Eva S. Rodansky

Novel Rho/MRTF/SRF inhibitors block matrix-stiffness and TGF-β-induced fibrogenesis in human colonic myofibroblasts

Background: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. Methods: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. Results: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. Conclusions:Targeting the Rho/MRTF/SRF mechanism with second-generation Rho/MRTF/SRF inhibitors may represent a novel approach to antifibrotic therapeutics.

Inhibition of Myocardin-Related Transcription Factor/Serum Response Factor Signaling Decreases Lung Fibrosis and Promotes Mesenchymal Cell Apoptosis

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

Background:Crohns disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression toward fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods:The stiffness of fresh ex vivo samples from normal human small intestine, Crohns disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, &agr;-smooth muscle actin staining, and gene expression. Results:Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures and between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased &agr;-smooth muscle actin protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase, and proinflammatory gene expression and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions:Matrix stiffness, representative of the pathologic stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways, suggesting that the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to auto-propagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease.

CARD-024, a vitamin D analog, attenuates the pro-fibrotic response to substrate stiffness in colonic myofibroblasts

Intestinal fibrosis is one of the major complications of Crohns disease (CD) for which there are no effective pharmacological therapies. Vitamin D deficiency is common in CD, though it is not known whether this is a contributing factor to fibrosis, or simply a consequence of the disease itself. In CD, fibrosis is mediated mainly by activated intestinal myofibroblasts during remodeling of extracellular matrix in response to wound healing. We investigated the effects of CARD-024 (1-alpha-hydroxyvitamin D5), a vitamin D analog with minimal hypercalcemic effects, on the pro-fibrotic response of intestinal myofibroblasts to two fibrogenic stimuli: TGFβ stimulation and culture on a physiologically stiff matrix. TGFβ stimulated a fibrogenic phenotype in Ccd-18co colonic myofibroblasts, characterized by an increase in actin stress fibers and mature focal adhesions, and increased αSMA protein expression, while CARD-024 repressed αSMA protein expression in a dose-dependent manner. Culture of colonic myofibroblasts on physiological high stiffness substrates induced morphological changes with increased actin stress fibers and focal adhesion staining, induction of αSMA protein expression, FAK phosphorylation, induction of fibrogenic genes, and repression of COX-2 and IL-1β. CARD-024 treatment repressed the stiffness-induced morphological features including stellate cell morphology and the maturation of focal adhesions. CARD-024 repressed the stiffness-mediated induction of αSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX-2/IL-1β inflammatory pathway. In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.

Intestinal organoids: a model of intestinal fibrosis for evaluating anti-fibrotic drugs.

BACKGROUND & AIMS Intestinal fibrosis is a critical complication of Crohns disease (CD). Current in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, while in vivo rodent models do not fully recapitulate human disease and have limited utility for large-scale screening. Here, we exploit recent advances in stem cell derived human intestinal organoids (HIOs) as a new human model of fibrosis in CD. METHODS Human pluripotent stem cells were differentiated into HIOs. We identified myofibroblasts, the key effector cells of fibrosis, by immunofluorescence staining for alpha-smooth muscle actin (αSMA), vimentin, and desmin. We examined the fibrogenic response of HIOs by treatment with transforming growth factor beta (TGFβ) in the presence or absence of the anti-fibrotic drug spironolactone. Fibrotic response was assayed by expression of fibrogenic genes (COL1A1 (collagen, type I, alpha 1), ACTA2 (alpha smooth muscle actin), FN1 (fibronectin 1), MYLK (myosin light chain kinase), and MKL1 (megakaryoblastic leukemia (translocation) 1)) and proteins (αSMA). RESULTS Immunofluorescent staining of organoids identified a population of myofibroblasts within the HIO mesenchyme. TGFβ stimulation of HIOs produced a dose-dependent pro-fibrotic response. Spironolactone treatment blocked the fibrogenic response of HIOs to TGFβ. CONCLUSIONS HIOs contain myofibroblasts and respond to a pro-fibrotic stimulus in a manner that is consistent with isolated human myofibroblasts. HIOs are a promising model system that might bridge the gap between current in vitro and in vivo models of intestinal fibrosis in IBD.

Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications

Background and Aims Intestinal fibrosis is a frequent complication in Crohns disease [CD]. The mouse Salmonella typhimurium model, due to its simplicity, reproducibility, manipulability, and penetrance, is an established fibrosis model for drug discovery and preclinical trials. However, the severity of fibrosis and mortality are host- and bacterial strain-dependent, thus limiting the original model. We re-evaluated the S. typhimurium model to optimise fibrosis and survival, using commercially available mouse strains. Methods Fibrotic and inflammatory markers were evaluated across S. typhimurium ΔaroA:C57bl/6 studies performed in our laboratory. A model optimisation study was performed using three commercially available mouse strains [CBA/J, DBA/J, and 129S1/SvImJ] infected with either SL1344 or ΔaroA S. typhimurium. Fibrotic penetrance was determined by histopathology, gene expression, and αSMA protein expression. Fibrosis severity, penetrance, and survival were analysed across subsequent CBA studies. Results Fibrosis severity and survival are both host- and bacterial strain-dependent. Marked tissue fibrosis and 100% survival occurred in the CBA/J strain infected with SL1344. Subsequent experiments demonstrated that CBA/J mice develop extensive intestinal fibrosis, characterised by transmural tissue fibrosis, a Th1/Th17 cytokine response, and induction of pro-fibrotic genes and extracellular matrix proteins. A meta-analysis of subsequent SL1344:CBA/J studies demonstrated that intestinal fibrosis is consistent and highly penetrant across histological, protein, and gene expression markers. As proof-of-concept, we tested the utility of the SL1344:CBA/J fibrosis model to evaluate efficacy of CCG-203971, a novel anti-fibrotic drug. Conclusion The S. typhimurium SL1344:CBA/J model is an optimised model for the study of intestinal fibrosis.

Su2037 Detecting Bowel Wall Fibrosis With Photoacoustic Imaging in an Animal Model of Crohn's Disease

Background/Aims: Optical coherence tomography (OCT) enables three-dimensional (3D) examination of the tissue architecture with micrometer resolution. In previous studies, endoscopic OCT was used to provide structural information correlated with histology for gastrointestinal pathologies. However, the utility of endoscopic OCT for investigating the pathology of the terminal ileum (TI) in patients with symptoms of inflammatory bowel disease (IBD), for example, has been very limited. Endoscopically, reaching the TI entails advancing the colonoscope through multiple sharp bends of the colon, which prevents smooth rotation of previous generations of proximally actuated probes. Recently, our group has developed ultrahigh speed endoscopic OCT technology with a micromotor imaging probe, which enables precise volumetric imaging by avoiding the non-uniform rotational distortion inherent in proximally actuated probes. In this study, we reported structural and vascular changes of TI with chronic inflammation using endoscopic OCT and OCT angiography (OCTA). Methods: This prototype endoscopic OCT system achieves an imaging speed of 600,000 A-scans per second and a frame rate of 400 frames per second. The diameter of the imaging probe was small enough to pass through the accessory port of the colonoscope, enabling coregistered biopsies. OCTA visualized microvasculature by measuring decorrelation between sequential OCT images to generate motion contrast from blood flow. The study was performed at the Veterans Affairs Boston Healthcare System (VABHS) with the approval from the institutional review boards from VABHS, Harvard Medical School, and MIT. Results: Figure 1 shows OCT images of a normal TI (Figs. 1(A, B)) and TI with chronic inflammation (Figs. 1(C, D)) as well as the coregistered en face OCTA (Fig. 1(E)) at the same depth as Fig. 1(C). Typical villous structure of normal TI can be observed in Fig. 1(A) at 100 μm depth, which was consistent with the colonoscopy findings (inset, Fig. 1(A)). The slight tilted orientation of the villi was caused by the gentle pressing of the imaging probe, which was observed along the cross-sectional OCT image as well (Fig. 1(B)). On the contrary, a loss of villous structure and nodular mucosal appearance were identified in Fig. 1(C) of TI with chronic inflammation, which was confirmed by the coregistered biopsy. In addition, a decreased imaging depth was observed in the cross-sectional OCT image (Fig. 1(D)). Fig 1(E) shows the coregistered en faceOCTA as Fig. 1(C) where increased vasculature with abnormal vascular branching was observed. Conclusions: This study demonstrates that ultrahigh speed endoscopic OCT technology and OCTA allows the visualization structural and vascular features of the TI with chronic inflammation. Acknowledgment: NIH R01CA075289-17, R44-CA101067-07, AFOSR FA9550-10-1-0551 and FA9550-12-1-0499.

Mo2034 Card-024, a Vitamin D Analog, Attenuates the PRO-Fibrotic Response to TGFβ or Substrate Stiffness in Colonic Myofibroblasts

Background and aims: Diversion colitis is a relatively common complication of colostomy involving the excluded colons segment. Although the pathogenesis of the chronic inflammatory process remains unclear, the lack of luminal nutrients is believed to play a major role in the development of the disease. Therefore, we tested whether essential colonocyte nutrients, such as butyrate or glutamine could attenuate the inflammation in a model of diversion colitis. Methods: Forty eight male Wistar SPF rats were randomly distributed in 4 groups of 12 rats each. Animals were submitted to a Hartmanns end colostomy, and treated with enemas containing glutamine, butyrate, or saline. Enemas were administered twice a week in the excluded segment of the colon from the 4th to the 12th week after the surgical procedure. Follow-up video-colonoscopy was performed every 4 weeks, during 12 weeks. Afterwards, animals of all groups, including a normal control group were sacrificed either on the 8th week or the 12th week. The effects of treatment were evaluated using videoendoscopic and histologic scores, Picrosirius, Periodic Acid of Schiff and TUNEL staining, and also measuring the production of IL-1β, TNF-α and TGF-β in organ cultures, by ELISA. Results: Colonoscopies of the diverted segment, on 8th and 12th weeks, showed mucosa with hyperemia, increased number of vessels, spontaneous bleeding and mucus discharge, with the peak of the inflammatory activity in the 8th week. Treatment with either glutamine or butyrate prevented a significant increase in both colonoscopic (P < 0.01) and histological (P < 0.01) inflammatory scores, and stabilized the densities of collagen fibers in tissue (P < 0.02), the number of goblet cells (P <0.04), and the rate of apoptosis within the epithelium (P < 0.04) to normal low values, compared to the high values of the untreated inflamed tissues. Butyrate and glutamine enemas significantly abrogated the increased production of IL-1β and TGF-β (butyrate), and also of TNF-α (butyrate and glutamine) from tissue explants of surgically diverted colon, and levels were kept within the normal ranges following treatment. Conclusion: Topical administration of either glutamine or butyrate provides mucosal protection against the development of inflammation in the diverted colonic segment after colostomy. These results strongly suggest that the lack of specific luminal nutrients can induce inflammation, and support the potential utilization of glutamine or butyrate enemas for the treatment and possibly the prevention of human diversion colitis.