expression by brain irradiation. Int. J. Radiat. Oncol. Biol. Phys. 33(3):

619-626.

Hopewell, J.W., and Young CM. 1978. Changes in the microcirculation of normal tissues after

irradiation. Int. J. Radiat. Oncol. Biol. Phys. 4(1-2): 53-58.

Hopkins, N., and McLoughlin, P. 2002. The structural basis of pulmonary hypertension in chronic

lung disease: remodelling, rarefaction or angiogenesis?. J. Anat. 201(4): 335-48.

Hughes-Davies, L., Sacks, D., Rescigno, J., Howard, S., and Harris, J. 1995. Serum cardiac troponin T

levels during treatment of early-stage breast cancer. J. Clin. Oncol. 13(10): 2582-2584.

Huss, J.M., and Kelly, D.P. 2004. Nuclear receptor signaling and cardiac energetics. Circ. Res. 95(6):

568–578.

Hussein, M.R., Abu-Dief, E.E., Kamel, E., Abou-El-Ghait, A.T., Abdulwahed, S.R., and Ahmad,

M.H. 2008. Melatonin and roentgen irradiation-induced acute radiation enteritis in Albino rats: An

animal model. Cell Biol. Int. 32(11): 1353-1361.

Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, E.B., Mueller-Dieckmann, C., et al.

1996. Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic

degradation of I kappa B-alpha. Cell. 86(5): 787-798.

Ivanov, V.K., Maksioutov, M.A., Chekin, S.Y., Petrov, A.V., Biryukov, A.P., Kruglova, Z.G., et al.

2006. The risk of radiation-induced cerebrovascular disease in Chernobyl emergency workers. Health

Phys. 90(3): 199-207.

Ivanová, M., Dovinová, I., Okruhlicová, L., Tribulová, N., Simončíková, P., Barteková, M., et al.

2012. Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix

metalloproteinases in rats. Acta Pharmacol. Sin. 33(4):459-469.

Jahroudi, N., Ardekani, A.M., and Greenberger, J.S. 1996. Ionizing radiation increases transcription of

the von Willebrand factor gene in endothelial cells. Blood 88(10): 3801-3814.

Janicki, J.S., Brower, G.L., Gardner, J.D., Forman, M.F., Stewart, J.A. Jr., Murray, D.B., et al. 2006.

Cardiac mast cell regulation of matrix metalloproteinase-related ventricular remodeling in chronic

pressure or volume overload. Cardiovasc. Res. 69(3): 657-665.

Jaworski, C., Mariani, J.A., Wheeler, G., and Kaye, D.M. 2013. Cardiac complications of thoracic

irradiation. J. Am. Coll. Cardiol. 61(23): 2319-2328.

Jelicks, L.A., and Gupta, R.K. 1994. Nuclear magnetic resonance measurement of intracellular sodium

in the perfused normotensive and spontaneously hypertensive rat heart. Am. J. Hypertens. 7(5): 429-

435.

Page 31 of 48



Draft

31

Jones, R., and Reid, L. 1995. Vascular remodeling in clinical and experimental pulmonary

hypertensions. In: Pulmonary Vascular Remodeling. Edited by Bishop JE, Reeves JT, Laurent GJ.

London: Portland Press Ltd., London, pp. 47–116.

Kawano, S., Kubota, T., Monden, Y., Tsutsumi, T., Inoue, T., Kawamura, N., et al. 2006. Blockade of

NF-kappaB improves cardiac function and survival after myocardial infarction. Am. J. Physiol. Heart

Circ. Physiol. 291(3): 1337-1344.

Kim, I., Moon, S.O., Kim, S.H., Kim, H.J., Koh, Y.S., and Koh, G.Y. 2001. Vascular endothelial

growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion

molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells.

J. Biol. Chem. 276(10): 7614-7620.

Konings, A.W., Hardonk, M.J., Wieringa, R.A., and Lamberts, H.B. 1975. Initial events in radiation

induced atheromatosis I. Activation of lysosomal enzymes. Strahlentherapie 150(4): 444-448.

Konings, A.W., Smit Sibinga, C.T., Aarnoudse, M.W., de Witt, S.S., and Lamberts, H.B. 1978. Initial

events in radiation-induced atheromatosis. Damage to intimal cells. Strahlentherapie 154(11): 795-

800.

Kozak, K.R., Hong, T.S., Sluss, P.M., Lewandrowski, E.L., Aleryani, S.L., Macdonald, S.M., et al.

2008. Cardiac blood biomarkers in patients receiving thoracic (chemo)radiation. Lung Cancer 62(3):

351-355.

Kruse, J.J., Zurcher, C., Strootman, E.G., Bart, C.I., Schlagwein, N., Leer, J.W., et al. 2001. Structural

changes in the auricles of the rat heart after local ionizing irradiation. Radiother. Oncol. 58(3): 303-

311.

Kruse, J.J.C.M., Floot, B.G.J., te Poele, J.A.M., Russell, N.S., and Stewart, F.A. 2009. Radiation-

induced activation of TGF-β signaling pathways in relation to vascular damage in mouse kidneys.

Radiat. Res. 171(2): 188-197.

Kubes, P., Suzuki, M., and Granger, D.N. 1991. Nitric oxide: an endogenous modulator of leukocyte

adhesion. Proc. Natl. Acad. Sci. U.S.A. 88(11): 4651-4655.

Kura, B., Yin, C., Frimmel, K., Krizak, J., Okruhlicova, L., Kukreja, R.C., et al. 2016. Changes of

microRNA-1, -15b and -21 levels in irradiated rat hearts after treatment with potentially

radioprotective drugs. Physiol. Res. 65(Suppl 1): S129-S137.

Kura, B., Babal, P., and Slezak, J. 2017. Implication of microRNAs in the development of radiation-

induced heart disease. Can. J. Physiol. Pharmacol. in press.

Kyrkanides, S., Moore, A.H., Olschowka, J.A., Daeschner, J.C., Williams, J.P., Hansen, J.T., et al.

2002. Cyclooxygenase-2 modulates brain inflammation-related gene expression in central nervous

system radiation injury. Brain Res. Mol. Brain Res. 104(2): 159-169.

Lagrand, W.K., Niessen, H.W., Wolbink, G.J., Jaspars, L.H., Visser, C.A., Verheugt, F.W., et al.

1997. C-reactive protein colocalizes with complement in human hearts during acute myocardial

infarction. Circulation 95(1): 97-103.

Page 32 of 48



Draft

32

Landry, D.B., Couper, L.L., Bryant, S.R., and Lindner, V. 1997. Activation of the NF-kappa B and I

kappa B system in smooth muscle cells after rat arterial injury. Induction of vascular cell adhesion

molecule-1 and monocytes chemoattractant protein-1. Am. J. Pathol. 151(4): 1085-1095.

Latronico, M.V., and Condorelli, G. 2009. MicroRNAs and cardiac pathology. Nat. Rev. Cardiol. 6(6):

419-29.

Lau, D.C., Dhillon, B., Yan, H., Szmitko, P.E., and Verma S. 2005. Adipokines: molecular links

between obesity and atheroslcerosis. Am. J. Physiol. Heart. Circ. Physiol. 288(5): 2031-2041.

Lauk, S. 1987. Endothelial alkaline phosphatase activity loss as an early stage in the development of

radiation-induced heart disease in rat. Radiat. Res. 110(1): 118-128.

Lauk, S., and Trott, K.R. 1988. Radiation induced heart disease in hypertensive rats. Int. J. Radiat.

Oncol. Phys. 14(1): 109-114.

Lauk, S., and Trott, K.R. 1990. Endothelial cell proliferation in the rat heart following local heart

irradiation. Int. J. Radiat. Biol. 57(5): 1017-1030.

Lawrence, D.A. 1996. Transforming growth factor-beta: a general review. Eur. Cytokine. Netw. 7(3):

363-374.

Lebrun, F., Francois, A., Vergnet, M., Lebaron-Jacobs, L., Gourmelon, P., and Griffiths, N.M. 1998.

Ionizing radiation stimulates muscarinic regulation of rat intestinal mucosal function. Am. J. Physiol.

275(6 Pt 1): G1333-1340.

Li, M., Marin-Muller, C., Bharadwaj, U., Chow, K.H., Yao, Q., and Chen, C. 2009. MicroRNAs:

control and loss of control in human physiology and disease. World. J. Surg. 33(4): 667–684.

Lopaschuk, G.D., Ussher, J.R., Folmes, C.D.L., Jaswal, J.S., and Stanley, W.C. 2010. Myocardial fatty

acid metabolism in health and disease. Physiol. Rev. 90(1): 207–258.

Loscalzo, J. 2001. Nitric oxide insufficiency, platelet activation, and arterial thrombosis. Circ. Res.

88(8): 756–762.

Mach, F., Schonbeck, U., and Libby, P. 1998. CD40 signaling in vascular cells: A key role in

atherosclerosis? Atherosclerosis. 137(Suppl. 1): 89-95.

Maeda, S. 1980. Pathology of experimental radiation pancarditis. I. Observation on radiation-induced

heart injuries following a single dose of x-ray irradiation to rabbit heart with special reference to its

pathogenesis. Acta. Pathol. Jpn. 30(1):59-78.

Matsumoto, H., Takahashi, A., and Ohnishi, T. 2004. Radiation-induced adaptive responses and

bystander effects. Biol. Sci. Space. 18(4): 247-254.

Maurer, M., Wedemeyer, J., Metz, M., Piliponsky, A.M., Weller, K., Chatterjea, D., et al. 2004. Mast

cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature 432(7016): 512-516.

Mège, A., Ziouèche, A., Pourel, N., and Chauvet, B. 2011. Radiation-related heart toxicity. Cancer

Radiother. 15(6-7): 495-503.

Page 33 of 48



Draft

33

Mezesova, L., Vlkovicova, J., Kalocayova, B., Jendruchova, V., Barancik, M., Fulop. M., et al. 2014.

Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma. Mol. Cell. Biochem. 388(1-2): 241-

247.

Moore, A.H., Olschowka, J.A., Williams, J.P., Paige, S.L., and O’Banion, M.K. 2004. Radiation-

induced edema is dependent on cyclooxygenase 2 activity in mouse brain. Radiat. Res. 161(2): 153-

160.

Moreira, O.C., Oliveira, V.H., Benedicto, L.B.F., Nogueira, C.M., Mignaco, J.A., Fontes, C.F.L., et al.

2008. Effects of gamma-irradiation on the membrane ATPases of human erythrocytes from

transfusional blood concentrates. Ann. Hematol. 87(2): 113-119.

Morgan, M.J., and Liu, Z.G. 2012. Crosstalk of reactive oxygen species and NF-κB signaling. Cell

Res. 21(1): 103-115.

Moulder, J.E. 2004. Post-irradiation approaches to treatment of radiation injuries in the context of

radiological terrorism and radiation accidents: a review. Int. J. Radiat. Biol. 80(1): 3-10.

Mukherjee, R., Herron, A.R., Lowry, A.S., Stroud, R.E., Stroud, M.R., Wharton, J.M., et al. 2006.

Selective induction of matrix metalloproteinases and tissue inhibitor of metalloproteinases in atrial and

ventricular myocardium in patients with atrial fibrillation. Am. J. Cardiol. 97(4): 532-537.

Muráriková, M., Ferko, M., Barteková, M., Waczulíková, I., Ledvényiová, V., and Ziegelhöffer, A.

2013. Increased resistance of diabetic mitochondria to ischemic-reperfusion myocardial injury.

Cardiology. Let. 22(3): 245-261.

Negre-Salvayre, A., Coatrieux, C., Ingueneau, C., and Salvayre, R. 2008. Advanced lipid peroxidation

end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for

the inhibitors. Br. J. Pharmacol. 153(1): 6-20.

Nellessen, U., Zingel, M., Hecker, H., Bahnsen, J., and Borschke, D. 2010. Effects of radiation therapy

on myocardial cell integrity and pump function: which role for cardiac biomarkers? Chemotherapy

56(2): 147-152.

Nijmeijer, R., Lagrand, W.K., Lubbers, Y.T., Visser, C.A., Meijer, C.J., Niessen, H.W., et al. 2003. C-

reactive protein activates complement in infarcted human myocardium. Am. J. Pathol. 163(1): 269-

275.

Nilsson, L., Halle´n, J., Atar, D., Jonasson, L., and Swahn, E. 2012. Early measurements of plasma

matrix metalloproteinase-2 predict infarct size and ventricular dysfunction in ST-elevation myocardial

infarction. Heart. 98(1): 31-36.

Niu, X.F., Smith, C.W., and Kubes, P. 1994. Intracellular oxidative stress induced by nitric oxide

synthesis inhibition increases endothelial cell adhesion to neutrophils. Circ. Res. 74(6): 1133-1140.

Okruhlicova, L., Knezl, V., Navarova, J., Sotnikova, R., Bernatova, I., Frimmel, K., et al. 2012.

Inflammation-induced structural alterations of endothelium contribute to increased susceptibility of the

heart to ischemia/reperfusion injury. Acta Physiol. 206(Suppl. 693): 208.

Olszewski, M.B., Groot, A.J., Dastych, J., and Knol, E.F. 2007. TNF trafficking to human mast cell

granules: mature chain-dependent endocytosis. J Immunol. 178(9): 5701-5709.

Page 34 of 48



Draft

34

Pacher, P., and Szabo, C. 2008. Role of the peroxynitritepoly( ADP-ribose) polymerase pathway in

human disease. Am. J. Pathol. 173(1): 2-13.

Pan, Z.W., Lu, Y.J., and Yang, B.F. 2010. MicroRNAs: a novel class of potential therapeutic targets

for cardiovascular diseases. Acta. Pharmacol. Sin. 31(1): 1-9.

Paris, F., Fuks, Z., Kang, A., Capodieci, P., Juan, G., Ehleiter, D., et al. 2001. Endothelial apoptosis as

the primary lesion initiating intestinal radiation damage in mice. Science. 293(5528): 293-297.

Peng, H.B., Libby, P., and Liao, J.K. 1995. Induction and stabilization of Iκ Bα by nitric oxide

mediates inhibition of NF-κB. J. Biol. Chem. 270(23): 14214-14219.

Pepys, M.B., and Hirschfield, G.M. 2003. C-reactive protein: a critical update. J. Clin. Invest. 111(12):

1805-1812.

Rabinovitch, M., Gamble, W., Nadas, A.S., Miettinen, O.S., and Reid, L. 1979. Rat pulmonary

circulation after chronic hypoxia: hemodynamic and structural features. Am. J. Physiol. 236(6): 818-

827.

Rajagopalan, S., Meng, X.P., Ramasamy, S., Harrison, D.G., and Galis, Z.S. 1996. Reactive oxygen

species produced by macrophage-derived foam cells regulate the activity of vascular matrix

metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J. Clin. Invest. 98(11):

2572-2579.

Ravingerová, T., Slezák, J., Tribulová, N., Džurba, A., Uhrík, B., Ziegelhöffer, A. 1999. Free oxygen

radicals contribute to high incidence of reperfusion-induced arrhythmias in isolated rat heart. Life Sci.

65(18-19): 1927-1930.

Ravingerová, T., Matejíková, J., Neckář, J., Andelová, E., Kolář, F. 2007. Differential role of

PI3K/Akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart. Mol.

Cell. Biochem. 297(1-2): 111-120.

Ravingerova, T., Adameova, A., Carnicka, S., Nemcekova, M., Kelly, T., Matejikova, J., et al. 2011.

The role of PPAR in myocardial response to ischemia in normal and diseased heart. Gen. Physiol.

Biophys. 30(4): 329-341.

Ravingerová, T., Carnická, S., Nemčeková, M., Ledvényiová, V., Adameová, A., Kelly, T., et al.

2012. PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial

protection against acute ischaemia–reperfusion injury: involvement of PI3K-Akt. Can. J. Physiol.

Pharmacol. 90(8): 1135-1144.

Razeghi, P., Young, M.E., Abbasi, S., and Taegtmeyer, H. 2001. Hypoxia in vivo decreases

peroxisome proliferator-activated receptor alpha-regulated gene expression in rat heart. Biochem.

Biophys. Res. Commun. 287(1): 5-10.

Ridnour, L.A., Thomas, D.D., Mancardi, D., Espey, M.G., Miranda, K.M., Paolocci, N., et al. 2004.

The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting

perspective on stressful biological situations. Biol. Chem. 385(1): 1-10.

Rutqvist, L.E., Lax, I., Fornander, T., and Johansson, H. 1992. Cardiovascular mortality in a

randomized trial of adjuvant radiation therapy versus surgery alone in primary breast cancer. Int. J.

Radiat. Oncol. Biol. Phys. 22(5): 887-896.

Page 35 of 48



Draft

35

Ryland, D., and Reid L. 1975. The pulmonary circulation in cystic fibrosis. Thorax. 30(3): 285-292.

Salameh, A., and Dhein, S. 2005. Pharmacology of Gap junctions .New pharmacological targets for

treatment of arrhythmia, seizure and cancer? Biochim. Biophys. Acta. 1719(1-2): 36-58.

Sambandam, N., Morabito, D., Wagg, C., Finck, B.N., Kelly, D.P., and Lopaschuk, G.D. 2006.

Chronic activation of PPAR-a is detrimental to cardiac recovery after ischemia. Am. J. Physiol. Heart

Circ. Physiol. 290(1): H87–H95.

Schick, P.K., Walker, J., Profeta, B., Denisova, L., and Bennett, V. 1997. Synthesis and secretion of

von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation.

Arterioscler. Thromb. Vasc. Biol. 17(4): 797-801.

Schultz-Hector, S. 1992. Radiation-induced heart disease: review of experimental data on dose

response and pathogenesis. Int. J. Radiat. Biol. 61(2): 149-160.

Schultz-Hector, S., and Balz, K. 1994. Radiation-induced loss of endothelial alkaline phosphatase

activity and development of myocardial degeneration: an ultrastructural study. Lab. Invest. 71(2): 252-

260.

Schultz-Hector, S., and Trott, K.R. 2007. Radiation-induced cardiovascular diseases: is the

epidemiologic evidence compatible with the radiobiologic data?. Int. J. Radiat. Oncol. Biol. Phys.

67(1): 10-18.

Schultz-Hector, S., Balz, K., Bohm, M., Ikehara, Y., and Rieke, L. 1993. Cellular localization of

endothelial alkaline phosphatase reaction product and enzyme protein in the myocardium.

J.Histochem. Cytochem. 41(12): 1813-1821.

Schultz-Hector, S., Bohm, M., Blochel, A., Dominiak, P., Erdmann, E., Muller-Schauenburg, W., et

al. 1992. Radiation-induced heart disease: morphology, changes in catecholamine synthesis and

content, beta-adrenoceptor density, and hemodynamic function in an experimental model. Radiat. Res.

129(3): 281-289.

Seddon, B., Cook, A., Gothard, L., Salmon, E., Latus, K., Underwood, S.R., et al. 2002. Detection of

defects in myocardial perfusion imaging in patients with early breast cancer treated with radiotherapy.

Radiother. Oncol. 64(1): 53-63.

Severs, N.J., Dupont, E., Coppen, S.R., Halliday, D., Inett, E., Baylis, D., et al. 2004. Remodelling of

gap junctions and connexin expression in heart disease. Biochim. Biophys. Acta. 1662(1-2): 138-148.

Siomek, A. 2012. NF-κB signaling pathway and free radical impact. Acta Biochim. Pol. 59(3): 323-

331.

Slezak, J., Barancik, M., Ravingerova, T., Carnicka, S., Frimmel, K., Ferko, M., et al. 2013a.

Mechanisms of early cardiac dysfunction after mediastinal irradiation. Proceedings of Advaces in

cardiovascular research: from bench to bedside. International symposium, Bratislava, Slovakia, May

23-26, 2013. p. 26.

Slezak, J., Barancik, M., Ravingerova, T., Tribulova, N., Kura, B., Lazou, A., et al. 2014. Molecular

mechanisms of myocardial irradiation injury and potential prevention targets. Proceedings of New

Frontiers in Basic Cardiovascular Research 2014: 11th Meeting of France - New EU Members,

Smolenice, Slovakia, June 15-18, 2014. p. 29.

Page 36 of 48



Draft

36

Slezak, J., Fogarassyova, M., and Barancik, M. 2013b. The role of matrix metalloproteinases in

responses of rat hearts after mediastinal irradiation. Proceedings of Advaces in cardiovascular

research: from bench to bedside. International symposium, Bratislava, Slovakia, May 23-26, 2013. p.

72.

Slezák, J., Kura, B., Frimmel, K., Zálešák, M., Ravingerová, T., Viczenczová, C., et al. 2016.

Preventive and therapeutic application of molecular hydrogen in situations with excessive production

of free radicals. Physiol. Res. 65(Suppl. 1): S11-28.

Slezak, J., Kura, B., Ravingerová, T., Tribulova, N., Okruhlicova, L., and Barancik, M. 2015.

Mechanisms of cardiac radiation injury and potential preventive approaches. Can. J. Physiol.

Pharmacol. 93(9): 737-753.

Slezak, J., Tribulova, N., Ivanova, M., Styk, J., Frimmel, K., Ravingerova, T., et al. 2011. Proton

radiation-induced cardiovascular toxicity: pathology and prevention. Exp. Clin. Cardiol. 16(Suppl. A):

28A.

Slezak, J., Tribulova, N., Ivanova, M., Styk, J., Frimmel, K., Ravingerova, T., et al. 2012. Chronic

ischemic cardiomyopathy induced by mediastinal irradiation: pathology and prevention. Proceedings

of Conference and advanced research workshop "Sudden cardiac death & cardioprotection, Timisoara,

Rom., September 6-9, 2012. p. 52.

Smeets, P.J., Planavila, A., van der Vusse, G.J., and van Bilsen, M. 2007. Peroxisome proliferator-

activated receptors and inflammation: take it to heart. Acta. Physiol. (Oxf). 191(3): 171–188.

Smith, R.E., Janjan, N., Kretzschmar, S., and Hackbarth, D. 1989. The effect of radiation therapy on

von Willebrand factor in patients with angiosarcoma. Radiother. Oncol. 16(4): 297-304.

Squire, I.B., Evans, J., Ng, L.L., Loftus, I.M., and Thompson, M.M. 2004. Plasma MMP-9 and MMP-

2 following acute myocardial infarction in man: correlation with echocardiographic and neurohumoral

parameters of left ventricular dysfunction. J. Card. Fail. 10(4): 328-333.

Stewart, F.A., Hoving, S., and Russell, N.S. 2010. Vascular damage as an underlying mechanism of

cardiac and cerebral toxicity in irradiated cancer patients. Radiat. Res. 174(6): 865-869.

Stewart, F.A., Seemann, I., Hoving, S., and Russell, N.S. 2013. Understanding radiationinduced

cardiovascular damage and strategies for intervention. Clin. Oncol. 25(10): 617-624.

Tatton, W., Chalmers-Redman, R., and Tatton, N. 2003. Neuroprotection by deprenyl and other

propargylamines: glyceraldehyde-3-phosphate dehydrogenase rather than monoamine oxidase. J.

Neural. Transm. 110(5): 509-515.

Tchernof, A., Nolan, A., Sites, C.K., Ades, P.A., and Poehlman, E.T. 2002. Weight loss reduces C-

reactive protein levels in obese postmenopausal women. Circulation 105(5): 564-569.

te Poele, J.A., van Kleef, E.M., van der Wal, A.F., Dewit, L.G., and Stewart, F.A. 2001. Radiation-

induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor

antagonist clopidogrel. Int. J. Radiat. Oncol. Biol. Phys. 50(5): 1332-1338.

Thompson, D., Pepys, M.B., and Wood S.P. 1999. The physiological structure of human C-reactive

protein and its complex with phosphocholine. Structure 7(2): 169-177.

Page 37 of 48



Draft

37

Topkara, V.K., and Mann, D.L. 2011. Role of MicroRNAs in Cardiac Remodeling and Heart Failure.

Cardiovasc. Drugs. Ther. 25(2): 171-182.

Touyz, R.M., and Schiffrin, E.L. 2006. Peroxisome proliferator-activated receptors in vascular

biology-molecular mechanisms and clinical implications. Vascul. Pharmacol. 45(1): 19-28.

Tribulova, N., Knezl, V., Okruhlicova, L., and Slezak, J. 2008. Myocardial gap junctions: targets for

novel approaches in the prevention of life-threatening cardiac arrhythmias. Physiol. Res. 57(2): S1-

S13.

Tribulova, N., Knezl, V., Shainberg, A., Seki, S., and Soukup, T. 2009. Thyroid hormones and cardiac

arrhythmias. Vascul. Pharmacol. 52(3-4): 102-112.

Triggle, C.R., Samuel, S.M., Ravishankar, S., Marei, I., Arunachalam, G., and Ding, H. 2012. The

endothelium: influencing vascular smooth muscle in many ways. Can. J. Physiol. Pharmacol. 90(6):

713-738.

Tsutsui, H., Kinugawa, S., and Matsushima, S. 2009. Mitochondrial oxidative stress and dysfunction

in myocardial remodelling. Cardiovasc. Res. 81(3): 449-456.

Valko, M., Morris, H., and Cronin, M.T. 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem.

12(12): 1161-1208.

Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., and Mazur, M. 2006. Free radicals, metals and

antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160(1): 1-40.

van der Meeren, A., Squiban, C., Gourmelon, P., Lafont, H., and Gaugler, M.H. 1999. Diferential

regulation by IL-4 and IL-10 of radiation-induced IL-6 and IL-8 production and ICAM-1 expression

by human endothelial cells. Cytokine. 11(11): 831-838.

van Kleef, E.M., te Poele, J.A., Oussoren, Y.G., Verheij, M., van de Pavert, I., Braunhut, S.J., et al.

1998. Increased expression of glomerular von Willebrand factor after irradiation of the mouse kidney.

Radiat. Res. 150(5): 528-534.

Vassalle, C., Pratali, L., Boni, C., Mercuri, A., and Ndreu, R. 2008. An oxidative stress score as a

combined measure of the pro-oxidant and anti-oxidant counterparts in patients with coronary artery

disease. Clin. Biochem. 41(14-15): 1162-1167.

Verheij, M., Dewit, L.G., Boomgaard, M.N., Brinkman, H.J., and van Mourik, J.A. 1994. Ionizing

radiation enhances platelet adhesion to the extracellular matrix of human endothelial cells by an

increase in the release of von Willebrand factor. Radiat. Res. 137(2): 202-207.

Viczenczova, C., Szeiffova Bacova, B., Egan Benova, T., Kura, B., Yin, C., Weismann, P., et al. 2016.

Myocardial connexin-43 and PKC signalling are involved in adaptation of the heart to irradiation-

induced injury: Implication of miR-1 and miR-21. Gen. Physiol. Biophys. 35(2): 215-222.

Volanakis, J.E., and Kaplan, M.H. 1971. Specificity of C-reactive protein for choline phosphate

residues of pneumococcal C-polysaccharide. Proc. Soc. Exp. Biol. Med. 136(2): 612-614.

Vos, J., Aarnoudse, M.W., Dijk, F., and Lamberts, H.B. 1983. On the cellular origin and development

of atheromatous plaques. A light and electron microscopic study of combined Xray and

Page 38 of 48



Draft

38

hypercholesterolemia-induced atheromatosis in the carotid artery of the rabbit. Virchows. Arch. B Cell

Pathol. Incl. Mol. Pathol. 43(1): 1-16.

Wang, J., Zheng, H., Ou, X., Fink, L.M., and Hauer-Jensen, M. 2002a. Deficiency of microvascular

thrombomodulin and upregulation of protease-activated receptor 1 in irradiated rat intestine: Possible

link between endothelial dysfunction and chronic radiation fibrosis. Am. J. Pathol. 160(6): 2063-2072.

Wang, N., Zhou, Z., Liao, X., and Zhang, T. 2009. Role of microRNAs in cardiac hypertrophy and

heart failure. IUBMB. Life. 61(6): 566-571.

Wang, W., Schulze, C.J., Suarez-Pinzon, W.L., Dyck, J.R., Sawicki, G., and Schulz, R. 2002b.

Intracellular action of matrix metalloproteinase-2 accounts for acute myocardial ischemia and

reperfusion injury. Circulation 106(12): 1543-1549.

Webb, C.S., Bonnema, D.D., Ahmed, S.H., Leonardi, A.H., McClure, C.D., Clark, L.L., et al. 2006.

Specific temporal profile of matrix metalloproteinase release occurs in patients after myocardial

infarction: relation to left ventricular remodeling. Circulation 114(10): 1020-1027.

Weintraub, N.L., Jones, W.K., and Manka, D. 2010. Understanding radiation-induced vascular

disease. J. Am. Coll. Cardiol. 55(12): 1237-1239.

Wilson, S.H., Caplice, N.M., Simari, R.D., Holmes, D.R.Jr., Carlson, P.J., and Lerman, A. 2000.

Activated nuclear factor-κB is present in the coronary vasculature in experimental

hypercholesterolemia. Atherosclerosis 148(1): 23-30.

Wondergem, J., van der Laarse, A., van Ravels, F.J., van Wermeskerken, A.M., Verhoeve, H.R., de

Graaf, B.W., et al. 1991. In vitro assessment of cardiac performance after irradiation using an isolated

working rat heart preparation. Int. J. Radiat. Biol. 59(4): 1053-1068.

Yue, T.L., Bao, W., Jucker, B.M., Gu, J.L., Romanic, A.M., Brown, P.J., et al. 2003. Activation of

peroxisome proliferator-activated receptor-α protects the heart from ischemia/reperfusion injury.

Circulation, 108(19): 2393–2399.

Yusuf, S.W., Sami, S., and Daher, I.N. 2011. Radiation-induced heart disease: a clinical update.

Cardiol. Res. Pract. 2011(2011): 317659.

Zhao, W., Diz, D.I., and Robbins, M.E. 2007. Oxidative damage pathways in relation to normal tissue

injury. Br. J. Radiol. 80(1): S23-S31.

Zhong, G.Z., Chen, F.R., Bu, D.F., Wang, S.H., Pang, Y.Z., and Tang, C.S. 2004. Cobalt-60 gamma

radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells. Life Sci.

74(25): 3055-3063.

Zhou, Q., Zhao, Y., Li, P., Bai, X., and Ruan, C. 1992. Thrombomodulin as a marker of radiation-

induced endothelial cell injury. Radiat. Res. 131(3): 285-289.

Ziegelhöffer, A., Kjeldsen, K., Bundgaard, H., Breier, A., Vrbjar, N., and Dzurba, A. 2000. Na,K

ATPase in the myocardium: molecular principles, functional and clinical aspects. Gen. Physiol.

Biophys. 19(1): 9-47.

Ziegelhöffer, A., Mujkošová, J., Ferko, M., Vrbjar, N., Ravingerová, T., Uličná, O., et al. 2012. Dual

influence of spontaneous hypertension on membrane properties and ATP production in heart and

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kidney mitochondria in rat: effect of captopril and nifedipine, adaptation and dysadaptation. Can. J.

Physiol. Pharmacol. 90(9): 1311-1323.

Fig. 1: Concentrations of MDA, C-reactive protein and TNF-α in rats after mediastinum irradiation. A -

Concentrations of MDA in rat’s serum after irradiation of mediastinum area. Rats were irradiatied with single

dose of 10 Gy (4 Gy/min). MDA was measured 2 days, 9 days and 6 weeks after irradiation. p < 0,05 (Slezak et

al. unpublished). B - C-reactive protein and TNF-α values in rat left ventricle six weeks after mediastinal

irradiation (total dose 25 Gy, 6-7 Gy/min). (unpublished results). * (p < 0,05)

Fig. 2: Transmission electron microscopy (TEM) of the myocardium 6 weeks after irradiation: A: Control

myocardium. capillaries (cap), well preserved myocardial cell nucleus (N), polymorphonuclear cell (Le), red

blood cell (RC), cardiac myocyte (cmc); B: destroyed capillary (arrow) with interrupted endothelial cell (cap);

C: capillary (cap), monocyte MO; D: higher magnification of two endothelial cells (EC) of capillaries. EC on

the left side is degenerated one with large vacuoles. EC on the right is activated exhibiting numerous ribosomes.

(Slezak et al. unpublished results) E:Cross section through ventricular myocardium at low magnification. Normal

capillary density in control rats (f- 0,98); F: Increased capillary density (f- 1,21) 6 weeks after 25 Gy irradiation

(6-7 Gy/min.). Arrows pointing to individual capillaries. (unpublished results).

Fig. 3: Representative immunoblot of vWF expression (A) and its percentage of densitometric quantification (B)

in left ventricle. Control and irradiated 25Gy. Results are mean ± SEM. (unpublished results). As a loading

control was used GAPDH.

Fig. 4: Reperfusion-induced arrhythmias and postischemic recovery of function in the hearts of control and 25

Gy irradiated rats. A: PVC – premature ventricular complexes. C – controls, Ir – irradiated. Data are Means ±

S.E.M. from 6 hearts per group.* - p<0.05 vs. controls. Unpublished results. B: LVDP – left ventricular

developed pressure after 30-min global ischaemia and 40-min reperfusion. C – controls, Ir – irradiated. Data are

Means ± S.E.M. from 6 hearts per group expressed as % of preischemic values. (unpublished results). * (p <

0,05)

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Fig. 5: PPARα mRNA expression in rat heart six weeks after 25 Gy irradiation (6-7 Gy/min.). (unpublished

results). * (p < 0,05)

Fig. 6: Effect of ionizing radiation on total mitochondrial Mg2+

-ATPase activity in the left ventricle of rat heart.

C- controls, IR- irradiated . (unpublished results). * (p < 0,05)

Fig. 7: Expression of miRNA-1, -15b and -21 in the rat hearts six weeks after irradiation (total 25 Gy, 6-7

Gy/min.). (unpublished results). * (p < 0,05)

Fig. 8: Effects of irradiation on plasma MMP-2 and MMP-9 protein levels and activities. The MMPs activities

analysed by zymography in plasma samples prepared from whole artery blood of control rats (C) and rats

exposed to irradiation (R). A- Western blot record showing the influence of irradiation on gelatinolytic activities

of MMPs. Zymographic analysis of plasma samples revealed bands corresponding to the gelatinolytic activities

of 72 kDa MMP-2 and MMP-9. B- Western blot record showing the influence of irradiation on MMP-9 protein

levels in plasma. C- Western blot record showing the influence of irradiation on MMP-2 protein levels in

plasma. C – control; R – irradiation. (unpublished results).

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Fig. 1: Concentrations of MDA, C-reactive protein and TNF-α in rats after mediastinum irradiation. A - Concentrations of MDA in rat’s serum after irradiation of mediastinum area. Rats were irradiatied with single

dose of 10 Gy (4 Gy/min). MDA was measured 2 days, 9 days and 6 weeks after irradiation. p < 0,05

(Slezak et al. unpublished). B - C-reactive protein and TNF-α values in rat left ventricle six weeks after mediastinal irradiation (total dose 25 Gy, 6-7 Gy/min). (unpublished results). * (p < 0,05)

150x135mm (300 x 300 DPI)

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Fig. 2: Transmission electron microscopy (TEM) of the myocardium 6 weeks after irradiation: A: Control myocardium. capillaries (cap), well preserved myocardial cell nucleus (N), polymorphonuclear cell (Le), red blood cell (RC), cardiac myocyte (cmc); B: destroyed capillary (arrow) with interrupted endothelial cell

(cap); C: capillary (cap), monocyte MO; D: higher magnification of two endothelial cells (EC) of capillaries. EC on the left side is degenerated one with large vacuoles. EC on the right is activated exhibiting numerous

ribosomes. (Slezak et al. unpublished results) E:Cross section through ventricular myocardium at low magnification. Normal capillary density in control rats (f- 0,98); F: Increased capillary density (f- 1,21) 6

weeks after 25 Gy irradiation (6-7 Gy/min.). Arrows pointing to individual capillaries. (unpublished results).

174x172mm (300 x 300 DPI)

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Fig. 3: Representative immunoblot of vWF expression (A) and its percentage of densitometric quantification (B) in left ventricle. Control and irradiated 25Gy. Results are mean ± SEM. (unpublished results). As a

loading control was used GAPDH.

135x111mm (96 x 96 DPI)

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Fig. 4: Reperfusion-induced arrhythmias and postischemic recovery of function in the hearts of control and 25 Gy irradiated rats. A: PVC – premature ventricular complexes. C – controls, Ir – irradiated. Data are Means ± S.E.M. from 6 hearts per group.* - p<0.05 vs. controls. Unpublished results. B: LVDP – left

ventricular developed pressure after 30-min global ischaemia and 40-min reperfusion. C – controls, Ir – irradiated. Data are Means ± S.E.M. from 6 hearts per group expressed as % of preischemic values.

(unpublished results). * (p < 0,05)

318x128mm (300 x 300 DPI)

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Fig. 5: PPARα mRNA expression in rat heart six weeks after 25 Gy irradiation (6-7 Gy/min.). (unpublished results). * (p < 0,05)

113x93mm (300 x 300 DPI)

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Fig. 6: Effect of ionizing radiation on total mitochondrial Mg2+ -ATPase activity in the left ventricle of rat heart. C- controls, IR- irradiated . (unpublished results). * (p < 0,05)

141x86mm (300 x 300 DPI)

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Fig. 7: Expression of miRNA-1, -15b and -21 in the rat hearts six weeks after irradiation (total 25 Gy, 6-7 Gy/min.). (unpublished results). * (p < 0,05)

118x95mm (300 x 300 DPI)

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Fig. 8: Effects of irradiation on plasma MMP-2 and MMP-9 protein levels and activities. The MMPs activities analysed by zymography in plasma samples prepared from whole artery blood of control rats (C) and rats

exposed to irradiation (R). A- Western blot record showing the influence of irradiation on gelatinolytic activities of MMPs. Zymographic analysis of plasma samples revealed bands corresponding to the

gelatinolytic activities of 72 kDa MMP-2 and MMP-9. B- Western blot record showing the influence of irradiation on MMP-9 protein levels in plasma. C- Western blot record showing the influence of irradiation on

MMP-2 protein levels in plasma. C – control; R – irradiation. (unpublished results).

202x183mm (300 x 300 DPI)

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