Detecting time periods of differential gene expression using Gaussian processes: an application to endothelial cells exposed to radiotherapy dose fraction.

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.

Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis.

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.

Alteration of the serum N-glycome of mice locally exposed to high doses of ionizing radiation.

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.

Non-homogenous intratumor ionizing radiation doses synergize with PD1 and CXCR2 blockade.

The efficacy and side effects of radiotherapy (RT) depend on parameters like dose and the volume of irradiated tissue. RT induces modulations of the tumor immune microenvironment (TIME) that are dependent on the dose. Low dose RT (LDRT, i.e., single doses of 0.5-2 Gy) has been shown to promote immune infiltration into the tumor. Here we hypothesize that partial tumor irradiation combining the immunostimulatory/non-lethal properties of LDRT with cell killing/shrinkage properties of high dose RT (HDRT) within the same tumor mass could enhance anti-tumor responses when combined with immunomodulators. In models of colorectal and breast cancer in immunocompetent female mice, partial irradiation (PI) with millimetric precision to deliver LDRT (2 Gy) and HDRT (16 Gy) within the same tumor induces substantial tumor control when combined with anti-PD1. Using flow cytometry, cytokine profiling and single-cell RNA sequencing, we identify a crosstalk between the TIME of the differentially irradiated tumor volumes. PI reshapes tumor-infiltrating CD8+ T cells into more cytotoxic and interferon-activated phenotypes but also increases the infiltration of pro-tumor neutrophils driven by CXCR2. The combination of the CXCR2 antagonist SB225002 with PD1 blockade and PI improves tumor control and mouse survival. Our results suggest a strategy to reduce RT toxicity and improve the therapeutic index of RT and immune checkpoint combinations.

Evidence of Alveolar Macrophage Metabolic Shift Following Stereotactic Body Radiation Therapy -Induced Lung Fibrosis in Mice.

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.

The TG-interacting factor TGIF1 regulates stress-induced proinflammatory phenotype of endothelial cells.

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.

Deep models of integrated multiscale molecular data decipher the endothelial cell response to ionizing radiation.

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.

Variation of 4 MV X-ray dose rate in fractionated irradiation strongly impacts biological endothelial cell response in vitro.

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 postirradiation). 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.

A role for endothelial alpha-mannosidase MAN1C1 in radiation-induced immune cell recruitment.

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.

Deciphering the Dynamic Molecular Program of Radiation-Induced Endothelial Senescence.

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.

Preclinical Model of Stereotactic Ablative Lung Irradiation Using Arc Delivery in the Mouse: Is Fractionation Worthwhile?

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.

Preclinical Model of Stereotactic Ablative Lung Irradiation Using Arc Delivery in the Mouse: Effect of Beam Size Changes and Dose Effect at Constant Collimation.

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.

Stereotactic Lung Irradiation in Mice Promotes Long-Term Senescence and Lung Injury.

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.

Lung Stereotactic Arc Therapy in Mice: Development of Radiation Pneumopathy and Influence of HIF-1α Endothelial Deletion.

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.

Applying a multiscale systems biology approach to study the effect of chronic low-dose exposure to uranium in rat kidneys.

Purpose: To examine the effects of low-dose exposure to uranium with a systems biology approach, a multiscale high-throughput multi-omics analysis was applied with a protocol for chronic exposure to the rat kidney. Methods: Male and female rats were contaminated for nine months through their drinking water with a nontoxic solution of uranyl nitrate. A multiscale approach enabled clinical monitoring associated with metabolomic and transcriptomic (mRNA and microRNA) analyses. Results: A sex-interaction effect was observed in the kidney, urine, and plasma metabolomes of contaminated rats. Moreover, urine and kidney metabolic profiles correlated and confirmed that the primary dysregulated metabolisms are those of nicotinate-nicotinamide and of unsaturated fatty acid biosynthesis. Upstream of the metabolic pathways, transcriptomic profiles of the kidney reveal gene activity focused on gene regulation mechanisms, cell signaling, cell structure, developmental processes, and cell proliferation. Examination of epigenetic post-transcriptional gene regulation processes showed significant dysregulation of 70 micro-RNAs. The multi-omics approach highlighted the activities of the cells' biological processes on multiple scales through analysis of gene expression, confirmed by changes observed in the metabolome. Conclusion: Our results showed changes in multi-omic profiles of rats exposed to low doses of uranium contamination, compared with controls. These changes involved gene expression as well as modifications in the transcriptome and the metabolome. The metabolomic profile confirmed that the main molecular targets of uranium in kidney cells are the metabolism of nicotinate-nicotinamide and the biosynthesis of unsaturated fatty acids. Additionally, gene expression analysis showed that the metabolism of fatty acids is targeted by processes associated with cell function. These results demonstrate that multiscale systems biology is useful in elucidating the most discriminative pathways from genomic to metabolomic levels for assessing the biological impact of this low-level environmental exposure, i.e. the exposome.

Temporal clustering analysis of endothelial cell gene expression following exposure to a conventional radiotherapy dose fraction using Gaussian process clustering.

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.

HIF-1α Deletion in the Endothelium, but Not in the Epithelium, Protects From Radiation-Induced Enteritis.

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.

Conditional Plasminogen Activator Inhibitor Type 1 Deletion in the Endothelial Compartment Has No Beneficial Effect on Radiation-Induced Whole-Lung Damage in Mice.

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.

Endothelial Hey2 deletion reduces endothelial-to-mesenchymal transition and mitigates radiation proctitis in mice.

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.

Radiation-induced changes in the glycome of endothelial cells with functional consequences.

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.