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.

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.

AIMTo 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.METHODSRats 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.RESULTSA 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.CONCLUSIONThis 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.

(1) Département de Protection de la santé de l’Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, Institut de Radioprotection et de Sûreté Nucléaire, IRSN, B.P N17, 92262 Fontenay-aux-roses Cedex ; (2) Department of Veterans Affairs Medical Center, Metabolism Section (111F), 4150 Clement Street, San Francisco, 94121 California, Etats-Unis ; (3) Laboratoire de Physiologie de la Nutrition (LPN), Unité Associée Université Paris-Sud/INRA, Bâtiment 447, Université Paris-sud, 91405 Orsay ; (4) Physiologie Comparée et Endocrinologie Moléculaire, UMR Université Blaise Pascal-CNRS 6547, 24 avenue des Landais 63177, Aubière Cedex.

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. Army 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 macropliages. 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 gem-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. (C) 2004 by Radiation Research Society.

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. (C) 2004 Elsevier Inc.

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.

Restenosis is characterized by vascular smooth muscle cell (VSMC) proliferation and accumulation of collagen III in a hypertrophic and disorganized extracellular matrix. Restenosis is prevented by antimitotic agents or irradiation but no significant progress has been made to control collagen expression deregulation. Previously, we have shown that a new family of biopolymers named RGTA (heparan mimetics elaborated by grafting on dextran of carboxylate, sulfate, and benzylamide units) stimulate in vivo tissue repair and reduce fibrosis in various models. Using VSMC in vitro (pig aortic VSMC irradiated with a 60Co source and labeled with [3H]Proline), we now show that gamma-irradiation reduced cell survival by 50% and collagen synthesis 6-fold with a major increase in the ratio of collagen III to collagen I biosynthesis taken as a fibrotic index. RGTA added to the cells enhanced their survival up to 80% and reduced collagen III/I ratio back to values found in normal vascular tissues. These results suggest that RGTA combined with gamma-radiation could be an efficient strategy against restenosis.

(1) Département de Protection de la santé de l’Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, Institut de Radioprotection et de Sûreté Nucléaire, IRSN, B.P N17, 92262 Fontenay-aux-roses Cedex ; (2) Department of Veterans Affairs Medical Center, Metabolism Section (111F), 4150 Clement Street, San Francisco, 94121 California, Etats-Unis ; (3) Laboratoire de Physiologie de la Nutrition (LPN), Unité Associée Université Paris-Sud/INRA, Bâtiment 447, Université Paris-sud, 91405 Orsay ; (4) Physiologie Comparée et Endocrinologie Moléculaire, UMR Université Blaise Pascal-CNRS 6547, 24 avenue des Landais 63177, Aubière Cedex.

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.

Autosomal dominant type IIa hypercholesterolaemia (ADH) is characterised by an elevation of total plasma cholesterol associated with increased LDL particles. Numerous different molecular defects have been identified in the LDL receptor (LDLR) and few specific mutations in the apolipoprotein B (APOB) gene resulting in familial hypercholesterolaemia and familial defective apoB-100 respectively. To estimate the respective contribution of LDLR, APOB and other gene defects in this disease, we studied 33 well characterised French families diagnosed over at least three generations with ADH through the candidate gene approach. An estimation of the proportions performed with the HOMOG3R program showed that an LDLR gene defect was involved in approximately 50% of the families (P = 0.001). On the other hand, the estimated contribution of an APOB gene defect was only 15%. This low estimation of ADH due to an APOB gene defect is further strengthened by the existence of only two probands carrying the APOB (R3500Q) mutation in the sample. More importantly and surprisingly, 35% of the families in the sample were estimated to be linked to neither LDLR nor APOB genes. These data were confirmed by the exclusion of both genes through direct haplotyping in three families. Our results demonstrate that the relative contributions of LDLR and APOB gene defects to the disease are very different. Furthermore, our results also show that genetic heterogeneity is, generally, underestimated in ADH, and that at least three major groups of defects are involved. At this point, the contribution of the recently mapped FH3 gene to ADH cannot be assessed nor its importance in the group of ‘non LDLR / non APOB’ families.

PURPOSE:To investigate regional cholesterol synthesis and kinetics following whole-body gamma-irradiation in the genetically hypercholesterolaemic RICO rat.MATERIALS AND METHODS:Male 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.RESULTS:A 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.CONCLUSIONS:The 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.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.

Isolated hepatocytes from rats that had been kept in a steady state of [3H]cholesterol were incubated in a salt medium with or without serum. The cells released esterified cholesterol into the incubation medium as lipoproteins. This secretion, 18.1 +/- 0.5 nmol/h per g of cells, was increased when the cells were incubated in a medium containing serum (46.3 +/- 4.9 nmol/h per g of cells). This secretion was strikingly enhanced by cholesterol feeding (1% in the diet, 30 days) to 323-620 nmol/h per g of cells, and inhibited by cycloheximide, colchicine or EDTA. After removal of EDTA and addition of calcium, the cholesterol ester secretion was restored. Free cholesterol of previously labelled high-density lipoproteins (HDL) was exchanged (t1/2 = 30 min) with that of liver cells and esterified. The esterification rate (25.8 +/- 2.5 nmol/h per g of cells) was increased by cholesterol feeding (1% in the diet, 8 days) to 63.2 +/- 2.8 nmol/h per g of cells. No cholesteryl ester hydrolysis was detected with the isolated liver cells. Consequently, it is suggested that the turnover of hepatic cholesteryl ester was caused mainly by secretion in lipoproteins.

FEBS LettersVolume 130, Issue 1 p. 113-118 Full-length articleFree Access Preparation of adrenocortical cell suspension highly responsive to acth or dibutylryl-cyclic AMP Effects of albumin, Ca2+ or stress Wieslaw H. Trzeciak, Wieslaw H. Trzeciak Laboratoire de Physiologie de la Nutrition, Université de Paris Sud, Bâtiment 447, 91405 Orsay Cédex, France Present address: Department of Physiological Chemistry, Institute of Physiological Sciences, Academy of Medicine, 6 Swiecickiego St, 60-781 Poznan, Poland Search for more papers by this authorDenis Mathé, Denis Mathé Laboratoire de Physiologie de la Nutrition, Université de Paris Sud, Bâtiment 447, 91405 Orsay Cédex, France Equipe de Recherche Associée au CNRS (ERA 0415); Formation de Recherche Associé à l'INSERM (U 231) Search for more papers by this author Wieslaw H. Trzeciak, Wieslaw H. Trzeciak Laboratoire de Physiologie de la Nutrition, Université de Paris Sud, Bâtiment 447, 91405 Orsay Cédex, France Present address: Department of Physiological Chemistry, Institute of Physiological Sciences, Academy of Medicine, 6 Swiecickiego St, 60-781 Poznan, Poland Search for more papers by this authorDenis Mathé, Denis Mathé Laboratoire de Physiologie de la Nutrition, Université de Paris Sud, Bâtiment 447, 91405 Orsay Cédex, France Equipe de Recherche Associée au CNRS (ERA 0415); Formation de Recherche Associé à l'INSERM (U 231) Search for more papers by this author First published: July 20, 1981 https://doi.org/10.1016/0014-5793(81)80677-1Citations: 5 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume130, Issue1July 20, 1981Pages 113-118 ReferencesRelatedInformation

FEBS LettersVolume 120, Issue 2 p. 183-186 Full-length articleFree Access Coenzyme a-dependent esterification of cholesterol in rat lung Denis Mathé, Denis Mathé Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland Present address: Laboratoire de Physiologie de la Nutrition, Université de Paris-Sud, 91405 Orsay Cedex, France Search for more papers by this authorGeorge S. Boyd, George S. Boyd Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, ScotlandSearch for more papers by this author Denis Mathé, Denis Mathé Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland Present address: Laboratoire de Physiologie de la Nutrition, Université de Paris-Sud, 91405 Orsay Cedex, France Search for more papers by this authorGeorge S. Boyd, George S. Boyd Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, ScotlandSearch for more papers by this author First published: November 03, 1980 https://doi.org/10.1016/0014-5793(80)80293-6Citations: 3 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume120, Issue2November 03, 1980Pages 183-186 ReferencesRelatedInformation