Cellular and Molecular Gastroenterology and Hepatology最新文献

Hepatic stellate cells (HSCs) and their activated derivatives, often referred to as myofibroblasts (MFs), play a key role in progression of chronic liver injuries leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Until recently, MFs were considered a homogenous cell type majorly due to lack of techniques that allow complex molecular studies at a single-cell resolution. Recent technical advancements in genetic lineage-tracing models as well as the exponential growth of studies with single-cell transcriptome and proteome analyses have uncovered hidden heterogeneities among the HSC and MF populations in healthy states as well as chronic liver injuries at the various stages of tissue deformation. The identification of different phenotypes along the HSC/MF axis, which either maintain essential liver functions (“good” HSCs), emerge during fibrosis (“bad” HSCs), or even promote hepatocellular carcinoma (“ugly” HSCs), may lay the foundation for targeting a particular MF phenotype as potential treatment for chronic liver injuries.

Background & Aims

Barrett’s esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%–74% of patients with Barrett’s esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5⁺ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett’s esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett’s-like metaplasia and dysplasia development.

Methods

We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRAS^G12D expression targeting SCJ LGR5⁺ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids.

Results

p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRAS^G12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRAS^G12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition).

Conclusions

p16 deletion in LGR5⁺ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRAS^G12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett’s high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett’s-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Background & Aims

The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration.

Methods

We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization.

Results

Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3β. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization.

Conclusions

Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells.

Background & Aims

There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL-1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL-1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction.

Results

Alcohol-fed Rage^ΔMye mice exhibited decreased nuclear factor kappa B signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. In addition, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL-1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, because it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induced a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis.

Conclusions

We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.