Stromal vascular fraction for the treatment of the radiation-induced gastrointestinal syndrome

International audienceAccidental or intentional radiation exposures have serious health consequences for exposed individuals and can affect a large number of people. Large volume irradiation at high irradiation doses induces multiple tissue lesions. The gastro-intestinal tract is particularly sensitive to irradiation and lethality. At dose more than 10 Gy results in diarrhea, dehydration, sepsis and intestinal bleeding with mortality within 10 post-exposure. Radiationinduced gastrointestinal syndrome (GIS) results from direct cytocidal effects on intestinal stem cells and crypt stroma impairing epithelial regeneration. Damaged intestinal epithelium significantly reduces the mucosal integrity and promotes systemic bacteria influx resulting in sepsis and death. Given the logistical hurdle and the urgency for treatment in large numbers of casualties, there is a tremendous need for effective therapeutic measures, even if implemented several days after radiation exposure. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, antiinflammatory, immunomodulatory, and regenerative properties. We examined whether SVF restores the irradiated intestinal cells niche and mitigates the GIS. At the day of abdominal irradiation (18Gy) mice were injected in systemic with SVF, obtained by enzymatic digestion of adipose tissue. Seven days post-irradiation, SVF treatment limited weight loss and inhibited intestinal permeability [1]. When injected before 24 hours post-irradiation, SVF limited the mortality. SVF has an anti-inflammatory effect in the intestine by repressing proinflammatory cytokines, accelerating the maturation of monocyte able to generate antiinflammatory macrophages. Immunohistological analyses of intestine showed that SVF treatment stimulated the regeneration of the epithelium by promoting numerous hyperproliferative zones. SVF restored the cell population in the intestinal stem cell compartment. The ex-vivo intestinal "organoid" model that mimics the clinical response confirmed that SVF treatment stimulated the intestinal stem cell compartment. With pleiotropic effects that contribute to limite radiation-induced lethality, SVF offers attractive prospects for the treatment of emergency GIS

BMP antagonists secreted by mesenchymal stromal cells improve colonic organoid formation: Application for the treatment of radiation-induced injury

International audienceRadiation therapy is crucial in the therapeutic arsenal to cure cancers, however non-neoplastic tissues around an abdominopelvic tumor can be damaged by ionizing radiation. In particular, the radio-induced death of highly proliferative stem/progenitors cells of the colonic mucosa could induce severe ulcers. The importance of sequelae for patients with gastrointestinal complications after radiotherapy and the absence of satisfactory management has opened the field to the testing of innovative treatments. The aim of this study was to use adult epithelial cells from the colon, to reduce colonic injuries in an animal model reproducing radiation damage observed in patients. We demonstrated that transplanted in vitro-amplified epithelial cells from colonic organoids (ECO) of C57/Bl6 mice expressing GFP, implant, proliferate, and differentiate in irradiated mucosa and reduce ulcer size. To improve the therapeutic benefit of ECO-based treatment with clinical translatability, we performed co-injection of ECO with mesenchymal stromal cells (MSC), cells involved in niche function and widely used in clinical trials. We observed in vivo an improvement of the therapeutic benefit and in vitro analysis highlighted that co-culture of MSC with ECO increases the number, proliferation, and size of colonic organoids. We also demonstrated, using gene expression analysis and siRNA inhibition, the involvement of BMP-antagonists in MSC-induced organoid formation. This study provides evidence of the potential of ECO to limit late radiation effects on the colon and opens perspectives on combined strategies to improve their amplification abilities and therapeutic effects

Stromal vascular fraction for the treatment of the radiation-induced gastrointestinal syndrome

International audienceIntroduction: Accidental or intended high doses radiation exposures have serious consequences for the health of exposed people and may impact a large number of people (as well as military than civil). Exposure of a large volume at high irradiation doses induces multiple tissue lesions grouped under the name of Acute Radiation Syndrome (ARS). The gastrointestinal tract is especially sensitive to irradiation. At dose more than 10 Gy results in diarrhea, dehydration, sepsis and intestinal bleeding with death within 10 to 15 days post-exposure. Radiation-induced gastrointestinal syndrome results from direct cytocidal effects on intestinal stem cells and crypt stromal impairing epithelial regeneration. Irradiation rapidly reduces the mucosal integrity and promotes systemic bacteria influx resulting in sepsis and death. In this context, there is an urgent need for effective, rapid and applicable therapeutic measures for a large number of victims. Adipose-derived stromal vascular fraction (ADSVF) is an easily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties. We examined whether ADSVF protect irradiated intestinal cells niche and mitigate gastrointestinal syndrome. Methods: At the day of the abdominal irradiation (18Gy) mice were injected in systemic by the stromal vascular fraction (ADSVF) (2.5 10 6 cells), obtained through enzymatic digestion of inguinal adipose tissue. Results: We found that, at 7 days post-irradiation, treatment with ADSVF limited the weight loss of mice and reduced the intestinal permeability. Immunohistological analyses in intestinal tissues revealed an increase of regenerating crypts, a restoration of the stromal compartment and an increase of Ki67 + proliferating cells in response to ADSVF treatment. In addition, the treatment reduced significantly the expression of cytokines associated to inflammatory response in intestine and normalized the splenic immune cells (CD4, CD8, CD19) populations and increased the C11b/Ly6Clow/Cx3cr1 population. Conclusion: Treatment in emergency of gastrointestinlal syndrome could be achieved by intravenous injection of ADSVF inducing regeneration of intestine

The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice

International audienceBackground: The intestine is particularly sensitive to moderate-high radiation dose and the development of gastrointestinal syndrome (GIS) leads to the rapid loss of intestinal mucosal integrity, resulting in bacterial infiltration, sepsis that comprise patient survival. There is an urgent need for effective and rapid therapeutic countermeasures. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, anti-inflammatory and regenerative properties. We studied the therapeutic impact of SVF and its action on the intestinal stem cell compartment. Methods: Mice exposed to the abdomina l radiation (18 Gy) received a sing l e intravenous injection of stroma l vascu l ar fraction (SVF) (2.5 x 106 ce l l s), obtained by enzymatic digestion of inguina l fat tissue, on the day of irradiation. Morta l ity was eva l uated as we l l as intestina l regeneration by histologica l ana l yses and absorption function. Results: The SVF treatment l imited the weight l oss of the mice and inhibited the intestina l permeabi l ity and morta l ity after abdomina l irradiation. Histo l ogica l ana l yses showed that SVF treatment stimu l ated the regeneration of the epithe l ium by promoting numerous en l arged hyperpro l iferative zones. SVF restored CD24+/lysozyme_ and Paneth ce l l popu l ations in the ISC compartment with the presence of Paneth Ki67+ ce l l s. SVF has an anti-infl ammatory effect by repressing proinfl ammatory cytokines, increasing M2 macrophages in the il eum and anti-infl ammatory monocyte subtypes CD11 b+Ly6clowCX3CR1hgh in the spl een. Conclusions: Through the p l eiotropic effects that contribute to l imiting radiation-induced l etha l ity, SVF opens up attractive prospects for the treatment of emergency GIS

Long-term evolution of the epithelial cell secretome in preclinical 3D models of the human bronchial epithelium

International audienceThe human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions

Autologous Bone Marrow Mesenchymal Stem Cells Improve the Quality and Stability of Vascularized Flap Surgery of Irradiated Skin in Pigs.

International audienceCutaneous radiation syndrome has severe long-term health consequences. Because it causes an unpredictable course of inflammatory waves, conventional surgical treatment is ineffective and often leads to a fibronecrotic process. Data about the long-term stability of healed wounds, with neither inflammation nor resumption of fibrosis, are lacking. In this study, we investigated the effect of injections of local autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery for skin necrosis, in a large-animal model. Three months after irradiation overexposure to the rump, minipigs were divided into three groups: one group treated by simple excision of the necrotic tissue, the second by vascularized-flap surgery, and the third by vascularized-flap surgery and local autologous BM-MSC injections. Three additional injections of the BM-MSCs were performed weekly for 3 weeks. The quality of cutaneous wound healing was examined 1 year post-treatment. The necrotic tissue excision induced a pathologic scar characterized by myofibroblasts, excessive collagen-1 deposits, and inadequate vascular density. The vascularized-flap surgery alone was accompanied by inadequate production of extracellular matrix (ECM) proteins (decorin, fibronectin); the low col1/col3 ratio, associated with persistent inflammatory nodules, and the loss of vascularization both attested to continued immaturity of the ECM. BM-MSC therapy combined with vascularized-flap surgery provided mature wound healing characterized by a col1/col3 ratio and decorin and fibronectin expression that were all similar to that of nonirradiated skin, with no inflammation, and vascular stability. In this preclinical model, vascularized flap surgery successfully and lastingly remodeled irradiated skin only when combined with BM-MSC therapy. Stem Cells Translational Medicine 2018:569-582

3D model of the bronchial epithelial barrier to study repeated exposure to xenobiotics: Application to silver nanoparticles

International audienceThere is still a lack of in vitro human models to evaluate the chronic toxicity of drugs and environmental pollutants. Here, we used a 3D model of the human bronchial epithelium to assess repeated exposures to xenobiotics. The Calu-3 human bronchial cell line was exposed to silver nanoparticles (AgNP) 5 times during 12 days, at the air-liquid interface, to mimic single and repeated exposure to inhaled particles. Repeated exposures induced a stronger induction of the metal stress response and a steady oxidative stress over time. A sustained translocation of silver was observed after each exposure without any loss of the epithelial barrier integrity. The proteomic analysis of the mucus revealed changes in the secreted protein profiles associated with the epithelial immune response after repeated exposures only. These results demonstrate that advanced in vitro models are efficient to investigate the adaptive response of human cells submitted to repeated xenobiotic exposures

Pravastatin Limits Radiation-Induced Vascular Dysfunction in the Skin

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)-/- mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS-/- mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage

Cellular therapy treatment of chronic radiation cystitis in rats

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