Denis Mathé

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

PURPOSEnTo 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.nnnMETHODS AND MATERIALSnSixteen 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.nnnRESULTSnRadiation 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(+)).nnnCONCLUSIONnThe 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.

Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion

In this study, we have evaluated the interactions between ionizing radiation and a matrix metalloproteinase (MMP) inhibitor. Using Matrigel invasion assays, we show that ionizing radiation induced a dose-dependent increase in the invasive phenotype of cultured B16 melanoma cells and that conditioned medium from these irradiated B16 cells promoted endothelial cell [human microvascular endothelial cells (HMEC)] invasiveness. To determine whether the radiation-induced changes in invasive phenotype could be due to changes in MMP activation, we have tested the ability of the MMP inhibitor Metastat to modulate the ionizing radiation–induced invasive phenotype using both an in vitro melanoma model and a mouse s.c. tumor model. In these studies, Metastat inhibited the ionizing radiation–induced invasive phenotype in cultured B16 cells and similarly inhibited the increase in HMEC invasion induced by conditioned medium from irradiated B16 cells. Conversely, ionizing radiation increased B16 MMP-2 activity and the conditioned medium from irradiated B16 induced HMEC MMP-2 activity. To further investigate the interaction between ionizing radiation and MMP activation, we then studied the effects of ionizing radiation on downstream effectors of the MMP system. We found that ionizing radiation induced vascular endothelial growth factor (VEGF) secretion by B16 melanoma cells and that this secretion was inhibited by Metastat. Similarly, conditioned medium from irradiated B16 was also able to increase VEGF secretion in HMECs. Moreover, ionizing radiation–induced melanoma cell invasiveness was partially inhibited by an anti-VEGF monoclonal antibody. In vivo, ionizing radiation plus concomitant Metastat yielded the greatest growth inhibition of melanoma s.c. tumors and this effect correlated with inhibition of angiogenesis as measured by both Doppler ultrasonography and platelet/endothelial cell adhesion molecule-1 staining. Finally, ionizing radiation modulated MMP-2, VEGF, and VEGF receptor expression in these tumor samples using immunohistochemistry. Taken together, these results suggest that there is an ionizing radiation–induced tumor survival pathway and a possible paracrine ionizing radiation–induced stimulatory pathway emanating from tumor cells toward the endothelial bed that is impeded when Metastat is given simultaneously. This model could provide in vivo evidence of the antitumor efficacy of combining a MMP inhibitor with ionizing radiation to target radiation-induced invasion and angiogenesis.

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.

Expression and activation of MMP -2, -3, -9, -14 are induced in rat colon after abdominal X-irradiation

Objective. Colonic response to single-dose irradiation is characterized by epithelial denudation followed by restitution. Extracellular matrix (ECM) remodeling is involved in both of these phases. The aim of this study was to characterize the contribution of matrix metalloproteinases (MMPs) and of their stimulatory and inhibitory pathways in radiation-induced ECM remodeling in colonic tissue. Material and methods. Rats were irradiated with single-dose 10 Gy X-rays to the abdomen. Activity, localization, and mRNA levels of MMPs and molecules involved in their activation and inhibition (plasmin/plasminogen; TIMPs), of inflammatory mediators (IL-1β, TNF-α) in the distal colon, 1, 3, and 7 days after irradiation were analyzed using a combination of approaches including zymography, immunohistochemistry, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. The main finding of this study is that radiation-induced alteration of the mucosal structure is concomitant with local increased expression and activation of MMP subtypes involved in basement membrane degradation (MMP-2, -3, and -9). We investigated MMP-2 activation pathways and found an early increase in mRNA levels of soluble inflammatory mediators (TNF-α and IL-1β). Furthermore, transcription and activity of MMP-2 activating molecules, such as MMP-14, and molecules involved in the plasminogen/plasmin system were found to increase during the denudation phase. Interestingly, induction of MMP inhibitors TIMP-1 and PAI-1 was observed during the restitution phase. MMP inhibitors may be able to stop acute wound healing response by inhibiting ECM degradation. Conclusions. This study brings new insights into ECM remodeling in the colon after exposure to ionizing radiation and highlights the role of MMP subtypes specialized in basement membrane degradation.

Heparan mimetic regulates collagen expression and TGF-β1 distribution in gamma-irradiated human intestinal smooth muscle cells

Radiation‐induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF‐β1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF‐βl distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a car‐boxymethylated and sulfated dextran (RG‐1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After 60Co irradiation (10 Gy), HISM were labeled with [3H] proline (±RG‐1503). Radiolabeled collagen I, III, and V were quantified by SDS‐PAGE. TGF‐β1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85‐fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF‐β1 was increased (up to twofold) in irradiated HISM. RG‐1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG‐1503 decreased the amount of TGF‐β1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF‐β1 distribution. The antifibrotic RG‐1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation‐induced intestinal fibrosis.—Alexakis, C., Guettoufi, A., Mestries, P., Strup, C., Mathé, D., Barbaud, C., Barritault, D., Caruelle, J.‐ P., Kern, P. Heparan mimetic regulates collagen expression and TGF‐β1 distribution in gamma‐irradiated human intestinal smooth muscle cells. FASEB J. 15, 1546–1554 (2001)

Involvement of primary afferent nerves after abdominal irradiation: consequences on ileal contractile activity and inflammatory mediator release in the rat.

In this study we analyzed the role of substance P (SP) from afferent nerves in ileum contractibility and in the release of inflammatory mediators (neurotensin, Il-1β, and TNF-α) in ileal mucosa and muscularis layers after a 10-Gy γ-irradiation of the abdomen. Six hours after irradiation, SP concentrations were lower than in control rats, and 3 days after irradiation SP-induced contractile activity was higher. Irradiation significantly increased the levels of neurotensin, Il-1β, and TNF-α in both layers. Pretreatment with capsaicin depleted afferent nerve endings of SP and reduced SP levels by about 50%. Capsaicin treatment reduced SP concentrations further, beyond the levels due to irradiation, thereby suggesting that all sources of SP are affected by irradiation. Capsaicin treatment prevented the irradiation from affecting SP-induced contractile response or increasing neurotensin levels. This finding suggests that SP released by afferent nerve endings controls these functions. Proinflammatory cytokine release was not reduced by capsaicin treatment.

‘In‐field’ and ‘out‐of‐field’ functional impairment during subacute and chronic phases of experimental radiation enteropathy in the rat

Purpose: To investigate subacute and chronic functional consequences of localized irradiation of rat small intestine on exposed and shielded segments (proximal and distal). Materials and methods: The surgical model of a scrotal hernia was used. The ileal loop was exposed to single doses of 18, 21 or 29.6u2009Gy X‐irradiation. Epithelial structure and transport capacity were followed 2 and 26 weeks post‐exposure. Results: Irradiated segments showed mucosal ulceration followed by transmural fibrosis. Transport capacity was impaired from 2 to 26 weeks. Subacute functional impairment was noticed in the proximal segment, without either morphological alteration or neutrophil influx. At 26 weeks, both proximal and distal segments showed impaired epithelial transport capacity, with neutrophil influx in the submucosa in cases of 21‐Gy exposure and in the submucosa and muscularis propria after 29.6u2009Gy. Conclusions: Radiation enteritis was characterized by functional impairment, within as well as outside, the irradiation field. During the subacute phase, the irradiated segment may be a source of mediators which might influence intestinal function outside the site of injury via the blood stream and/or enteric nervous system. The development of an intestinal occlusion syndrome during the chronic phase might be responsible for intestinal dysfunction but it does not rule out a possible inflammatory process developing in the shielded parts of the small intestine.

A substance P receptor antagonist (FK 888) modifies gut alterations induced by ionizing radiation

PURPOSEnWe previously reported disturbances of ileal substance P (SP) levels and of characteristics of specific receptors after ionizing radiation associated with disorders of intestinal motility. The aim of this study was to investigate the effect of a SP receptor blockade by FK 888 on gut SP levels and contractile properties after rat irradiation.nnnMATERIALS AND METHODSnRats were exposed to 6 Gy whole-body gamma-irradiation and injected 1 h post-irradiation with FK 888 for 3 days (0.1 mg/kg/day). Plasma and ileal SP concentrations, ileal muscle SP receptor binding and SP-induced contractions in isolated ileum were investigated 3 and 14 days post-irradiation and FK 888 treatment.nnnRESULTSnIrradiation induced an increase of total SP binding site number at day 3 (1.3-fold) and day 14 (1.6-fold). FK 888 had no effect on SP receptor characteristics in irradiated animals. In contrast, FK 888 treatment caused a reduction of endogenous ileal SP level in mucosal (-29%) and muscularis (-40%) layers at day 3 and these decreases were greater at day 14, -88% in mucosal and -61% in muscularis layers. FK 888 treatment decreased efficacy of ileal contraction in both the control and irradiated rat but surprisingly it increased potency at day 3 and decreased it at day 14 in the irradiated rat.nnnCONCLUSIONSnThe findings demonstrate that a SP receptor antagonist could be effective on intestine contractility alteration induced several days after ionizing radiation exposure but not at 3 days after irradiation.

Regional cholesterol synthesis in the intestinal mucosa of the genetically hypercholesterolaemic RICO rat: kinetic study following whole-body gamma-irradiation

PURPOSEnTo investigate regional cholesterol synthesis and kinetics following whole-body gamma-irradiation in the genetically hypercholesterolaemic RICO rat.nnnMATERIALS AND METHODSnMale RICO rats were fed a semi-purified diet for 1 month. At 10 weeks old they were exposed to gamma-irradiation (4 Gy, 1.5 Gy/min) together with controls. At intervals from 1-8 days after irradiation an intraperitoneal administration of [1-14C] acetate was given in order to estimate cholesterogenesis in mucosal cells located at different sites in the small intestine. The protein and DNA contents of the different enterocytes isolated along the crypt/villus axis in four equal parts of the intestine were also determined.nnnRESULTSnA marked decrease of the mean quantities of cholesterol, DNA or protein in mucosa was seen 1 and 2 days after irradiation, showing the loss of 30-40% of the intestinal epithelium. An overshoot of the cell amount was observed after 4 days with a return to basal values by 8 days after irradiation. The kinetic and topological evolution of cholesterol radioactivity, which reflects in situ cholesterol synthesis, showed a typical gradient in controls and at 8 days after irradiation. Cholesterogenesis decreased from the first to the third quarter of the small intestine (duodenum to proximal ileum), and then increased in the fourth quarter (distal ileum). In all segments of the small intestine, cholesterogenesis decreased from crypt cells to villus tip. At days 1 and 2 the gradient of cholesterogenesis on the villus was abolished. A slow recovery was seen from day 4 with a strong overshoot of cholesterol synthesis in crypt cells in every part of the small intestine.nnnCONCLUSIONSnThe RICO rat is a useful model for studying the effect of irradiation on regional cholesterogenesis in intestinal mucosa. Cholesterol synthesis in crypt cells was lowered 1 and 2 days after irradiation, over-expressed after 4 days and subsequently returned to its normal level.