Novel radiation targets in the endothelium and heart muscle Venkata Ramesh Yentrapalli

Novel radiation targets in the endothelium and
heart muscle
Venkata Ramesh Yentrapalli
Centre for Radiation Protection Research
Department of Molecular Biosciences,
The Wenner-Gren Institute, Stockholm University, Sweden
Stockholm, 2013
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© Venkata Ramesh Yentrapalli
ISBN 978-91-7447-718-4 (Page 1-59)
All previously published papers are reproduced by permissions of the publishers
Printed in Sweden by Universitetsservice AB, Stockholm 2013
Distributor: Stockholm University Library
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To my parents
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ABSTRACT
Worldwide, people are being exposed to natural and man-made sources of radiation.
Epidemiological studies have shown an increased risk of vascular diseases in populations that
have been exposed to ionizing radiation. Vascular endothelium is implicated as one of the
targets for radiation leading to the development of cardiovascular diseases. However, the
molecular mechanisms behind the development of radiation-induced cardiovascular disease
in acute or chronic exposed people are not fully elucidated. The hypothesis that chronic low
dose rate ionizing radiation accelerates the onset of senescence of primary human umbilical
vein endothelial cells has been tested in papers I and II presented in this thesis. In vitro
studies show that, when exposed to continuous low dose rate gamma radiation these cells
enter premature senescence much earlier than non-irradiated control cells. Quantitative
proteomic analysis using isotope coded protein labeling coupled to LC-ESI-mass
spectrometry and followed by protein network analysis identified changes in senescencerelated biological pathways including cytoskeletal organisation, cell-cell communication and
adhesion, and inflammation influenced by radiation. Moreover, the role of PI3K/Akt/mTOR
pathway was implicated during the senescence process. Thus, chronic low dose rated
endothelial senescence may contribute to increased risk of radiation-induced cardiovascular
disease.
Paper III analyse the long-term effects of local high doses of radiation to the heart using a
mouse model. The results from proteomic and bioinformatics analysis indicated that an
impaired activity of the peroxisome proliferator-activated receptor-alpha (PPARA) is
involved in mediating the radiation response. Ionizing radiation markedly changed the
phosphorylation and ubiquitination status of PPARA. This was reflected by the decreased
expression of PPARA target genes involved in energy metabolism and mitochondrial
respiratory chain. This in vivo study suggests that alteration of cardiac metabolism
contributes to the impairment of heart structure and function after radiation.
Taken together, these in vitro and in vivo studies provide novel information on the pathways
in heart and endothelial cells that are affected over longer periods of time by ionizing
radiation.
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CONTENTS
PAGE NO
ABSTRACT
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LIST OF PAPERS
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ABBREVIATIONS
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INTRODUCTION
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1. Ionizing radiation
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1.1. General aspects of ionizing radiation
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1.2. Effects of ionizing radiation
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1.3. Epidemiology of radiation-induced non-cancerous health effects
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2. Cardiovascular system
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2.1. Cardiovascular diseases
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2.2. Radiation-induced cardiovascular damage
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2.3. Radiation and endothelial dysfunction
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3. Senescence
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3.1. Replicative senescence
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3.2. Characteristic features of senescence
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3.3. Stress-induced premature senescence
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3.4. Senescence-associated pathways
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3.5. Oxidative stress and radiation-induced senescence
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OBJECTIVES OF THE THESIS
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MATERIALS AND METHODS
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RESULTS AND DISCUSSION
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Paper I
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Paper II
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Paper III
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SUMMARY AND FUTURE PERSPECTIVES
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ACKNOWLEDGEMENTS
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REFERENCES
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LIST OF PAPERS
The doctoral thesis is based on the following three publications.
Paper I
Yentrapalli R, Azimzadeh O, Barjaktarovic Z, Sarioglu H, Wojcik A, Harms-Ringdahl M,
Atkinson MJ, Haghdoost S, Tapio S. Quantitative proteomic analysis reveals induction of
premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose
rate gamma-radiation. Proteomics. 2013 Apr;13(7):1096-107.
Paper II
Yentrapalli R, Azimzadeh O, Sriharshan A, Malinowsky K, Merl J, Wojcik A, HarmsRingdahl M, Atkinson MJ, Becker K-F, Haghdoost S and Tapio S. The PI3K/Akt/mTOR
pathway is a key player in the premature senescence of primary human endothelial cells
exposed to chronic radiation. PLoS One. 2013 Aug 1;8(8):e70024.
Paper III
Azimzadeh O, Sievert W, Sarioglu H, Yentrapalli R, Barjaktarovic Z, Sriharshan A, Ueffing
M, Janik D, Aichler M, Atkinson MJ, Multhoff G, Tapio S. PPAR Alpha: A Novel Radiation
Target in Locally Exposed Mus musculus Heart Revealed by Quantitative Proteomics. J
Proteome Res. 2013 Jun 7;12(6):2700-14.
Papers I, II and III are reproduced with permission from the publishers.
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LIST OF PAPERS
The following original publication is not included in this thesis
Azimzadeh O, Scherthan H, Yentrapalli R, Barjaktarovic Z, Ueffing M, Conrad M, Neff F,
Calzada-Wack J, Aubele M, Buske C, Atkinson MJ, Hauck SM, Tapio S. Label-free protein
profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate
mitochondrial impairment after ionizing radiation. J Proteomics. 2012 Apr 18;75(8):2384-95.
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ABBREVIATIONS
8-Oxo-dG
8-Oxo-7, 8-dihydro-2'-deoxyguanosine
8-Oxo-dGTP 8-Oxo-7, 8-dihydro-2'-deoxyguanosine triphosphate
CVD
Cardiovascular disease
DAVID
Database for Annotation, Visualization, and Integrated Discovery
DNA
Deoxyribonucleic acid
DSB
Double-strand breaks
EIF
Eukaryotic initiation factor
ERK
Extracellular signal-regulated kinases
ESI
Electron spray ionisation
GO
Gene Ontology
Gy
Gray
hMTH1
Human MutT homologue 1
HR
Homologous recombination
HUVEC
Human umbilical vein endothelial cells
ICPL
Isotope coded protein labelling
IPA
Ingenuity pathway analysis
LC
Liquid chromatography
LET
Linear energy transfer
Min
Minutes
mTOR
Mammalian target of rapamycin
MS
Mass spectrometry
NHEJ
Non-homologous end-joining
PAGE
Polyacrylamide gel electrophoresis
PANTHER
Protein Analysis through Evolutionary Relationship
PI3K
Phosphoinositide 3-kinase
PPARA
Peroxisome proliferator-activated receptor alpha
Rho GDI
Rho GDP dissociation inhibitor 1
RNA
Ribonucleic acid
RPPA
Reverse phase protein array
ROS
Reactive oxygen species
SA-β-gal
Senescence-associated beta-galactosidase
SDS
Sodium dodecyl sulphate
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SIPS
Stress-induced premature senescence
STRING
Search Tool for the Retrieval of Interacting Genes/Proteins
TBST
Tris-buffered saline-tween 20
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INTRODUCTION
People are being chronically exposed to natural radiation as well as to man-made sources of
radiation. High levels of natural background radiation are found in some regions of the globe
such as the southwest coast of Kerala in India, Yangjiang province in China, Ramsar in Iran
and Gaurapari in Brazil (1, 2). Residents living in these high background radiation areas
receive a high life time dose due to chronic low-level dose of radiation from environmental
radioactive elements. In addition to terrestrial sources, cosmic radiation adds to natural
radiation in our environment. Moreover, accidents in nuclear power industries including
scenarios of Fukushima Daiichi (Japan), Chernobyl (Ukraine) and atomic bomb explosions
(Hiroshima and Nagasaki) disperse radioactive materials into the environment. Man-made
sources of ionizing radiation are widely used as diagnostic tools and therapeutic agents in
treatment of diseases. The cumulative exposure to ionizing radiation has the potential to
cause harmful health effects leading to chronic diseases.
1. Ionizing radiation
1.1. General aspects of ionizing radiation
Radiation is defined as the physical process where particles or electromagnetic waves pass
through a medium or space. Ionizing radiation consists of either photon-radiation (gamma
rays and x-rays) or fast moving sub-atomic particles (beta particles, neutrons, etc.). Gamma
rays consist of electromagnetic energy in the form of photons emitted by radioactive nuclides
such as caesium-137. Also cosmic radiation is one of the sources of gamma radiation.
Gamma rays can penetrate biological tissues and cause ionization of atoms and molecules.
Gamma rays as well as x-rays are commonly used for medical and technological purposes.
The quantity of energy deposited by ionizing radiation in a defined mass of material is termed
the absorbed dose and is measured in J/kg and the unit name is Gray (Gy). The deposition of
energy through ionization of atoms and molecules causes chemical changes. Linear Energy
Transfer (LET) is defined as the energy per unit length transferred to material when an
ionizing particle/wave travels through it. It is measured in keV µm-1 and the value varies with
different types of radiation from a few keV µm-1 (diagnostic X-rays) to >1000 keV for heavy
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ions. A radiation track is characterised by energy depositions occurring in clusters along the
trajectories of charged particles. The penetration of gamma-rays into the tissue is much
deeper than that of alpha particles (3). The deposition of energy and the subsequent damage
induced by gamma-rays is spread throughout the tissue, whereas alpha particles deposit more
energy along its track causing high local damage.
1.2. Effects of ionizing radiation
The energy deposition releases ionization products along the track in a random manner.
These interact with other molecules to cause damage/modification to all molecular
components such as deoxy-ribonucleic acid (DNA), proteins and lipids (4). Direct radiation
damage is caused by direct ionizations of the DNA when the track crosses close to DNA
strand, which in this case may lead to single or double strand breaks (DSB) (5). Indirect
damage to DNA is mediated by radiation produced free radicals in the medium that diffuse to
DNA and react locally (6, 7).
Ionizing radiation can induce several types of DNA lesions including single strand breaks and
DSB, base damage, base loss and more complex combinations (also called locally multiple
damaged sites) (3). Severity of lesions depends on the energy deposition in time and space of
radiation. The majority of low LET radiation effects arise indirectly by production of free
radicals whereas high LET radiation induces a higher density of ionizations and excitations
(direct effect) along the track, causing multiple lesions at the sites of DNA (8, 9). DNA
lesions are repaired by specific repair mechanisms including base excision repair, mismatch
repair, nucleotide excision repair and DSB repair (10).
Among DNA lesions, DSB are considered as most biologically important as they could be
lethal (11) and two distinct double-strand break repair mechanisms are present in the cell:
homologous recombination (HR) and non-homologous end-joining (NHEJ) (12). HR is
considered as a precise repair mechanism due to copying information from the undamaged
homologous double strand of DNA. In contrast, NHEJ uses no sequence homology and is
prone to error. Damage to bases and sugars also results in the breakage of strands, all of
which disrupt structural integrity of the DNA. These modified bases and single strand breaks
are recognized and restored by base excision repair process (13). The fidelity and speed of
repair depends on the complexity of radiation induced damage (8, 14). Erroneous DNA repair
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may lead to the occurrence of mutations, neoplastic transformation, premature senescence
and cell death (14, 15).
Additionally, a broad range of molecular mechanisms are also providing evidence to
understand the radiation induced effects. At the level of the organism, radiation induced long
term consequences may at least in part be systemic due to the action of cytokines,
chemokines, continuous generation of free radicals (16), protein modification (4), non-coding
RNA regulation (17) and other local mediators released from damaged cells that lead to
alterations of the surrounding cells (18). However, the complete cellular/molecular
mechanism behind radiation induced cellular or tissue effects are not fully understood.
1.3. Epidemiology of radiation-induced non-cancerous health
effects
The evidence from epidemiological studies highlights cancer as one of the major health
hazards of ionizing radiation in the low and medium dose range. Recently, data are accruing
showing that the risk of non-cancerous diseases such as cardiovascular disease (CVD),
cataracts, respiratory and digestive diseases etc. are also significantly increased by radiation
(19, 20).
The life span studies on survivors of the atomic bomb explosions in Hiroshima and Nagasaki
show that these populations have developed an excess risk of non-cancer diseases (21-23).
The dose-response relationship for the risk of solid cancers seems to be linear down to at least
100 mGy (24), but much less is known about the shape of the dose-response curve for noncancer effects. It has been suggested that radiation-induced non-cancerous diseases can take
many decades to develop after radiation exposure (25). Cardiovascular diseases, especially
heart disease and stroke, are the major types of non-cancer effects among Japanese atomicbomb survivors with a significantly elevated risk at dose levels higher than 0.5 Gy (26).
Due to the increased use of radiation in diagnostic or occupational situations, the number of
people exposed to radiation is increasing. In the context of radiotherapy, increased risk of
non-cancer effects were observed after radiotherapy for breast cancer (27), peptic ulcers (28)
and Hodgkin disease (29). In addition, cohort studies on occupationally exposed radiation
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workers show increased risk of mortality from circulatory disease (30) as was shown for the
Workers of the Mayak Production Association that were exposed to chronic radiation (31,
32). However, the studies on long-term chronic low dose radiation-induced circulatory
disease still remain elusive. More data on biological effects of chronic doses and different
dose rates are also needed for risk estimations of adverse health effects during space travel
(33, 34).
2. Cardiovascular system
The cardiovascular system comprises the heart and blood vessels. Endothelial cells form a
unique and single-layered lining of the luminal side of the blood and lymphatic vessels. The
endothelium forms an interface between the circulating blood, lymph system and underlying
tissues. This disseminated organ possesses several vital functions including formation of new
blood vessels in a process called angiogenesis, regulation of perfusion, fluid and solute
exchange between tissue and blood, haemostasis (35), coagulation and inflammatory
response (36). These versatile, multifunctional properties make a proper functioning of the
endothelium crucial for health; endothelial damage or senescence leads to multiple vascular
diseases (37-39). Thus, endothelium plays a key role in physiological as well as pathological
processes (38, 40).
2.1. Cardiovascular disease
CVD is one of the major causes of the mortality, accounting for one-third of deaths
worldwide (41) and has also become a global economic problem in developed and
developing countries. The major risk factors include behavioural factors such as consumption
of tobacco, alcohol, and fatty nutrients; metabolic (raised lipid levels, obesity) and other risk
factors such as hypertension, advanced age, gender and family history (42). The major forms
of vascular disease are coronary heart disease and stroke. Atherosclerosis- a disease of the
arteries- is characterised by development of plaques, thickenings of artery wall as a result of
the accumulation of fatty materials such as cholesterol and triglyceride. Rupturing of plaques
leads to the formation of a thrombus that rapidly reduces or stops the blood flow and results
in death of the local tissue fed by the artery. Thus, atherosclerosis may lead to heart attacks
and stroke. Coronary heart disease is a late manifestation of atherosclerotic changes (43).
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Factors inducing atherosclerotic plaque formation represent the principal cause of CVD
mortality and morbidity.
2.2. Radiation-induced cardiovascular damage
Radiation-induced CVD is seen as a long-term effect of radiation (44). Cardiovascular
pathologies associated with radiation include myocardial infarct, congestive heart disease,
pericarditis, vascular abnormalities, atherosclerosis, valvular heart disease, arrhythmias etc.
(45). The incidence of cardiovascular disease among radiation exposed populations is
primarily influenced by general cardiovascular risk factors - environmental, life style, genetic
and other risk factors.
The cohort study in Canada on industrial, medical and dental workers exposed to radiation
showed a trend of increased mortality due to CVD (46). An increased risk of CVD was
observed in occupational studies on nuclear workers (47, 48) and chronically exposed
plutonium plant workers (32). Increase of circulatory and arteriosclerotic heart diseases have
been observed among US nuclear power industry workers exposed to chronic low dose
ionizing radiation (49). Radiation-related excess of CVD mortality and morbidity was
observed in life span studies among Japanese atomic bomb survivors (25). Epidemiological
evidence has established a link between cardiovascular disease and exposure of the heart and
major vessels to radiation doses above 0.5 Gy. For lower doses the evidences for a
detrimental effect are not conclusive. One of the suggested mechanisms triggered by a lowdose exposure could be endothelial dysfunction (50).
Radiation-induced CVD is of concern for radiotherapy patients. A substantial risk of CVD
mortality by myocardial infarctions and ischemic heart disease was observed after
radiotherapy for Hodgkin disease (51, 52). High doses of ionizing radiation ranging from 3 to
17 Gy that were used to treat left sided breast cancer patients have been associated with longterm risk of cardiac pathology such as diffused fibrotic injury to the pericardium and
myocardium (53, 54). Exposure of heart to a mean dose of 4.9 Gy during radiotherapy for
breast cancer showed an increased risk of coronary heart disease within a few years in a
population-based case-control study in Sweden and Denmark (27). Long term follow up of
cancer survivors have confirmed the association between coronary heart disease and high
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local doses (5-18 Gy) of ionizing radiation applied to treat peptic ulcers (28) and childhood
cancer (45).
Studies using mouse models have indicated that ionizing radiation (14 Gy) may be an
independent factor able to induce heart pathologies (55). Seemann et al. showed the changes
in the cardiac function, structural damage to myocardium and functional changes of micro
vascular endothelial cells in the dose (2, 8 and 16 Gy) and time dependent manners (56).
Experimental studies using a local heart dose of 2 Gy showed persistent changes after 40
weeks in the mitochondrial metabolism and cardiac cytoskeletal structure (57). Proteomic
studies at 5 and 24 hour after total body irradiation (3 Gy) indicated an early biological
response resulting from oxidative stress in the heart (58) thus indicating an increased risk of
cardiovascular effects after radiation exposure. In spite of epidemiological and biological
evidence demonstrating the damaging effect on the cardiovascular system, the mechanisms
still remain elusive.
2.3. Radiation and endothelial dysfunction
Epidemiological studies have suggested that vascular damage may be involved in radiationinduced CVD at doses from 2 Gy (37, 59). The delayed injury in several tissues after
radiotherapy has been considered to be a consequence of vascular damage, most often
affecting the microvessels (60). Using an ApoE-deficient mouse model, Hoving et al.
demonstrated by histopathological methods that radiation (> 5 Gy) caused damage to the
vasculature of the heart (61). Endothelial dysfunction is one of the contributors to vascular
diseases and adverse cardiovascular events (62). Consequently, endothelium is being
considered as one of the targets for radiation-induced CVD damage (63-65).
Endothelial damage was observed in human coronary arteries and cardiac microvasculature
after high doses (44). Proteomic studies after gamma irradiation ranging from 200 mGy to 10
Gy have revealed radiation-induced oxidative stress in endothelial cell lines affecting
metabolic pathways (66, 67), cytoskeletal structure (68, 69) and pro-inflammatory processes
(70). Furthermore, angiogenesis has been shown to be inhibited by radiation (15 Gy); a
process requiring endothelial cell proliferation (71). Experimental findings in rats identified
the significant loss of the enzyme alkaline phosphatase activity in endothelial cells (72).
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These functional and structural changes in endothelial cells could contribute to the
development of radiation-induced CVD (72).
Dysfunction of endothelial cells is involved in inflammatory processes, emphasized by
increased adhesiveness of leukocytes and platelets. Further alterations in the permeability of
endothelium could result in transmigration and activation of leukocytes which is associated
with chronic events of atherosclerotic plaque formation as shown in figure 1. Recognition of
chronic events such as transendothelial migration of leukocytes was observed in endothelium
after radiation (69, 73, 74). It has been shown that endothelial cell senescence plays an
important role in the inflammatory response and also in the initiation and development of
atherosclerotic plaques (75-77). Histological examination of the endothelium after
radiotherapy has shown increase in the senescence-associated beta galactosidase (SA-β-gal)
activity and also provides evidence for radiation-induced premature senescence in the
aetiology of CVD (78).
Atherosclerotic plaque formation can lead to complications such as myocardial infarction and
stroke (79). Significantly increased risk for arteriosclerotic diseases was observed among
U.S. nuclear power industry workers exposed to chronic low dose radiation (49). As
endothelial senescence can lead to progression of radiation-induced CVD, it is important to
investigate the effect of chronic low dose rates and the underlying mechanisms behind
radiation-induced premature senescence in endothelial cells.
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Figure 1. Initial cellular events in the progression of atherosclerosis. Endothelial dysfunction is
associated with the increased expression of adhesion molecules such as integrins. Further,
inflammatory events follow adhesion and transmigration of leukocytes leading to atherosclerotic
plaque formation in the blood vessel.
3. Senescence
3.1. Replicative senescence
Replicative senescence was first described in cultured fibroblasts by Leonard Hayflick and
Paul Moorhead in 1961 (80). The limited ability of mammalian cells to proliferate is
dependent on the type of cells and the maximum number of cell divisions in vitro is called the
Hayflick limit. Senescence is a complex biological process observed both under in vitro and
in vivo conditions. Senescence is considered to play a major role in aging and age-related
diseases (81). Endothelial senescence is associated with cardiovascular pathologies,
cerebrovascular diseases and atherosclerotic lesions (75, 76, 82).
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3.2. Characteristic features of senescence
Endothelial cells express increasing morphological abnormalities and functional changes
during senescence. A senescent endothelial cell shows the characteristic features of enlarged,
flat cell morphology with an increased cell volume (83) due to vacuolisation and increased
granularity. Senescent endothelial cells display increased activity of SA-β-gal (84). In
addition, endothelial senescence is associated with significant changes in gene expression
(85, 86), impairment in cell-cell communication, cell junction (87) and protein expression
(88). Senescent endothelial cells secrete pro-inflammatory cytokines such as interleukins (IL1 and IL-6) (89), chemokines and growth factors (90). The increased amount of extracellular
proteins (91) and phenotypical alterations (92) clearly distinguish endothelial senescent cells
from early passage cells. The phenotypic changes of endothelial cells occurring due to
senescence result in many pathophysiological consequences (92), for example atherosclerosis
(77).
A characteristic feature contributing to replicative senescence is an irreversible shortening of
telomeres. Telomeres consist of several thousands of repetitive sequences of guanine-rich
residues at the 3' end of the DNA. Their presence at chromosome ends maintains integrity
and stability of the genome. The length of telomeres decreases discretely after each cellular
replication; thus the length of telomeres is considered as a ‘molecular clock’ reflecting the
replicative history of a primary cell (93). The protein p53 is a mediator of replicative
senescence that is associated with shortened telomeres (94, 95).
3.3. Stress-induced premature senescence
Endothelial senescence can occur prematurely in response to different types of external
stressors. This process is independent of the telomere shortening and is thus physiologically
distinct from replicative senescence. This is called stress-induced premature senescence
(SIPS). Figure 2 illustrates different stressors that alone or in combination can induce
premature senescence such as lack of growth nutrients, suboptimal cell culture conditions
(96, 97), oncogenes activation, agents causing DNA damage such as ionizing radiation (98)
as well as UV radiation; and chemicals such as tertiary-butyl-hydroperoxide (92, 99, 100).
Oxidative stress-accelerated endothelial senescence has been observed in atherosclerotic
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patients associated with cardiovascular risk factors such as hypertension (101). In addition,
both gene and protein expression of endothelial cells are shown to be altered by ionizing
radiation (102). While senescence is induced by many factors, a number of characteristic
features are shown to be common including cell cycle arrest, morphological transformation,
induction of SA-β-gal or senescence associated secretory factors (103).
Figure 2. Factors and consequences associated with endothelial senescence. Multiple types of
stress-inducing factors (shown on the left side) induce premature senescence in endothelial cells. The
consequences observed in the senescent phenotype are shown on the right side.
3.4. Senescence-associated pathways
Both replicative and stress-induced senescent cells share some characteristic features such as
proliferative arrest, accumulation of cell cycle inhibitors, morphological changes and SA-βgal activity at pH 6.0. Pathways characterised with activation of p53→p21 or p16, all known
as cell cycle inhibitors, are associated with senescence (104). The shortening of telomeres
will become critical when telomeres fail to form complexes with sheltering proteins causing
telomere dysfunction, DNA damage and activation of p53 pathway (105). Accumulation of
DNA damage in response to various stressors also activates p53-p21 pathway similarly to
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telomere-dependent senescence (106). Moreover, induction of p16 levels has been observed
in senescent cells (107). A schematic representation of molecular pathways of senescence is
shown in figure 3.
The expression of cell cycle inhibitors in endothelial cells are also modulated by intracellular
signaling cascade phosphoinositide 3-kinases (PI3K)/Akt pathway (106, 108). These
pathways are known to play an important role in senescence of human umbilical vein
endothelial cells (HUVEC) (109). A downstream target of PI3K/Akt, mammalian target of
rapamycin (mTOR), is allied to proliferation of endothelial cells and angiogenesis (110-112).
Moreover, the upstream networks that lead to mTOR regulate several cellular activities
including transcription, translation, cell size, ribosomal biogenesis, and cytoskeletal
organization (113). Downstream effector proteins of mTOR: p70 ribosomal S6 kinase and
eukaryotic initiation factor (EIF) - 4E binding protein 1 play a crucial role in the regulation of
translation and protein synthesis. Cellular growth is also regulated by mTOR signaling, and it
is known to be associated with cellular senescence (114).
Figure 3. Schematic diagram of growth arrest pathways leading to endothelial senescence. Stress
inducing factors activates the molecular pathways either p53→p21 or p16 cell cycle inhibitors.
Growth arrest leads to endothelial senescence and further pathophysiological consequences.
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3.5. Oxidative stress and radiation-induced senescence
Endogenous reactive oxygen species (ROS) originates mainly from mitochondria as a normal
by-product of cellular respiration and is rapidly removed. Excess ROS react with intracellular
targets leading to peroxidation of lipids, hydroxylation of proteins and DNA base damage
(115). ROS can also react with 7, 8-dihydro-2'-deoxyguanosine triphosphate in the nucleotide
pool and give rise to 8-oxo-7, 8-dihydro-2'-deoxyguanosine triphosphate (8-oxo-dGTP). The
human MutT homologue 1 (hMTH1) enzyme prevents incorporation of 8-oxo-dGTP into the
DNA during DNA synthesis by hydrolysing it to 8-oxo-dGMP. 8-oxo-dGMP will be further
dephosphorylated to 8-Oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxo-dG) and then excreted
from the cells to extracellular fluids such as blood and urine in vivo or cell culture medium in
vitro (116) as shown in figure 4. Thus, extracellular 8-oxo-dG is being considered as a
sensitive biomarker for oxidative stress (117).
Figure 4. Formation and excretion of 8-oxo-dG. 8-oxo-dGTP will be formed in the nucleotide pool
from dGTP due to oxidative stress. It is converted into 8-oxo-dG as a result of the action of the
enzymes hMTH1 and 8-oxo-dGMPase and is then excreted into the extracellular medium. Modified
from Michaels et al. (118).
A number of antioxidant defence mechanisms are acting to neutralise the ROS and to protect
cells from damage (119). However, if the production of ROS exceeds the levels of the
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available antioxidants, oxidative stress is induced which may result in oxidized base damage
and increased risk of genotoxicity. In endothelial cell oxidative stress and the levels of free
radicals have been shown to correlate with senescence (120). Accumulated levels of 8hydroxydeoxyguanosine in the DNA was reported in cellular senescence due to increased
oxidative stress (121). It has been suggested that oxidative stress can cause accumulation of
single stranded breaks in DNA (122) which may trigger senescence. Oxidative stress coupled
to mitochondrial dysfunction is considered as a major stimulus for senescence (123).
Ionizing radiation itself induces oxidative stress, DNA damage and mitochondrial
dysfunction (124), and these are associated with premature senescence. Indeed, exposure to
high doses of ionizing radiation 5 or 15 Gy inhibits cell proliferation and induces premature
senescence in endothelial cells (125, 126). Similarly, 5 days after exposure to acute radiation
dose of 2, 4 and 8 Gy, a dose dependent increase of SA-β-gal activity was observed in
vascular endothelial cells (126). Endothelial senescence has been observed in vascular lesions
of radiotherapy patients exposed to acute doses (127).
So far, induction of senescence-like phenotype was studied as a function of radiation dose but
studies on the effect of the dose rate have been scarce. Mitchel et al. investigated the
importance of the dose-rate (1 mGy/min and 150 mGy/min) of irradiation on the
development and progression of atherosclerotic plaques in ApoE-deficient mice (128). They
reported that many parameters such as dose, dose rate, and age of mice influenced the
progression of atherosclerosis. The higher dose rate given at young ages could be both
protective and detrimental. However, the role of low dose rate ionizing radiation on induction
of endothelial senescence still remains unclear. Therefore, studies on low-dose-rate radiationinduced premature senescence will be helpful in revealing biological mechanisms of vascular
diseases in chronically exposed populations.
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OBJECTIVES OF THE THESIS
Radiation protection research is challenged to provide more precise risk estimates for chronic
radiation-induced cardiovascular diseases and a better mechanistic understanding the long
term effect of ionizing radiation on endothelium and the cardiac tissue.
The aims of the studies presented here were:
a) To determine whether chronic low dose rate radiation exposure premature endothelial
senescence and if the proteomic analysis could provide a mechanistic understanding of the
process.
b) To study long term effects of acute doses of radiation on cardiac tissue and through a
proteomic approach elucidate the possible molecular mechanism leading to cardiovascular
disease.
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MATERIALS AND METHODS
The materials and methods for these studies are described in detail in the attached
publications. A brief description of the methods used is outlined below.
Animals
Animal studies using mice C57BL/6 mice were performed in accordance with the German
Animal Welfare Law, approved by the institutional review committee and the government of
Upper Bavaria (Certificate number 211-2531-54/01).
Acute irradiation of mice
9-week old male mouse cardiac tissue was locally irradiated in vivo with an acute dose of 20220 kV X-rays with a dose rate of 3 Gy/min. Control mice were sham irradiated.
Chronic irradiation of HUVEC
Chronic irradiations of HUVEC were performed at a low-dose rate in a specially designed
and custom-built incubator equipped with a gamma radiation source (Cs137) as shown in
figure 5. Chronic exposures at dose rates of 1.4 mGy/h or 2.4 mGy/h or 4.1 mGy/h were
investigated. Chronic irradiation of HUVEC was carried out until the cells lost their
proliferative capacity. During sub-culturing of cells the irradiation was paused for between 30
min and 1 hour. Sham irradiated cells were grown in an identical incubator, but without
exposure to radiation.
HUVEC culture and growth kinetics
HUVEC exhibit several properties intermediate between different types of endothelial cells
such as microvascular and macrovascular endothelial cells (129-133). Yet, HUVEC are a
good model system to study CVD, based on their retained ability to form vascular tubules in
vitro (134). HUVEC can be expanded up to a minimum of 21 cumulative population
doublings by repeated sub-culturing. The HUVEC cells in these studies were from a single
donor and from an early passage (P2) and were chronically irradiated.
26
Figure 5. Specially designed, custom-made incubator to irradiate cells. Incubator is fitted with a
gamma radiation source (Cs137) to expose cells to ionizing radiation at low dose rate.
Senescence associated-β-galactosidase staining
Beta-galactosidase is an enzyme active at pH 4.0 and is present in normal cells in the
lysosome. Beta-galactosidase is involved in the cleavage of disaccharide galactoside to
monosaccharide subunits. Senescent cells contain the beta-galactosidase enzyme active at pH
6.0 (84, 135). This is a cytoplasmic form appearing due to increased expression of lysosomal
beta-galactosidase protein (84). Senescent endothelial cells can be visualised histochemically
under light microscope after senescence-associated beta-galactosidase (SA-beta gal) staining
(136, 137).
27
Proteomics
The extensive improvements in the proteomic field provide tools for the identification and
quantification of proteins. Here, we applied mass spectrometry (MS) based techniques to
investigate proteome of HUVEC or cardiac tissue. The proteome has been defined as the
entire set of proteins expressed by a cell, organism, or tissue at a particular time or
conditions.
Isotope coded protein labeling
Proteins were isolated from cells or tissue and their concentration was measured by Bradford
assay (138). Equal amount of proteins from different experimental conditions were alkylated
and denatured independently. Proteins were labeled using isotope coded protein labeling
(ICPL) approach; either ICPL - duplex [1-(12C61H4)-nicotinoyloxy-succinimide (ICPL-0) and
1-(13C62H4)-nicotinoyloxy-succinimide (ICPL-6)] or ICPL-triplex [ICPL-0, 1-(12C62D4)nicotinoyloxy-succinimide (ICPL-4) and ICPL-6] approach, depending on the number of
different experimental conditions. Proteins were precipitated by acetone and solubilized in
Laemmli sample buffer. Further, proteins were separated by 12% sodium dodecyl sulphate
(SDS) - Polyacrylamide gel electrophoresis (PAGE) to decrease the level of proteome
complexity based on their molecular weight as shown in figure 6. The gel was stained
overnight with Coomassie Brilliant Blue.
Mass spectrometry analysis
After staining, the gel lane was cut into slices and the separated proteins of each slice were
subjected to in-gel digestion with trypsin (139). Enzymatically digested peptides were
resolved by reversed phase chromatography operated on a nano-high performance liquid
chromatography (LC) coupled to electron spray ionisation (ESI)-MS/MS. The obtained
MS/MS spectra were searched against the Ensembl human database (Version: 2.4) using the
Mascot search engine (version 2.3.02; Matrix Science) or Ensembl Mus musculus database,
depending on the source of protein. Data processing for the identification and quantitation of
ICPL labelled proteins was performed using Proteome Discoverer version 1.3.0.339 (Thermo
Scientific). Relative abundance was obtained by isotope labelling (140) and spike-in standard
proteins were used to obtain absolute protein abundance (141).
28
Figure 6. General outline of sample preparation using isotope-coded protein labelling duplex
methodology coupled to mass spectrometry. The extracted protein samples from sham and
irradiated HUVEC were labelled with different stable isotopes. The protein samples were denatured
and alkylated and mixed. Separation of proteins based on molecular size was carried out using SDSPAGE electrophoresis. The digested peptides were investigated by LC-ESI-MS/MS analysis. The
spectra were analysed and identified by MASCOT software. Further, data were processed using
Proteome Discoverer software version 1.3 and protein interaction studies to predict the Gene
Ontology and network analysis.
29
Bioinformatics analysis
A) Protein-protein interaction analysis
The pathways involved in the radiation response were identified by several software tools
(142). Ingenuity Pathway Analysis (IPA) (Ingenuity® Systems, http://www.ingenuity.com)
provides information about canonical pathways, protein networks and delivers graphical
representation of up- and down-regulated proteins (143). Network analysis also provides
information about unidentified proteins allowing either direct or indirect relationships within
the network. IPA uses pre-existing libraries, the protein-protein interaction being
experimentally proved. In addition, open source software, Search Tool for the Retrieval of
Interacting Genes/Proteins (STRING) (http://string-db.org/) (144) coupled to open access
Reactome database (http://www.reactome.org/) (145) was utilized for data analysis and
visualization of protein-protein interactions and networks. UniProt Knowledgebase
(http://www.uniprot.org/) was applied for protein ID, name or description information.
B) Functional classification
The open access softwares, Protein Analysis through Evolutionary Relationship (PANTHER)
(http://www.pantherdb.org/) (146) and Database for Annotation, Visualization, and Integrated
Discovery (DAVID) (http://david.abcc.ncifcrf.gov/) (147), provide systematic and integrative
analysis of proteins. Therefore, a large number of deregulated proteins can be categorized
according to cellular component, biological process and molecular function, based on Gene
Ontology (GO).
Immunoblot analysis
Western blots were performed to validate deregulated proteins from the proteomic approach.
Briefly, the cell pellet was dissolved with lysis buffer and total cellular protein concentration
was determined by Bradford reagent (138). Proteins (15-20 µg) were separated on one
dimensional-SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was
washed with Tris buffered saline-tween 20 (TBST) buffer 3 times, 10 min, then incubated in
a blocking solution for 1 hour, followed by primary antibody incubation overnight at 4 °C.
The membrane was washed with TBST and incubated with horseradish peroxidase
conjugated secondary antibody against primary antibody, for 1 hour at room temperature.
30
Membrane was exposed to chemiluminescence solution and bands were quantified using
either TotalLab TL100 (www.totallab.com) or Image Quant software.
Reverse phase protein array
Reverse phase protein array (RPPA) allows the quantitative measurement of protein
expression levels in a micro-scaled dot-blot platform. Protein extraction from HUVEC was
performed using EXB Plus buffer (148) and concentration was determined by Bradford assay
according to instructions by the manufacturer (BioRad, CA). RPPA protocol is similar to that
of immunoblotting. The protein samples were arrayed on nitrocellulose-coated glass slides in
several dilutions. Thus, protein-antibody signal intensity can be analyzed in the linear
dynamic range (149). The nitrocellulose slides were incubated with peroxidase solution for 5
min. and then washed with TBST solution for 3 times, 10 min. The glass slides were
incubated in blocking solution for 1 hour followed by washing with TBST and then incubated
with primary antibody solution overnight. Slides were washed with TBST and incubated with
appropriate secondary antibodies. Several slides were arrayed in parallel by staining with
Sypro Ruby in order to normalise the antibody signals to the total amount of protein. The
slides were developed and images were scanned (Scanjet 3770, Hewlett-Packard, Hamburg,
Germany) and saved. The obtained images were further analysed by using Micro-vigene
software.
31
RESULTS AND DISCUSSION
Paper I
Title: “Quantitative proteomic analysis reveals induction of premature senescence in
human umbilical vein endothelial cells exposed to chronic low-dose rate gamma
radiation”
An increased risk for circulatory diseases is indicated in epidemiological studies of radiation
exposed individuals. Damage to vasculature is considered as a strong target candidate for the
etiological mechanism due to the high level of radiation sensitivity of endothelial cells. We
suggest that exposure to chronic low dose radiation results in an accelerated senescence and
impaired functioning of endothelial cells, leading to circulatory diseases. The aim of this
study was to test the hypothesis that chronic low dose rate (4.1 mGy/h) gamma irradiation
induces premature senescence on primary HUVEC and to elucidate the mechanisms involved
through a proteomic approach.
The sham-irradiated HUVEC were cultured until the senescence was reached at 20.5 ± 1.4
(mean ± SEM) cumulative population doublings. The chronically irradiated HUVEC (4.1
mGy/h) showed a significantly reduced cumulative population doublings of 7.5 ± 1.0 (mean
± SEM), constituting 61% of the control. This indicated that replicative capacity was
significantly diminished under chronic irradiation conditions. The irradiated cells underwent
premature senescence as verified by the morphological changes and cytosolic SA-ß-gal
staining. Similar frequency of SA-ß-gal positive stained cells was reported in HUVEC
exposed to acute ionizing radiation, 4 Gy (126).
Proteomic analysis was performed from the cell lysates of week 1, week 3 and week 6
corresponding to cumulative doses 0.69 Gy, 2.07 Gy and 4.13 Gy, respectively. The number
of differentially regulated proteins during this time period was 31 (week 1), 61 (week 3) and
54 (week 6). Affected protein networks from IPA indicated a shift from maintenance of
cellular function to cell morphology and movement with the progressing senescence. Chronic
radiation induced senescence correlated with deregulation of proteins involved in cell-cell
and cell-matrix interactions showing the effect on endothelial permeability, angiogenesis and
morphology of the cell (87). In this study, HUVEC cells underwent senescent associated
32
morphological changes at cumulative dose of 4.13 Gy. Similarly, acute doses of ionizing
radiation have shown morphological changes and modulation of cytoskeleton (150). The
radiation induced alterations of Rho GDP dissociation inhibitor (Rho GDI), CD44,
intracellular adhesion molecule and vimentin observed in our study suggested the remodeling
of cytoskeleton and potential cellular integrity disruption after chronic ionizing radiation.
We have observed alterations in proteins involved in biological processes such as
proliferation, protein biosynthesis, oxidative stress and inflammation. The deregulated
proteins from week 6 include reticulocalbin, alpha actinin, CD44 and plasminogen activator
inhibitor 1 were reported previously in replicative senescent and stress-induced premature
senescent cells (88). In good agreement with previously published data about replicative
senescence (106), the immunoblotting showed the activation of p53 and up regulation of
cyclin-dependent protein kinase inhibitor, p21 in response to a cumulative dose of 4.13 Gy.
The p53 activation is known to be induced by ionizing radiation (150), as well as DNA
damage and oxidative stress leading to senescence (120).
In summary, low dose rate chronic radiation induces premature senescence of HUVEC as
verified by SA-ß-gal expression, activation of p53, up-regulation of p21, morphological and
protein changes at cumulative dose of 4 Gy.
Main findings in paper I:
•
This study showed for the first time that low dose rate (4.1 mGy/h) chronic
induces premature senescence in HUVEC.
•
Senescent associated changes were observed for a cumulative dose of 4 Gy.
•
Quantitative proteomics analysis showed changes in the proteome that relate to
cytoskeletal disorganization, protein biosynthesis, inflammation and oxidative
stress.
33
Paper II
Title: “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of
primary human endothelial cells exposed to chronic radiation”
In paper I, we proved that exposure to chronic low dose rate radiation (4.1 mGy/h) results in
an accelerated senescence and hypothetically an impaired endothelial cell function. The aim
of this second study was to investigate if even lower dose rates of gamma radiation could
accelerate premature senescence in HUVEC and if so, are the biological pathways affected
similar to that observed in response to the dose rate of 4.1 mGy/h.
Therefore, HUVEC were cultured under chronic exposure to 2.4 and 1.4 mGy/h. The
HUVEC chronically irradiated with a dose rate of 2.4 mGy/h showed a significantly reduced
cumulative population doubling of 11±0.6 (mean ± SEM) compared to sham-irradiated cells
of 20.5 ± 1.4 (mean ± SEM). Further, the cells irradiated with the 2.4 mGy/h dose rate
showed an early increased number of cytosolic SA-ß-gal stain cells. In contrast, a dose rate of
1.4 mGy/h neither inhibits the growth nor influences the number of senescent cells measured
by SA-β-gal staining.
Moreover, cell cycle regulator proteins p53, p21 and p16 expression were examined by
immunoblot at week 1, week 6 and week 10. Accumulation of cell cycle inhibitor protein p21
was observed at week 10 irrespective of dose rates. The persistent up-regulation of p21 has
also been observed in mouse models exposed to acute/fractionated ionizing radiation (151).
Also for the dose rate 2.4 mGy/h premature senescence was induced at a cumulative dose of
4 Gy. For HUVEC chronically exposed to 1.4 mGy/h loss of cell proliferation or premature
senescence could not be verified as they did not differ significantly from the sham exposed
control cells. It should be noted however, that at the end of the chronic exposure to 1.4
mGy/h a cumulative dose of 4 Gy had been reached.
Moreover, senescence processes were examined by assessing the expression of senescence
related pathways with reverse phase protein array analysis at week 1, week 6 and week 10.
The ratio of phosphorylated-total extracellular signal-regulated kinases (ERK) 44/42 was
reduced only with 2.4 mGy/h dose rate at cumulative dose of 4 Gy. Additionally, based on
previous tested dose rate (4.1 mGy/h) we assessed Rho GDI which is associated with cellular
34
adhesion and migration. In accordance with the 4.1 mGy/h dose rate (paper I), Rho-GDI was
altered in both tested chronic dose rates but independent of accumulated dose. ERK and Rho
are involved in cytoskeletal organization and decreased ERK activity is observed in
replicative senescence (152). Therefore, our study shows radiation induced cellular
senescence is characterized by disrupted cytoskeletal organization, loss of cell-cell junction,
cell morphology and migration (87).
Importantly, the down regulation of PI3K/Akt/mTOR signaling pathway was observed at
week 10 for HUVEC exposed to 1.4 and as well as 2.4 mGy/h (106). Inhibition of PI3K/Akt
signaling pathway was known previously in induction premature senescence with elevated
expression of p21 (153). Subsequently, PI3K/Akt regulates their downstream target mTOR
and its inhibition was observed under oxidative stress and DNA damage (154). Global
proteome changes were assessed more in detail at week 10 by ICPL triplex methodology. Of
those 2607 identified, 130 proteins (1.4 mGy/h) and 270 proteins (2.4 mGy/h) were
differentially regulated. This proteomic study confirms the decreased levels of EIF-4E, RPS6,
EIF-3 and many ribosomal proteins at week 10 exposed to 2.4 mGy/h (4 Gy). Whereas, EIF4E, RPS6 are not regulated with 1.4 mGy/h. All these proteins are downstream targets of
mTOR pathway affecting translation process. This may explain why the inhibitory effect on
cellular growth and proliferation was not so marked when the lower dose rate was tested.
In line with previous studies we found alteration of proteins in mitochondrial metabolism and
endoplasmic reticulum indicative of increased radiation induced oxidative stress (124), a
major stimulus for the induction of senescence (123). Therefore this study shows the
influence of chronic dose rate induced stress on induction of senescence.
In summary these results suggest that premature senescence in HUVEC induced by low dose
rate chronic exposure has a threshold dose (4 Gy) as well as a threshold for the dose rate
(>1.4 mGy/). This study has also highlighted the PI3K/Akt/mTOR signaling pathway in
senescence induced by ionizing radiation.
Main findings in paper II:
•
Low dose rate chronically exposed HUVEC will enter premature senescence if
the accumulated dose exceeds 4 Gy before the cells enter replicative senescence
35
•
Radiation-induced premature senescence is mediated through p21 dependent
pathway.
•
Proteomic and RPPA studies showed PI3K/Akt/mTOR signaling in progression
of senescence.
36
Paper III
Title: “PPAR alpha: a novel radiation target in locally exposed Mus musculus heart
revealed by quantitative proteomics”
The damage of the cardiac endothelial cells and myocardium has been indicated in radiationinduced CVD. Radiation therapy patients who have received high doses of ionizing radiation
to the thorax show a markedly increased risk of cardiac morbidity and mortality decades after
the treatment. However, the mechanism leading to radiation-related heart disease is unclear.
The goal of this study was to investigate radiation-induced cardiac damage in general and in
particular the role of peroxisome proliferator-activated receptor alpha (PPARA) in this
process a.
C57BL/6 mice received 8 or 16 Gy local heart irradiation with X-ray at the age of 9 weeks.
The mice were sacrificed 16 weeks after irradiation and the cardiac tissue was analysed using
proteomics, transcriptomics, immunoblotting, immunohistochemistry and bioinformatics. The
number of differentially regulated proteins in 8 Gy and 16 Gy irradiated hearts was 54 and
116, respectively with 33 proteins in common. The majority of deregulated proteins were
involved in fatty acid metabolism, mitochondrial respiratory chain, antioxidant defence and
structural organisation.
Bioinformatics analysis predicted the inactivation of PPARA and mitochondrial transcription
factor A in cardiac tissue. Although protein expression of PPARA was down regulated at
only 8 Gy, the post translational modifications – phosphorylation and ubiquitination are up
regulated along with activation of ERK 44/42, an upstream regulator of PPARA (155). This
will in turn result in disassembly of PPARA complex and leads to reduced transcription of
PPARA target genes. Therefore, gene expression profiling revealed down-regulation of 17
and 10 genes out of 84 PPARA signaling related genes at 8 and 16 Gy, respectively. Majority
of regulated genes are involved in fatty acid metabolism. In addition, elevated free fatty acids
and high density lipoproteins in serum, correlates the impaired PPARA activity in cardiac
tissue due to irradiation.
Proteomic data suggested that cardiac injury is hallmarked by oxidative stress response and
disturbed energy metabolism. Electron microscopic studies showed radiation-induced
37
damage in the mitochondrial structure and size. The reduced levels of the subunits of
complexes I, III, V suggest impairment of mitochondrial function. This can leads to enhanced
production of ROS contributing to increased oxidative stress. It has been shown that PPARA
is implicated in anti-inflammatory role in cardiac tissue (156) and in agreement with this, we
detected mild infiltration of the myocardium with CD-45 positive inflammatory cells at 16
weeks with less significance than earlier observation 40 weeks after 16 Gy irradiation (56).
Our immunoblot analysis showed radiation-induced oxidative stress resulting in protein
modifications including carbonylation and nitrosylation. PPARA null mice showed
remarkable oxidative and nitrosative modification of contractile proteins contributing to
cardiac dysfunction and diffuse fibrosis (157). These findings are consistent with observed
reduction of heart systolic and diastolic volumes in C57BL/6 mice 20 weeks after exposure to
8 and 16 Gy (56).
Our proteomic data showed differential regulation of apolipoprotein A I, gelsolin, decorin,
collagen and fibrinogen associated with different types of amyloidosis. PPARA is known to
be involved in inflammatory response by activation or inhibition of amyloid (158). It was
suggested that vascular leakage contributed in the deposition of amyloid in extracellular
matrix of myocardium resulting in the death of mouse after 40 weeks post irradiation.
In summary, this study suggests that cardiac energy metabolism, structure and function are
impaired after local high-dose radiation. Radiation induced impairment of PPARA activity
could contribute to the pathogenesis of cardiac disease. Further studies on cellular and animal
models may provide evidence of radiation effects on PPARA activity.
Main findings in paper III:
•
High doses of radiation cause reduction of myocardial lipid metabolism,
mitochondrial dysfunction, enhanced inflammation and structural damage.
•
Radiation-induced impairment of cardiac metabolism is associated with reduced
PPARA activity.
•
ERK phosphorylation event regulates PPARA activity.
38
SUMMARY AND FUTURE PERSPECTIVES
Epidemiological studies show an association between both acute and chronic radiation
exposure and long term cardiovascular effects. The present study was designed to better
understand the long term effect of ionizing radiation on endothelium (Paper I and II) and the
cardiac tissue (Paper III).
Our studies have demonstrated that chronic low dose rate gamma radiation induces premature
senescence in primary HUVEC. Senescence was observed when the accumulated dose
reached the value of around 4 Gy, with dose rates of either 4.1 or 2.4 mGy/h. In case of the
dose rate 1.4 mGy/h, the cumulative population doublings were similar to the control cells
before senescence was reached although the cumulative dose at this point was around 4 Gy.
Our study (unpublished data) also showed significant increased levels of 8-oxo-dG in the
medium with only 4.1 mGy/h dose rate at a cumulative dose 4 Gy. Therefore, the cumulative
dose alone cannot be correlated to the induction of SIPS. These finding emphasize that the
SIPS for HUVEC is both dose and dose rate dependent.
This study highlights the important role of PI3K/Akt/mTOR pathway inhibition in the
induction of senescence. Moreover, the proteomic studies presented show the effect of
chronic radiation on multiple signal transduction pathways involved in endothelial
dysfunction. These include translation process, cellular adhesion, cell-cell communication
and cytoskeletal organisation. The alterations of these processes are previously implicated as
characteristic of senescent endothelial cells. Although practical significance of this study
remains to be confirmed by in vivo research, increased understanding of the mechanisms
leading to endothelial senescence may provide a basis for preventive measures for CVD seen
in populations chronically exposed to low dose rate radiation.
There is a considerable evidence for increased risk of cardiovascular disease among people
exposed to fractionated radiation doses (159). Further studies are necessary to investigate the
effects of clinically relevant fractionated doses associated with vascular endothelial
senescence. The induction of endothelial senescence after acute radiation (4 Gy) has been
shown by SA-β-gal staining (126). Whether the identical molecular pathways are shared
between acute and chronic dose radiation-induced senescence should be investigated.
39
A sound functioning of the cardiac tissue depends on the undisturbed interplay between the
endothelium (cardiac vasculature) and myocardium (cardiac muscle). The long term effects
of high doses ionizing radiation on heart have been well documented in radiotherapy. Our
study on murine locally irradiated heart, with a clinically relevant acute gamma doses (8 and
16 Gy), revealed impaired lipid metabolism and oxidative phosphorylation mainly due to the
inactivation of PPARA. This study showed that high doses of radiation significantly changed
the phosphorylation and ubiquitination status of PPARA as a consequence of the activation of
ERK pathway. These posttranslational modifications events result in inactivation of PPARA
and subsequently persistent alteration of cardiac metabolism contributes to the cardiac
structural and functional damage after radiation. These findings showed the series of cardiac
tissue disordering including cardiac toxicity, inflammation and cardiac extracellular matrix
remodelling. Vascular leakage and inflammation, diffuse amyloidosis and even sudden death
in mice have been reported over longer period of time after locally heart exposure to 16 Gy
(56).
Our future studies will focus on how radiation-induced vascular dysfunction and particularly
endothelial senescence could influence the cardiac metabolism by reducing the flow of
nutrients through blood. The simultaneous analysis of heart tissue and endothelial cells
isolated from heart vessels of irradiated mice will help to understand the interaction between
these two essential components of the cardiovascular system.
40
ACKNOWLEDGEMENTS
I wish to express sincere gratitude to my supervisor Dr. Siamak Haghdoost for supervision
of thesis. Thank you for giving the opportunity to do my doctoral studies at your department
and for guidance, advice and delightful support during these years.
Dr. Soile Tapio, I would like to express my sincere gratitude for her supervision, scientific
discussions, valuable suggestions, endless support and inspiration towards my work and
obviously for introducing me to the field of Radiation Proteomics. It has been a great pleasure
to work with you.
I am profoundly grateful to Prof. Dr. Mats Harms-Ringdahl, Prof. Dr. Mike Atkinson and
Prof. Dr. Andrzej Wojcik for valuable suggestions, support, co-operation and helpful
collaboration.
Dr. Omid Azimzadeh: for being such a wonderful colleague and providing extensive support
in proteomics. Your knowledge and support have helped me a lot during my Ph.D. Thank you
for introducing me to FFPE.
My special thanks to Prof. Karl-Friedrich Becker for being an external examiner and for
suggestions particularly in performing RPPA experiments. I would also like to acknowledge
Katharina Malinowsky, Institute of Pathology, Technische Universität München, Germany
for analyzing RPPA.
I am thankful to Dr. Zarko Barjaktarovic, Stefan Kempf, Mayur Bakshi, Stefanie
Winkler and Dr. Arundhathi Sriharshan for their knowledge transfer in proteomics and
fruitful group discussions. A special thanks to all my colleagues at Institute of Radiation
Biology, Helmholtz Zentrum Munich, and Germany.
A special thanks for my colleagues Traimate Sangsuwan, Sara Shakeri Manesh and
Ainars Bajinskis for their support in my research and personal work during my time in the
Helmholtz Zentrum, Munich. My hearted whole thanks to the Centre for Radiation Protection
Research group present and past members. Siv, Elina Staaf, Karl, Asal and Sara.
41
My gratitude to kind administrative persons Eva Petersson, Eva Eyton and Solvejg
Schröder.
I am thankful for the support of a grant from the European Community's Seventh Framework
Programme (EURATOM) contract n° 249689 (DoReMi) and from the Swedish Radiation
Safety Authority.
To my beloved wife Sanisetty Manasa, family - Yentrapalli Suresh and Subbaratnamma,
and friends - Sunil Rayappa, Selva, Jayaprabha, Bamini, Sriharan; Thank you all for your
kind support.
42
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