Human Colonic Myofibroblasts Research Articles

In vitro evaluation of the cytotoxicity and modulation of mechanisms associated with inflammation induced by perfluorooctanesulfonate and perfluorooctanoic acid in human colon myofibroblasts CCD-18Co

Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the most notable members of an emerging class of persistent organic pollutants (POPs), poly- and perfluoroalkyl acids (PFASs). In this study, the CCD-18Co myofibroblasts were selected as a cell model to investigate the cytotoxic effects of PFOS and PFOA. The aim was to perform an in vitro evaluation of the ability of these compounds to induce cytotoxicity and modulate mechanisms associated with inflammation as measured by (i) colon fibroblasts viability, (ii) colon fibroblasts proliferation, and (iii) IL-6 production. The data provided in this study suggest that PFOS and PFOA can have cytotoxic potential and modulate processes associated with intestinal inflammation such as myofibroblasts proliferation and IL-6 production at concentrations similar to those detected in vivo. Our results also highlight the influence of culture serum concentration in cytotoxic in vitro studies, which should be considered in future toxicity studies involving PFOS and PFOA. The results contribute to a better knowledge of the effects of PFOS and PFOA in human cells, a phenomenon still not fully examined.

Intraperitoneal application of phospholipids for the prevention of postoperative adhesions: a possible role of myofibroblasts

BackgroundPeritoneal adhesions, organized as fibrous bands after abdominal surgery, are related with considerable morbidity and repeated hospitalization. Phospholipids, natural constituents of the peritoneal fluid, seem to display excellent antiadhesive properties. The aim of this study was to investigate whether intraperitoneal application of phospholipids is capable of reducing postoperative adhesions and the possible underlying mechanisms. Materials and methodsTwenty male Wistar rats were subjected to a midline laparotomy and a standard peritoneal and cecum abrasion trauma. Before laparotomy closure, a bolus of 3 mL of phospholipids (12 mg/mL) or NaCl (placebo) was given intraperitoneally. Seven days later, the quality and the quantity of adhesions, as well as serum proinflammatory and/or profibrotic mediators, were blindly assessed. Human colonic subepithelial myofibroblasts were isolated from normal controls and cultured with transforming growth factor-β1 (TGFβ1, 5 ng/mL) in the presence of phospholipids (30–300 μg/mL). Collagen production in culture supernatants and migratory activity of myofibroblasts were also assessed. ResultsPhospholipids reduced intra-abdominal adhesions (P < 0.001), with respect to their intensity and area, and serum levels of cytokines (interleukin 1β, interleukin 6, platelet-derived growth factor-1, and TGFβ1) compared with placebo-treated rats. Stimulation of myofibroblasts with TGFβ1 significantly increased (P < 0.001) the basic collagen production. The presence of phospholipids significantly reduced (P < 0.001) both the TGFβ1 induced and the basic collagen production. Using a wound healing assay, phospholipids were found to reduce the basic and the TGFβ1-induced migration of myofibroblasts in a concentration-dependent manner. ConclusionsIntraperitoneal phospholipids might be involved in the prevention of postoperative adhesions formation via the reduction of proinflammatory and/or profibrotic mediators and by inhibiting fibrogenic properties of mesenchymal cells.

Triptolide ameliorates colonic fibrosis in an experimental rat model.

Triptolide is known to exert anti-inflammatory and immunomodulatory activities; however, its impact on intestinal fibrosis has not been previously examined. Based on our previous studies of the suppressive activity of triptolide on human colonic subepithelial myofibroblasts and the therapeutic efficacy of triptolide in Crohn’s disease, it was hypothesized that triptolide may have beneficial effects on intestinal fibrosis. In the present study, colonic fibrosis was induced in rats by 6 weekly repeated administration with a low-dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and was then treated with triptolide or PBS daily (control) simultaneously. Extracellular matrix (ECM) deposition in the colon was examined with image analysis of Masson Trichrome staining. Total collagen levels in colonic homogenates were measured by a Sircol assay. Collagen Iα1 transcripts and collagen I protein were measured ex vivo in the isolated colonic subepithelial myofibroblasts by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis, respectively. The results indicated that triptolide decreased ECM deposition and collagen production in the colon, and inhibited collagen Iα1 transcripts and collagen I protein expression in the isolated subepithelial myofibroblasts of the rats with colonic fibrosis. In conclusion, triptolide ameliorates colonic fibrosis in the experimental rat model, suggesting triptolide may be a promising compound for inflammatory bowel disease treatment.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2–ASK1 apoptosis pathways and oxidative stress is mitigated by Srx–Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration.

The glucagon-like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, or endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and liver, particularly in hepatocytes. In two mice models of trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared with control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of inflammatory bowel disease patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mouse liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation dampens its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extraintestinal functions of GLP-2.

Novel Rho/MRTF/SRF Inhibitors Block Matrix-stiffness and TGF-β–Induced Fibrogenesis in Human Colonic Myofibroblasts

Ras homolog gene family, member A (RhoA)/Rho-associated coiled-coil forming protein kinase signaling is a key pathway in multiple types of solid organ fibrosis, including intestinal fibrosis. However, the pleiotropic effects of RhoA/Rho-associated coiled-coil forming protein kinase signaling have frustrated targeted drug discovery efforts. Recent recognition of the role of Rho-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factor A (MRTF-A) suggest a novel locus for pharmacological intervention. Because RhoA signaling is mediated by both physical and biochemical stimuli, we examined whether pharmacological inhibition of RhoA or the downstream transcription pathway of MRTF-A/SRF could block intestinal fibrogenesis in 2 in vitro models. In this study, we demonstrate that inhibition of RhoA signaling blocks both matrix-stiffness and transforming growth factor beta-induced fibrogenesis in human colonic myofibroblasts. Repression of alpha-smooth muscle actin and collagen expression was associated with the inhibition of MRTF-A nuclear localization. CCG-1423, a first-generation Rho/MRTF/SRF pathway inhibitor, repressed fibrogenesis in both models, yet has unacceptable cytotoxicity. Novel second-generation inhibitors (CCG-100602 and CCG-203971) repressed both matrix-stiffness and transforming growth factor beta-mediated fibrogenesis as determined by protein and gene expression in a dose-dependent manner. Targeting the Rho/MRTF/SRF mechanism with second-generation Rho/MRTF/SRF inhibitors may represent a novel approach to antifibrotic therapeutics.

Regulation of eotaxin-3/CC chemokine ligand 26 expression by T helper type 2 cytokines in human colonic myofibroblasts

Eotaxins induce the trafficking of eosinophils to the sites of inflammation via CC chemokine receptor 3 (CCR3). In this study, we investigated eotaxin-3/CC chemokine ligand 26 (CCL26) expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for eotaxin-3 expression in human colonic myofibroblasts. Eotaxin-3 mRNA and protein expression was evaluated by real time-polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Eotaxin-3 mRNA expression was elevated significantly in the active lesions of ulcerative colitis (UC) patients. Significant elevations were also observed in the active lesions of Crohn's disease (CD) patients, but this was significantly lower than that detected in the active UC lesions. There were no significant increases in the inactive lesions of UC or CD patients. Colonic myofibroblasts were identified as a major source of eotaxin-3 in the colonic mucosa, and interleukin (IL)-4 and IL-13 enhanced eotaxin-3 mRNA and protein expression significantly in these cells. There was a significant positive correlation between mucosal eotaxin-3 and IL-4 mRNA expression in the active lesions of IBD patients. The IL-4- and IL-13-induced eotaxin-3 mRNA expression was regulated by the signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signalling (SOCS)1-mediated pathways. Interferon (IFN)-γ acts as a negative regulator on the IL-4- and IL-13-induced eotaxin-3 expression via STAT-1 activation. Eotaxin-3 expression was elevated specifically in the active lesions of IBD, in particular UC. Eotaxin-3 derived from colonic myofibroblasts may play an important role in the pathophysiology of UC.

TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR

BackgroundEnhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.Methods18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10mm cell culture dishes and were used from passages 10–14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 μM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors.ResultsExposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR.ConclusionSynergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.

Epithelial expression of interleukin-37b in inflammatory bowel disease

Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses.

Anti-melanin-concentrating hormone treatment attenuates chronic experimental colitis and fibrosis

Fibrosis represents a major complication of several chronic diseases, including inflammatory bowel disease (IBD). Treatment of IBD remains a clinical challenge despite several recent therapeutic advances. Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide shown to regulate appetite and energy balance. However, accumulating evidence suggests that MCH has additional biological effects, including modulation of inflammation. In the present study, we examined the efficacy of an MCH-blocking antibody in treating established, dextran sodium sulfate-induced experimental colitis. Histological and molecular analysis of mouse tissues revealed that mice receiving anti-MCH had accelerated mucosal restitution and lower colonic expression of several proinflammatory cytokines, as well as fibrogenic genes, including COL1A1. In parallel, they spared collagen deposits seen in the untreated mice, suggesting attenuated fibrosis. These findings raised the possibility of perhaps direct effects of MCH on myofibroblasts. Indeed, in biopsies from patients with IBD, we demonstrate expression of the MCH receptor MCHR1 in α-smooth muscle actin(+) subepithelial cells. CCD-18Co cells, a primary human colonic myofibroblast cell line, were also positive for MCHR1. In these cells, MCH acted as a profibrotic modulator by potentiating the effects of IGF-1 and TGF-β on proliferation and collagen production. Thus, by virtue of combined anti-inflammatory and anti-fibrotic effects, blocking MCH might represent a compelling approach for treating IBD.

Celecoxib induces proliferation and Amphiregulin production in colon subepithelial myofibroblasts, activating erk1–2 signaling in synergy with EGFR

The COX-2 inhibitor Celecoxib, tested in phase III trials for the prevention of sporadic colon adenomas, reduced the appearance of new adenomas, but was unable to affect the incidence of colon cancer. Moreover the 5years follow-up showed that patients discontinuing Celecoxib treatment had an increased incidence of adenomas as compared to the placebo arm. In the APC(min/+) mouse model short term treatment with Celecoxib reduced gut adenomas, but a prolonged administration of the drug induced fibroblast activation and intestinal fibrosis with a final tumor burden. The way Celecoxib could directly activate human colon myofibroblasts (MF) has not yet been investigated.We found that MF are activated by non toxic doses of Celecoxib. Celecoxib induces erk1–2 and Akt phosphorylation within 5′. This short term activation is apparently insufficient to cause phenotypic changes, but the contemporary triggering of EGFR causes an impressive synergic effect inducing MF proliferation and the neo-expression and release of Amphiregulin (AREG), a well known EGFR agonist involved in colon cancer progression. As a confirm to these observations, the erk inhibitor U0126 and the EGFR inhibitors Tyrphostin and Cetuximab were able to contrast AREG induction.Our data provide evidence that Celecoxib directly activates MF empowering EGFR signaling. According to these results the association with EGFR (or erk1–2) inhibitors could abolish the off-target activity of Celecoxib, possibly extending the potential of this drug for colon cancer prevention.

Tacrolimus (FK506) suppresses TNF-α-induced CCL2 (MCP-1) and CXCL10 (IP-10) expression via the inhibition of p38 MAP kinase activation in human colonic myofibroblasts

In order to investigate the molecular mechanisms underlying the immunosuppressive effects of tacrolimus (FK506) on intestinal inflammation, we examined whether FK506 effects cytokine/chemokine secretion in human colonic myofibroblasts. Human colonic myofibroblasts were isolated from normal human colonic tissue. The mRNA and protein expression for human CCL2 and CXCL10 were analyzed by real-time PCR and ELISA, respectively. p38 MAP kinase activation was evaluated by western blotting. Tacrolimus (1 µM) suppressed tumor necrosis factor (TNF)-α-induced CCL2 and CXCL10 mRNA expression, but did not modulate TNF-α-induced interleukin (IL)-6 or CXCL8 mRNA expression. Dose-dependent, inhibitory effects of tacrolimus on CCL2 and CXCL10 expression were observed at the mRNA and protein levels. Significant inhibitory effects of tacrolimus were observed at concentrations as low as 0.5 µM for CCL2 and 0.1 µM for CXCL10, respectively. TNF-α-induced CCL2 and CXCL10 expression depended on p38 MAP kinase activation, and tacrolimus strongly inhibited the TNF-α-induced phosphorylation of p38 MAP kinase. Tacrolimus did not affect interferon (IFN)-γ-induced signaling transducer and activator of transcription (STAT)-1 phosphorylation, nor did it modulate CXCL10 mRNA and protein expression. In conclusion, tacrolimus suppressed CCL2 and CXCL10 expression in human colonic myofibroblasts. These inhibitory effects of tacrolimus may play key roles in the therapeutic effects of colonic inflammation in inflammatory bowel disease (IBD) patients.

Proinflammatory cytokines induce crosstalk between colonic epithelial cells and subepithelial myofibroblasts: Implication in intestinal fibrosis

Background and aimsColonic epithelial cells and adjacent subepithelial myofibroblasts are important counterparts in the pathogenesis of intestinal inflammation and fibrosis. We investigated the possible crosstalk between them, whilst focusing on the mucosal inflammation pathways that potentially trigger intestinal fibrosis. MethodsWe studied the effects of proinflammatory cytokines (IL-1α, TNF-α, IFN-γ) on human colonic epithelial cell lines and the effects of epithelial cell-conditioned media on primary human colonic subepithelial myofibroblasts isolated from normal controls or patients with inflammatory Crohn's disease along with the corresponding 18CO cell line. Readouts included production of TGF-β and TIMP-1, total collagen synthesis, matrix metalloproteinases MMP-2 and MMP-9 and myofibroblast migration/mobility. ResultsProinflammatory cytokines upregulated TGF-β and TIMP-1 in colonic epithelial cells. Conditioned medium from these epithelial cell cultures induced production of MMP-9 and collagen and inhibited the migration/mobility of subepithelial myofibroblasts. MMP-9 production depended on endothelin receptor A signalling on responding myofibroblasts. Collagen up-regulation was independent of TGF-β, CTGF, TF and endothelin. Subepithelial myofibroblasts isolated from Crohn's disease patients had similar responses to those isolated from normal controls, with the exception of higher basal collagen production. ConclusionsOur study indicates that colonic epithelial cells may respond to an inflammatory milieu by inducing myofibroblast functions similar to those observed during intestinal fibrosis.

Tu1824 TNF-a Induces Up-Regulation of EGFR Expression and Signaling in Human Colonic Myofibroblasts

Tu1824 TNF-a Induces Up-Regulation of EGFR Expression and Signaling in Human Colonic Myofibroblasts

Tu1825 Protease-Activated Receptor 2 Exerts a Tonic Anti-Apoptotic Effect in HT-29 Colonic Epithelial Cells

Backround: The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, which has been strongly associated with intestinal repair and the development of several human cancers. Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 10-14. 18Co cells were exposed to TNF-α (10 ng/ml) and EGF (5 ng/ ml) under varying conditions. EGFR protein expression, EGFR tyrosine phosphorylation at Y845 and Y1068, p42/44 MAPK phosphorylation, and COX-2 expression were assessed by Western blot analysis. Live cell imaging was performed using Quantum dot technology. Experiments were also performed using primary myofibroblasts harvested from human colon tissue. Results: Treatment of 18Co cells with TNF-α led to a time-dependent increase in cell surface EGFR expression, an effect that was completely prevented by inhibition of protein synthesis with cycloheximide. Stimulation of up-regulated EGFR with EGF or HBEGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a synergistic increase in COX-2 expression compared to 18Co cells treated with EGF andHB-EGF alone. Importantly, TNF-α also increased EGFR expression in primarymyofibroblasts isolated from human colon tissue. Conclusions: Our results imply that TNF-α induced a significant upregulation of EGFR in human colonic myofibroblasts that is associated with enhanced signaling and increased functional activity (ERK, COX-2). Induction of EGFR expression by TNF-α provides a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes IBD, and which may contribute to a microenvironment that predisposes to colitis-associated cancer.

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC)

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance (1H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to –N+(CH3)3 groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to –CH2 groups and terminal –CH3 groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid–crystalline phase (Lα). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C–O–P–O–C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.

Synergistic Cox-2 Expression Induced By LPA and TNF-Alpha Involves Transactivation of the EGF Receptor in Human Colonic Myofibroblasts

Synergistic Cox-2 Expression Induced By LPA and TNF-Alpha Involves Transactivation of the EGF Receptor in Human Colonic Myofibroblasts

Spironolactone and Colitis: Increased Mortality in Rodents and in Humans

Crohn's disease causes intestinal inflammation leading to intestinal fibrosis. Spironolactone is an antifibrotic medication commonly used in heart failure to reduce mortality. We examined whether spironolactone is antifibrotic in the context of intestinal inflammation. In vitro, spironolactone repressed fibrogenesis in transforming growth factor beta (TGF-β)-stimulated human colonic myofibroblasts. However, spironolactone therapy significantly increased mortality in two rodent models of inflammation-induced intestinal fibrosis, suggesting spironolactone could be harmful during intestinal inflammation. Since inflammatory bowel disease (IBD) patients rarely receive spironolactone therapy, we examined whether spironolactone use was associated with mortality in a common cause of inflammatory colitis, Clostridium difficile infection (CDI). Spironolactone use during CDI infection was associated with increased mortality in a retrospective cohort of 4008 inpatients (15.9% vs. 9.1%, n = 390 deaths, P < 0.0001). In patients without liver disease, the adjusted odds ratio (OR) for inpatient mortality associated with 80 mg spironolactone was 1.99 (95% confidence interval [CI]: 1.51-2.63) In contrast to the main effect of spironolactone mortality, multivariate modeling revealed a protective interaction between liver disease and spironolactone dose. The adjusted OR for mortality after CDI was 1.96 (95% CI: 1.50-2.55) for patients without liver disease on spironolactone vs. 1.28 (95% CI: 0.82-2.00) for patients with liver disease on spironolactone when compared to a reference group without liver disease or spironolactone use. We propose that discontinuation of spironolactone in patients without liver disease during CDI could reduce hospital mortality by 2-fold, potentially reducing mortality from CDI by 35,000 patients annually across Europe and the U.S.

Tissue factor–thrombin signaling enhances the fibrotic activity of myofibroblasts in systemic sclerosis through up‐regulation of endothelin receptor A

The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)-thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ET(A) ) were investigated. Collagen production and migratory activity of HCMFs were further assessed. HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ET(A) expression. ET(A) overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. Tissue factor-thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET(A) expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.