Extracellular Vesicles in Hepatobiliary Health and Disease

Abstract

Extracellular vesicles (EVs) are membrane-bound nanoparticles released by cells and are an important means of intercellular communication in physiological and pathological states. We provide an overview of recent advances in the understanding of EV biogenesis, cargo selection, recipient cell effects, and key considerations in isolation and characterization techniques. Studies on the physiological role of EVs have relied on cell-based model systems due to technical limitations of studying endogenous nanoparticles in vivo . Several recent studies have elucidated the mechanistic role of EVs in liver diseases, including nonalcoholic fatty liver disease, viral hepatitis, cholestatic liver disease, alcohol-associated liver disease, acute liver injury, and liver cancers. Employing disease models and human samples, the biogenesis of lipotoxic EVs downstream of endoplasmic reticulum stress and microvesicles via intracellular activation stress signaling are discussed in detail. The diverse cargoes of EVs including proteins, lipids, and nucleic acids can be enriched in a disease-specific manner. By carrying diverse cargo, EVs can directly confer pathogenic potential, for example, recruitment and activation of monocyte-derived macrophages in NASH and tumorigenicity and chemoresistance in hepatocellular carcinoma. We discuss the pathogenic role of EVs cargoes and the signaling pathways activated by EVs in recipient cells. We review the literature that EVs can serve as biomarkers in hepatobiliary diseases. Further, we describe novel approaches to engineer EVs to deliver regulatory signals to specific cell types, and thus use them as therapeutic shuttles in liver diseases. Lastly, we identify key lacunae and future directions in this promising field of discovery and development. © 2023 American Physiological Society. Compr Physiol 13:4631-4658, 2023.

Figure 1

Biogenesis of extracellular vesicles. Microvesicles are formed directly from plasma membrane budding by sequestering cytosolic cargo. Exosomes arise via endosomal maturation into multivesicular bodies (MVB). MVBs contain intraluminal vesicles generated by invagination of the limiting membrane and eventually released at the plasma membrane as exosomes.

Figure 2

Methods of extracellular vesicle isolation. Various methods of EV isolation are suited for different scenarios based on required yield, purity, and specificity for an EV subtype. Often, separation from non-EV lipoproteins in biological fluids that have similar size and density is also required. Some examples depicted are (A) precipitation, for example, with polyethylene glycol (B) size exclusion chromatography (C) differential centrifugation (dUC) (D) density gradient ultracentrifugation, and (E) immunoaffinity-based capture.

Figure 3

Small EVs from hepatocyte cell line. Cell culture supernatant was fixed and processed for immunogold-based detection of cytochrome P450 2E1 (Cyp2E1, smaller particle size) and asialoglycoprotein 2 (Asgr2, larger particle size) to demonstrate the detection of hepatocyte-derived EVs by this technique. Magnification 150k ×, scale bar 100 nm.

Figure 4

Role of extracellular vesicles in nonalcoholic steatohepatitis. EVs mediate multi-organ cross-talk—between the liver, adipose tissue, and bone marrow; as well as multi-cellular cross-talk—between hepatocytes, non-parenchymal cells in the liver, and immune cells. Lipotoxic hepatocytes activate intracellular stress responses including endoplasmic reticulum (ER) stress-mediated activation of inositol requiring enzyme 1 α (IRE1α), activation of serine threonine Rho- kinase 1 (ROCK1) and caspase 3, and mixed lineage kinase 3 (MLK3) which increase the formation and release of EVs. These EVs are heterogeneous and carry diverse cargo such as proteins including Vanin, TRAIL, CXCL10, nucleic acids including microRNAs (mir-128–3p, miR-223) and mitochondrial DNA (mtDNA), and lipids such as sphingosine 1 phosphate (S1P) which serve ligands for a multitude receptor-mediated or epigenetic regulatory signaling pathways in recipient cells. Though factors targeting subsets of EVs to specific recipient cells remain unknown, studies demonstrate that specific cell types respond to certain cargoes. For example, lipotoxic ER stress in hepatocytes can increase release of EVs containing ceramide-derived S1P which is a chemoattractant to proinflammatory circulating macrophages expressing S1P receptors. Lipotoxic EVs also contain CXC motif chemokine ligand 10 (CXCL10), TNFα-related apoptosis-inducing ligand (TRAIL), mtDNA which engages CXC motif chemokine receptor 3 (CXCR3), TRAIL-receptor (TRAIL-R), and Toll-like receptor 9 (TLR9), respectively. In contrast, hepatocyte uptake via the low-density lipoprotein receptor (LDL-R), of miR-223 enriched EVs from neutrophils may have an anti-inflammatory effect in NASH. Vanin-enriched hepatocyte-derived EVs and miR-128–3p enriched EVs increase hepatic stellate cell activation. Integrin beta 1 (ITGB1) in hepatocyte-derived EVs is internalized by circulating monocytes to increase their adhesion to liver sinusoidal endothelial cells (LSEC). Adipose tissue-derived EVs containing monocyte chemotactic protein 1 (MCP1), interleukin 6 (IL6), and cluster of differentiation 36 (CD36) influence hepatocyte insulin resistance and lipid metabolism. Cells and tissues are denoted in bold, and the biological effect is italicized.

Figure 5

Role of extracellular vesicles in primary tumors of the liver. In hepatocellular carcinoma (HCC) as well as cholangiocarcinoma (CCA), EVs are implicated in cross-talk between cancer cells and other cell types inhabiting the tumor microenvironment. In HCC, tumor cells mediate a feed-forward loop with tumor cells by downregulating expression of the vacuolar protein sorting 4 homolog A (VPS4A) that promotes secretion of EVs with bioactive cargo such as microRNA (miRs) with oncogenic potential or long noncoding RNA (lincRNA-regulator of reprogramming) which confer chemoresistance. HCC-derived EVs mediate diverse processes such as angiogenesis via miR-155 containing EVs that activate heat shock proteins 70 on endothelial cells, epithelial-mesenchymal transition via miR-1247–3p that activate β-integrin on fibroblasts, and stellate cell activation via Twist1. Conversely, cancer-associated fibroblast (CAF) derived EVs carrying interleukins (IL6/8) promote HCC metastatic potential via tranglutaminase2 signaling, while miR-320a reduces tumor progression by suppressing PBX homeobox 3 (PBX3) signaling. Crosstalk with immune cells occurs via tumor-derived exosomes containing high mobility group box 1 (HMGB1) that mediate immune escape by binding to toll-like receptors 2/4 on B cells, and heat shock proteins that activate cytotoxic natural killer (NK) cells after exposure to chemotherapy. Tumor-associated macrophage-derived exosomes that contain integrinαMβ2 and TGFβ boost the migratory potential of HCC by activating matrix metalloproteinase-9 signaling and stellate cell activation respectively. In cholangiocarcinoma, EVs containing circular RNAs (circ-CCAC1) disrupt endothelial barrier integrity by downregulating intercellular junction proteins such as occludin and promote angiogenesis. CCA-EVs promote tumor stroma formation by inducing fibroblast differentiation of mesenchymal stem cells, and conversely CAF-derived EVs carrying miR-195 inhibit CCA growth. Cell types are denoted in bold, and the biologic effect is italicized.