An inducible model for genetic manipulation and fate-tracing of PDGFRß-expressing fibrogenic cells in the liver.

Myofibroblasts are the source of extracellular matrix protein during liver fibrogenesis. Fibroblasts, hepatic stellate cells (HSCs) and vascular smooth muscle cells are mesenchymal subpopulations in the liver that are characterized by the expression of PDGFRß and contribute to the pool of these myofibroblasts. Conditional knockout models are important to better understand the function of specific liver cell populations including mesenchymal cells. While there is a limited number of constitutive mouse models for liver mesenchymal cell specific transgene expression, there is no established model for inducible gene targeting in HSCs or PDGFRß-expressing mesenchymal cell populations in the liver. To address this, we investigated whether the tamoxifen inducible PDGFRß-P2A-CreERT2 mouse can be used as a reliable tool to specifically express transgens in liver mesenchymal cells. Our data demonstrate, that PDGFRß-P2A-CreERT2 specifically and efficiently marks over 90% of retinoid positive HSCs in healthy and fibrotic liver in mice upon tamoxifen injection, and that those cells give rise to Col1a1-expressing myofibroblasts in different models of liver fibrosis. Together with a negligible background recombination of only about 0.33%, this confirms that the PDGFRß-P2A-CreERT2 mouse is nearly as efficient as established constitutive LratCre and PDGFRß-Cre mouse models for recombination in HSCs, and that it is a powerful model for mesenchymal liver cell studies that require an inducible Cre approach.

Pulmonary Arterial Hypertension and Consecutive Right Heart Failure Lead to Liver Fibrosis.

Hepatic congestion occurs in patients with right heart failure and can ultimately lead to liver fibrosis or cardiac cirrhosis. Elevated pulmonary arterial pressure is found in patients with hepatic congestion. However, whether pulmonary arterial hypertension (PAH) can be a cause of liver fibrosis is unknown. The aim of this study was to investigate whether rats in the SuHx model with severe PAH develop liver fibrosis and to explore the mechanisms of congestive hepatic fibrosis both in rats and humans. To achieve this, PAH was induced in six to eight-week old male Sprague Dawley rats by a single subcutaneous injection of the VEGFR 2 inhibitor SU5416 and subsequent hypoxia for 3 weeks, followed by a 6-week period in room air. SuHx-exposed rats developed severe PAH, right ventricular hypertrophy (RVH), and consecutive right ventricular failure. Cardiac magnetic resonance imaging (MRI) and histological analysis revealed that PAH rats developed both hepatic congestion and liver fibrosis. Gene set enrichment analysis (GSEA) of whole liver RNA sequencing data identified a hepatic stellate cell specific gene signature in PAH rats. Consistently, tissue microarray from liver of patients with histological evidence of hepatic congestion and underlying heart disease revealed similar fibrogenic gene expression patterns and signaling pathways. In conclusion, severe PAH with concomitant right heart failure leads to hepatic congestion and liver fibrosis in the SU5416/hypoxia rat PAH model. Patients with PAH should therefore be screened for unrecognized liver fibrosis.

The purinergic P2Y14 receptor links hepatocyte death to hepatic stellate cell activation and fibrogenesis in the liver.

Fibrosis contributes to ~45% of deaths in western countries. In chronic liver disease, fibrosis is a major factor determining outcomes, but efficient antifibrotic therapies are lacking. Although platelet-derived growth factor and transforming growth factor-ß constitute key fibrogenic mediators, they do not account for the well-established link between cell death and fibrosis in the liver. Here, we hypothesized that damage-associated molecular patterns (DAMPs) may link epithelial cell death to fibrogenesis in the injured liver. DAMP receptor screening identified purinergic receptor P2Y14 among several candidates as highly enriched in hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Conversely, P2Y14 ligands uridine 5'-diphosphate (UDP)-glucose and UDP-galactose were enriched in hepatocytes and were released upon different modes of cell death. Accordingly, ligand-receptor interaction analysis that combined proteomic and single-cell RNA sequencing data revealed P2Y14 ligands and P2Y14 receptor as a link between dying cells and HSCs, respectively. Treatment with P2Y14 ligands or coculture with dying hepatocytes promoted HSC activation in a P2Y14-dependent manner. P2Y14 ligands activated extracellular signal-regulated kinase (ERK) and Yes-associated protein (YAP) signaling in HSCs, resulting in ERK-dependent HSC activation. Global and HSC-selective P2Y14 deficiency attenuated liver fibrosis in multiple mouse models of liver injury. Functional expression of P2Y14 was confirmed in healthy and diseased human liver and human HSCs. In conclusion, P2Y14 ligands and their receptor constitute a profibrogenic DAMP pathway that directly links cell death to fibrogenesis.

Tumor restriction by type I collagen opposes tumor-promoting effects of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.