Expression of hepatocyte growth factor and its receptor c-met, correlates with severity of pathological injury in experimental alcoholic liver disease.

Expression of hepatocyte growth factor (HGF) and its receptor, c-met is up-regulated in various forms of liver injury. This study evaluated the relationship between HGF and c-met expression and pathological changes in experimental alcoholic liver disease. Rats (5 per group) were fed ethanol and a diet containing saturated fat corn oil or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in controls. In a second set of experiments, Kupffer cells, endothelial cells and hepatocytes were isolated from rats in each group. Pathological evaluation and analysis of HGF and c-met expression were performed in liver and the different cell types. Increased expression of HGF and c-met expression was detected in the liver of rats showing necroinflammatory changes. The Kupffer and endothelial cells were primarily responsible for the increase in HGF, c-met expression was seen only in hepatocytes. Thus, up-regulation of HGF and c-met occurred in the presence of the necrosis and inflammation suggesting that HGF may be acting to protect against liver injury or accelerate the regenerative process

Severe polyposis in Apc1322T mice is associated with submaximal Wnt signalling and increased expression of the stem cell marker Lgr5

Background and aims: Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, ApcR850X (Min) and. Apc1322T (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. Methods: Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. Results: As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. Conclusions: The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth

Targeted Induction of Endoplasmic Reticulum Stress Induces Cartilage Pathology

Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS–causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF–mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress–inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix proteins

DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo.

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic

Putative direct and indirect Wnt targets identified through consistent gene expression changes in APC-mutant intestinal adenomas from humans and mice

In order to identify new genes with differential expression in early intestinal tumours, we performed mRNA (messenger ribonucleic acid) expression profiling of 16 human and 63 mouse adenomas. All individuals had germline APC mutations to ensure that tumorigenesis was driven by ‘second hits’ at APC. Using stringent filtering to identify changes consistent between humans and mice, we identified 60 genes up-regulated and 151 down-regulated in tumours. For 22 selected genes—including known Wnt targets—expression differences were confirmed by qRT–PCR (quantitative reverse transcription polymerase chain reaction). Most, but not all, differences were also present in colorectal carcinomas. In situ analysis showed a complex picture. Expression of up-regulated genes in adenomas was usually uniform/diffuse (e.g. ITGA6) or prominent in the tumour core (e.g. LGR5); in normal tissue, these genes were expressed at crypt bases or the transit amplifying zone. Down-regulated genes were often undetectable in adenomas, but in normal tissue were expressed in mesenchyme (e.g. GREM1/2) or differentiated cells towards crypt tops (e.g. SGK1). In silico analysis of TCF4-binding motifs showed that some of our genes were probably direct Wnt targets. Previous studies, mostly focused on human tumours, showed partial overlap with our ‘expression signature’, but 37 genes were unique to our study, including TACSTD2, SEMA3F, HOXA9 and IER3 (up-regulated), and TAGLN, GREM1, GREM2, MAB21L2 and RARRES2 (down-regulated). Combined analysis of our and published human data identified additional genes differentially expressed in adenomas, including decreased BMPs (bone morphogenetic proteins) and increased BUB1/BUB1B. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective: Barrett's oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods: Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results: Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Conclusions: Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus