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Even though X-ray beams are widely used in medical diagnosis or radiotherapy, the comparisons of their dose rates are scarce. We have recently demonstrated in vitro (clonogenic assay, cell viability, cell cycle, senescence) and in vivo (weight follow-up of animals and bordering epithelium staining of lesion), that for a single dose of irradiation, the relative biological effectiveness (RBE) deviates from 1 (up to twofold greater severe damage at the highest dose rate depending on the assay) when increasing the dose rate of high energy X-ray beams. To further investigate the impact of the dose rate on RBE, in this study, we performed in vitro fractionated irradiations by using the same two dose rates (0.63 and 2.5 Gy.min−1) of high-energy X-rays (both at 4 MV) on normal endothelial cells (HUVECs). We investigated the viability/mortality, characterized radiation-induced senescence by using flow cytometry and measured gene analysis deregulations on custom arrays. The overall results enlighten that, in fractionated irradiations when varying the dose rate of high-energy X-rays, the RBE of photons deviates from 1 (up to 2.86 for viability/mortality experiments performed 21 days postirradiation). These results strengthen the interest of multiparametric analysis approaches in providing an accurate evaluation of the outcomes of irradiated cells in support of clonogenic assays, especially when such assays are not feasible. [ABSTRACT FROM AUTHOR]

Human cancer is characterised by complex molecular aberrations which result in a wide variety of clinical manifestations. B-cell chronic lymphocytic leukaemia (B-CLL) is particularly diverse, both in terms of molecular changes and clinical course, and consequently our understanding of the pathology of this disease is generally poor. Furthermore, the heterogeneity of this tumour type coupled with the absence of an obvious genetic "hallmark", such as gain of oncogene function or loss of suppressor-gene function, has led many investigators to question whether B-CLL is a single disease entity. In most cases, B-CLL does not show specific reciprocal chromosomal translocations as found in other haemopoietic malignant diseases. The genomic instability of B-CLL results in numerous different types of chromosomal losses and gains, giving rise to unsettled karyotypes among individuals with this disease. Nevertheless, genetic data imply that B-CLL is a single disease characterised by a common gene-expression profile and by the existence of specific subtypes that may have clinical correlates in patients. [ABSTRACT FROM AUTHOR]

Purpose: Radiation-induced pneumopathy is the main dose-limiting factor in cases of chest radiation therapy. Macrophage infiltration is frequently observed in irradiated lung tissues and may participate in lung damage development. Radiation-induced lung fibrosis can be reproduced in rodent models using whole thorax irradiation but suffers from limits concerning the role played by unexposed lung volumes in damage development., Methods and Materials: Here, we used an accurate stereotactic body radiation therapy preclinical model irradiating 4% of the mouse lung. Tissue damage development and macrophage populations were followed by histology, flow cytometry, and single-cell RNA sequencing. Wild-type and CCR2 KO mice, in which monocyte recruitment is abrogated, were exposed to single doses of radiation, inducing progressive (60 Gy) or rapid (80 Gy) lung fibrosis., Results: Numerous clusters of macrophages were observed around the injured area, during progressive as well as rapid fibrosis. The results indicate that probably CCR2-independent recruitment and/or in situ proliferation may be responsible for macrophage invasion. Alveolar macrophages experience a metabolic shift from fatty acid metabolism to cholesterol biosynthesis, directing them through a possible profibrotic phenotype. Depicted data revealed that the origin and phenotype of macrophages present in the injured area may differ from what has been previously described in preclinical models exposing large lung volumes, representing a potentially interesting trail in the deciphering of radiation-induced lung damage processes., Conclusions: Our study brings new possible clues to the understanding of macrophage implications in radiation-induced lung damage, representing an interesting area for exploration in future studies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vitro modeling of the different steps of immune cell recruitment is essential to decipher the role of endothelial cells in this process. Here, we present a protocol for the assessment of human monocyte transendothelial migration using a live cell imaging system. We describe steps for culture of fluorescent monocytic THP-1 cells and chemotaxis plate preparation with HUVEC monolayers. We then detail real-time analysis using the IncuCyte® S3 live-cell imaging system, image analysis, and assessment of transendothelial migration rates. For complete details on the use and execution of this protocol, please refer to Ladaigue et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Radiation therapy damages tumors and normal tissues, probably in part through the recruitment of immune cells. Endothelial high-mannose N-glycans are, in particular, involved in monocyte-endothelium interactions. Trimmed by the class I α-mannosidases, these structures are quite rare in normal conditions. Here, we show that the expression of the endothelial α-mannosidase MAN1C1 protein decreases after irradiation. We modeled two crucial steps in monocyte recruitment by developing in vitro real-time imaging models. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion and enhances transendothelial migration of monocytes. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Purpose: Radiation-induced cellular senescence is a double-edged sword, acting as both a tumor suppression process limiting tumor proliferation, and a crucial process contributing to normal tissue injury. Endothelial cells play a role in normal tissue injury after radiation therapy. Recently, a study observed an accumulation of senescent endothelial cells (ECs) around radiation-induced lung focal lesions following stereotactic radiation injury in mice. However, the effect of radiation on EC senescence remains unclear because it depends on dose and fractionation, and because the senescent phenotype is heterogeneous and dynamic., Methods and Materials: Using a systems biology approach in vitro, we deciphered the dynamic senescence-associated transcriptional program induced by irradiation., Results: Flow cytometry and single-cell RNA sequencing experiments revealed the heterogeneous senescent status of irradiated ECs and allowed to deciphered the molecular program involved in this status. We identified the Interleukin-1 signaling pathway as a key player in the radiation-induced premature senescence of ECs, as well as the endothelial-to-mesenchymal transition process, which shares strong hallmarks of senescence., Conclusions: Our work provides crucial information on the dynamics of the radiation-induced premature senescence process, the effect of the radiation dose, as well as the molecular program involved in the heterogeneous senescent status of ECs., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The vascular endothelium is a hot spot in the response to radiation therapy for both tumors and normal tissues. To improve patient outcomes, interpretable systemic hypotheses are needed to help radiobiologists and radiation oncologists propose endothelial targets that could protect normal tissues from the adverse effects of radiation therapy and/or enhance its antitumor potential. To this end, we captured the kinetics of multi-omics layers-i.e. miRNome, targeted transcriptome, proteome, and metabolome-in irradiated primary human endothelial cells cultured in vitro . We then designed a strategy of deep learning as in convolutional graph networks that facilitates unsupervised high-level feature extraction of important omics data to learn how ionizing radiation-induced endothelial dysfunction may evolve over time. Last, we present experimental data showing that some of the features identified using our approach are involved in the alteration of angiogenesis by ionizing radiation., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Lung stereotactic body radiation therapy is characterized by a reduction in target volumes and the use of severely hypofractionated schedules. Preclinical modeling became possible thanks to rodent-dedicated irradiation devices allowing accurate beam collimation and focal lung exposure. Given that a great majority of publications use single dose exposures, the question we asked in this study was as follows: in incremented preclinical models, is it worth using fractionated protocols or should we continue focusing solely on volume limitation? The left lungs of C57BL/6JRj mice were exposed to ionizing radiation using arc therapy and 3 × 3 mm beam collimation. Three-fraction schedules delivered over a period of 1 week were used with 20, 28, 40, and 50 Gy doses per fraction. Lung tissue opacification, global histological damage and the numbers of type II pneumocytes and club cells were assessed 6 months post-exposure, together with the gene expression of several lung cells and inflammation markers. Only the administration of 3 × 40 Gy or 3 × 50 Gy generated focal lung fibrosis after 6 months, with tissue opacification visible by cone beam computed tomography, tissue scarring and consolidation, decreased club cell numbers and a reactive increase in the number of type II pneumocytes. A fractionation schedule using an arc-therapy-delivered three fractions/1 week regimen with 3 × 3 mm beam requires 40 Gy per fraction for lung fibrosis to develop within 6 months, a reasonable time lapse given the mouse lifespan. A comparison with previously published laboratory data suggests that, in this focal lung irradiation configuration, administering a Biological Effective Dose ≥ 1000 Gy should be recommended to obtain lung fibrosis within 6 months. The need for such a high dose per fraction challenges the appropriateness of using preclinical highly focused fractionation schedules in mice., 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 Bertho, Dos Santos, Braga-Cohen, Buard, Paget, Guipaud, Tarlet, Milliat and François.)

Purpose: Stereotactic body radiation therapy is a therapeutic option offered to high surgical risk patients with lung cancer. Focal lung irradiation in mice is a new preclinical model to help understand the development of lung damage in this context. Here we developed a mouse model of lung stereotactic therapy using arc delivery and monitored the development of lung damage while varying the beam size and dose delivered., Methods and Materials: C57BL/6JRj mice were exposed to 90 Gy focal irradiation on the left lung using 1-mm diameter, 3 × 3 mm 2 , 7 × 7 mm 2 , or 10 × 10 mm 2 beam collimation for beam size effect and using 3 × 3 mm 2 beam collimation delivering 20 to 120 Gy for dose effect. Long-term lung damage was monitored with micro-computed tomography imaging with anatomopathologic and gene expression measurements in the injured patch and the ipsilateral and contralateral lungs., Results: Both 1-mm diameter and 3 × 3 mm 2 beam collimation allow long-term studies, but only 3-mm beam collimation generates lung fibrosis when delivering 90 Gy. Dose-effect studies with constant 3-mm beam collimation revealed a dose of 60 Gy as the minimum to obtain lung fibrosis 6 months postexposure. Lung fibrosis development was associated with club cell depletion and increased type II pneumocyte numbers. Lung injury developed with ipsilateral and contralateral consequences such as parenchymal thickening and gene expression modifications., Conclusions: Arc therapy allows long-term studies and dose escalation without lethality. In our dose-delivery conditions, dose-effect studies revealed that 3 × 3 mm 2 beam collimation to a minimum single dose of 60 Gy enables preclinical models for the assessment of lung injury within a 6-month period. This model of lung tissue fibrosis in a time length compatible with mouse life span may offer good prospects for future mechanistic studies., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Lung cancer will be treated more frequently using stereotactic body radiation therapy, and preclinical research to model long-term toxicity of ablative doses of radiation is crucial. Stereotactic lung irradiation of a small volume can induce radiation pneumonitis and fibrosis in normal tissues., Methods and Materials: Senescence has been reported to contribute to lung fibrosis, and we investigated in vivo the effects of ablative doses of ionizing radiation on senescence-associated processes. The left lung of p16 INK4a -LUC knock-in mice was exposed to a single dose or fractionated radiation doses in a millimetric volume using a small animal radiation research platform., Results: Single or fractionated ablative radiation induces acute and very long-term p16 INK4a activation in the irradiated lung target volume associated with lung injury. We observed a panel of heterogeneous senescent cells including pneumocytes, macrophages, and endothelial cells that accumulated around the radiation-induced lung focal lesion, suggesting that different senescent cell types may contribute to radiation injury., Conclusions: This work provides important information on the long-term effects of ablative radiation doses in the normal lung and strongly suggests that stress-induced senescence is involved in stereotactic body radiation therapy-induced late fibrosis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Based on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their energies are scarce. We therefore assessed in vitro the effects of low- and high-energy X-rays using Human umbilical vein endothelial cells (HUVECs) by performing clonogenic assay, measuring viability/mortality, counting γ-H2AX foci, studying cell proliferation and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. Taken together, excepted for γ-H2AX foci counts, these experiments systematically show more adverse effects of high energy X-rays, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay.

Purpose: Stereotactic body radiation therapy offers good lung local tumor control by the administration of a high dose per fraction in small volumes. Stereotactic body radiation therapy preclinical modeling is now possible, and our aim was to develop a model of focal irradiation of the mouse lung and to investigate the impact of conditional hypoxia-inducible factor 1α (HIF-1α) deletion in the endothelium on radiation-induced tissue damage., Methods and Materials: The Small Animal Radiation Research Platform was used to create a mouse model of focal irradiation of the lung using arc therapy. HIF-1α conditional deletion was obtained by crossing mice expressing Cre recombinase under the endothelial promoter VE-cadherin (VECad-Cre +/+ mice) with HIF-1α floxed mice., Results: Lung stereotactic arc therapy allows thoracic wall sparing and long-term studies. However, isodose curves showed that neighboring organs received significant doses of radiation, as revealed by ipsilateral lung acute red hepatization and major gene expression level modifications. Conditional HIF-1α deletion reduced acute lung edema and tended to diminish neutrophil infiltrate, but it had no impact on long-term global tissue damage., Conclusions: Arc therapy for focal high-dose irradiation of mouse lung is an efficient model for long-term studies. However, irradiation may have a strong impact on the structure and function of neighboring organs, which must be considered. HIF-1α conditional deletion has no beneficial impact on lung damage in this irradiation schedule., (Copyright © 2019. Published by Elsevier Inc.)

The vascular endothelium is considered as a key cell compartment for the response to ionizing radiation of normal tissues and tumors, and as a promising target to improve the differential effect of radiotherapy in the future. Following radiation exposure, the global endothelial cell response covers a wide range of gene, miRNA, protein and metabolite expression modifications. Changes occur at the transcriptional, translational and post-translational levels and impact cell phenotype as well as the microenvironment by the production and secretion of soluble factors such as reactive oxygen species, chemokines, cytokines and growth factors. These radiation-induced dynamic modifications of molecular networks may control the endothelial cell phenotype and govern recruitment of immune cells, stressing the importance of clearly understanding the mechanisms which underlie these temporal processes. A wide variety of time series data is commonly used in bioinformatics studies, including gene expression, protein concentrations and metabolomics data. The use of clustering of these data is still an unclear problem. Here, we introduce kernels between Gaussian processes modeling time series, and subsequently introduce a spectral clustering algorithm. We apply the methods to the study of human primary endothelial cells (HUVECs) exposed to a radiotherapy dose fraction (2 Gy). Time windows of differential expressions of 301 genes involved in key cellular processes such as angiogenesis, inflammation, apoptosis, immune response and protein kinase were determined from 12 hours to 3 weeks post-irradiation. Then, 43 temporal clusters corresponding to profiles of similar expressions, including 49 genes out of 301 initially measured, were generated according to the proposed method. Forty-seven transcription factors (TFs) responsible for the expression of clusters of genes were predicted from sequence regulatory elements using the MotifMap system. Their temporal profiles of occurrences were established and clustered. Dynamic network interactions and molecular pathways of TFs and differential genes were finally explored, revealing key node genes and putative important cellular processes involved in tissue infiltration by immune cells following exposure to a radiotherapy dose fraction., Competing Interests: The authors have declared that no competing interests exist.

Altered by ionising radiation, the vascular network is considered as a prime target to limit normal tissue damage and improve tumour control in radiotherapy (RT). Irradiation damages and/or activates endothelial cells, which then participate in the recruitment of circulating cells, especially by overexpressing cell adhesion molecules, but also by other as yet unknown mechanisms. Radiation-induced lesions are associated with infiltration of immune-inflammatory cells from the blood and/or the lymph circulation. Damaged cells from the tissues and immune-inflammatory resident cells release factors that attract cells from the circulation, leading to the restoration of tissue balance by fighting against infection, elimination of damaged cells and healing of the injured area. In normal tissues that surround the tumours, the development of an immune-inflammatory reaction in response to radiation-induced tissue injury can turn out to be chronic and deleterious for the organ concerned, potentially leading to fibrosis and/or necrosis of the irradiated area. Similarly, tumours can elicit an immune-inflammation reaction, which can be initialised and amplified by cancer therapy such as radiotherapy, although immune checkpoints often allow many cancers to be protected by inhibiting the T-cell signal. Herein, we have explored the involvement of vascular endothelium in the fate of healthy tissues and tumours undergoing radiotherapy. This review also covers current investigations that take advantage of the radiation-induced response of the vasculature to spare healthy tissue and/or target tumours better.

Purpose: The main objective of radiobiology is to establish links between doses and radiation-induced biological effects. In this context, well-defined dosimetry protocols are crucial to the determination of experimental protocols. This work proposes a new dosimetry protocol for cell irradiation in a SARRP and shows the importance of the modification of some parameters defined in dosimetry protocol for physical dose and biological outcomes., Materials and Methods: Once all parameters of the configuration were defined, dosimetry measurements with ionization chambers and EBT3 films were performed to evaluate the dose rate and the attenuation due to the cell culture medium. To evaluate the influence of changes in cell culture volume and/or additional filtration, 6-well plates containing EBT3 films with water were used to determine the impact on the physical dose at 80 kV. Then, experiments with the same irradiation conditions were performed by replacing EBT3 films by HUVECs. The biological response was assessed using clonogenic assay., Results: Using a 0.15 mm copper filter lead to a variation of +1% using medium thickness of 0.104 cm to -8% using a medium thickness of 0.936 cm on the physical dose compare to the reference condition (0.313 cm). For the 1 mm aluminum filter, a variation of +8 to -40% for the same medium thickness conditions has been observed. Cells irradiated in the same conditions showed significant differences in survival fraction, corroborating the effects of dosimetric changes on physical dose., Conclusions: This work shows the importance of dosimetry in radiobiology studies and the need of an accurate description of the dosimetry protocol used for irradiation.

Purpose: To investigate whether the endothelial pool of plasminogen activator inhibitor type 1 (PAI-1) plays a role in the development of radiation-induced lung damage, as previously demonstrated in the intestine., Methods and Materials: Human lung microvascular endothelial cells were exposed to 10 Gy irradiation so as to study their ability to acquire an "activated" phenotype. Mice in which the Cre-Lox strategy was used to produce PAI-1 deletion specifically in the endothelial compartment were exposed to 17 Gy whole-thorax irradiation and followed up for 2, 13, and 23 weeks after irradiation., Results: Human lung microvascular endothelial cells had an activated phenotype after radiation exposure, overexpressed PAI-1, and underwent endothelial-to-mesenchymal transition. In mice, knockout of PAI-1 in the endothelium had no beneficial effect on radiation-induced lung damage and showed a tendency to worsen acute lesions., Conclusions: As opposed to the intestine, the endothelial pool of PAI-1 does not play a determinant role in the development of radiation-induced lung damage. The therapeutic value of PAI-1 inhibition in lung radiation injury may be associated with other types of cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Aims: Radiation therapy in the pelvic area is associated with side effects that impact the quality of life of cancer survivors. Interestingly, the gastrointestinal tract is able to adapt to significant changes in oxygen availability, suggesting that mechanisms related to hypoxia sensing help preserve tissue integrity in this organ. However, hypoxia-inducible factor (HIF)-dependent responses to radiation-induced gut toxicity are unknown. Radiation-induced intestinal toxicity is a complex process involving multiple cellular compartments. Here, we investigated whether epithelial or endothelial tissue-specific HIF-1α deletion could affect acute intestinal response to radiation., Methods: Using constitutive and inducible epithelial or endothelial tissue-specific HIF-1α deletion, we evaluated the consequences of epithelial or endothelial HIF-1α deletion on radiation-induced enteritis after localized irradiation. Survival, radiation-induced tissue injury, molecular inflammatory profile, tissue hypoxia, and vascular injury were monitored., Results: Surprisingly, epithelium-specific HIF-1α deletion does not alter radiation-induced intestinal injury. However, irradiated VECad-Cre +/- HIF-1α FL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1α FL/FL mice., Conclusions: We demonstrate in vivo that HIF-1α impacts radiation-induced enteritis and that this role differs according to the targeted cell type. Our work provides a new role for HIF-1α and endothelium-dependent mechanisms driving inflammatory processes in gut mucosae. Results presented show that effects on normal tissues have to be taken into account in approaches aiming to modulate hypoxia or hypoxia-related molecular mechanisms.

As it is altered by ionizing radiation, the vascular network is considered as a prime target in limiting normal tissue damage and improving tumor control in radiation therapy. Irradiation activates endothelial cells which then participate in the recruitment of circulating cells, especially by overexpressing cell adhesion molecules, but also by other as yet unknown mechanisms. Since protein glycosylation is an important determinant of cell adhesion, we hypothesized that radiation could alter the glycosylation pattern of endothelial cells and thereby impact adhesion of circulating cells. Herein, we show that ionizing radiation increases high mannose-type N-glycans and decreases glycosaminoglycans. These changes stimulate interactions measured under flow conditions between irradiated endothelial cells and monocytes. Targeted transcriptomic approaches in vitro in endothelial cells and in vivo in a radiation enteropathy mouse model confirm that genes involved in N- and O-glycosylation are modulated by radiation, and in silico analyses give insight into the mechanism by which radiation modifies glycosylation. The endothelium glycome may therefore be considered as a key therapeutic target for modulating the chronic inflammatory response observed in healthy tissues or for participating in tumor control by radiation therapy.

The current study evaluated the role of Hey2 transcription factor in radiation-induced endothelial-to-mesenchymal transition (EndoMT) and its impact on radiation-induced tissue damage in mice. Phenotypic modifications of irradiated, Hey2 siRNA- and Hey2 vector plasmid-transfected human umbilical vein endothelial cells (HUVECs) resembling EndoMT were monitored by qPCR, immunocytochemistry and western blots. Subsequently, in mice, a Cre-LoxP strategy for inactivation of Hey2 specifically in the endothelium was used to study the biological consequences. Total body irradiation and radiation proctitis were monitored to investigate the impact of conditional Hey2 deletion on intestinal stem cells and microvascular compartment radiosensitivity, EndoMT and rectal damage severity. We found that EndoMT occurs in irradiated HUVECs with concomitant Hey2 mRNA and protein increase. While Hey2 silencing has no effect on radiation-induced EndoMT in vitro, Hey2 overexpression is sufficient to induce phenotypic conversion of endothelial cells. In mice, the conditional deletion of Hey2 reduces EndoMT frequency and the severity of rectal tissue damage. Our data indicate that the reduction in mucosal damage occurs through decline in stem/clonogenic epithelial cell loss mediated by microvascular protection. EndoMT is involved in radiation proctitis and this study demonstrates that a strategy based on the reduction of EndoMT mitigates intestinal tissue damage.

The pathophysiological mechanism involved in side effects of radiation therapy, and especially the role of the endothelium remains unclear. Previous results showed that plasminogen activator inhibitor-type 1 (PAI-1) contributes to radiation-induced intestinal injury and suggested that this role could be driven by an endothelium-dependent mechanism. We investigated whether endothelial-specific PAI-1 deletion could affect radiation-induced intestinal injury. We created a mouse model with a specific deletion of PAI-1 in the endothelium (PAI-1KO(endo)) by a Cre-LoxP system. In a model of radiation enteropathy, survival and intestinal radiation injury were followed as well as intestinal gene transcriptional profile and inflammatory cells intestinal infiltration. Irradiated PAI-1KO(endo) mice exhibited increased survival, reduced acute enteritis severity and attenuated late fibrosis compared with irradiated PAI-1(flx/flx) mice. Double E-cadherin/TUNEL labeling confirmed a reduced epithelial cell apoptosis in irradiated PAI-1KO(endo). High-throughput gene expression combined with bioinformatic analyses revealed a putative involvement of macrophages. We observed a decrease in CD68(+)cells in irradiated intestinal tissues from PAI-1KO(endo) mice as well as modifications associated with M1/M2 polarization. This work shows that PAI-1 plays a role in radiation-induced intestinal injury by an endothelium-dependent mechanism and demonstrates in vivo that the endothelium is directly involved in the progression of radiation-induced enteritis.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

The endothelial-to-mesenchymal transition (EndoMT) is a crucial cellular process during heart development necessary to the formation of cardiac valves. This embryonic process reappears in several pathological situations, such as vascular injury or organ fibrosis of various etiologies, as a mediator of extracellular matrix-producing cells. Because radiation induces both vascular damage and fibrosis, we investigated whether radiation exposure induces EndoMT in primary human intestinal microvascular endothelial cells (HIMECs) and whether EndoMT contributes to radiation-induced rectal damage in humans and in a preclinical model of radiation proctitis in mice. Irradiated HIMECs show phenotypic hallmarks of radiation-induced endothelial cell activation in vitro. Moreover, HIMECs undergo changes in molecular expression pattern compatible with EndoMT, with up-regulation of mesenchymal markers and down-regulation of endothelial markers via transforming growth factor/Smad pathway activation. In vivo, EndoMT readily occurs in the human rectum after radiation therapy for rectal adenocarcinoma. Finally, EndoMT was observed in rectal mucosal and submucosal microvessels in a preclinical model of radiation proctitis in Tie2-green fluorescent protein reporter-expressing mice all along radiation proctitis development, also associated with transforming growth factor/Smad pathway activation. In conclusion, radiation-induced cell activation and tissue inflammation constitute a setting that fosters the phenotypic conversion of endothelial cells into mesenchymal cells. Therefore, EndoMT is identified as a potential participant in radiation-induced gut damage and may represent an interesting therapeutic target in cases of radiation-induced pelvic disease., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Motivation: Identifying the set of genes differentially expressed along time is an important task in two-sample time course experiments. Furthermore, estimating at which time periods the differential expression is present can provide additional insight into temporal gene functions. The current differential detection methods are designed to detect difference along observation time intervals or on single measurement points, warranting dense measurements along time to characterize the full temporal differential expression patterns., Results: We propose a novel Bayesian likelihood ratio test to estimate the differential expression time periods. Applying the ratio test to systems of genes provides the temporal response timings and durations of gene expression to a biological condition. We introduce a novel non-stationary Gaussian process as the underlying expression model, with major improvements on model fitness on perturbation and stress experiments. The method is robust to uneven or sparse measurements along time. We assess the performance of the method on realistically simulated dataset and compare against state-of-the-art methods. We additionally apply the method to the analysis of primary human endothelial cells under an ionizing radiation stress to study the transcriptional perturbations over 283 measured genes in an attempt to better understand the role of endothelium in both normal and cancer tissues during radiotherapy. As a result, using the cascade of differential expression periods, domain literature and gene enrichment analysis, we gain insights into the dynamic response of endothelial cells to irradiation., Availability and Implementation: R package 'nsgp' is available at www.ibisc.fr/en/logiciels_arobas., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The threat of a large scale radiological emergency, where thousands of people may require fast biological dosimetry for the purpose of triage, makes it necessary to search for new, high throughput biological dosimeters. The authors tested an assay based on the quantitative analysis of selected proteins in peripheral blood serum. They were particularly interested in testing proteins that are specific to irradiation of skin, as these can be used in cases of partial body exposure. Candidate proteins were identified in an earlier study with mice, where skin of the animals was exposed to different doses of radiation and global expression of serum proteins was analyzed. Eight proteins were found, the expression of which showed a consistent dose-response relationship. Human analogues of these proteins were identified, and their expression was measured in peripheral blood serum of 16 breast cancer patients undergoing external beam radiotherapy. The proteins were Apolipoprotein E; Apolipoprotein H; Complement protein 7; Prothrombinase; Pantothenate Kinase 4; Alpha-2-macroglobulin; Fetuin B and Alpha-1-Anti-Chymotrypsin. Measurements were carried out in blood samples collected prior to exposure (control), on the day after one fraction (2 Gy), on the day after five fractions (10 Gy), on the day after 10 fractions (20 Gy), and 1 mo after 23-25 fractions (total dose of 46-50 Gy). Multivariate analysis was carried out, and a multinomial logistic regression model was built. The results indicate that the combined analysis of Apolipoprotein E, Factor X, and Pantothenate Kinase 4 allows discriminating between exposure to 2 Gy and lower and between 10 Gy and higher. The discrimination is possible up to 1 mo after exposure.

The finding of new diagnostic and prognostic markers of local radiation injury, and particularly of the cutaneous radiation syndrome, is crucial for its medical management, in the case of both accidental exposure and radiotherapy side effects. Especially, a fast high-throughput method is still needed for triage of people accidentally exposed to ionizing radiation. In this study, we investigated the impact of localized irradiation of the skin on the early alteration of the serum proteome of mice in an effort to discover markers associated with the exposure and severity of impending damage. Using two different large-scale quantitative proteomic approaches, 2D-DIGE-MS and SELDI-TOF-MS, we performed global analyses of serum proteins collected in the clinical latency phase (days 3 and 7) from non-irradiated and locally irradiated mice exposed to high doses of 20, 40 and 80 Gy which will develop respectively erythema, moist desquamation and necrosis. Unsupervised and supervised multivariate statistical analyses (principal component analysis, partial-least square discriminant analysis and Random Forest analysis) using 2D-DIGE quantitative protein data allowed us to discriminate early between non-irradiated and irradiated animals, and between uninjured/slightly injured animals and animals that will develop severe lesions. On the other hand, despite a high number of animal replicates, PLS-DA and Random Forest analyses of SELDI-TOF-MS data failed to reveal sets of MS peaks able to discriminate between the different groups of animals. Our results show that, unlike SELDI-TOF-MS, the 2D-DIGE approach remains a powerful and promising method for the discovery of sets of proteins that could be used for the development of clinical tests for triage and the prognosis of the severity of radiation-induced skin lesions. We propose a list of 15 proteins which constitutes a set of candidate proteins for triage and prognosis of skin lesion outcomes.

Skin exposure to ionizing radiation affects the normal wound healing process. We investigated the beneficial effects of a pharmacological treatment with geranylgeranylacetone (GGA) on keratinocytes using in vitro scratch wound injury assay in nonirradiated and irradiated conditions. Irradiation affected the wound closure of keratinocytes 24 h after scratch injury, whereas re-epithelialization was markedly accelerated after GGA treatment when compared to nontreated keratinocytes. We demonstrated that GGA treatment increased migration of human epidermal keratinocytes and this migratory property was not related to RhoA signaling. Interestingly, Western blot analysis revealed that GGA treatment down-regulated caspase 3 active form expression and up-regulated the activated phenotype by inducing both keratin 6 (K6) expression and interleukin-1β (IL-1β) release without modification of the differentiate phenotype. Finally, the proteomic profiling was performed on keratinocytes, showing that global protein changes occurred after irradiation of keratinocytes treated or untreated with GGA.

Exposure of the skin to ionizing radiation leads to characteristic reactions that will often turn into a pathophysiological process called the cutaneous radiation syndrome. The study of this disorder is crucial to finding diagnostic and prognostic bioindicators of local radiation exposure or radiation effects. It is known that irradiation alters the serum proteome content and potentially post-translationally modifies serum proteins. In this study, we investigated whether localized irradiation of the skin alters the serum glycome. Two-dimensional differential in-gel electrophoresis of serum proteins from a man and from mice exposed to ionizing radiation showed that potential post-translational modification changes occurred following irradiation. Using a large-scale quantitative mass-spectrometry-based glycomic approach, we performed a global analysis of glycan structures of serum proteins from non-irradiated and locally irradiated mice exposed to high doses of γ-rays (20, 40, and 80 Gy). Non-supervised descriptive statistical analyses (principal component analysis) using quantitative glycan structure data allowed us to discriminate between uninjured/slightly injured animals and animals that developed severe lesions. Decisional statistics showed that several glycan families were down-regulated whereas others increased, and that particular structures were statistically significantly changed in the serum of locally irradiated mice. The observed increases in multiantennary N-glycans and in outer branch fucosylation and sialylation were associated with the up-regulation of genes involved in glycosylation in the liver, which is the main producer of serum proteins, and with an increase in the key proinflammatory serum cytokines IL-1β, IL-6, and TNFα, which can regulate the expression of glycosylation genes. Our results suggest for the first time a role of serum protein glycosylation in response to irradiation. These protein-associated glycan structure changes might signal radiation exposure or effects.

All tissues can be damaged by ionizing radiation. Early biomarkers of radiation injury are critical for triage, treatment and follow-up of large numbers of people exposed to ionizing radiation after terrorist attacks or radiological accident, and for prediction of normal tissue toxicity before, during and after a treatment by radiotherapy. The comparative proteomic approach is a promising and powerful tool for the discovery of new radiation biomarkers. In association with multivariate statistics, proteomics enables measurement of the level of hundreds or thousands of proteins at the same time and identifies set of proteins that can discriminate between different groups of individuals. Human serum and plasma are the preferred samples for the study of normal and disease-associated proteins. Extreme complexity, extensive dynamic range, genetic and physiological variations, protein modifications and incompleteness of sampling by two-dimensional electrophoresis and mass spectrometry represent key challenges to reproducible, high-resolution, and high-throughput analyses of serum and plasma proteomes. The future of radiation research will possibly lie in molecular networks that link genome, transcriptome, proteome and metabolome variations to radiation pathophysiology and serve as sensors of radiation disease. This chapter reviews recent advances in proteome analysis of serum and plasma as well as its applications to radiation biology and radiation biomarker discovery for both radiation exposure and radiation tissue toxicity.

Erythema was observed on the skin of the first patients treated with radiation therapy. It is in particular to reduce this erythema, one feature of tissue inflammation, that prescribed dose to the tumor site started to be fractionated. It is now well known that radiation exposure of normal tissues generates a sustained and apparently uncontrolled inflammatory process. Radiation-induced inflammation is always observed, often described, sometimes partly explained, but still today far from being completely understood. The thing with the gut and especially the gut mucosa is that it is at the frontier between the external milieu and the organism, is in contact with a plethora of commensal and foreign antigens, possesses a dense-associated lymphoid tissue, and is particularly radiation sensitive because of a high mucosal turnover rate. All these characteristics make the gut mucosa a strong responsive organ in terms of radiation-induced immunoinflammation. This paper will focus on what has been observed in the normal gut and what remains to be done concerning the immunoinflammatory response following localized radiation exposure.

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)(-/-) mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS(-/-) mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage.

Early biomarkers of radiation injury are critical for triage, treatment, and follow-up of large numbers of people exposed to ionising radiation after terrorist attacks or nuclear accident. Operational monoparametric protein or amino acid biomarkers (amylase, Flt3-Ligand, citrulline) can help for the diagnostic of radiation exposure or injury. However, these biomarkers are not sufficient for a fast and accurate triage, and if individuals are assessed more than 48 h after exposure. The comparative proteomic approach represents a promising powerful tool for the discovery of new radiation biomarkers. In association with multivariate statistics, proteomic enables to measure the level of hundreds or thousands of proteins at the same time and identifies sets of proteins that can discriminate different groups of individuals.

Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.