Jocelyne Aigueperse

Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome

Recent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi‐organ failure syndrome following irradiation in a non‐human primate model.

Pravastatin Inhibits the Rho/CCN2/Extracellular Matrix Cascade in Human Fibrosis Explants and Improves Radiation-Induced Intestinal Fibrosis in Rats

Purposes: Intestinal complications after radiotherapy are caused by transmural fibrosis and impair the quality of life of cancer survivors. Radiation fibrosis was considered permanent and irreversible, but recently, its dynamic nature was shown, providing new opportunities for the development of antifibrotic therapies. Among these new targets, we identified the Rho/ROCK pathway and thought to investigate whether pravastatin treatment inhibits Rho pathway activation and elicits an antifibrotic action. Experimental Design: Rho and ROCK activities were monitored in human explants presenting radiation fibrosis remodeling after incubation with pravastatin. Subsequent modulation of CCN2, type I collagen, and fibronectin expression were assessed ex vivo and in intestinal smooth muscle cells derived from radiation enteropathy. Then, the therapeutic relevance of the antifibrotic action of pravastatin was explored in vivo in a rat model of chronic radiation fibrosis (19 Gy X-rays) treated with 30 mg/kg/d pravastatin in the drinking water. Results: The results obtained with human explants show that pravastatin specifically inhibits Rho activity in submucosal mesenchymal cells. Pravastatin also elicits ROCK inhibition, and subsequent CCN2 production in human explants and smooth muscle cells isolated from radiation enteropathy. Inhibition of type I collagen and fibronectin does occur, showing that pravastatin modulates the secretory phenotype of mesenchymal cells. Lastly, curative pravastatin administration improves radiation enteropathy in rats. This structural improvement is associated with decreased deposition of CCN2 and subsequent decreased extracellular matrix deposition. Conclusion: Targeting established fibrosis with pravastatin is an efficient and safe antifibrotic strategy in radiation-induced enteropathy, and is easily transferable into the clinic.

Fibrogenic signals in patients with radiation enteritis are associated with increased connective tissue growth factor expression

PURPOSE To investigate the expression of a new fibrogenic cytokine the connective tissue growth factor (CTGF) in intestinal radiation fibrosis and to characterize the mesenchymal cell subtypes involved in CTGF synthesis and collagen deposition. METHODS AND MATERIALS Sixteen patients with radiation enteritis that occurred after radiotherapy for pelvic malignancies and 6 with histologically normal bowel entered the study. Immunohistochemistry, Western blot analysis, and real-time reverse transcriptase-polymerase chain reaction were performed to study CTGF expression, along with other known markers of radiation fibrosis: the pro-fibrogenic cytokine transforming growth factor (TGF)-beta1 and phenotypic markers of the fibroblast differentiation the alpha-sm actin (A), vimentin (V), and desmin (D). Finally, the collagen accumulation was measured by Sirius red staining and colorimetric assay. RESULTS Radiation enteritis was characterized by increased collagen content within the intestinal wall. CTGF immunoreactivity, protein, and mRNA level were increased in radiation enteritis compared with the healthy bowel. On the contrary, no increase of the TGF-beta1 mRNA level was observed in radiation enteritis compared with healthy bowel, and the level of TGF-beta protein was slightly increased in radiation enteritis. A co-localization of CTGF immunoreactivity and collagen deposition was found in the extracellular matrix and subtypes of activated mesenchymal cells with a fibroblast phenotype (V(+)/D(-)/A(-)) and myofibroblast phenotype (V(+)/D(-/+)/A(+)). CONCLUSION The increased level of CTGF protein and mRNA associated with the accumulation of fibroblasts/myofibroblasts and collagen deposition were parts of the fibrogenic signals involved in the persistence of late intestinal radiation fibrosis.

Gene Expression Profile in Human Late Radiation Enteritis Obtained by High-Density cDNA Array Hybridization

Abstract Vozenin-Brotons, M-C., Milliat, F., Linard, C., Strup, C., François, A., Sabourin, J-C., Lasser, P., Lusinchi, A., Deutsch, E., Girinsky, T., Aigueperse, J., Bourhis, J. and Mathé, D. Gene Expression Profile in Human Late Radiation Enteritis Obtained by High-Density cDNA Array Hybridization. Radiat. Res. 161, 299–311 (2004). Late radiation enteritis is a sequela of radiation therapy to the abdomen. The pathogenic process is poorly understood at the molecular level. cDNA array analysis was used to provide new insights into the pathogenesis of this disorder. Gene profiles of six samples of fibrotic bowel tissue from patients with radiation enteritis and six healthy bowel tissue samples from patients without radiation enteritis were compared using membrane-based arrays containing 1314 cDNAs. Results were confirmed with real-time RT-PCR and Western blot analysis. Array analysis identified many differentially expressed genes involved in fibrosis, stress response, inflammation, cell adhesion, intracellular and nuclear signaling, and metabolic pathways. Increased expression of genes coding for proteins involved in the composition and remodeling of the extracellular matrix, along with altered expression of genes involved in cell- to-cell and cell-to-matrix interactions, were observed mainly in radiation enteritis samples. Stress, inflammatory responses, and antioxidant metabolism were altered in radiation enteritis as were genes coding for recruitment of lymphocytes and macrophages. The Rho/HSP27 (HSPB1)/zyxin pathway, involved in tissue contraction and myofibroblast transdifferentiation, was also altered in radiation enteritis, suggesting that this pathway could be related to the fibrogenic process. Our results provide a global and integrated view of the alteration of gene expression associated with radiation enteritis. They suggest that radiation enteritis is a dynamic process involving constant remodeling of each structural component of the intestinal tissue, i.e. the mucosa, the mesenchyme, and blood vessels. Functional studies will be necessary to validate the present results.

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-β1

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.

Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage

Abstract Van der Meeren, A., Mouthon, M-A., Vandamme, M., Squiban, C. and Aigueperse, J. Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage. Radiat. Res. 161, 549–559 (2004). Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.

Modulation of gut substance P after whole-body irradiation : A new pathological feature

Exposure to ionizing radiation induces gastrointestinal dysfunction and inflammatory reactions. The present study carried out in the rat, focuses on substance P, an inflammatory mediator implicated in the control of intestinal motility. We have investigated the effects of gamma irradiation on plasma and tissue substance P levels, ileal smooth muscle activity, and properties of specific receptors. Plasma and ileal (mucosa and muscle) substance P concentrations were measured by radioimmunoassay. At doses ranging from 1 to 8 Gy, plasma substance P levels increased in a dose-dependent manner up to four days after irradiation. Ileal mucosal concentration decreased rapidly 1 hr after a 6-Gy irradiation as compared to controls. A second class of binding sites appeared three days after 6 Gy irradiation. In addition, substance P contractile effects measured on isolated ileum showed a fourfold decrease of EC50, three days after 6 Gy irradiation. These results indicated that gamma irradiation induced an increase of plasma levels concomitant with a modification of gastrointestinal substance P specific binding sites and contractile activity.

Exposure to ionizing radiation modifies neurally-evoked electrolyte transport and some inflammatory responses in rat colon in vitro.

PURPOSE To characterize the responsiveness of the colon to neural stimulation following acute exposure of rats to gamma-radiation and to correlate observed changes to a number of parameters. MATERIALS AND METHODS Rats were exposed to 5 or 10 Gy 137Cs gamma-radiation or not (sham-irradiated) and studied at 1, 3 and 7 days after irradiation. Stripped segments of colon were mounted in Ussing chambers for measurements of neurally-evoked electrolyte transport (electrical field stimulation). Colonic tissue was also taken for biochemical (tissue 5-hydroxytryptamine, histamine, leukotriene B4, nitric oxide synthase) and histological analyses (mast cells). RESULTS In irradiated rats both proximal and distal colon were hyporesponsive to electrical field stimulation at 1 and 3 days, but had recovered by 7 days. In the distal colon, carbachol responses were attenuated 1 day after 10 Gy. Mast cells, tissue histamine and leukotriene B4 synthesis were significantly reduced at all time points but no changes were seen in 5-HT or inducible NOS activity. CONCLUSIONS Rat colon becomes hyporesponsive to neural stimuli post-irradiation. The response initially (1 and 3 days) correlates with decreased mast cells and histamine, but not at 7 days.

In vivo alterations of fluid and electrolyte fluxes in rat colon by gamma irradiation.

Colonic function in rats was investigated up to14 days following exposure to whole-body gammairradiation (8 Gy) using a combination of in vivo and invitro approaches. Water and electrolyte fluxes were measured in vivo under anesthesia by insertionof an agarose cylinder into the descending colon.Short-circuit current responses (Isc; basal,agonist-stimulated) of distal colon were measured invitro as were mannitol and sodium fluxes. Water andelectrolyte absorption (Na, Cl) was markedly reduced atfour days after irradiation but returned to normal atseven days. Potassium secretion was increased from one to seven days after exposure. There were nodifferences in basal Isc, Na, or mannitolfluxes at four days but responses to secretagogues(5-hydroxytryptamine, forskolin, carbachol) wereattenuated. No morphological alterations were associatedwith these functional modifications.

Absorption of uranium through the entire gastrointestinal tract of the rat.

The aim was to determine the gastrointestinal segments preferentially implicated in the absorption of uranium. The apparent permeability to uranium (233U) was measured ex vivo in Ussing chambers to assess uranium passage in the various parts of the small and large intestines. The transepithelial electrical parameters (potential difference, short-circuit current, transepithelial resistance and tissue conductance) were also recorded for each segment. Determination of in vivo uranium absorption after in-situ deposition of 233U in digestive segments (buccal cavity, ileum and proximal colon) and measurements of uranium in peripheral blood were then made to validate the ex vivo results. In addition, autoradiography was performed to localize the presence of uranium in the digestive segments. The in vivo experiments indicated that uranium absorption from the digestive tract was restricted to the small intestine (with no absorption from the buccal cavity, stomach or large intestine). The apparent permeability to uranium measured with ex vivo techniques was similar in the various parts of small intestine. In addition, the experiments demonstrated the existence of a transcellular pathway for uranium in the small intestine. The study indicates that uranium absorption from the gastrointestinal tract takes place exclusively in the small intestine, probably via a transcellular pathway.