Extracellular vesicles in fatty liver promote a metastatic tumor microenvironment.

Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.

Experimental Workflow for Preclinical Studies of Human Antifibrotic Therapies.

Chronic liver diseases accompanied by liver fibrosis have caused significant morbidity and mortality in the world with increasing prevalence. Nonetheless, there are no approved antifibrotic therapies. Although numerous preclinical studies showed satisfactory results in targeting fibrotic pathways, these animal studies have not led to success in humans. In this chapter, we summarize the experimental approaches currently available, including in vitro cell culture models, in vivo animal models, and new experimental tools relevant to humans, and discuss how we translate laboratory results to clinical trials. We will also address the obstacles in transitioning promising therapies from preclinical studies to human antifibrotic treatments.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro.

Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.