Endothelial Zeb2 preserves the hepatic angioarchitecture and protects against liver fibrosis.

AIMS: Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. METHODS AND RESULTS: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarization'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. CONCLUSION: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

In vitro Three-Dimensional Sprouting Assay of Angiogenesis using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

Pericytes in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by multi-systemic vascular dysplasia affecting 1 in 5000 people worldwide. Individuals with HHT suffer from many complications including nose and gastrointestinal bleeding, anemia, iron deficiency, stroke, abscess, and high-output heart failure. Identification of the causative gene mutations and the generation of animal models have revealed that decreased transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) signaling and increased vascular endothelial growth factor (VEGF) signaling activity in endothelial cells are responsible for the development of the vascular malformations in HHT. Perturbations in these key pathways are thought to lead to endothelial cell activation resulting in mural cell disengagement from the endothelium. This initial instability state causes the blood vessels to response inadequately when they are exposed to angiogenic triggers resulting in excessive blood vessel growth and the formation of vascular abnormalities that are prone to bleeding. Drugs promoting blood vessel stability have been reported as effective in preclinical models and in clinical trials indicating possible interventional targets based on a normalization approach for treating HHT. Here, we will review how disturbed TGF-ß and VEGF signaling relates to blood vessel destabilization and HHT development and will discuss therapeutic opportunities based on the concept of vessel normalization to treat HHT.