Dataset Information

Effect of ABT-263 on Intestinal Fibrosis in Human Myofibroblasts, Human Intestinal Organoids, and the Mouse Salmonella typhimurium Model.

ABSTRACT:

Background

Intestinal fibrosis and subsequent intestinal obstruction are common complications of Crohn's disease (CD). Current therapeutics combat inflammation, but no pharmacological therapy exists for fibrostenotic disease. Pathological persistence of activated intestinal myofibroblasts is a key driver of fibrosis in CD. In other organ systems, BH-3 mimetic drugs that affect Bcl-2 apoptotic pathways induce apoptosis in activated myofibroblasts and reduce fibrogenic gene expression, thereby reducing fibrosis.

Methods

We evaluated the proapoptotic and antifibrotic efficacy of several classes of BH-3 mimetics in 2 in vitro fibrogenesis models. The candidate molecule, ABT-263, was advanced to a 3-dimensional human intestinal organoid (HIO) model. Finally, the therapeutic efficacy of ABT-263 was evaluated in the mouse Salmonella typhimurium intestinal fibrosis model.

Results

The BH-3 mimetics induced apoptosis, repressed fibrotic protein expression, and reduced fibrogenic gene expression in normal human intestinal myofibroblasts. The BH-3 mimetics that target Bcl-2 and Bcl-xl demonstrated the greatest efficacy in vitro. The ABT-199 and ABT-263 induced apoptosis and ameliorated fibrogenesis in the in vitro myofibroblast models. In the HIO model, ABT-263 inhibited fibrogenesis and induced apoptosis. In the mouse S. typhimurium model, dose-dependent reduction in macroscopic pathology, histological inflammation, inflammatory and fibrotic gene expression, and extracellular matrix protein expression indicated ABT-263 may reduce intestinal fibrosis.

Conclusions

In vitro, the antifibrotic efficacy of BH-3 mimetics identifies the Bcl-2 pathway as a druggable target and BH-3 mimetics as putative therapeutics. Reduction of inflammation and fibrosis in the mouse intestinal fibrosis model by ABT-263 indicates BH-3 mimetics as potential, novel antifibrotic therapeutics for Crohn's disease.

SUBMITTER: Johnson LA

PROVIDER: S-EPMC9017142 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundIntestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.MethodsChronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases.ResultsMice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts.ConclusionsIn summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Project description:Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.