mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Lipid Polyunsaturated Fatty Acid Chains in Mouse Kidneys Were Increased within 5 min of a Single High Dose Whole Body Irradiation.

To understand the ultra-early reaction of normal organ lipids during irradiation, we investigated the response of lipids, including polyunsaturated fatty acid (PUFA) chains, which are particularly susceptible to damage by ROS, in mice's kidneys, lungs, brains, and livers within 5 min of single high-dose irradiation. In this study, we set up three groups of C56BL/6 male mice and conducted whole-body irradiation with 0 Gy, 10 Gy, and 20 Gy single doses. Kidney, lung, brain, and liver tissues were collected within 5 min of irradiation. PUFA-targeted and whole lipidomic analyses were conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that PUFA chains of kidney phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG) significantly increased within 5 min of 10 Gy and 20 Gy irradiation. The main components of increased PUFA chains in PC and PE were C18:2, C20:4, and C22:6, and in TG the main component was C18:2. The kidney lipidomes also showed significant changes from the perspective of lipid species, mainly dominated by an increase in PC, PE, TG, and signal lipids, while lipidomes of the lung, brain, and liver were slightly changed. Our results revealed that acute PUFA chains increase and other lipidomic changes in the kidney upon whole-body irradiation within 5 min of irradiation. The significantly increased lipids also showed a consistent preference for possessing PUFA chains. The lipidomic changes varied from organ to organ, which indicates that the response upon irradiation within a short time is tissue-specific.

Archetypal Arg169Cys mutation in NOTCH3 does not drive the pathogenesis in cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy via a loss-of-function mechanism.

BACKGROUND AND PURPOSE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy, the most common heritable small vessel disease of the brain, is caused by dominant mutations in the NOTCH3 receptor that stereotypically lead to age-dependent Notch3ECD deposition in the vessels. NOTCH3 loss of function has been demonstrated for few mutations. However, whether this finding applies to all mutations and whether a loss-of-function mechanism drives the manifestations of the disease remain yet unknown. This study investigated the in vivo functionality of the Arg169Cys archetypal mutation. METHODS: We used mice with constitutive or conditional reduction of NOTCH3 activity, mice harboring the Arg169Cys mutation at the endogenous Notch3 locus (Notch3Arg170Cys), and mice overexpressing the Arg169Cys NOTCH3 mutant (TgPAC-Notch3R169C) on either a Notch3 wild-type or a null background. NOTCH3 activity was monitored in the brain arteries by measuring the expression of NOTCH3 target genes using real-time polymerase chain reaction. Notch3ECD deposits were assessed by immunohistochemistry. Brain parenchyma was analyzed for vacuolation and myelin debris in the white matter and infarcts. RESULTS: We identified a subset of genes appropriate to detect NOTCH3 haploinsufficiency in the adult. Expression of these genes was unaltered in Notch3Arg170Cys mice, despite marked Notch3ECD deposits. Elimination of wild-type NOTCH3 did not influence the onset and burden of white matter lesions in 20-month-old TgPAC-Notch3R169C mice, and 20-month-old Notch3-null mice exhibited neither infarct nor white matter changes. CONCLUSIONS: These data provide strong evidence that cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy can develop without impairment of NOTCH3 signaling and argue against a loss of NOTCH3 function as a general driving mechanism for white matter lesions in cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy.

Transcriptome analysis for Notch3 target genes identifies Grip2 as a novel regulator of myogenic response in the cerebrovasculature.

OBJECTIVE: Notch3 is critically important for the structure and myogenic response of distal arteries, particularly of cerebral arteries. However, signaling pathways acting downstream of Notch3 remain largely unknown. METHODS AND RESULTS: Transcriptome analysis using tail arteries of Notch3-null mice identified a core set of 17 novel Notch3-regulated genes confirmed in tail or brain arteries. Postnatal deletion of RBP-Jκ in smooth muscle cells recapitulated the structural, functional, and molecular defects of brain arteries induced by Notch3 deficiency. Transient in vivo blockade of the Notch pathway with γ-secretase inhibitors uncovered, in addition to Notch3, 6 immediate responders, including the voltage-gated potassium channel Kv1.5, which opposes to myogenic constriction of brain arteries, and the glutamate receptor-interacting protein 2 (Grip2) with no previously established role in the cerebrovasculature. We identified a vascular smooth muscle cell isoform of Grip2. We showed that Notch3-RBP-Jκ specifically regulates this isoform. Finally, we found that cerebral arteries of Grip2 mutant mice, which express an N-terminally truncated Grip2 protein, exhibited selective attenuation of pressure-induced contraction. CONCLUSIONS: Our data provide insight into how Notch3 signals in the brain arteries, establishing the postnatal requirement of smooth muscle RBP-Jκ in this context. Notch3-regulated transcriptome provides potential for modulating myogenic response in the cerebrovasculature.

A point cloud segmentation framework for image-based spatial transcriptomics.

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated and experimental datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and in-situ cell type calling. ComSeg is available as a documented and open source pip package at https://github.com/fish-quant/ComSeg .

Differential Remodeling of the Oxylipin Pool After FLASH Versus Conventional Dose-Rate Irradiation In Vitro and In Vivo.

PURPOSE: The products of lipid peroxidation have been implicated in human diseases and aging. This prompted us to investigate the response to conventional (CONV) versus FLASH irradiation of oxylipins, a family of bioactive lipid metabolites derived from omega-3 or omega-6 polyunsaturated fatty acids through oxygen-dependent non-enzymatic as well as dioxygenase-mediated free radical reactions. METHODS AND MATERIALS: Ultrahigh performance liquid chromatography coupled to tandem mass spectrometry was used to quantify the expression of 37 oxylipins derived from eicosatetraenoic, eicosapentaenoic and docosahexaenoic acid in mouse lung and in normal or cancer cells exposed to either radiation modality under precise monitoring of the temperature and oxygenation. Among the 37 isomers assayed, 14-16 were present in high enough amount to enable quantitative analysis. The endpoints were the expression of oxylipins as a function of the dose of radiation, normoxia versus hypoxia, temperature and post-irradiation time. RESULTS: In normal, normoxic cells at 37°C radiation elicited destruction and neosynthesis of oxylipins acting antagonistically on a background subject to rapid remodeling by oxygenases. Neosynthesis was observed in the CONV mode only, in such a way that the level of oxylipins at 5 minutes after FLASH irradiation was 20-50% lower than in non-irradiated and CONV-irradiated cells. Hypoxia mitigated the differential CONV versus FLASH response in some oxylipins. These patterns were not reproduced in tumor cells. Depression of specific oxylipins following FLASH irradiation was observed in mouse lung at 5 min following irradiation, with near complete recovery in 24 hours and further remodeling at one week and two months post-irradiation. CONCLUSIONS: Down-regulation of oxylipins was a hallmark of FLASH irradiation specific of normal cells. Temperature effects suggest that this process occurs via diffusion-controlled, bimolecular recombination of a primary radical species upstream from peroxyl radical formation and evoke a major role of the membrane composition and fluidity in response to the FLASH modality.

The AsiDNA™ decoy mimicking DSBs protects the normal tissue from radiation toxicity through a DNA-PK/p53/p21-dependent G1/S arrest.

AsiDNA™, a cholesterol-coupled oligonucleotide mimicking double-stranded DNA breaks, was developed to sensitize tumour cells to radio- and chemotherapy. This drug acts as a decoy hijacking the DNA damage response. Previous studies have demonstrated that standalone AsiDNA™ administration is well tolerated with no additional adverse effects when combined with chemo- and/or radiotherapy. The lack of normal tissue complication encouraged further examination into the role of AsiDNA™ in normal cells. This research demonstrates the radioprotective properties of AsiDNA™. In vitro, AsiDNA™ induces a DNA-PK/p53/p21-dependent G1/S arrest in normal epithelial cells and fibroblasts that is absent in p53 deficient and proficient tumour cells. This cell cycle arrest improved survival after irradiation only in p53 proficient normal cells. Combined administration of AsiDNA™ with conventional radiotherapy in mouse models of late and early radiation toxicity resulted in decreased onset of lung fibrosis and increased intestinal crypt survival. Similar results were observed following FLASH radiotherapy in standalone or combined with AsiDNA™. Mechanisms comparable to those identified in vitro were detected both in vivo, in the intestine and ex vivo, in precision cut lung slices. Collectively, the results suggest that AsiDNA™ can partially protect healthy tissues from radiation toxicity by triggering a G1/S arrest in normal cells.

An interactive murine single-cell atlas of the lung responses to radiation injury.

Radiation Induced Lung Injury (RILI) is one of the main limiting factors of thorax irradiation, which can induce acute pneumonitis as well as pulmonary fibrosis, the latter being a life-threatening condition. The order of cellular and molecular events in the progression towards fibrosis is key to the physiopathogenesis of the disease, yet their coordination in space and time remains largely unexplored. Here, we present an interactive murine single cell atlas of the lung response to irradiation, generated from C57BL6/J female mice. This tool opens the door for exploration of the spatio-temporal dynamics of the mechanisms that lead to radiation-induced pulmonary fibrosis. It depicts with unprecedented detail cell type-specific radiation-induced responses associated with either lung regeneration or the failure thereof. A better understanding of the mechanisms leading to lung fibrosis will help finding new therapeutic options that could improve patients' quality of life.

Lung Organotypic Slices Enable Rapid Quantification of Acute Radiotherapy Induced Toxicity.

To rapidly assess healthy tissue toxicities induced by new anti-cancer therapies (i.e., radiation alone or in combination with drugs), there is a critical need for relevant and easy-to-use models. Consistent with the ethical desire to reduce the use of animals in medical research, we propose to monitor lung toxicity using an ex vivo model. Briefly, freshly prepared organotypic lung slices from mice were irradiated, with or without being previously exposed to chemotherapy, and treatment toxicity was evaluated by analysis of cell division and viability of the slices. When exposed to different doses of radiation, this ex vivo model showed a dose-dependent decrease in cell division and viability. Interestingly, monitoring cell division was sensitive enough to detect a sparing effect induced by FLASH radiotherapy as well as the effect of combined treatment. Altogether, the organotypic lung slices can be used as a screening platform to rapidly determine in a quantitative manner the level of lung toxicity induced by different treatments alone or in combination with chemotherapy while drastically reducing the number of animals. Translated to human lung samples, this ex vivo assay could serve as an innovative method to investigate patients' sensitivity to radiation and drugs.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

A Comprehensive Analysis of the Relationship Between Dose Rate and Biological Effects in Preclinical and Clinical Studies, From Brachytherapy to Flattening Filter Free Radiation Therapy and FLASH Irradiation.

PURPOSE: For many years, the effect of dose rate (DR) was considered negligible in external beam radiation therapy (EBRT) until very-high DR (>10 Gy/min) became possible and ultrahigh DR (>40 Gy/s) showed dramatic protection of normal tissues in preclinical experiments. We propose a critical review of preclinical and clinical studies to investigate the biological and clinical effects of DR variation in the range covering brachytherapy to flattening filter free EBRT and FLASH. METHODS AND MATERIALS: Preclinical and clinical studies investigating biological and clinical DR effects were reviewed extensively. We also conducted an in silico study to assess the effect of pulse DR (DRp), taking into account the mean time between 2 tracks during the pulse. RESULTS: Preclinical studies have shown that an increase in DR in the range of 0.01 to 20 Gy/min (not including ultralow or ultrahigh DR) resulted in decreased survival of both normal and tumor cells. This effect was attributed primarily to increasingly unrepaired "sublethal" DNA damage with increasing the DR. However, the models and irradiation conditions have often been very different from one radiobiological study to another. Moreover, the physical parameters on the spatial and temporal microstructure of the beam were not considered systematically. In particular, the DRp was rarely mentioned. The in silico studies showed that for the same average DR, increasing DRp induced an increase of mean track rates. These results could explain the presence of more complex damage when the DRp was increased within the range of DR considered, in relation to the time-dependent probability of accumulating unrepaired, "sublethal" DNA lesions in close proximity. CONCLUSIONS: Knowledge of the beam microstructure is critical to understanding the biological impact and the clinical outcomes of radiation at the DR commonly used in radiation therapy.

FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence.

PURPOSE: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. EXPERIMENTAL DESIGN: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc-/- mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. RESULTS: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc-/- mice harboring critically short telomeres and lack of telomerase activity. CONCLUSIONS: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

The European Joint Research Project UHDpulse - Metrology for advanced radiotherapy using particle beams with ultra-high pulse dose rates.

UHDpulse - Metrology for advanced radiotherapy using particle beams with ultra-high pulse dose rates is a recently started European Joint Research Project with the aim to develop and improve dosimetry standards for FLASH radiotherapy, very high energy electron (VHEE) radiotherapy and laser-driven medical accelerators. This paper gives a short overview about the current state of developments of radiotherapy with FLASH electrons and protons, very high energy electrons as well as laser-driven particles and the related challenges in dosimetry due to the ultra-high dose rate during the short radiation pulses. We summarize the objectives and plans of the UHDpulse project and present the 16 participating partners.

Simulation and experimental validation of a prototype electron beam linear accelerator for preclinical studies.

PURPOSE: Measurements and Monte-Carlo simulations were carried out to model the dose distribution of a prototype electron beam linear accelerator (Kinetron LINAC) to determine the dose to organs in small animal irradiations experiments. Dose distributions were simulated using the GATE8.0/Geant4.10.3 Monte-Carlo platform, and measured in air and solid water phantoms using a commercial scintillating screen detector and new EBT-XD Gafchromic films. METHODS: The LINAC is able to produce 4.5 MeV electron beams at dose-rates ranging from Gy/min to thousands of Gy/s, and is used to study the radiobiological effects of very-high dose-rates that have been shown to protect normal tissues from radiation toxicity. Numerical simulations and experimental dosimetric characterisation of this electron accelerator were performed with the Monte-Carlo toolkit and various detectors. Absolute dose distributions in solid water were measured and compared with simulations. Realistic electron irradiation conditions were simulated in voxelised mice CT images. 3D dose distributions and dose-volume histograms in lungs of mice were simulated and analyzed. RESULTS: Measured and calculated depth-dose profiles for several beam configurations (energy and dose-rate) were compared. Beam emittance was validated by comparing measured and calculated beam sizes along the central axis in air: the deviation for all conditions was less than 1 mm. A good agreement was obtained between experimental dose distributions and the results obtained with simulations (<2% dose differences for lateral and depth-dose profiles). CONCLUSIONS: The method presented here, relying on few free parameters, can be adapted to very-high dose-rate electron irradiation to support the analysis of preclinical research experiments.

Activating NOTCH3 mutation in a patient with small-vessel-disease of the brain.

The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the NOTCH3 receptor. NOTCH3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal NOTCH3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of NOTCH3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and NOTCH3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of NOTCH3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical NOTCH3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders NOTCH3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of NOTCH3 mutations and that NOTCH3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.

Experimental Set-up for FLASH Proton Irradiation of Small Animals Using a Clinical System.

PURPOSE: Recent in vivo investigations have shown that short pulses of electrons at very high dose rates (FLASH) are less harmful to healthy tissues but just as efficient as conventional dose-rate radiation to inhibit tumor growth. In view of the potential clinical value of FLASH and the availability of modern proton therapy infrastructures to achieve this goal, we herein describe a series of technological developments required to investigate the biology of FLASH irradiation using a commercially available clinical proton therapy system. METHODS AND MATERIALS: Numerical simulations and experimental dosimetric characterization of a modified clinical proton beamline, upstream from the isocenter, were performed with a Monte Carlo toolkit and different detectors. A single scattering system was optimized with a ridge filter and a high current monitoring system. In addition, a submillimetric set-up protocol based on image guidance using a digital camera and an animal positioning system was also developed. RESULTS: The dosimetric properties of the resulting beam and monitoring system were characterized; linearity with dose rate and homogeneity for a 12 × 12 mm2 field size were assessed. Dose rates exceeding 40 Gy/s at energies between 138 and 198 MeV were obtained, enabling uniform irradiation for radiobiology investigations of small animals in a modified clinical proton beam line. CONCLUSIONS: This approach will enable us to conduct FLASH proton therapy experiments on small animals, specifically for mouse lung irradiation. Dose rates exceeding 40 Gy/s were achieved, which was not possible with the conventional clinical mode of the existing beamline.