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Purpose 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. Material and methods 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. Results 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 post-irradiation). Conclusion 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.

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.Using a systems biology approach in vitro, we deciphered the dynamic senescence-associated transcriptional program induced by irradiation.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.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.

Summary 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., Graphical abstract, Highlights • Irradiation promotes cell death, senescence, and impaired angiogenesis in HUVECs • Multi-omics analyses enable tracking pathways from early to late dysfunction • Dynamic modeling allows accurate learning of essential dysfunction checkpoints • Loss-/gain-of-function experiments identify key players of angiogenesis in vitro, Radiation biology; Systems biology; Omics; Proteomics; Metabolomics; Transcriptomics

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.

Introduction et but de l’etude La cystite radique chronique est une pathologie consecutive d’une irradiation pelvienne caracterisee, par une inflammation chronique evoluant parfois vers une fibrose, avec des symptomes tels que des douleurs et des saignements. Des etudes precedentes sur la rectite radique ont montre que les cellules souches mesenchymateuses peuvent moduler l’inflammation chronique et la fibrose apres l’irradiation. Materiel et methodes Notre etude etait divisee en deux parties, la modelisation de la cystite radique chronique chez le rat puis l’effet du traitement par les cellules souches mesenchymateuses. La cystite radique chronique a ete induit par une irradiation localisee de l’integralite de la vessie a l’aide du Small Animal Radiation Research Platform (SARRP). Une gamme de doses de 20 a 80 Gy a ete delivree et un suivi de 3 a 12 mois apres l’irradiation a ete realise. Pour le traitement nous avons retenu la dose de 40 Gy, suivie 4 mois apres l’irradiation d’une injection intraveineuse de 5 millions de cellules souches mesenchymateuses, administree toutes les deux semaines (trois injections en tout). Un suivi physiologique, histologique et moleculaire a ete realise pendant 12 mois apres l’irradiation. Resultats et analyse statistique Les resultats ont montre l’apparition de la cystite radique chronique des 6 mois apres l’irradiation, avec une inflammation chronique, une hematurie, une desorganisation de l’urothelium et une fibrose, augmentant avec le temps. L’analyse transcriptomique a indique un profil en faveur d’une inflammation chronique par rapport au temoin non irradie, avec la surexpression des genes CCL5 (5 fois ; valeur p Conclusion Le traitement par les cellules souches mesenchymateuses semble diminuer l’hyperplasie par rapport au temoin irradie. Il permet egalement de retarder l’hematurie, de diminuer les lesions vasculaires et l’hyperplasie. L’analyse de l’effet des cellules souches mesenchymateuses sur l’inflammation et la regeneration de l’urothelium est en cours.

International audience; Purpose The fluorescent in situ hybridization (FISH) technique, which easily detects reciprocal translocations, is currently used to estimate doses in retrospective biological dosimetry, after suspected accidental overexposure to ionizing radiation (IR). This study of 42 cases aimed to verify the appropriateness of this assay for radiation dose reconstruction, compared to the dicentric assay, and to evaluate other limitations. Material and methods We labeled chromosomes 2, 4, and 12 by 3-color FISH painting to detect translocations on lymphocytes of patients with suspected past IR overexposure. Result Translocation dose estimation showed doses significantly different from 0 Gy in 25 of the 42 cases. The lowest positive dose measured was 0.3 Gy. Several months after IR exposure, the doses measured by translocation and dicentric assays are quite similar. For a year, dose estimation by translocation assay becomes more relevant as dicentric frequency starts to decrease, coming close to 0 for more than a year after the exposure. The persistence of translocations enabled us to corroborate an overexposure 44 years earlier. Interpretation of the observed translocation yield requires the knowledge of the patient’s other radiation exposures. A dose assessment by this biomarker is relevant only if the radiation exposure is confirmed. Conclusions This technique is appropriate for corroborating a former IR exposure of individuals. When the radiation dose is greater than 1 Gy, the translocations in complex exchanges must be considered. Another relevant point is the use of an appropriate background yield of translocations. The dose assessment, however, also depends on exposure to various genotoxic agents besides IR. If no evidence about the existence of radiation exposure is available, dose assessment is not useful. For this reason, report only the translocation frequency and its comparison with the background yield by age class is preferable. © 2018 Informa UK Limited, trading as Taylor and Francis Group.

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.C57BL/6JRj mice were exposed to 90 Gy focal irradiation on the left lung using 1-mm diameter, 3 × 3 mmBoth 1-mm diameter and 3 × 3 mmArc therapy allows long-term studies and dose escalation without lethality. In our dose-delivery conditions, dose-effect studies revealed that 3 × 3 mm

International audience; Purpose: Lung cancer will be treated more frequently using stereotactic body radiation therapy, and preclinical research tomodel long-term toxicity of ablative doses of radiation is crucial. Stereotactic lung irradiation of a small volume can induceradiation 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 ofablative doses of ionizing radiation on senescence-associated processes. The left lung of p16$^{INK4a}$ -LUC knock-in mice wasexposed 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 lungtarget 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 senescentcell types may contribute to radiation injury.Conclusions: This work provides important information on the long-term effects of ablative radiation doses in the normal lungand strongly suggests that stress-induced senescence is involved in stereotactic body radiation therapyeinduced late fibrosis.

International audience; 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. © 2019

Introduction et but de l’etude La cystite radique chronique est une maladie parfois consecutive des radiotherapies pelviennes. Elle est caracterisee par une inflammation chronique, des lesions vasculaires et une hypoxie des tissus evoluant vers la fibrose. En clinique, la cystite radique chronique entraine des douleurs, des saignements, de l’incontinence et dans les cas les plus graves des fistules. Il n’existe pas de traitement curatif. Nous proposons de tester une therapie cellulaire utilisant les cellules stromales mesenchymateuses comme nouvelle approche therapeutique. Nos etudes precedentes sur la rectite radique ont montre que les cellules stromales mesenchymateuses peuvent moduler l’inflammation chronique et la fibrose tissulaire apres irradiation. Materiel et methodes Notre etude est divisee en deux parties, la modelisation de la cystite radique chronique puis l’effet d’un traitement par les cellules stromales mesenchymateuses. La modelisation preclinique de la cystite radique chronique chez le rat a ete realisee par irradiation localisee guidee par l’imagerie scanographique de la vessie entre 20 et 80 Gy avec un suivi de 3 a 15 mois apres l’irradiation. L’evolution de la cystite radique chronique a ete suivie au niveau de l’expression des genes, des proteines, des parametres histologiques et fonctionnels. Resultats et analyse statistique La mesure des parametres urinaires a revele une hematurie transitoire augmentant avec le temps et la dose d’irradiation sans diminution du volume urinaire jusqu’a 6 mois. L’analyse transcriptomique indiquait une inflammation aigue a 3 mois (augmentation de l’expression des genes codant pour le chemokine [C-C motif] ligand [CCL]-2, CCL5, le tumour necrosis factor alpha [TNFα] et l’interleukine [IL]-1β). Elle etait accompagnee d’une regeneration tissulaire et vasculaire (augmentation de l’expression genique de l’epidermal growth factor [EGF] et du vascular endothelial growth factor [VEGF]) couplee a un remodelage de la matrice extracellulaire avec l’augmentation de l’expression genique de la metalloprotease MMP2, des inhibiteurs TIMP1/2/4, des collagenes Col1α2, Col3α1 et du proteoglycane Cspg4. A 6 mois, une deuxieme phase d’inflammation (augmentation de l’expression des genes codant pour CCL5, IL1β, IL6 et l’hypoxia-inducible factor 1-alpha [HIF1α]) a ete observee. Elle etait correlee avec une desorganisation de l’urothelium mais sans regeneration tissulaire et vasculaire, ni de remodelage de la matrice extracellulaire. Conclusion Ces premiers resultats sont en faveur de la mise en place d’une cystite radique chronique 6 mois apres l’irradiation, caracterisee par une inflammation chronique, des signes d’hypoxie, d’hematurie et de desorganisation de l’urothelium, sans diminution du volume urinaire. L’analyse de la cinetique sur des temps plus longs permettra de caracteriser l’evolution vers une cystite radique chronique confirmee accompagnee de fibrose. La seconde phase de l’etude est en cours pour evaluer si le traitement par les cellules stromales mesenchymateuses pourrait limiter la fibrogenese en inhibant les voies inflammatoires et en augmentant l’angiogenese. Nos resultats fourniront des donnees concernant le potentiel antifibrotique des cellules stromales mesenchymateuses et le traitement de la cystite radique par les cellules stromales mesenchymateuses.

International audience; Bacgrounf and aimChronic radiocystitis (CRC) is a pathology resulting from irradiation of the pelvic area without long term effective treatment. CRC is characterized by chronic inflammation progressing to fibrosis, fistulas and cystectomy in the most severe cases. Our laboratory have previously demonstrated that MSC treatment reverse similar damages in irradiated colon. Furthermore in a clinical phase 1/2 treatments using MSCs for hemorrhagic cystitis, which is pathology similar to CRC, was sucessfull (Ringden O et al., 2007). Based on these previous results, our objective is to evaluate whether adipose derived mesenchymal stem cells (MSCs) could be an innovative treatment of CRC. Methods, results and conclusionPreclinical modeling of CRC in rats (Sprague Dawley) was established by irradiating the entire bladder with a single dose of 40 Gray using the Small Animal Radiation Research Platform (SARRP, figure 1)). At four months after irradiation, animals received a treatment consisting in tree intravenous injections of 5 million of MSCs every two weeks. After irradiation and treatment, a physiological, histological and molecular follow-up was performed on 14 months (figure 2). Results have shown, without MSC treatment, an initiation of CRC at 6 months, with chronic inflammation, hypoxia, hematuria, disorganization of the urothelium and fibrosis. Analysis of urinary parameters has revealed hematuria increasing with time. Transcriptomic analysis indicates chronic inflammation (IL1β, CCL2, IL6) and hypoxia (HIF1α). Histological observations reveal a disorganization of the urothelium with loss of superficial cells and fibrosis (figure 3). Study is in progress to evaluate whether MSC treatment could limite fibrogenesis by inhibiting the inflammatory pathways and increasing angiogenesis, in our model CRC preclinical model. Our results will provide data regarding the anti-fibrotic potential of MSCs and could support their use in the treatment of CRC.Ringden O, Uzunel M, Sundberg B, et al. Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia 2007: 21: 2271–2276.

International audience; 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. © 2015 American Society for Investigative Pathology.

International audience; 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. © 2017 Elsevier Inc.

Introduction et but de l’etude La cystite radique chronique est une pathologie consecutive d’une irradiation de la zone pelvienne. Elle se caracterise par une inflammation chronique evoluant parfois vers une fibrose et dans les cas les plus graves vers des fistules avec des symptomes comme des douleurs et des saignements. Il n’existe pas de traitement efficace et nous proposons de tester les cellules souches mesenchymateuses comme nouvelle methode therapeutique. Nos etudes anterieures sur la rectite radique ont demontre que les souches mesenchymateuses permettent de reverser l’inflammation chronique et la fibrose apres irradiation. Materiel et methodes La modelisation preclinique de la cystite radique chronique chez le rat a ete mise en place par une irradiation localisee guidee par l’image de la vessie de 20 a 40 Gy avec un suivi de trois a six mois apres l’irradiation. Des analyses d’expression de genes et proteines ainsi que des parametres histologiques et fonctionnels ont ete realises. Resultats et analyse statistique L’analyse des parametres urinaires a permis de mettre en evidence des hematuries passageres mais pas de diminution du volume urinaire au cours des 6 mois. L’analyse transcriptomique a indique un profil en faveur de l’inflammation chronique (interleukine [IL]1β, chimiokine[CCL2], IL6) et de l’hypoxie (hypoxia-inducible factor 1-alpha [HIF1α]) a 6 mois mais pas de profil profibrotique (αSMA). Les observations histologiques ont revele une desorganisation de l’urothelium a 6 mois, avec une diminution de son epaisseur et des lesions vasculaires mais pas de fibrose, ce qui est coherent avec les resultats d’expression des genes. Conclusion Ces premiers resultats attestent de la pertinence de l’etude en montrant une initiation de la cystite radique chronique a 6 mois avec une inflammation chronique, des signes d’hypoxie, des hematuries et une desorganisation de l’urothelium, sans diminution du volume urinaire. L’analyse de la cinetique sur des temps plus tardifs permettra de caracteriser l’evolution vers la fibrose et d’avoir une cystite radique chronique etablie. Dans un second temps nous mettrons en place le traitement de cette pathologie par souches mesenchymateuses.

Objectif de l’etude La radiotherapie stereotaxique permet l’irradiation de petits volumes avec une precision millimetrique. La radiotherapie stereotaxique est utilisee comme alternative a la chirurgie pour traiter les tumeurs pulmonaires non a petites cellules. Malgre la reduction du volume de tissu sain irradie, les patients voient se developper des effets secondaires : des pneumopathies radiques et des fibroses pulmonaires. Aujourd’hui, le manque de donnees radiobiologiques aux fortes doses par fraction reste une problematique. L’objectif est donc d’etudier les consequences de l’irradiation stereotaxique pulmonaire. Materiel et methode Le Small Animal Radiation Research Platform (SARRP) est un systeme de micro-irradiation guidee par l’image et couple a un systeme de planification des traitements. Notre objectif est d’acquerir in vivo des donnees radiopathologiques, considerant l’effet du volume irradie, de la dose totale delivree ainsi que du fractionnement. Les premieres irradiations ont permis d’observer l’effet du volume irradie grâce a l’utilisation de 4 collimateurs : 10 × 10 mm2, 7 × 7 mm2, 3 × 3 mm2 et ϕ1 mm, pour une dose unique delivree par arctherapie, centree sur le poumon gauche. Resultats L’analyse histologique montre une reaction de cicatrisation impliquant un epaississement des septa alveolaires, de la fibrose, un œdeme alveolaire, des foyers de macrophages et une diminution des cellules de Clara. Les irradiations pour l’etude de l’effet de la dose sont en cours. La contention et le repositionnement des souris pour l’irradiation fractionnee est au point et un premier protocole de trois fractions de 20 Gy a ete teste sur quelques animaux. In vitro, afin de determiner la reponse cellulaire aux fortes doses par fraction, differentes lignees cellulaires pulmonaires sont irradiees suivant cinq protocoles de fractionnement et analysees. Conclusion Cette etude permettra de comprendre la reponse des differents types cellulaires pulmonaires aux fortes doses par fraction. La modelisation preclinique associee aux donnees radiobiologiques permettent de mieux comprendre les risques et les effets de la radiotherapie stereotaxique sur les tissus sains.

International audience; Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization. We therefore assessed the effect of a therapeutic strategy based on EPC administration in the healing of radiation-induced damage. To improve cell therapy for clinical use, we used pretreatment with ephrin B2-Fc (Eph-B2-Fc) and/or coadministration with smooth muscle progenitor cells. At day 3, EPCs promoted dermal wound healing in both nonirradiated and irradiated mice by 1.2- and 1.15-fold, respectively, compared with animals injected with phosphate-buffered saline. In addition, EPCs also improved skin-blood perfusion and capillary density in both irradiated and nonirradiated mice compared with PBS-injected animals. We also demonstrated that activation with Eph-B2-Fc increased wound closure by 1.6-fold compared with unstimulated EPCs in nonirradiated mice. Interestingly, the beneficial effect of Eph-B2-Fc was abolished in irradiated animals. In addition, we found that Eph-B2-Fc stimulation did not improve EPC-induced vascular permeability or adhesiveness compared to unstimulated EPCs. We hypothesized that this effect was due to high oxidative stress during irradiation, leading to inhibition of EPCs' beneficial effect on vascular function. In this line, we demonstrated that, in irradiated conditions, N-acetyl-L-cysteine treatment restored the beneficial effect of EPC stimulation with Eph-B2-Fc in the wound healing process. In conclusion, stimulation by Eph-B2-Fc improved the beneficial effect of EPCs in physiological conditions and irradiated conditions only in association with antioxidant treatment. Additionally, cotherapy was beneficial in pathological conditions. © 2015 Cognizant Comm. Corp.

International audience; Background Radiation damage to the normal gut is a dose-limiting factor in the application of radiation therapy to treat abdominal and pelvic cancers. All tissue cell types react in concert to orchestrate an acute inflammatory reaction followed by a delayed chronic scarring process. Osteopontin (OPN) is a matricellular protein known to be involved in various physiological but also pathological processes such as tissue inflammation and fibrosis. Aims The aim of our study was to determine whether OPN knockout influences the severity of radiation proctitis and to investigate the role of OPN in the development of radiation-induced gut damage. Results Here we show that human radiation proctitis is associated with increased immunostaining of the intracellular and extracellular/matrix-linked isoforms of OPN. Moreover, endothelial cells in vitro and rectal tissue in a preclinical model of radiation proctitis in mice both respond to radiation exposure by a sustained increase in OPN mRNA and protein levels. Genetic deficiency of OPN did not influence radiation-induced rectal damage and was associated with significantly decreased animal survival. The acute and late radiation injury scores were similar in OPN-null mice compared with their control littermates. Conclusion This study shows that in our model and given the pleiotropic actions of OPN in tissue inflammation and fibrosis, further studies are necessary to understand the precise roles of OPN in radiation-induced proctitis and to determine whether OPN is a useful therapeutic tool in prevention of radiation-induced intestinal tissue injury. © 2015, Springer Science+Business Media New York.

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-1KOendo) 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-1KOendo mice exhibited increased survival, reduced acute enteritis severity and attenuated late fibrosis compared with irradiated PAI-1flx/flx mice. Double E-cadherin/TUNEL labeling confirmed a reduced epithelial cell apoptosis in irradiated PAI-1KOendo. 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-1KOendo 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.

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 Contact: markus.heinonen@ibisc.fr or florence.dalche@ibisc.fr Supplementary information: Supplementary data are available at Bioinformatics online.

International audience; The endothelium contributes to the control of the tissue inflammatory response following stress and in particular after exposure to ionizing radiation. We previously showed that the TG-interacting factor 1 (TGIF1) plays a role in radiation-induced normal tissue injury. In this study we hypothesized that this protein could play a role in inflammation. The role of TGIF1 in the stress-induced proinflammatory phenotype was investigated in human endothelial cells. In HUVECs ionizing radiation induces TGIF1 expression as well as a proinflammatory phenotype associated with up-regulation of IL-6, IL-8, CXCL1, MIP-2, and MCP-1. TGIF1 overexpression enhances the radiation-induced proinflammatory phenotype whereas TGIF1 silencing limits both the TNF-α- and radiation-induced overexpression of proinflammatory cytokines. Interestingly, in vivo, in radiation-induced intestinal inflammation in mice, TGIF1 genetic deficiency is associated with a reduced radiation-induced overexpression of proinflammatory molecules. In HUVECs, TNF-α-and radiation-induced NF-κB pathway activation is not influenced by TGIF1 expression, whereas TGIF1 knockdown inhibits both TNF-α- and radiation-induced p38 MAPK pathway activation. This study demonstrates that TGIF1 plays a role in TNF-α-and radiation-induced inflammation and suggests that it could be a target in limiting this event in the vascular compartment. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

International audience; Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism. © 2012 Hneino et al.

Purpose: To assess the frequency and the functional characteristics of one major component of immune tolerance, the CD4+FoxP3+ regulatory T cells (Tregs) in a mouse model of abdominal irradiation. Methods and Materials: Mice were exposed to a single abdominal dose of γ-radiation (10 Gy). We evaluated small intestine Treg infiltration by Foxp3 immunostaining and the functional suppressive activity of Tregs isolated from mesenteric lymph nodes. Results: Foxp3 immunostaining showed that radiation induced a long-term infiltration of the intestine by Tregs (levels 5.5 times greater than in controls). Co-culture of Tregs from mesenteric lymph nodes with CD4+ effector cells showed that the Tregs had lost their suppressive function. This loss was associated with a significant decrease in the levels of Foxp3, TGF-β, and CTLA-4 mRNA, all required for optimal Treg function. At Day 90 after irradiation, Tregs regained their suppressive activity as forkhead box P3 (Foxp3), transforming growth factor beta (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression returned to normal. Analysis of the secretory function of mesenteric lymph node Tregs, activated in vitro with anti-CD3/anti-CD28 Abs, showed that this dysfunction was independent of a defect in interleukin-10 secretion. Conclusion: Radiation caused a long-term accumulation of function-impaired Foxp3+CD4+ Tregs in the intestine. Our study provides new insights into how radiation affects the immune tolerance in peripheral tissues. © 2011 Elsevier Inc.

cited By 16; The purpose of this study was to evaluate the in vivo dose-response relation of chromosome aberration formation and distribution in a context of localised and fractionated radiotherapy. Cytogenetic analysis was applied to eight patients, all treated for the same tumour localisation; the same localisation was used to prevent the variability usually observed between patients treated with radiotherapy and to allow the corresponding roles of the size of irradiation field and of the dose rate to be studied. The yield of dicentrics, centric rings and fragments was measured in blood samples taken before treatment, during the course of radiotherapy and up to 6 months after. After the first fraction of radiotherapy, we observed that the whole-body dose estimated from the yield of dicentrics and rings was higher (0.35±0.2 Gy) than the calculated equivalent whole-body dose (0.07±0.04 Gy). By contrast, the partial-body dose derived from the Qdr (quotient of dicentrics and rings) model was estimated to be 2.2±0.3 Gy, which agreed quite well with the dose delivered to the tumour (2.1±0.1 Gy). We also found a correlation between the yield of induced chromosome aberrations and the target field size (p=0.014). U-value analysis showed that the distribution of dicentrics and rings was overdispersed, despite the fractionation of the exposure, and a positive correlation between the U-value and the dose rate was observed (p=0.017). Overall, these results suggest that the proportion of undamaged lymphocytes could increase with the dose rate. © 2010 The British Institute of Radiology.

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.

International audience; Intestinal radiation injury is a dose-limiting factor in radiation therapy for abdominal and pelvic cancers. Because transforming growth factor-β1 is a key mediator involved in radiation-induced damage, we hypothesized that its target gene, plasminogen activator inhibitor type 1 (PAI-1), is an essential mediator of intestinal radiation toxicity. In a model of radiation enteropathy, survival was monitored and intestinal radiation injury was assessed in both wild-type (Wt) and PAI-1 knockout mice. Immunohistochemical labeling of PAI-1 was also assessed in patients treated with preoperative radiotherapy for rectal adenocarcinoma. Finally, the molecular mechanisms involved in radiation-induced PAI-1 expression were investigated. We found that PAI-1 -/- mice exhibited increased survival and better intestinal function compared with Wt mice. Intestinal radiation injury was attenuated in irradiated PAI-1 -/- mice compared with irradiated Wt mice, and irradiation increased blood cell-endothelial cell interactions in Wt but not PAI-1 -/- mice. In vivo, radiation-induced intestinal damage in mice, as well as in patients treated with radiotherapy, was associated with the up-regulation of PAI-1 in the endothelium. In vitro, irradiation increased PAI-1 expression in endothelial cells by a p53/Smad3-dependent mechanism. Together, these data demonstrate that PAI-1 plays a critical role in radiation-induced intestinal damage, suggesting that PAI-1 is an attractive target for preventing or reducing the side effects of radiation therapy. Copyright © American Society for Investigative Pathology.

Normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Intestinal radiation toxicity is characterized by mucosal injury, inflammation, vascular activation followed by development of progressive vascular fibrosis/sclerosis and radiation enteritis. The endothelium is known to play a critical role in radiation-induced intestinal injury. Previous studies showed that endothelial cell (EC) apoptosis plays a central role in early radiation-induced intestinal injury. Recently, we demonstrated that plasminogen activator inhibitor type 1 (PAI-1) is an essential mediator of late intestinal radiation toxicity. PAI-1 knockout mice (PAI-1 -/-) are protected against intestinal radiation-induced damage with increased survival and better intestinal function compared with wild type mice (Wt). However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damages. We hypothesized that PAI-1 could contribute to the radiosensitivity of the endothelium in acute phases of radiation enteropathy. In vitro, irradiation stimulates PAI-1 expression (mRNA and protein) in EC 4 hours to 48 hours after irradiation. Moreover, FACS analyses (Sub-G1 and AnnexinV) and caspase assay showed that apoptosis is rapidly induced 4h to 24h after irradiation. These results suggest that PAI-1 could play a key role in radiation-induced EC cell death. To prove that PAI-1 is involved, molecular modulation of PAI-1 expression was performed using an expression vector and a RNA interference strategy. In EC overexpressing PAI-1 (pCMV PAI-1), radiation-induced apoptosis is increased 6h after 10Gy irradiation compared with EC transfected with a control vector. 48 hours after PAI-1 siRNA transfection, PAI-1 mRNA and protein levels are decreased by 85%. Sub-G1 and Annexin V flow cytometry analyses showed that radiation-induced apoptosis is decreased in siRNA-PAI-1 transfected EC. These preliminary results show that PAI-1 plays a role in the radiosensitivity of EC. To prove the relevance of our in vitro results, radiation-induced EC apoptosis in vivo was monitored in a model radiation enteropathy. After exposure of an intestinal segment to 19 Gy radiation, acute intestinal radiation injury are assessed in Wt and PAI-1 -/- mice 4 hours to 3 days after irradiation. Radiation injury score (RIS) is monitored and radiation-induced EC apoptosis is followed by double immunolabelling TUNEL or caspase-3 / CD31. RIS is reduced in PAI-1 -/- mice compared to Wt mice suggesting that acute EC apoptosis is reduced in PAI-1 -/- mice. This current and ongoing work should allow to determine the putative role of PAI-1 in radiation-induced endothelial cells apoptosis and consequently in intestinal radiation injury.

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.

Purpose To compare translocation rate using either M-FISH or FISH-3 in two patients treated for head and neck cancer, with a view to retrospective dosimetry. Materials and methods Translocation analysis was performed on peripheral blood lymphocyte cultures from blood samples taken at different times during the radiotherapy (0 Gy, 12 Gy and 50 Gy) and a few months after the end of the treatment (follow-up). Results Estimated translocation yield varied according to the FISH technique used. At 50 Gy and follow-up points, the translocation yields were higher with FISH-3 than with M-FISH. This difference can be attributed to three events. First, an increase in complex aberrations was observed for 50 Gy and follow-up points compared with 0 Gy and 12 Gy points. Second, at the end of treatment for patient A, involvement of chromosomes 2, 4, 12 in translocations was less than expected according to the Lucas formula. Third, a clone bearing a translocation involving a FISH-3 painted chromosome was detected. Conclusions More translocations were detected with M-FISH than with FISH-3, and so M-FISH is expected to improve the accuracy of chromosome aberration analyses in some situations.

This case report describes a patient suffering from accidental cutaneous radiation syndrome. Clinical symptoms were characterized by the presence of moist epidermal denudation over approximately 8% of the body surface without signs of necrosis 88 days after radiation exposure. The skin transcriptional profile was obtained and provides a comprehensive overview of the changes in gene expression associated with skin wound healing after irradiation. In particular, our data show a specific set of genes, i.e. SOD1, GPX1, TDX1, TDX2 and HSP60, implicated in the redox control of normal skin repair after radiation exposure, whereas HOX1 and HOX2 were involved in the pathological skin repair. A reduction in the antioxidant capacity of the irradiated tissue concomitant with a progressive establishment of an uncontrolled inflammatory response was noted. Our data corroborate the hypothesis that ROS modulation is a key element of the healing response after cutaneous exposure to radiation and that the collapse of skin antioxidant status interferes directly with wound healing in skin after radiation exposure. Thus a better understanding of the molecular events through which oxidative stress modulates the healing response could result in a more rational therapeutic approach to the pathological process induced after exposure of skin to radiation. © 2007 by Radiation Research Society.

Background: Ionising radiation-induced reactive oxygen species (ROS) overproduction induces keratinocyte alterations and constitutes one of the most common effects after therapeutic γ-irradiation. ROS production is controlled by a complex enzymatic system. Objective: The aim of our study is to analyse the role of radiation-induced oxidative stress in keratinocytes death by apoptosis. We hypothesized that keratinocyte capacity to hamper radiation-induced ROS generation may control their radiosensitivity. Methods: For this purpose, an original human skin explant model was developed and two types of human epidermal cells were used: primary keratinocytes NHEK and spontaneous non-tumourigenic cell line HaCaT. Results: cDNA-arrays analysis was performed 24 h after a 20 Gy γ-radiation and revealed down-regulation of genes involved in oxidative stress control and the apoptosis process. This was confirmed by alterations in catalase, GPx and SOD enzymatic activities. This redox modulation was concomitant to the down-regulation of anti-apoptotic genes and up-regulation of some pro-apoptotic genes (caspase 10, ubiquitin C). Interestingly TUNEL labelling revealed an increase in the number of apoptotic cells. We also demonstrated a differential inducibility of the cell antioxidant network in two keratinocyte lines, which results in a differential cellular level of ROS, explaining their different radiosensitivities. Conclusion: Keratinocytes apoptosis is partly dependent on ROS production after exposure to γ-rays. In addition, the differential radiosensitivity of keratinocytes is linked to different oxidative stress responses. © 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Purpose: To compare the efficiency of different cytogenetic tools in estimating the doses received by four people involved in the Lilo accident and to monitor the dose estimate over 4.5 years. Materials and methods: Several young Georgian frontier guards handled at least one of the 12 Caesium sources found in a former Russian military camp. Overexposure lasted from July 1996 to May 1997. The Institute for Radiological Protection and Nuclear Safety (IRSN) obtained blood samples taken at several intervals post-exposure from the four most highly-exposed people. Dose estimation was performed using dicentric and translocation scoring. Results: The first dose estimations performed by dicentric scoring gave whole-body doses ranging from 0.4 to 1.3 Gy. Overexposure was complex and several mathematical models were used to take this complexity into account. This could provide information concerning the circumstances of overexposure. Concerning follow-up, the yield of dicentrics decreased by about 50% in the first 4 months following the end of overexposure whereas translocations were stable over the period of analysis. Conclusion: It has been useful to compare cytogenetic results with clinical results. The results presented here reveal good stability of translocations. However the first dose estimation was not attempted until 6 months after the last exposure. © 2006 Taylor & Francis.

The Institute of Radiation Protection and Nuclear Safety (IRSN) organized a biological dosimetry international intercomparison with the purpose of comparing (i) dicentrics yield produced in human lymphocytes; (ii) the gamma and neutron dose estimate according to the corresponding laboratory calibration curve. The experimental reactor SILENE was used with different configurations: bare source 4 Gy, lead shield 1 and 2 Gy and a 60Co source 2 Gy. An increasing variation of dicentric yield per cell was observed between participants when there were more damages in the samples. Doses were derived from the observed dicentric rates according to the dose-effect relationship provided by each laboratory. Differences in dicentric rate values are more important than those in the corresponding dose values. The doses obtained by the participants were found to be in agreement with the given physical dose within 20%. The evaluation of the respective gamma and neutron dose was achieved only by four laboratories, with some small variations among them. © Oxford University Press 2004; all rights reserved.