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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.
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Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Radioterapia , Teorema de Bayes , Células Cultivadas , Relação Dose-Resposta à Radiação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Neoplasias/radioterapia , Distribuição Normal , Fatores de TempoRESUMO
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.
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Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Proctite/metabolismo , Lesões por Radiação/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Transição Epitelial-Mesenquimal , Fibrose/metabolismo , Fibrose/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Proctite/genética , Proctite/patologia , Regulação para Cima/efeitos da radiaçãoRESUMO
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-1ß, 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.
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Proteínas Sanguíneas/metabolismo , Queimaduras/sangue , Fígado/efeitos da radiação , Polissacarídeos/sangue , Processamento de Proteína Pós-Traducional , Lesões Experimentais por Radiação/sangue , Pele/efeitos da radiação , Adulto , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Queimaduras/etiologia , Queimaduras/genética , Sequência de Carboidratos , Eletroforese em Gel Bidimensional , Raios gama/efeitos adversos , Regulação da Expressão Gênica , Glicômica , Glicosilação , Humanos , Interleucina-1beta/sangue , Interleucina-6/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polissacarídeos/química , Análise de Componente Principal , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/genética , Pele/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Necrose Tumoral alfa/sangueRESUMO
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.
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Proteínas Sanguíneas/análise , Exposição Ambiental , Proteoma/análise , Lesões por Radiação/sangue , Animais , Biomarcadores/sangue , Humanos , Espectrometria de Massas , Metabolômica/métodos , Análise Multivariada , Proteômica/métodos , Lesões por Radiação/diagnóstico , Radiação IonizanteRESUMO
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.
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Monócitos , Migração Transendotelial e Transepitelial , Humanos , Movimento Celular , Células Endoteliais , QuimiotaxiaRESUMO
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.
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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.
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Senescência Celular , Células Endoteliais , Animais , Células Endoteliais/patologia , Camundongos , Fenótipo , Transdução de SinaisRESUMO
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.
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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.
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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 mm2, 7 × 7 mm2, or 10 × 10 mm2 beam collimation for beam size effect and using 3 × 3 mm2 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 mm2 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 mm2 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.
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Pulmão/efeitos da radiação , Radiocirurgia/efeitos adversos , Animais , Bronquiolite/etiologia , Contagem de Células , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Fibrose , Pulmão/patologia , Masculino , Camundongos , Análise de SobrevidaRESUMO
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 p16INK4a-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 p16INK4a 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.
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Senescência Celular/efeitos da radiação , Lesão Pulmonar/patologia , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Relação Dose-Resposta à Radiação , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Lesão Pulmonar/diagnóstico por imagem , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Camundongos , Tomografia Computadorizada por Raios XRESUMO
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.
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Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Pulmonares/radioterapia , Pulmão/efeitos da radiação , Órgãos em Risco/efeitos da radiação , Radiocirurgia/métodos , Radioterapia de Intensidade Modulada/métodos , Animais , Tomografia Computadorizada de Feixe Cônico , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Deleção de Genes , Hibridização Genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Integrases/metabolismo , Pulmão/diagnóstico por imagem , Camundongos , Órgãos em Risco/diagnóstico por imagem , Fenótipo , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/efeitos da radiação , Edema Pulmonar/prevenção & controle , Fibrose Pulmonar/diagnóstico por imagem , Doses de Radiação , Pneumonite por Radiação/diagnóstico por imagem , Radiocirurgia/efeitos adversos , Radioterapia de Intensidade Modulada/efeitos adversos , Corrida/fisiologia , Seleção ArtificialRESUMO
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.
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Histonas/efeitos da radiação , Fótons/efeitos adversos , Eficiência Biológica Relativa , Raios X/efeitos adversos , Sobrevivência Celular/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Marcadores Genéticos/efeitos da radiação , Histonas/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Transferência Linear de Energia , Estudo de Prova de ConceitoRESUMO
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.
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Endotélio Vascular/fisiopatologia , Imunidade/efeitos da radiação , Lesões por Radiação/fisiopatologia , Radioterapia/efeitos adversos , Apoptose/efeitos da radiação , Endotélio Vascular/imunologia , Endotélio Vascular/efeitos da radiação , Humanos , Radioterapia (Especialidade)RESUMO
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.
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Fracionamento da Dose de Radiação , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Transcriptoma/efeitos da radiação , Análise por Conglomerados , Humanos , Família Multigênica , Distribuição Normal , Fenótipo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
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.
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Radiometria/instrumentação , Desenho de Equipamento , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Raios XRESUMO
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-1αFL/FL mice present with lower radiation-induced damage, showed a preserved vasculature, reduced hypoxia, and reduced proinflammatory response compared with irradiated HIF-1αFL/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.
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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.
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Endotélio Vascular/metabolismo , Transição Epitelial-Mesenquimal , Pulmão/metabolismo , Pulmão/efeitos da radiação , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Animais , Movimento Celular , Endotélio Vascular/efeitos da radiação , Técnicas de Inativação de Genes/métodos , Humanos , Pulmão/citologia , Macrófagos , Camundongos , Camundongos Knockout , Neutrófilos , Inibidor 1 de Ativador de Plasminogênio/deficiência , Inibidor 1 de Ativador de Plasminogênio/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transição Epitelial-Mesenquimal/genética , Deleção de Genes , Proctite/etiologia , Lesões por Radiação/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Células Cultivadas , Transição Epitelial-Mesenquimal/efeitos da radiação , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Camundongos , Fenótipo , Proctite/metabolismo , Proctite/patologia , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Proteínas Repressoras/metabolismo , TranscriptomaRESUMO
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.