The Protein Kinase A Inhibitor KT5720 Prevents Endothelial Dysfunctions Induced by High-Dose Irradiation.

High-dose irradiation can trigger numerous endothelial dysfunctions, including apoptosis, the overexpression of adhesion molecules, and alteration of adherens junctions. Altogether, these endothelial dysfunctions contribute to the development of tissue inflammation and organ damage. The development of endothelial dysfunctions may depend on protein phosphorylation by various protein kinases, but the possible role of protein kinase A (PKA) has not been investigated so far, and efficient compounds able to protect the endothelium from irradiation effects are needed. Here we report the beneficial effects of the PKA inhibitor KT5720 on a panel of irradiation-induced endothelial dysfunctions in human pulmonary microvascular endothelial cells (HPMECs). High-dose X-irradiation (15 Gy) triggered the late apoptosis of HPMECs independent of the ceramide/P38 MAP kinase pathway or p53. In contrast, the treatment of HPMECs with KT5720 completely prevented irradiation-induced apoptosis, whether applied before or after cell irradiation. Immunostainings of irradiated monolayers revealed that KT5720 treatment preserved the overall integrity of endothelial monolayers and adherens junctions linking endothelial cells. Real-time impedance measurements performed in HPMEC monolayers confirmed the overall protective role of KT5720 against irradiation. Treatment with KT5720 before or after irradiation also reduced irradiation-induced ICAM-1 overexpression. Finally, the possible role for PKA in the development of endothelial dysfunctions is discussed, but the potency of KT5720 to inhibit the development of a panel of irradiation-induced endothelial dysfunctions, whether applied before or after irradiation, suggests that this compound could be of great interest for both the prevention and treatment of vascular damages in the event of exposure to a high dose of radiation.

Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

In Situ Identification of Both IL-4 and IL-10 Cytokine-Receptor Interactions during Tissue Regeneration.

Cytokines secreted by individual immune cells regulate tissue regeneration and allow communication between various cell types. Cytokines bind to cognate receptors and trigger the healing process. Determining the orchestration of cytokine interactions with their receptors on their cellular targets is essential to fully understanding the process of inflammation and tissue regeneration. To this end, we have investigated the interactions of Interleukin-4 cytokine (IL-4)/Interleukin-4 cytokine receptor (IL-4R) and Interleukin-10 cytokine (IL-10)/Interleukin-10 cytokine receptor (IL-10R) using in situ Proximity Ligation Assays in a regenerative model of skin, muscle and lung tissues in the mini-pig. The pattern of protein-protein interactions was distinct for the two cytokines. IL-4 bound predominantly to receptors on macrophages and endothelial cells around the blood vessels while the target cells of IL-10 were mainly receptors on muscle cells. Our results show that in situ studies of cytokine-receptor interactions can unravel the fine details of the mechanism of action of cytokines.

The immunosuppressant drug Cyclosporin A aggravates irradiation effects in endothelial cells.

The immunosuppressant drug Cyclosporin A (CsA) has been widely used to prevent the development of Graft-versus-Host Disease (GvHD) that can occur after transplantation, including allogeneic graft after accidental high-dose irradiation in humans. Here, we show that CsA alone stimulates ICAM-1 overexpression in human pulmonary microvascular endothelial cells (HPMECs) through Toll-Like Receptor 4 (TLR4) and NF-κB activation. In HPMECs, CsA treatment significantly worsened the overexpression of ICAM-1 induced by high-dose irradiation (15 Gy). This additive effect of CsA was also observed when ICAM-1 overexpression was induced by another pathway (Ca2+ entry) in macrovascular endothelial cells. In addition, CsA triggered apoptosis as well as rearrangement of the actin cytoskeleton and adherens junctions (VE-Cadherin) in microvascular endothelial monolayers. High-dose irradiation triggered similar deleterious effects in endothelial monolayers and, again, CsA treatment strongly aggravated the effects of irradiation. Altogether, these results suggest that post-transplant CsA treatment may exacerbate the deleterious effects of irradiation on the endothelium.

Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ.

Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ.

TRPV4 channel activation induces the transition of venous and arterial endothelial cells toward a pro-inflammatory phenotype.

The Transient Receptor Potential Vanilloid 4 (TRPV4) of endothelial cells contributes to many important functions including the regulation of Ca2+ homeostasis, cell volume, endothelial barrier permeability, and smooth muscle tone. However, its role in the transition of endothelial cells toward a pro-inflammatory phenotype has not been studied so far. Using both arterial and venous endothelial cells, we first show that the pharmacological activation of TRPV4 channels with GSK1016790A, a potent TRPV4 agonist, triggers robust and sustained Ca2+ increases, which are blocked by both TRPV4 antagonists HC067047 and RN9893. TRPV4 activation also triggers the actin cytoskeleton and adherens junction (VE-Cadherin) rearrangement in both arterial and venous endothelial cells and leads to rapid decreases of trans-endothelial electrical resistance. In addition to its effect on endothelial barrier integrity, TRPV4 activation selectively increases ICAM-1 surface expression in arterial and venous endothelial cells, due to the stimulation of ICAM-1 gene expression through the NF-κB transcription factor. TRPV4 channel activation also induced apoptosis of venous and arterial endothelial cells, while TRPV4 blockade reduced apoptosis, even in the absence of TRPV4 activation. As altered barrier integrity, increased adhesion molecule expression and apoptosis are hallmarks of the pro-inflammatory state of endothelial cells, our results indicate that TRPV4 channel activity can induce the transition of both venous and arterial endothelial cells toward a pro-inflammatory phenotype.

Characterization of macrophages, giant cells and granulomas during muscle regeneration after irradiation.

Macrophages play a fundamental role in the different stages of muscle regeneration although the precise mechanisms involved are not entirely understood. Here we investigated the types of macrophages and cytokines that appeared in muscles after local gamma irradiation of mini-pigs that underwent no subsequent treatment or received three successive adipose tissue-derived stem cell (ASC) injections. Although some variability was observed among the three animals included in each study group, a general picture emerged. No macrophages appeared in control muscles from regions that had not been irradiated nor in muscles from irradiated regions derived from two animals. A third irradiated, but untreated animal, with characteristic muscle fibrosis and necrosis due to irradiation, showed invasion of M2 macrophages within small muscle lesions. In contrast, among the three ASC-treated and irradiated animals, one of them had completely recovered normal muscle architecture at the time of sampling with no invading macrophages, muscle from a second one contained mostly M1 macrophages and some M2-like macrophages whereas muscle from a third one displayed granulomas and giant cells. ASC treatment was associated with the presence of similar levels of pro-inflammatory cytokines within the two animals in the process of muscle regeneration whereas the levels of IL-4 and IL-10 expression were distinct from one animal to another. Microspectrofluorimetry and in situ hybridization revealed strong expression of TGF-ß1 and TNFα in regenerating muscle. Overall, the data confirm the critical role of macrophages in muscle regeneration and suggest the involvement of a complex network of cytokine expression for successful recovery.

Hybridization-chain-reaction is a relevant method for in situ detection of M2d-like macrophages in a mini-pig model.

Macrophages are a heterogeneous population of cells with an important role in innate immunity and tissue regeneration. Based on in vitro experiments, macrophages have been subdivided into five distinct subtypes named M1, M2a, M2b, M2c, and M2d, depending on the means of their activation and the cell surface markers they display. Whether all subtypes can be detected in vivo is still unclear. The identification of macrophages in vivo in the regenerating muscle could be used as a new diagnostic tool to monitor therapeutic strategies for tissue repair. The use of classical immunolabeling techniques is unable to discriminate between different M2 macrophages and a functional characterization of these macrophages is lacking. Using in situ hybridization coupled with hybridization-chain-reaction detection (HCR), we achieved the identification of M2d-like macrophages within regenerating muscle and applied this technique to understand the role of M2 macrophages in the regeneration of irradiated pig-muscle after adipose tissue stem cell treatment. Our work highlights the limits of immunolabeling and the usefulness of HCR analysis to provide valuable information for macrophage characterization.

Bringing Radiation Exposures and Associated Health Risks into Perspective-Development of an App.

The NATO HFM 291 research task group (RTG) on "Ionizing Radiation Bioeffects and Countermeasures" represents a group of scientists from military and civilian academic and scientific institutions primarily working in the field of radiobiology. Among other tasks, the RTG intends to extend their work on risk estimation and communication to bridge the gap in appropriate judgment of health risks given a certain radiation exposure. The group has no explicit psychological background but an expertise in radiobiology and risk assessment. The group believes that, as one of the essential first steps in risk communication, it is required to put radiation risk into perspective. Radiation risk requires a weight in comparison to already-known risks. What we envision is to Compare Radiation exposure-associated health Risks (CRRis App) with daily life health risks caused by other common exposures such as cigarette smoking, driving a car, etc. Within this paper, we provide (1) an overview of health risks after radiation exposure, (2) an explanation of the task and concept of an envisioned CRRis App, (3) an overview of existing software tools related to this issue, (4) a summary of inputs and discussions with experts in the field of radiation protection and risk communication during the ConRad conference, and finally, (5) identification of the next steps in the development of the App.

MSC-Derived Extracellular Vesicles: New Emergency Treatment to Limit the Development of Radiation-Induced Hematopoietic Syndrome?

Nuclear accidents or acts of terrorism involving radioactive sources might lead to mass casualties irradiation. The hematopoietic system is one of the most critical and radiation-sensitive tissues because the limited life span of blood cells requires the continuous division of hematopoietic stem cells (HSCs) into the bone marrow. The radiation-induced hematopoietic syndrome, RI-HS, is an impairment of the hematopoiesis that will result in pancytopenia of various degrees. In fact, treatment with granulocyte-colony stimulating factor (G-CSF) is considered as a valuable adjunct to treatment controls in some irradiated patients. Nevertheless, these overexposed patients with bone marrow suppression have minimal medullary territories that do not allow complete recovery of hematopoiesis but lead to significant immunoreactivity following allogeneic hematopoietic stem cell transplantation (HSCT). The high morbidity and mortality of these overexposed patients is a reminder of the lack of effective treatment for hematopoietic syndrome. During the last 20 y, a therapeutic approach for mesenchymal stem cells (MSC) has been proposed for the management of accidentally irradiated victims. Many preclinical animal studies have shown that MSC, mainly by their secretory activity, in particular extracellular vesicles (EVs), contribute to the control of inflammation and promote regeneration of tissues by accelerating angiogenesis and re-epithelialization processes. Therefore, we investigated the potential effect of EVs on the reduction of early bone marrow ionization toxicity, early anti-apoptotic therapy, and vascular protection in the RI-HS model. The main purpose is to propose an innovative treatment of non-patient-specific RI-HS emergency treatment in order to limit allogeneic HSC.

First Insights Into the M2 Inflammatory Response After Adipose-Tissue-Derived Stem Cell Injections in Radiation-Injured Muscles.

The cutaneous radiation syndrome is the clinical consequence of local high-dose irradiation. It is characterized by extensive inflammation, necrosis, and poor revascularization of the skin, resulting in muscle inflammation and fibrosis. Based on these physiopathological processes, subcutaneous injections of adipose-tissue-derived stem/stromal cells have shown favorable effects on skin-wound healing in a minipig model of cutaneous radiation syndrome, in which muscle fibrosis persisted. Since fibrosis is mainly due to the inflammatory processes that often affect underlying tissues as well, the beneficial effects of intramuscular injections of adipose-tissue-derived stem/stromal cells on tissue recovery were evaluated. The polarization of the inflammatory response of irradiated muscle in a minipig model of cutaneous radiation syndrome was determined after acute local irradiation with 50 Gy gamma rays in a preliminary study (six minipigs). Analysis of the main inflammatory cytokines of the inflammatory response M1 (IL-1-beta and IL-6) and M2 (IL-10 and TGF-beta) by western blotting and in situ hybridization, as well as analysis of CD80/CD206 M1/M2 macrophage-specific markers by immunohistochemistry on minipig muscle samples, was performed 76 d after irradiation. The treatment of irradiated muscles with autologous adipose-tissue-derived stem/stromal cells led to an increase in IL-10 and TGF-beta, being associated with an increase in CD68+/CD206+ cells in this area. This highlights a polarization of M2 in the inflammatory response and indicates that adipose-tissue-derived stem/stromal cells may direct the irradiated tissues' inflammatory response towards a proregenerative outcome.

French Policies for Victim Management During Mass Radiological Accidents/Attacks.

In the current international context, emergency medical services have to be prepared for chemical, biological, radiological, and nuclear events. Emergency response to radiological or nuclear events requires coordination between many components: the fire brigade, emergency medical services (including nurses and physicians), police, hospitals, etc. To optimize efficiency, victim management in France is governed by specific policies and planned responses. This plan for radiological/nuclear event response is inspired by military chemical, biological, radiological, and nuclear victim management in the operational theatre and is based on extraction (removal to a safe environment), first triage, decontamination, second triage, treatment, substance identification, and training. It is also supported by specific equipment. Prehospital victim management in case of nuclear and radiological accident or attack will be described, as well as French-specific supplies. This response plan is constantly evolving due to the complexity of radiological and nuclear events.

Contribution of INTRAMUSCULAR Autologous Adipose Tissue-Derived Stem Cell Injections to Treat Cutaneous Radiation Syndrome: Preliminary Results.

Cutaneous radiation syndrome caused by high dose located irradiation is characterized by delayed symptoms, incomplete wound healing, and poor revascularization. Subcutaneous adipose tissue derived stromal/stem cells have been shown to improve skin repair in a minipig model of cutaneous radiation syndrome despite a subcutaneous defect being a consequence of radio-induced muscular fibrosis. Based on the pro-myogenic potential of stromal/stem cells, a new protocol combining subcutaneous and intramuscular injections was evaluated in a preliminary study. Six female minipigs were locally irradiated at the dose of 50 Gy using a Co source (0.6 Gy min) and randomly divided into two groups. Three animals received the vehicle (phosphate-buffer-saline solution) and three animals received three injections of 75 × 10 adipose tissue derived stromal/stem cells each time (day 25, 46, and 66 post-irradiation). Pigs were euthanized on day 76 post-irradiation before development of clinical skin symptoms. All minipigs exhibited a homogeneous skin evolution. Macroscopic observation of irradiated muscles showed prominent fibrosis and necrosis areas in controls as opposed to adipose tissue-derived stromal/stem cells injected animals. Moreover, muscle biopsy analysis highlighted a recruitment of myofibroblasts (Immune Reactive Score: p < 0.01), an interleukin 10 secretion and a muscle regeneration pathway activation after intramuscular injections of adipose tissue-derived stromal/stem cells (western-blot: respectively, 200-fold change difference and twofold higher in treated animals). Globally, these preliminary data suggest that intramuscular injections of adipose tissue-derived stromal/stem cells improve muscle regeneration in the cutaneous-radiation syndrome. Further work is ongoing to evaluate this therapeutic strategy on a larger animal number with a longer clinical follow-up.

Transient gene therapy to treat cutaneous radiation syndrome: development in a minipig model.

Cutaneous radiation syndrome is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Adipocyte derived stem cells injection may improve tissue regeneration through secreted factors. Thus mesenchymal stem cells secretome optimization, using transient transfection, may represent a new strategy to treat this syndrome. Sonic hedgehog, a secreted protein involved in cell proliferation and angiogenesis, has been chosen as a first candidate. Here preliminary results are reported of the therapeutic potential of transient gene therapy to cure cutaneous radiation syndrome in a minipig model. Adipocyte derived stem cells were transiently transfected by electroporation with a plasmid coding for Sonic Hedgehog. Göttingen minipigs were locally irradiated using a (60)Co gamma source at the dose of 50 Gy and received Phosphate Buffer Salin (controls: n = 8), stem cells (50 × 106 each time, n = 5) or transfected stem cells (25±7 × 106 each time, n = 1). All controls exhibited a homogeneous clinical evolution of cutaneous radiation syndrome with final necrosis (day 91). In stem cell injected minipigs, an ultimate wound healing was observed in four out of five grafted animals (day 130 ± 28, complete in two of them) (historical results). The Sonic hedgehog animal, albeit injected with a lower number of transfected stem cells, presented a very similar evolution of skin healing without necrosis or uncontrolble pain. Globally this preliminary report suggests that local injection of Sonic Hedgehog transfected adipocyte derived stem cells may improve wound healing. Thus work is ongoing to evaluate this therapeutic strategy on a larger number of animals.

Application of adipocyte-derived stem cells in treatment of cutaneous radiation syndrome.

Cutaneous radiation syndrome caused by local high dose irradiation is characterized by delayed outcome and incomplete healing. Recent therapeutic management of accidentally irradiated burn patients has suggested the benefit of local cellular therapy using mesenchymal stem cell grafting. According to the proposed strategy of early treatment, large amounts of stem cells would be necessary in the days following exposure and hospitalization, which would require allogeneic stem cells banking. In this context, the authors compared the benefit of local autologous and allogeneic adipocyte-derived stem cell injection in a large animal model. Minipigs were locally irradiated using a 60Co gamma source at a dose of 50 Gy and divided into three groups. Two groups were grafted with autologous (n = 5) or allogeneic (n = 5) adipocyte-derived stem cells four times after the radiation exposure, whereas the control group received the vehicle without cells (n = 8). A clinical score was elaborated to compare the efficiency of the three treatments. All controls exhibited local inflammatory injuries leading to a persistent painful necrosis, thus mimicking the clinical evolution in human victims. In the autologous adipocyte-derived stem cells group, skin healing without necrosis or uncontrollable pain was observed. In contrast, the clinical outcome was not significantly different in the adipocyte-derived stem cell allogeneic group when compared with controls. This study suggests that autologous adipocyte-derived stem cell grafting improves cutaneous radiation syndrome wound healing, whereas allogeneic adipocyte derived stem cells do not. Further studies will establish whether manipulation of allogeneic stem cells will improve their therapeutic potential.

Autologous adipocyte derived stem cells favour healing in a minipig model of cutaneous radiation syndrome.

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a (60)Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50 × 10(6) ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/- 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS.