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.

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-1beta, TNF-alpha) 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-alpha and IL-1beta). 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.

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.

Induction of CTGF by TGF-beta1 in normal and radiation enteritis human smooth muscle cells: Smad/Rho balance and therapeutic perspectives.

BACKGROUND AND PURPOSE: Transforming Growth Factor beta1 (TGF-beta1) and its downstream effector Connective Tissue Growth Factor (CTGF/CCN2), are well known fibrogenic activators and we previously showed that the Rho/ROCK pathway controls CTGF expression in intestinal smooth muscle cells isolated from patients with delayed radiation enteritis. The aim of the present work was to investigate the balance between Smad and Rho signalling pathways in the TGF-beta1 CTGF induction and modulation of radiation-induced fibrogenic differentiation after addition of pravastatin, an inhibitor of Rho isoprenylation. PATIENTS AND METHODS: Primary human smooth muscle cells isolated from normal (N-SMC) or radiation enteritis (RE-SMC) biopsies were incubated with TGF-beta1 (10 ng/ml). Induction of CTGF, as well as nucleo-cytoplasmic distribution of phospho-Smad2/3, Smad2/3 and Smad4 were analysed by Western blot and immunocytochemistry. Smad DNA binding was assessed by EMSA and Rho activation was measured by pull-down assay. RESULTS: After TGF-beta1 addition, Smads were translocated to the nucleus in both cell types. Nuclear accumulation of Smad as well as their DNA-binding activity were higher in N-SMC than in RE-SMC, whereas the opposite was observed for Rho activation, suggesting a main involvement of Rho pathway in sustained fibrogenic differentiation. This hypothesis was further supported by the antifibrotic effect observed in vitro after cell treatment with pravastatin (i.e. decreased expression of CTGF, TGF-beta1 and Collagen Ialpha2). CONCLUSIONS: Our results suggest that TGF-beta1-induced CTGF transactivation mainly depends on the Smad pathway in N-SMC, whereas in RE-SMC, Smad and Rho pathways are involved. Inhibition of Rho activity by pravastatin alters fibrogenic differentiation in vitro which opens up new therapeutic perspectives.

Expression of matrix metalloproteinases and tissue inhibitor metalloproteinases increases in X-irradiated rat ileum despite the disappearance of CD8a T cells.

AIM: To investigate their expression and activity in the rat ileum after exposure to ionizing radiation along with that of the cellular effectors and molecular mediators involved in the regulation of MMPs. METHODS: Rats were exposed to a single 10-Gy dose of X-rays delivered to the abdomen. A combination of methods, such as zymography, immunohistochemistry and real time reverse transcriptase-polymerase chain reaction, were used to localize and quantify MMPs and the molecules involved in MMP activating and inhibitory pathways (plasmin/plasminogen, TIMPs), CD8+, as well as inflammatory (interleukin (IL)-1beta, IL-8, tumor necrosis factor-alpha, TNF-alpha) and fibrogenic mediators (transforming growth factor-beta1-3) within ileal tissue at 1, 3, and 7 d after irradiation. RESULTS: A marked increase in MMP-2 and -14 mRNA and protein levels associated with an increased activity of MMP-2 was observed in irradiated ileal tissue. MMP-2 and -14 expression was mainly observed in inflammatory, epithelial, and mesenchymal cells, whereas a slight increase in MMP-3 expression was detected in the few infiltrating macrophages at d 1 after irradiation. Conversely, MMP-1, -7, and -9 mRNA levels were not found to be affected by abdominal irradiation. Irradiation was found to induce disappearance of CD8+ cells. Furthermore, we have observed that TNF-alpha and IL-1beta protein levels increased 6 h after irradiation, whereas those of IL-8 only increased after 3 d and was concomitant with neutrophil infiltration. In addition, the expressions of molecules involved in MMP activating and inhibitory pathways (urokinase-type plasminogen activator and tissue-type plasminogen activator; TIMP-1, TIMP-2, and plasminogen activator-inhibitor-1) were found to be increased after abdominal irradiation. CONCLUSION: This study showed that abdominal irradiation induces an acute remodeling of the ileum associated with an increased expression of MMPs and TIMPs that do not involve CD8+ T cells but involve mesenchymal and epithelial cells, although to a lesser extent, and probably even soluble inflammatory and fibrogenic mediators.

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis.

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

Gene expression profile in human late radiation enteritis obtained by high-density cDNA array hybridization.

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.

Acute induction of inflammatory cytokine expression after gamma-irradiation in the rat: effect of an NF-kappaB inhibitor.

PURPOSE: The pathologic changes within the intestinal muscle layer may be at the origin of the cytokines that account for acute radiation-induced inflammation. We were specifically interested in evaluating the efficacy of an inhibitor of nuclear transcription factor kappa B (NF-kappaB) activation that is involved in regulating cytokine expression. METHODS AND MATERIALS: Cytokine expression was analyzed in the ileal muscularis layer by reverse transcriptase-polymerase chain reaction (RT-PCR) at 3 h, 6 h, and 3 days after a 10-Gy gamma whole-body irradiation of rats. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally (30 mg/kg) 15 min before irradiation and once a day for 3 days. RESULTS: Interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNA increased at 3 h and 6 h after irradiation, and expression of IL-6 and IL-8 was elevated at 3 days. On the other hand, levels of the anti-inflammatory cytokine IL-10 were markedly lower on Day 3. Overexpression of IL-6 on Day 3 was combined with upregulation of the IL-6 receptors (gp130/gp80) and suppressor of cytokine signaling-3 (SOCS3) genes. CAPE treatment did not significantly change IL-1beta or TNF-alpha expressions in the irradiated rats; it increased IL-10 expression at 6 h but had no effect on it on Day 3. CAPE treatment inhibited the radiation-induced expression of IL-6, IL-6 receptors (IL-6rs), and SOCS3 at 3 days. CONCLUSION: In vivo, irradiation induced a cascade of inflammatory responses that involved the transcription factor NF-kappaB; this inflammation was reduced by CAPE treatment.

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-1beta, and TNF-alpha) in ileal mucosa and muscularis layers after a 10-Gy gamma-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-1beta, and TNF-alpha 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.

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.