Search

Background and Purpose: Cardiotoxicity is a well-known adverse effect of radiation therapy. Measurable abnormalities in the heart function indicate advanced and often irreversible heart damage. Therefore, early detection of cardiac toxicity is necessary to delay and alleviate the development of the disease. The present study investigated long-term serum proteome alterations following local heart irradiation using a mouse model with the aim to detect biomarkers of radiation-induced cardiac toxicity. Materials and Methods: Serum samples from C57BL/6J mice were collected 20 weeks after local heart irradiation with 8 or 16 Gy X-ray; the controls were sham-irradiated. The samples were analyzed by quantitative proteomics based on data-independent acquisition mass spectrometry. The proteomics data were further investigated using bioinformatics and ELISA. Results: The analysis showed radiation-induced changes in the level of several serum proteins involved in the acute phase response, inflammation, and cholesterol metabolism. We found significantly enhanced expression of proinflammatory cytokines (TNF-α, TGF-β, IL-1, and IL-6) in the serum of the irradiated mice. The level of free fatty acids, total cholesterol, low-density lipoprotein (LDL), and oxidized LDL was increased, whereas that of high-density lipoprotein was decreased by irradiation. Conclusions: This study provides information on systemic effects of heart irradiation. It elucidates a radiation fingerprint in the serum that may be used to elucidate adverse cardiac effects after radiation therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Azimzadeh, von Toerne, Subramanian, Sievert, Multhoff, Atkinson and Tapio.)

Extracellular vesicles (EVs) are freely circulating nano/micrometer-sized membrane-bound vesicles released by various cell types. Their cargo consists of proteins, lipids, metabolites, and different types of RNA molecules reflecting the origin of the secreting cell type or tissue. Since the EV cargo is constantly changing in response to pathological status or different environmental stressors, it has been extensively studied in the quest for biomarkers, especially in the cancer research. Mass spectrometry (MS)-based proteome analysis is a powerful tool to elucidate the protein cargo in EVs. This chapter describes and characterizes three MS-compatible lysis methods, namely by using urea, guanidium hydrochloride, and radioimmunoprecipitation buffer for isolating proteins from EVs.

Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.

Extracellular vesicles (EVs) are cell-derived membrane-bound organelles that have generated interest as they reflect the physiological condition of their source. Mass spectrometric (MS) analyses of protein cargo of EVs may lead to the discovery of biomarkers for diseases. However, for a comprehensive MS-based proteomics analysis, an optimal lysis of the EVs is required. Six methods for the protein extraction from EVs secreted by the head and neck cell line BHY were compared. Commercial radioimmunoprecipitation assay (RIPA) buffer outperformed the other buffers investigated in this study (Tris-SDS, Tris-Triton, GuHCl, urea-thiourea, and commercial Cell-lysis buffer). Following lysis with RIPA buffer, 310 proteins and 1469 peptides were identified using LTQ OrbitrapXL mass spectrometer. Among these, 86% of proteins and 72% of peptides were identified in all three replicates. In the case of other buffers, Tris-Triton identified on average 277 proteins, Cell-lysis buffer 257 proteins, and Tris-SDS, GuHCl and urea-thiourea each 267 proteins. In total, 399 proteins including 74 of the top EV markers (Exocarta) were identified, the most of the latter (73) using RIPA. The proteins exclusively identified using RIPA represented all Gene Ontology cell compartments. This study suggests that RIPA is an optimal lysis buffer for EVs in combination with MS., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidemiological data from radiotherapy patients show the damaging effect of ionizing radiation on heart and vasculature. The endothelium is the main target of radiation damage and contributes essentially to the development of cardiac injury. However, the molecular mechanisms behind the radiation-induced endothelial dysfunction are not fully understood. In the present study, 10-week-old C57Bl/6 mice received local X-ray heart doses of 8 or 16 Gy and were sacrificed after 16 weeks; the controls were sham-irradiated. The cardiac microvascular endothelial cells were isolated from the heart tissue using streptavidin-CD31-coated microbeads. The cells were lysed and proteins were labeled with duplex isotope-coded protein label methodology for quantification. All samples were analyzed by LC-ESI-MS/MS and Proteome Discoverer software. The proteomics data were further studied by bioinformatics tools and validated by targeted transcriptomics, immunoblotting, immunohistochemistry, and serum profiling. Radiation-induced endothelial dysfunction was characterized by impaired energy metabolism and perturbation of the insulin/IGF-PI3K-Akt signaling pathway. The data also strongly suggested premature endothelial senescence, increased oxidative stress, decreased NO availability, and enhanced inflammation as main causes of radiation-induced long-term vascular dysfunction. Detailed data on molecular mechanisms of radiation-induced vascular injury as compiled here are essential in developing radiotherapy strategies that minimize cardiovascular complications.

Formalin-fixed, paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the formalin fixation proteins undergo degradation and cross-linking, making conventional protein analysis technologies challenging. Cross-linking is even more challenging when quantitative proteome analysis of FFPE tissue is planned. The use of conventional protein labeling technologies on FFPE tissue has turned out to be problematic as the lysine residue labeling targets are frequently blocked by the formalin treatment. We have established a qualitative and quantitative proteomics analysis technique for FFPE tissues that combines label-free proteomic analysis with optimized protein extraction and separation conditions.

Rapidly developing postgenome research has made proteins an attractive target for biological analysis. The well-established term of proteome is defined as the complete set of proteins expressed in a given cell, tissue or organism. Unlike the genome, a proteome is rapidly changing as it tends to adapt to microenvironmental signals. The systematic analysis of the proteome at a given time and state is referred to as proteomics. This technique provides information on the molecular and cellular mechanisms that regulate physiology and pathophysiology of the cell. Applications of proteome profiling in radiation research are increasing. However, the large-scale proteomics data sets generated need to be integrated into other fields of radiation biology to facilitate the interpretation of radiation-induced cellular and tissue effects. The aim of this review is to introduce the most recent developments in the field of radiation proteomics.

Epidemiological studies establish that children and young adults are especially susceptible to radiation-induced cardiovascular disease (CVD). The biological mechanisms behind the elevated CVD risk following exposure at young age remain unknown. The present study aims to elucidate the long-term effects of ionizing radiation by studying the murine cardiac proteome after exposure to low and moderate radiation doses. NMRI mice received single doses of total body (60)Co gamma-irradiation on postnatal day 10 and were sacrificed 7 months later. Changes in cardiac protein expression were quantified using isotope-coded protein label and tandem mass spectrometry. We identified 32, 31, 66, and 34 significantly deregulated proteins after doses of 0.02, 0.1, 0.5, and 1.0 Gy, respectively. The four doses shared 9 deregulated proteins. Bioinformatics analysis showed that most of the deregulated proteins belonged to a limited set of biological categories, including metabolic processes, inflammatory response, and cytoskeletal structure. The transcription factor peroxisome proliferator-activated receptor alpha was predicted as a common upstream regulator of several deregulated proteins. This study indicates that both adaptive and maladaptive responses to the initial radiation damage persist well into adulthood. It will contribute to the understanding of the long-term consequences of radiation-induced injury and developmental alterations in the neonatal heart.

Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cell-cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease.

Radiobiological archives have not been overwhelmed by applications to use their stored tissue materials, in part because of lack of technologies to conduct quantitative analysis of the tissues. Over the last decade, advances in methodology have made it possible to routinely extract both DNA and RNA from archival materials. The quantitative analysis of gene expression by reverse transcription real-time PCR (QRT-PCR), and of gene copy number by array-based CGH (aCGH), are now firmly established methods for the study of tissue samples stored as paraffin blocks. More recent developments in proteomic technology have enabled the extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissue samples. These protein extracts have been used to produce quantitative data by Western blotting and even to identify proteins through LC/MS analysis. The development of applications using gel-based proteomics (2D/MS) is still a future challenge. This report gives an overview of the methodology already applicable on FFPE tissue, as well as the novel technologies to be used in the future research. Our goal is to collect existing animal and human tissue samples for use by the radiation biology community, to test new technologies for preparation of material from tissue samples, and to actively pursue materials arising from ongoing research.

Simple Summary: Epidemiological evidence supports an increased risk of cardiovascular diseases (CVD) at doses and dose rates much lower than previously believed. However, the molecular mechanism involved in cardiac damage after chronic irradiation has not been fully elucidated. Here, we studied the proteome of mice hearts chronically irradiated with a very low dose (1 mGy/day) or with a low dose (20 mGy/day) for 300 days. Changes in the proteome were further validated by immunoblotting, enzyme activity assays, immunohistochemistry, and targeted transcriptomics. The data presented here demonstrate that chronic low-dose total body irradiation induces biological changes in mouse cardiac tissue that resemble the effects of premature cardiac ageing. A comprehensive understanding of the molecular mechanisms of CVD following chronic low dose irradiation will enable the identification of target molecules or pathways which may be used in bioassays for measuring endpoints relevant to radiation risk assessment. Recent epidemiologic studies support an association between chronic low-dose radiation exposure and the development of cardiovascular disease (CVD). The molecular mechanisms underlying the adverse effect of chronic low dose exposure are not fully understood. To address this issue, we have investigated changes in the heart proteome of ApoE deficient (ApoE−/−) C57Bl/6 female mice chronically irradiated for 300 days at a very low dose rate (1 mGy/day) or at a low dose rate (20 mGy/day), resulting in cumulative whole-body doses of 0.3 Gy or 6.0 Gy, respectively. The heart proteomes were compared to those of age-matched sham-irradiated ApoE−/− mice using label-free quantitative proteomics. Radiation-induced proteome changes were further validated using immunoblotting, enzyme activity assays, immunohistochemistry or targeted transcriptomics. The analyses showed persistent alterations in the cardiac proteome at both dose rates; however, the effect was more pronounced following higher dose rates. The altered proteins were involved in cardiac energy metabolism, ECM remodelling, oxidative stress, and ageing signalling pathways. The changes in PPARα, SIRT, AMPK, and mTOR signalling pathways were found at both dose rates and in a dose-dependent manner, whereas more changes in glycolysis and ECM remodelling were detected at the lower dose rate. These data provide strong evidence for the possible risk of cardiac injury following chronic low dose irradiation and show that several affected pathways following chronic irradiation overlap with those of ageing-associated heart pathology. [ABSTRACT FROM AUTHOR]

Purpose:Epidemiological studies indicate that radiation doses as low as 0.5 Gy increase the risk of cardiovascular disease decades after the exposure. The aim of the present study was to investigate whether this radiation dose causes late molecular alterations in endothelial cells that could support the population-based data. Materials and methods:Human coronary artery endothelial cells were irradiated at 0.5 Gy (X-ray) and radiation-induced changes in the proteome were investigated after different time intervals (1, 7 and 14 d) using ICPL technology. Key changes identified by proteomics and bioinformatics were validated by immunoblotting and ELISA. Results:The radiation-induced alteration of the endothelial proteome was characterized by sustained perturbation of Rho GDP-dissociation inhibitor (RhoGDI) and nitric oxide (NO) signalling pathways. At later time-points, this was accompanied by reduced proteasome activity, enhanced protein carbonylation indicating augmented oxidative stress, and senescence. Conclusions:These molecular changes are indicative of long-term premature endothelial dysfunction and provide a mechanistic framework to the epidemiological data showing increased risk of cardiovascular disease at 0.5 Gy. [ABSTRACT FROM PUBLISHER]

Purpose:Multiple cell types secrete exosome-like extracellular vesicles (ELVs) to the extracellular environment. Pathological conditions can produce characteristic changes to the vesicle cargo. We investigated if ionizing radiation is capable of inducing changes in the protein and microRNA (miRNA) cargo of ELVs. Materials and methods:Whole blood samples from healthy donors were irradiated with 2 Gy gamma rays and then peripheral blood mononuclear cells and plasma were separated from residual blood and co-cultivated for 24 h. The released ELVs were collected by differential ultracentrifugation from irradiated and non-irradiated samples. microRNAs and proteins were quantified by qPCR and label-free proteomics. Results:Here we report a first characterization of radiation-induced changes in the protein and miRNA cargo of ELVs isolated from plasma. Proteome analysis of ELVs identified 214 proteins, of which nine significantly changed their abundance after irradiation. The radiation-induced down-regulation of afamin and serpine peptidase F1 was confirmed by immunoblotting. miRNA expression profiling identified 58 different exosomal miRNAs, the expression of miR-204-5p, miR-92a-3p and miR-31-5p was significantly increased in ELVs from irradiated samples. Conclusions:This study provides evidence that radiation-induced changes occur in the protein and miRNA cargo of plasma ELVs. These data imply a novel systemic communication pathway between irradiated and non-irradiated cells and tissues. [ABSTRACT FROM PUBLISHER]

Purpose:Ionizing radiation induces cardiovascular disease, the endothelium being the main target. The exact mechanism of the damage is unclear but the involvement of multiple signaling pathways is probable. Reversible lysine acetylation is a posttranslational protein modification that regulates activity across a broad range of signaling pathways. The aim of this study was to determine if a low radiation dose results in acetylome alteration in endothelial cells. Materials and methods:Human coronary artery endothelial cell line was irradiated with Cs-137 gamma-rays (0.5 Gy) and proteomics analysis was performed using enriched acetylated peptides and all peptides. Data were validated using immunoblotting, deacetylase activity assay, and RhoA activity assay. Results:Nearly a hundred proteins were found to have an altered acetylation status 24 h after irradiation, primarily due to an overall decrease in acetylation. The expression of specific deacetylases was significantly increased, coinciding with an enhancement in global deacetylase activity. Proteins changed in their acetylation status belonged to several pathways including protein synthesis, cytoskeleton-related processes, protein folding and calcium signaling. The predicted changes in the RhoA/actin cytoskeleton pathway were validated by immunoassay. Conclusions:This study shows that protein acetylation is an important mediator of radiation responses in human cardiac coronary endothelial cells. Increased knowledge of the endothelial response to radiation is crucial for the development of normal tissue-sparing modalities during radiation therapy. [ABSTRACT FROM PUBLISHER]

Background: The harmful consequences of in utero irradiation on learning and memory have been recognised but the molecular mechanisms behind the damage are still unknown. Results: Using a mass spectrometry-based approach, we investigated the long-term changes in the global cortical and hippocampal proteome 6 months after 0.1, 0.5 and 1.0 Gy in utero X-ray irradiation delivered on embryonic day 11 in male C57Bl/6 J offspring. We noted alterations in several signalling pathways involved in cognition, the transcription factor cAMP response element-binding protein (CREB) playing a central role. Immunoblotting of CREB and phosphorylated CREB (Ser133) showed an altered expression profile at all doses in the hippocampus and at 0.5 and 1.0 Gy in the cortex. The greatest reduction in the phospho-CREB level was seen at 1.0 Gy in the hippocampus. It was accompanied by enhanced expression of postsynaptic density protein 95 (PSD95), suggesting effect on synaptic plasticity in neuronal dendrites. Conclusions: As the CREB signalling pathway plays a crucial role in neuronal plasticity and long-term memory formation in the brain, the radiation-induced alterations of this pathway seen here are in good agreement with the cognitive dysfunction seen in in utero irradiated populations. These data contribute to a deeper biological understanding of molecular mechanisms behind the long-term damage induced by relatively low doses of ionising radiation during gestation. [ABSTRACT FROM AUTHOR]

Background / purpose of the study Epidemiological evidence suggests that low doses of ionising radiation (≤1.0 Gy) produce persistent alterations in cognition if the exposure occurs at a young age. The mechanisms underlying such alterations are unknown. We investigated the long-term effects of low doses of total body gamma radiation on neonatally exposed NMRI mice on the molecular and cellular level to elucidate neurodegeneration. Results Significant alterations in spontaneous behaviour were observed at 2 and 4 months following a single 0.5 or 1.0 Gy exposure. Alterations in the brain proteome, transcriptome, and several miRNAs were analysed 6-7 months post-irradiation in the hippocampus, dentate gyrus (DG) and cortex. Signalling pathways related to synaptic actin remodelling such as the Rac1- Cofilin pathway were altered in the cortex and hippocampus. Further, synaptic proteins MAP-2 and PSD-95 were increased in the DG and hippocampus (1.0 Gy). The expression of synaptic plasticity genes Arc, c-Fos and CREB was persistently reduced at 1.0 Gy in the hippocampus and cortex. These changes were coupled to epigenetic modulation via increased levels of microRNAs (miR-132 / miR-212, miR-134). Astrogliosis, activation of insulingrowth factor / insulin signalling and increased level of microglial cytokine TNFα indicated radiation-induced neuroinflammation. In addition, adult neurogenesis within the DG was persistently negatively affected after irradiation, particularly at 1.0 Gy. Conclusion These data suggest that neurocognitive disorders may be induced in adults when exposed at a young age to low and moderate cranial doses of radiation. This raises concerns about radiation safety standards and regulatory practices. [ABSTRACT FROM AUTHOR]

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation. [ABSTRACT FROM AUTHOR]

Radiation-induced cardiovascular disease is associated with metabolic remodeling in the heart, mainly due to the inactivation of the transcription factor peroxisome proliferator-activated receptor alpha (PPARα), thereby inhibiting lipid metabolic enzymes. The objective of the present study was to investigate the potential protective effect of fenofibrate, a known agonist of PPARα on radiation-induced cardiac toxicity. To this end, we compared, for the first time, the cardiac proteome of fenofibrate- and placebo-treated mice 20 weeks after local heart irradiation (16 Gy) using label-free proteomics. The observations were further validated using immunoblotting, enzyme activity assays, and ELISA. The analysis showed that fenofibrate restored signalling pathways that were negatively affected by irradiation, including lipid metabolism, mitochondrial respiratory chain, redox response, tissue homeostasis, endothelial NO signalling and the inflammatory status. The results presented here indicate that PPARα activation by fenofibrate attenuates the cardiac proteome alterations induced by irradiation. These findings suggest a potential benefit of fenofibrate administration in the prevention of cardiovascular diseases, following radiation exposure. [ABSTRACT FROM AUTHOR]

Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor. [ABSTRACT FROM AUTHOR]

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues. [ABSTRACT FROM AUTHOR]

In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg−1) and irradiated (whole-body, 100 mGy or 200 mGy, 137Cs) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose. [ABSTRACT FROM AUTHOR]

Chronic exposure to low-dose ionizing radiation is associated with an increased risk of cardiovascular disease. Alteration in energy metabolism has been suggested to contribute to radiation-induced heart pathology, mitochondrial dysfunction being a hallmark of this disease. The goal of this study was to investigate the regulatory role of acetylation in heart mitochondria in the long-term response to chronic radiation. ApoE-deficient C57Bl/6J mice were exposed to low-dose-rate (20 mGy/day) gamma radiation for 300 days, resulting in a cumulative total body dose of 6.0 Gy. Heart mitochondria were isolated and analyzed using quantitative proteomics. Radiation-induced proteome and acetylome alterations were further validated using immunoblotting, enzyme activity assays, and ELISA. In total, 71 proteins showed peptides with a changed acetylation status following irradiation. The great majority (94%) of the hyperacetylated proteins were involved in the TCA cycle, fatty acid oxidation, oxidative stress response and sirtuin pathway. The elevated acetylation patterns coincided with reduced activity of mitochondrial sirtuins, increased the level of Acetyl-CoA, and were accompanied by inactivation of major cardiac metabolic regulators PGC-1 alpha and PPAR alpha. These observations suggest that the changes in mitochondrial acetylation after irradiation is associated with impairment of heart metabolism. We propose a novel mechanism involved in the development of late cardiac damage following chronic irradiation. [ABSTRACT FROM AUTHOR]

Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: Radiotherapy of thoracic and chest-wall tumours increases the long-term risk of radiation-induced heart disease. The aim of this study was to investigate the long-term effect of local heart irradiation on cardiac mitochondria. Methods: C57BL/6 and atherosclerosis-prone ApoE−/− mice received local heart irradiation with a single X-ray dose of 2Gy. To investigate the low-dose effect, C57BL/6 mice also received a single heart dose of 0.2Gy. Functional and proteomic alterations of cardiac mitochondria were evaluated after 40weeks, compared to age-matched controls. Results: The respiratory capacity of irradiated C57BL/6 cardiac mitochondria was significantly reduced at 40weeks. In parallel, protein carbonylation was increased, suggesting enhanced oxidative stress. Considerable alterations were found in the levels of proteins of mitochondria-associated cytoskeleton, respiratory chain, ion transport and lipid metabolism. Radiation induced similar but less pronounced effects in the mitochondrial proteome of ApoE−/− mice. In ApoE−/−, no significant change was observed in mitochondrial respiration or protein carbonylation. The dose of 0.2Gy had no significant effects on cardiac mitochondria. Conclusion: This study suggests that ionising radiation causes non-transient alterations in cardiac mitochondria, resulting in oxidative stress that may ultimately lead to malfunctioning of the heart muscle. [Copyright &y& Elsevier]

Abstract: Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p ≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. [Copyright &y& Elsevier]

Abstract: Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4h and 24h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment. [Copyright &y& Elsevier]