Shah, Vijay H.

Extracellular vesicles (EVs) are nanometer‐sized, membrane‐bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell‐to‐cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano‐shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (H epatology 2016;64:2219‐2233).

Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of endothelial deletion of Rbpj from birth. Two‐week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto‐systemic shunts. These mutant mice presented with severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin‐B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis, albeit milder than that observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks. Conclusions: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302‐1316)

The capillary network irrigating the liver is important not only for nutrient and oxygen delivery, but also for the signals distributed to other hepatic cell types necessary to maintain liver homeostasis. During development, endothelial cells are a key component in liver zonation. In adulthood, they maintain hepatic stellate cells and hepatocytes in quiescence. Their importance in pathobiology is highlighted in liver regeneration and chronic liver diseases, where they coordinate paracrine cell behavior. During regeneration, liver sinusoidal endothelial cells induce hepatocyte proliferation and angiogenesis. During fibrogenesis, they undergo morphological and functional changes, which are reflected by their role in hepatic stellate cell activation, inflammation, and distorted sinusoidal structure. Therapeutic strategies to target angiocrine signaling are in progress but are in the early stages. Here, we offer a short synthesis of recent studies on angiocrine signaling in liver homeostasis, regeneration, and fibrogenesis.

Abstract Introduction: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under TGF-β stimulation, hepatic stellate cells (HSCs), which are liver specific pericytes, transdifferentiate into myofibroblasts that promote tumor implantation and growth in the liver. The regulation of this HSC activation process in the liver however remains poorly understood. In this study, we tested a hypothesis that actin binding protein vasodilator-stimulated phosphoprotein (VASP), previously implicated in focal adhesion and migration of HSCs, may regulate the TGF-β mediated HSC activation process by interacting with and regulating TGF-β receptors. Methods: VASP shRNA and siRNA were used to knockdown VASP in primary human HSCs and LX2, an immortalized HSC cell line. TGF-β activation of HSCs was quantitated by immunofluorescence staining (IF) for alpha-smooth muscle actin (α-SMA) and Western blot analysis (WB) for α-SMA, fibronectin and P-SMAD2. Total TGF-β receptor levels were determined by WB, and plasma membrane TβRII was quantitated by biotinylation of cell surface proteins approach. Protein subcellular localization was investigated by IF and protein interactions were determined by double IF and co-immunoprecipitation (IP). Regulation of stellate cell VASP by HSC/tumor interactions was investigated by IF on human colorectal liver metastases and the role of stellate cell VASP in tumor growth was studied in a tumor/HSC coimplantation mouse model. Results: TGF-β1 induced more than 60% of human primary HSCs into myofibroblasts as determined by α-SMA IF. Two different VASP shRNAs significantly reduced this effect of TGF-β1 on HSC activation (P<0.05). The effect of VASP knockdown on HSC activation was confirmed in LX2 cells as well as using a VASP siRNA approach. VASP knockdown increased total TGF receptor protein levels with no effects on SMAD2 and 3 protein levels. VASP interacted with TβRII as evidenced by double IF and co-IP. Biotin-labeling of cell surface proteins and IF revealed that VASP knockdown reduced TβRII protein level at the plasma membrane and that VASP overexpression increased TβRII at the plasma membrane. Concurrently, VASP knockdown reduced plasma membrane localization of Rab11a, a small GTPase that marks recycling endosomes and is essential for recycling of plasma membrane receptors. VASP/Rab11 interactions were confirmed by IF and co-IP. Furthermore, we found that VASP knockdown HSCs significantly reduced tumor growth in mice as compared to control HSCs in a HSC/tumor coimplantation model. In patients, metastasis of colorectal cancer cells in liver sinusoids induced upregulation of VASP and α-SMA in adjacent HSCs as revealed by IF on biopsies of human colorectal liver metastases. Conclusions: Our studies demonstrate a requirement of VASP in the TGF-β dependent HSC activation in the tumor microenvironment and ensuing tumor growth. VASP interacts with Rab11 and regulates Rab11 positive recycling endosomal function thereby promoting TβRII recycling back to the plasma membrane in HSCs. VASP in the tumor microenvironment thus presents a therapeutic target for reducing tumor implantation and metastatic growth in the liver. Citation Format: Kangsheng Tu, Jiachu Li, Chunsheng Liu, Vijay H. Shah, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes TGF-beta mediated myofibroblastic activation by regulating recycling of TGF-beta receptor II to the plasma membrane. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A70. doi:10.1158/1538-7445.CHTME14-A70

Abstract Cancer-associated fibroblasts (CAFs), the most abundant cells in the tumor microenvironment (TME), are a key source of the extracellular matrix (ECM) that constitutes the desmoplastic stroma. Through remodeling of the reactive tumor stroma and paracrine actions, CAFs regulate cancer initiation, progression, and metastasis, as well as tumor resistance to therapies. The CAFs found in stroma-rich primary hepatocellular carcinomas (HCC) and liver metastases of primary cancers of other organs predominantly originate from hepatic stellate cells (HSTC), which are pericytes associated with hepatic sinusoids. During tumor invasion, HSTCs transdifferentiate into myofibroblasts in response to paracrine signals emanating from either tumor cells or a heterogeneous cell population within the hepatic tumor microenvironment. Mechanistically, HSTC-to-myofibroblast transdifferentiation, also known as, HSTC activation, requires cell surface receptor activation, intracellular signal transduction, gene transcription, and epigenetic signals, which combined ultimately modulate distinct gene expression profiles that give rise to and maintain a new phenotype. The current review defines a paradigm that explains how HSTCs are activated into CAFs to promote liver metastasis. Furthermore, a focus on the most relevant intracellular signaling networks and epigenetic mechanisms that control HSTC activation is provided. Finally, we discuss the feasibility of targeting CAF/activated HSTCs, in isolation or in conjunction with targeting cancer cells, which constitutes a promising and viable therapeutic approach for the treatment of primary stroma-rich liver cancers and liver metastasis. Mol Cancer Res; 13(4); 604–12. ©2014 AACR.