As described earlier, EVs may play a pathogenic role in cell-cell signalling and mediation of hepatocellular damage. EVs are small (≤1.5 µM) particles (See Figure 1.15 for details on size and mechanism of EV production) which are physiologically involved in normal cell to cell communication (Paolicelli et al. 2019). However, they arise from the PM of activated or damaged cells, and exhibit exposed anionic phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) on their surface (Shet 2008). EVs also bear surface membrane antigens reflecting their cells of origin (see Table 1.6 for summary of some cell markers used to characterise EVs).
The process of EV release involves cleavage of the cell cytoskeleton and is mediated by caspase 3. Several groups, including studies from the host laboratory, have demonstrated that Ca2+ dependent proteases are involved in EV generation (Teoh et al.
2014, Ajamieh et al. 2015). These enzymes include calpain and gelsolin, both of which cleave actin filaments; this allows for activation of scramblase leading to cytoskeletal eversion. The changes to the cellular cytoskeleton also result in phospholipid reorganisation, with migration of PS and PE to the outer cellular surface, allowing EVs to bud off (Figure 1.15).
Within the last 10 years, the role of EVs has emerged as a complex one. It is now known that they are heterogenous particles, and potentially contain a multitude of biologically active macromolecules (see Figure 1.16). The exact composition of EVs is largely dependent on parental cell type and the mechanism for their formation. Not withstanding this, most EVs contain:
1. DNA, including mitochondrial DNA (Tatischeff et al. 1998)
2. RNAs (Eg: miRNA, coding mRNA, non-coding, as well as RNA-inducing silencing
complexes (RISC) (Silva and Melo 2015, Iavello et al. 2016)
3. Proteins: heat shock proteins, growth factors, tetraspanins. The later includes a
number of CD proteins listed in Table 1.6 (De Maio 2011, Chiasserini et al. 2014).
4. Lipid-enriched bilayer (compared with parent cell), comprised of FC,
sphingomyelin, PS and bioactive eiconsanoids and prostaglandins, as well as lipid rafts (Koumangoye et al. 2011, Fais et al. 2016). Importantly, cholesterol plays an important role in EV release (Llorente et al. 2007) and uptake (Mulcahy et al. 2014).
phagocytosis, lipid raft internalisation, and receptor-mediated uptake. Once internalised, the EV cargo (Figure 1.16) is released into the target cell where it can, depending on the exact cargo composition, induce a number of function effects, including hyper- inflammation (Szabo and Momen-Heravi 2017).
Figure 1.15 Types of extracellular vesicles (EVs) and mechanisms of their release.
(A) Exosomes, the smallest EVs, measure 40-100 nm (size of an average virus) and are generated by either the classical and/or direct pathways. The classical pathway (as represented by pink arrows) involves invagination of plasma membrane to form multivesicular bodies (MVBs). These coalesce within endosomal vesicles and fuse with the PM releasing extracellular exosomes. The direct pathway is simpler (blue arrows). Here exosomes bleb off the PM surfaces into the extracellular space. Importantly all EVs harbour markers of the cell of origin; this allows for qualitative analysis using assay techniques (see Table 1.6 for several examples of cell-specific markers). (B) Microparticles (MPs) are larger (0.1-1 µm; roughly the size of cocci bacteria) and are generated by phosphatidyl serine (PS) translocation from the inner to the outer PM surface. This reorganises the cytoskeleton causing MP release. (C) Apoptotic bodies are larger 1-4 µm EVs, formed from activation of apoptotic pathways (especially mitochondrial dysfunction and leakage). Here, DNA fragmentation, PM asymmetry, and disorganisation of cytoskeleton proteins generates the apoptotic fragments. Image adapted from Lemoinne et al. (2014).
Figure 1.16 Typical extracellular vesicle composition.
Details of EV composition are described in Section 1.7.4, Szabo and Momen-Heravi (2017) recently published a review detailing EV involvement in viral and alcoholic liver disease, as well as NAFLD (image adapted from their review).
Abbreviations: AA, amino acids; DNA, deoxyribosnucleic acid; FC, free cholesterol, HSP90, heat shock protein 90; lncRNA, long noncoding RNA; MHC, major histocompatibility complex class; mRNA, messenger RNA; miRNA, microRNA; RABs, Ras-related proteins.
Exposing primary rat hepatocytes or HepG2 cells (a well-differentiated immortalised human hepatoma cell line) to palmitic or stearic acid results in EV release (Povero et al.
2013). Povero and colleagues (2013) subsequently demonstrated that these isolated EVs contain several microRNAs (miR) and can recruit, as well as activate HSCs. In their quiescent state, HSCs store vitamin A in the peri-sinusoid. This inactivated phenotype is governed by peroxisome proliferator-activated receptor (PPAR)- expression (Hazra et al. 2004). The EVs derived from FFA-damaged hepatocytes suppress HSC PPAR- expression, thereby activating HSC expression of pro-fibrotic genes, including collagen type-1 and alpha smooth muscle actin (Povero et al. 2013).
Importantly, to the author’s knowledge, the relationship between cholesterol lipotoxicity in hepatocytes and EV formation has not been explored. It is a major strand of the research conducted in this thesis.
Asialoglycoprotein receptor-ß Hepatocytes WB Ise et al. 2001, Severgnini et al. 2012, Shi et al. 2013 VE-
cadherin/CD144 Sinusoidal endothelia cells (SES) FACS/WB
Lalor et al. 2006,
Goldman et al. 2014
Vascular cell
adhesion molecule SES
WB Holmen et al. 2005, Lalor et al. 2006 E-Selectin SES WB Daneker et al. 1998, Holmen et al. 2005, Lalor et al. 2006 CD41 Platelets FACS Mitjavila-Garcia et al. 2002, Bagamery et al. 2005, van Velzen et al.
2012
Pselectin/CD62P Activated platelets FACS/WB
Murakami et al. 1996,
Jy et al. 1999, Lu et al.
2011
Tissue
factor/CD142 Activated platelets FACS/WB Panasiuk et al. 2007
CD1d tetramer NK T cells FACS
Miyagi et al. 2003, Stenstrom et al. 2005,
Montoya et al. 2007
CD8 CD8 T cells FACS Storek et al. 1998,
Ondoa et al. 2005
Ly6G Neutrophils FACS Rose et al. 2012
CD15 Neutrophils FACS Pillay et al. 2013
F4/80 KC/macrophages FACS Zhang et al. 2008, Rose
et al. 2012
CD14 Monocytes/macrophages/myeloid
dendritic cells FACS Pillay et al. 2013 Abbreviations: CD, cluster of differentiation; EV, extracellular vesicles; FACS, fluorescence-activated cell sorting; KC, Kupffer cell; Ly6G, lymphocyte antigen 6 complex locus G6D; NK, natural killer cell; SES, sinusoidal endothelial cell; WB, western blot.
1.8 Recent conceptual changes in NASH
Following the completion of the research outlined in this thesis, there have been new and emerging concepts of regarding the pathogenesis and treatment of NASH. This short review will focus on two topics, namely contemporary research regarding the role
of the microbiome in NASH pathogenesis, and novel treatment options based on potential molecular targets in NASH.