Intercellular crosstalk of hepatic stellate cells in liver fibrosis: New insights into therapy

Journal Pre-proof Intercellular crosstalk of hepatic stellate cells in liver fibrosis: new insights into therapy Xuanyan Cai, Jiajia Wang, Jincheng Wang, Qian Zhou, Bo Yang, Qiaojun He, Qinjie Weng

PII:

S1043-6618(19)32419-3

DOI:

https://doi.org/10.1016/j.phrs.2020.104720

Reference:

YPHRS 104720

To appear in:

Pharmacological Research

Received Date:

29 October 2019

Revised Date:

8 January 2020

Accepted Date:

20 February 2020

Please cite this article as: Cai X, Wang J, Wang J, Zhou Q, Yang B, He Q, Weng Q, Intercellular crosstalk of hepatic stellate cells in liver fibrosis: new insights into therapy, Pharmacological Research (2020), doi: https://doi.org/10.1016/j.phrs.2020.104720

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Title: Intercellular crosstalk of hepatic stellate cells in liver fibrosis: new insights into therapy

Author: Xuanyan Cai a, Jiajia Wang a, Jincheng Wang a, Qian Zhou c, Bo Yang a, Qiaojun He a, b, Qinjie Weng a, b, *.

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Affiliation:

Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China

Center for Drug Safety Evaluation and Research, Zhejiang University, Hangzhou

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b

310058, PR China

Department of Pharmacy, Hangzhou Medical College, Hangzhou 310053, PR China

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c

Qinjie Weng, PhD.

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Corresponding author:

Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of

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Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China Tel: +86-13819494249; Fax: +86-0571-8820-6087

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E-mail: [email protected]

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Graphical abstract

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Abstract

Liver fibrosis is a dynamic wound-healing process characterized by the net

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accumulation of extracellular matrix. There is no efficient antifibrotic therapy other than liver transplantation to date. Activated hepatic stellate cells (HSCs) are the major

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cellular source of matrix-producing myofibroblasts, playing a central role in the

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initiation and progression of liver fibrosis. Paracrine signals from resident and inflammatory cells such as hepatocytes, liver sinusoidal endothelial cells, hepatic macrophages, natural killer/natural killer T cells, biliary epithelial cells, hepatic progenitor cells, and platelets can directly or indirectly regulate HSC differentiation and activation. Intercellular crosstalk between HSCs and those “responded” cells has been 2

a critical event involved in HSC activation and fibrogenesis. This review summarizes recent advancement regarding intercellular communication between HSCs and other “responded cells” during liver fibrosis and experimental models of intercellular crosstalk systems, and provides novel ideas for potential antifibrotic therapeutic strategy.

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Key words: liver fibrosis, hepatic stellate cells, intercellular crosstalk, antifibrotic

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therapy

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1. Introduction Liver fibrosis is a wound-healing response to the continuous action of various injury factors, characterized by the net accumulation of extracellular matrix (ECM), or scar [1]. A variety of chronic stimuli can cause liver fibrosis, including chronic viral infection (hepatitis B virus or hepatitis C virus), toxic damage, alcohol abuse (longstanding excessive alcohol consumption), non-alcoholic fatty liver disease /non-

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alcoholic steatohepatitis (NASH), autoimmune liver diseases, and metabolic and

genetic diseases [2-5]. If left untreated, liver fibrosis can progress to cirrhosis associated

with a series of lethal complications, such as functional liver failure, portal hypertension,

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ascites, hepatic encephalopathy, renal and cardiac disturbances, and even hepatocellular

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carcinoma (HCC) [6-8]. Cirrhosis is a critical risk factor for liver cancer and a challenge for human health, resulting in 1.16 million deaths annually worldwide [9]. The direct

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and indirect costs for treating cirrhosis exceed $12 billion each year in the United States

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alone [10]. Although some studies have indicated that liver fibrosis is reversible and cirrhosis may regress in some cases, there is no effective antifibrotic therapy [11, 12].

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Currently, the only available therapeutic approaches entail eliminating chronic stress and liver transplantation [13, 14]. Therefore, it is urgent to identify the vital mechanism

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underlying liver fibrosis and develop more efficient antifibrotic therapies to improve the quality of life in patients with liver fibrosis. HSCs, nonparenchymal cells (NPCs) localized in the perisinusoidal space of Disse, are publicly regarded as the major cellular source of matrix-producing myofibroblasts following their transdifferentiation into proliferative and fibrogenic myofibroblast-like 4

cells in a process termed “activation” [15]. Preventing the activation of HSCs can reduce deposition of ECM proteins, and slow down or even reverse liver fibrosis. Therefore, HSCs represent a primary target for antifibrotic therapy. In fact, HSC activation is a dynamic process that mainly depends on the interaction with other “responded” cells, which are mainly composed of hepatocytes, liver sinusoidal endothelial cells (LSECs), hepatic macrophages, natural killer/natural killer T

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(NK/NKT) cells, biliary epithelial cells (BECs), hepatic progenitor cells (HPCs) and platelets, which can precisely “talk” to each other [16]. In the interactive network,

chemokines and molecular signals released from those cells directly or indirectly affect

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the state of HSCs, namely, “quiescent” or “activated” HSCs regulate the progression,

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aggravation, and resolution of liver fibrosis. Understanding the intercellular crosstalk between HSCs and those “responded” cells and clarifying the role of their interaction

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at different stages of fibrosis progression are critical for discovering novel antifibrotic

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options.

This review mainly summarizes recent advancement in intercellular crosstalk

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between HSCs and other “responded cells” in the pathogenesis of liver fibrosis, focusing on the role of their interaction on HSC activation. It also outlines the common

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experimental models of intercellular crosstalk systems used in this field, which provides a deeper understanding of the molecular mechanisms underlying liver fibrosis and novel ideas for effective antifibrotic therapy. 2. HSCs as precursors of myofibroblasts HSCs are derived from embryonic mesothelial cells and reside in the virtual 5

subendothelial space between hepatocytes and endothelial cells (ECs), where small or soluble molecules exchange in bidirectional transfer. In the normal liver, HSCs are quiescent and represent 5–8% of the total number of liver cells; they are known for vitamin A (retinoid) storage in their cytoplasmic droplets [17, 18]. The vitamin A lipid droplet is a conspicuous feature for identification during HSC isolation. Desmin and some neural markers such as glial fibrillary acidic protein and synaptophysin, are also

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common identification markers [19]. Following liver fibrosis from any cause, quiescent

HSCs become activated, gradually reducing the vitamin A storage capacity, proliferating, developing contractile function, secreting excessive ECM proteins, and

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releasing a series of pro-inflammatory (e.g., interleukin 6 [IL-6]) and profibrogenic

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factors (e.g. transforming growth factor beta [TGF-β]) [20]. Activated HSCs transdifferentiate into myofibroblast-like cells, which are key sources of excess ECM

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in liver fibrosis and can be identified by a set of proteins including α-smooth muscle

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actin (α-SMA), collagens (1a1, 3a1), vimentin, osteopontin, lysyl oxidases (LOX) and tissue inhibitor of metalloproteinase 1 (TIMP1) [21, 22]. It is now widely believed that

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activated HSCs are the main contributors to the pool of fibrogenic myofibroblasts, contributing 82–96% of myofibroblasts in various types of chronic liver diseases, and

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are crucial components of the fibrotic response during fibrogenesis [23]. Generally, HSC activation comprises two phases: initiation and perpetuation (Fig. 1). If fibrosis begins to regress, activated HSCs may progress to a potential third stage, resolution, where they may undergo apoptosis, become senescent, or revert to a quiescent phenotype [4, 24]. In the initiation phase, HSCs receive molecular signals from 6

neighboring cells that secrete profibrogenic cytokines and growth factors, and then they manifest profibrogenic transcriptional and secretory properties. When activated, HSCs proliferate and constantly secrete ECM proteins, leading to ECM accumulation and scar tissue formation. They are in a process of perpetuation. The perpetuation of HSCs is a closely orchestrated process that comprises a number of cell-cell interactions and wound-healing responses.

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3. Intercellular crosstalk in the activation of HSCs

Studies have elucidated the complex dynamic process of HSC activation through

multiple signaling pathways from other “responded” cells during liver fibrosis as

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summarized below (Fig. 2).

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3.1. Hepatocytes

Hepatocytes are the most abundant cell type in liver and are predisposed to damage

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to the liver, such as damage from alcohol, viral infection, and toxic compounds, leading

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to acute or chronic liver disease. In response to liver injury, hepatocytes release a series of key signals such as reactive oxygen species (ROS) [25], hedgehog (Hh) [26, 27],

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nucleotides [28], fatty accumulation [29], damage-associated molecular patterns (DAMPs) [30], eliciting inflammation and fibrosis. Damage-induced hepatocyte cell

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death responses in various forms including apoptosis, necrosis, necroptosis and autophagic cell death can directly affect HSC activation [31]. It has been reported that HSCs

are

able

to

phagocytose

hepatocyte

apoptotic

bodies,

inducing

transdifferentiation of HSCs into myofibroblasts along with a series of profibrogenic responses [32, 33]. Blocking the “phagocytosis” of HSCs, or building a “screen” 7

between HSCs and hepatocytes may be an effective strategy. Galectin-3 can regulate phagocytosis-mediated HSC activation, and inhibiting galectin-3 in animal models has shown to significantly decrease fibrosis [34]. GR-MD-02 (Galectin Therapeutics), a galectin-3 inhibitor, is currently under evaluation in phase IIB trials for patients with compensated NASH cirrhosis (NCT02462967) [35]. Additionally, hepatocytes are also capable of pyroptosis, a newly discovered programmed death. The NOD-like receptor

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family pyrin domain containing 3 (NLRP3) inflammasome activation triggers hepatocyte pyroptosis, resulting in activation of HSCs with collagen deposition in the liver [30].

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Some important inflammatory mediators released from damaged hepatocytes

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might directly or indirectly promote HSC activation and fibrosis. Inactivation of hepatocyte nuclear factor 1α (HNF1α), an important transcription factor in hepatocytes,

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enlarges liver injury and results in hepatic dysfunction [36, 37]. Inhibition of HNF1α

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in hepatocytes leads to the elevation of IL-6 and TGF-1, inducing the activation of HSCs; However, activated HSCs further suppress the expression of HNF1α in

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hepatocytes by releasing TNF-α and IL-6 [38]. Exosomes from damaged hepatocytes can mediate toll-like receptor 3 (TLR3) activation and enhance expression of IL-17A,

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IL-1 and IL-23 in HSCs, triggering IL-17A production by T cells and exacerbating liver fibrosis [39]. Abnormal expression of proteins in hepatocytes is related to HSC activation during liver fibrosis. Hepatocyte-derived high-mobility group box-1 (HMGB1) participates in fibrosis progression by activating phosphorylated MAPK/ERK kinase 8

1/2 (p-MEK1/2), phosphorylated extracellular signal-related protein kinases 1 and 2 (pERK1/2) and phosphorylated c-Jun, and signal receptor advanced glycation end products (RAGE) in HSCs, ultimately increasing collagen type I production [40]. Based on the important impact of HMGB1 on HSC activation, researchers have found some promising treatments for liver fibrosis related to the RAGE/HMGB1 axis or HMGBrelated signaling pathways, such as nilotinib and quercetin [41-44]. Furthermore, the

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upregulation of spleen tyrosine kinase (SYK) and platelet-derived growth factor receptor α (PDGFRα) in hepatocytes can directly shift its expression in HSCs,

regulating HSC activation and proliferation [45-48]. In light of current research, key

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proteins involved in the interaction between hepatocytes and HSCs may be potent

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therapeutic targets in the future. 3.2. LSECs

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LSECs form the wall of liver sinusoids and represent approximately 15–20% of

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liver cells, but only 3% of liver volume [49]. In physiological conditions, LSECs represent a permeable barrier for the bidirectional transfer of small or soluble molecules

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between the blood and the space of Disse [22]. Meanwhile, LSECs also interact with hepatocytes and HSCs for protein, lipid, and glucose metabolism [50]. In a normal liver,

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paracrine production of vascular endothelial growth factor (VEGF) by hepatocytes and HSCs stimulates LSECs to produce nitric oxide (NO), which is essential for maintaining the LSEC phenotype [51]. Following liver injury, LSECs release different angiocrine signals to balance liver regeneration and fibrosis, ultimately maintaining liver homeostasis. When stimulated by acute liver injury or partial hepatectomy, 9

upregulated VEGF promotes the expression of hepatocyte growth factor (HGF) by LSECs and resident sinusoidal progenitor cells, which in turn elicits liver regeneration [52]. In contrast, activation of fibroblast growth factor receptor 1 (FGFR1) in LSECs by chronic injury drives a profibrotic angiocrine response to HSCs, thereby provoking fibrosis [53]. In the progress of liver fibrosis, damaged LSECs gradually lose fenestration with

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the basement membrane, becoming capillarized, which is one of the main pathological

changes of liver fibrosis. Capillarized LSECs lose their hepatoprotective property and the capacity to inactivate HSCs, thus promoting intrahepatic vasoconstriction and liver

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fibrosis [50]. Activated HSCs contract and deposit excessive ECM in the space of Disse,

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leading to the loss of endothelial fenestration and dysfunction of LSECs. Thus, a self-

hepatic fibrosis.

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perpetuating cycle connects and stimulates HSCs and LSECs, further contributing to

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Current research indicates that normally differentiated LSECs are gatekeepers of fibrosis through maintaining HSC quiescence and promoting reversion of activated

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HSCs to quiescence via a VEGF-stimulated-NO-dependent/independent pathway [54, 55]. Upregulation of the transcription factor kruppel-like factor 2 (KLF2) induced by

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statins in LSECs increases endothelial NO synthase expression, conferring vasoprotection and HSC deactivation [56]. Interestingly, recent data support the fact that LSECs promote HSC activation through the liberation of cellular microvesicles. Exosome-packaged sphingosine kinase 1 (SK1) derived from ECs promotes HSC migration and AKT phosphorylation through fibronectin/integrin-dependent exosome 10

adherence and dynamin-dependent exosome internalization [57]. This research opens a new avenue to understand the sinusoidal communications between LSECs and HSCs. Considering the cellular role of LSECs in HSCs, LSEC protection may be an effective strategy for attenuating fibrosis progression. Currently, some compounds have shown good ability to maintain the typical phenotype of LSECs and promising results for fibrosis [58-61]. BAY 60-2770, an activator of soluble guanylate cyclase, can restore

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the differentiation of LSECs and lead to quiescence of HSCs without directly affecting HSCs, preventing progression of thioacetamide-induced cirrhosis in rats [61].

Amusingly, Liu and colleagues [62] utilized fibrotic microniches (FμNs), recapitulating

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the interaction of HSCs and LSECs, to understand the response to antiangiogenic drugs

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at different stages of liver fibrosis. These drugs were only found to be effective for early-stage fibrosis, and the exact mechanism is unclear. Therefore, future studies are

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needed to discover the key event in the crosstalk of HSCs and LSECs at different stages

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of fibrosis, which will assist in drug designed and precise intervention strategies targeting stage-specific disease progression.

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3.3. Immune cells

Immune cells have diverse functions to ensure homeostasis, antimicrobial defense

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and proper metabolism in the liver. Ample studies have shown that immune signaling pathways play important roles in hepatic fibrogenesis. Immune cells secrete inflammatory cytokines or chemokines in response to inflammatory or metabolic stimuli, which directly or indirectly affects hepatic injury. Importantly, bidirectional interaction between immune cells and HSCs regulates positively or negatively the 11

pathogenesis of liver fibrosis. 3.3.1. Hepatic macrophages Hepatic macrophages hold dual functions in initiating, perpetuating and even restricting inflammation in the pathogenesis of liver fibrosis. Traditionally, according to their ontogeny, hepatic macrophages can be broadly divided into liver-resident macrophages, termed Kupffer cells (KCs), and blood/bone marrow-derived

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macrophages, termed monocyte derived macrophages [63].

KCs, attached to the sinusoidal endothelial layer, can be activated by circulating

diverse stimuli of blood and secreting various cytokines including TGF-1, TNF-,

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MCP-1, chemokines (e.g., CCL3 and CCL5), and other soluble mediators, eliciting a

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physiological response to other liver cells [64, 65]. Currently, there is clear evidence that KCs bilaterally regulate the activation of HSCs. Pradere and colleagues [66]

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demonstrated that KCs contribute to HSC survival in an NF-κB-dependent manner.

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Consistently, inhibition of NF-κB by sulfasalazine stimulates HSC apoptosis, exerting a potential antifibrotic role [67]. Leptin possesses profibrogenic activity in the liver,

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which is related to the direct effect on KCs with the release of TGF-1 and connective tissue growth factor (CTGF) to mediate HSC activation [68]. Suppression of

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macrophage infiltration can also directly inhibit activation of HSCs and liver fibrogenesis[69]. On the other hand, KCs can negatively mediate the fibrogenic response of HSCs on an H2O2-IL-6-dependent mechanism [70]. Based on current studies, it is not known why KCs have two different responses to HSCs. It is necessary to elucidate the key triggers between KC activity and HSC activation in future works, 12

which will benefit precision intervention of KCs on liver fibrosis. Researchers have found that some important proteins can regulate the cooperation between KCs and HSCs [71-73], which can be promising therapeutic targets for liver fibrosis. Oncostatin M (OSM), a member of the IL-6 family cytokines, induces collagen production in HSCs depending on the cellular source of fibrogenic cytokines from hepatic macrophages [72]. Blocking macrophage regulation on OSM inhibits collagen

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secretion of HSCs, leading to decreased fibrosis.

Massive monocyte-derived macrophages can be recruited to the injured liver, and a part of them can differentiate into resident macrophages, contributing to liver injury

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progression. Generally, infiltrating monocytes are divided into two major subsets: Ly-

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6Chi monocytes and Ly-6Clo monocytes. Ly-6Chi monocytes mainly express inflammatory cytokines or chemokine receptors, such as C-C motif chemokine

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receptors (e.g., CCR1, CCR2 and CCR5), exhibiting a pro-inflammatory phenotype.

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Whereas the Ly-6Clo monocytes express more scavenging receptors, such as CX3CR1, and the high expression of matrix degrading metalloproteinases (MMPs) (e.g., MMP-

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9, MMP-12, MMP-13), representing a fibrolytic subset in resolving fibrosis [74]. Karlmark and colleagues [75] claimed that Ly-6Chi monocytes, acting as precursors of

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CD11b+F4/80+ intrahepatic macrophages, directly activate HSCs by producing TGF1. CCR1 and CCR5 are upregulated in experimental mouse models of fibrogenesis. CCR1 and CCR5-deficient mice displayed reduced macrophage infiltration in the injured liver, thereby inhibiting HSC activation and fibrogenesis [76]. Additionally, the increased recruitment of Ly-6Chi monocytes after CCl4 treatment is CCR213

dependent. Inhibiting CCRs has been a drug research hotspot in liver fibrosis. The dual CCR2/CCR5 inhibitor, Cenicriviroc (Allergan), is being evaluated for its antifibrotic efficacy in phase III clinical trials (NCT 03028740) [74, 77, 78]. It is commonly believed that CX3CR1 controls the differentiation of Ly-6Clo monocytes [79, 80]. CX3CR1-deficient mice display prolonged inflammatory monocyte infiltration by activating anti-apoptotic BCL-2 expression, promoting HSC activation and resulting

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in enhanced liver fibrosis [79, 81]. Interestingly, a phenotypic shift between LyC6hi and LyC6lo can determine the development of fibrosis. Bárcena and colleagues [82] found that CD5L can promote a phenotypic shift in monocytes from LyC6hi to LyC6lo,

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regulating HSC activation and attenuating CCl4-induced fibrosis. Furthermore, a

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recent study uncovered a novel scar-associated TREM2+CD9+ macrophage sub (SAMФ), differentiating from circulating monocytes, can promote primary human

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HSC proliferation, which may be a potential therapeutic target cell in the future [9].

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Bone marrow-derived macrophages (BMDMs) have a nonnegligible role in modulating the immune microenvironment in the fibrotic liver [83-85]. M1-polarized

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BMDMs can recruit significantly more F4/80+ endogenous macrophages and NK cells to promote HSC apoptosis through their paracrine function. Therefore, M1-polarized

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BMDMs have a better output in the therapy of hepatic fibrosis compared to the M0 or M2 BMDMs [85]. As a bridge of inflammation to HSC activation, macrophages have been potential effective targets for fibrosis treatment. Researchers are working tirelessly to develop effective drugs targeting macrophages, and some are making their way into clinical 14

trials. Curcumin, an anti-inflammatory and anti-oxidant natural compound, has been widely reported to inhibit KC activation and Ly6Chi monocyte infiltration, thereby reducing HSC activation and protecting against liver fibrosis [86, 87]. Amine oxidase copper-containing 3 (AOC3) positively regulates the lymphocyte recruitment signal in the liver [88]. A pharmacological inhibitor of AOC3, BI 1467335 (Boehringer), has been tested in phase II trials for patients with NASH (NCT 03166735) [35].

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Additionally, autologous macrophage therapy, a potential effective antifibrotic cell

therapy, has shown safety and feasibility for cirrhosis patients in the first-in-human study. This macrophage therapy is currently under evaluation in a phase II randomized

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controlled trial (ISRCTN 10368050) for an antifibrotic ability test [9].

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3.3.2. NK/NKT cells

NK cells reside in the liver sinusoid and represent the major liver lymphocyte

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population [89]. Many studies have indicated that NK cells have an antifibrotic function

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through killing activated HSCs and producing interferon-γ (IFN-γ) in mice and humans [90-92]. NKp46 is one of the major NK activating receptors expressed by NK cells.

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Absence of NKp46 can exacerbate liver fibrosis directly through the killing of primary murine and human HSCs [93]. The upregulated retinoic acid (RA) in activated HSCs

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can sensitize early activated HSCs to NK cell death [94]. However, RA can also suppress NK cell activity via induction of TGF-β and suppression of cytokine signaling 1 to inhibit IFN-γ signaling during advanced liver injury [95]. Notably, NK cells preferentially kill senescent-activated HSCs, facilitating the resolution of fibrosis [96]. Generally, NKT cells can be divided into two distinct subpopulations: type I NKT 15

cells (express a semi-invariant T cell receptors [TCRs], comprising 95% of liver NKT cells) and type II NKT cells (express more diverse TCRs) [97]. NKT cells share similar phenotypic and functional characteristics with NK cells, but have both antifibrotic and profibrotic properties in regulating liver fibrosis. Similar to NK cells, NKT cells can directly kill activated HSCs and produce IFN-γ to have inhibitory effects on liver fibrosis [98]. Despite this, NKT cells promote HSC activation and liver fibrosis by

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producing inflammatory cytokines such as IL-4 and IL-13 [99]. NKT cell-conditioned medium stimulates primary HSCs to become myofibroblastic, and CD1d-deficient mice

lacking NKT cells develop less fibrosis in NASH-related fibrogenesis [100]. Different

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from the regulation models mentioned above, IL-30 can recruit NKT cells to the injured

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liver and selectively remove activated HSCs, indirectly modulating intercellular communication in NKT cells and HSCs [101-103].

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3.4. BECs

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BECs, also known as cholangiocytes, are epithelial cells arranged along the biliary tract. They are comprised of a complex three-dimensional conduction network

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extending from the small intrahepatic bile duct to the extrahepatic bile duct. Generally, BECs are primary sites of damage in liver for primary biliary cirrhosis and primary

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sclerosing cholangitis [104]. Moreover, cholangiocyte senescence has been a hallmark in the progression of liver fibrosis, where cholangiocytes secrete a number of profibrogenic and chemotactic factors including CTGF, PDGF, TGF-β1, MCP-1, and IL-6 to attract HSCs [105]. Proliferating cholangiocytes are a major source of the profibrogenic CTGF during biliary fibrosis [106] and directly attract lobular 16

myofibroblastic HSCs into the portal tracts through releasing PDGF. Additionally, cholangiocytes modulate mesenchymal-epithelial interactions through producing Hh ligands, directly promoting the growth of myofibroblast cells [107]. As we understand that cholangiocyte proliferation and paracrine have stimulating effects on HSC activation, the targeting proliferating cholangiocytes or the disruption of cholangiocyte secretion of profibrogenic factors will be potential anti-fibrosis therapies. Over and

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above these interference strategies, targeting some key regulators (e.g., substance P,

osteopontin) that indirectly promote collagen secretion and inhibit HSC senescence by regulating cholangiocytes function may be another hotspot for further research [108,

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109].

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3.5. HPCs

HPCs are positioned within the terminal bile ductile and are quiescent in the

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healthy liver. With a strong potential for a heterogeneous multipotent transient

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amplifying cell population (oval cells), HPCs play an irreplaceable role in regeneration of the damaged liver [110, 111]. Kordes and colleagues [112] found that CD133+ HSCs

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could differentiate into endothelial or hepatocyte lineages, and recognize them as progenitor cells. Subsequently, the research team found that transplanted HSCs could

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form progenitor cells in rats, and cultured HSCs transiently adopted a similar expression profile to that of HPCs during differentiation, showing the inseparable relationship between HPCs and HSCs [113]. Combined with the present research, HPCs represent an intermediate stage for differentiating HSCs in liver regeneration, where HSCs can differentiate into HPCs through mesenchymal-to-epithelial transition (MET) [113-115]. 17

Along with their differentiation into HPCs, HSCs can further regulate HPC differentiation via a series of signaling axes such as the TGF-β1/Jagged1 pathway [116], NF-κB-inducible NO synthase signaling [117]. There is currently little research on fibrogenesis and regression; further investigation is required. 3.6. Platelets Platelets are derived from mature megakaryocytes (MKs) through cytoplasmic

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fragmentation consisting of long pseudopodial elongations that break in blood flow

[118]. It has been reported that platelets can accumulate at the site of injury liver in

pathological conditions, such as liver cirrhosis [119], viral hepatitis [120], cholestasis

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[121], ischemia/reperfusion [122, 123], and the residual liver after hepatectomy [124],

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playing important roles in hemostasis [125], wound healing [126], and liver regeneration [127-129]. In fibrotic liver, platelets can be directly adjacent to collagen

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fibrils [119, 129]. Platelets and platelet-derived ATP can suppress HSC activation and

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reduce type I collagen production via the adenosine-cAMP signaling pathway [130]. In addition, platelets release many important moderators including PDGF [131], TGF-β1

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[132], HGF [133, 134], VEGF [135], and epidermal growth factor (EGF) [136], which are intrinsically involved in fibrogenesis. Inhibiting the production of these moderators

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from platelets, such as MOR8457, a high-affinity monoclonal antibody against PDGFB, can reduce the level of HSC activation and collagen deposition in liver fibrosis [137]. Therefore, antiplatelet therapy, such as anti-PDGF-B treatment, has been a potential strategy to treat fibrosis in patients.

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4. Experimental models of intercellular crosstalk system To better decipher the key signaling network of intercellular crosstalk, a series of established experimental systems (in vitro, ex vivo and in vivo ) are used to try to mimic the complex cell-cell interactions involved in HSC activation and liver fibrosis (Table 1). Moreover, these models are comprehensive and precise to screen putative antifibrotic compounds, evaluate potential drug efficacy and toxicity, and greatly

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accelerate the development of antifibrotic strategies. Co-culture systems, joining at least two cell types, are the main in vitro models for exploring the role of intercellular

crosstalk. Ex vivo models, represented by PCLSs and liver organoids, attempt to

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simulate the hepatic microcirculatory architecture, and have shown great appeal to

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researchers in recent years. Animal models can dynamically display the pathological process and partly reflect the major pathological features in humans, which is an

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4.1. In vitro models

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indispensable tool for researchers to study liver fibrosis.

For in-depth investigation of the mechanisms driving HSC activation and liver

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fibrosis, in vitro models are powerful and indispensable. Generally, primary HSCs and a variety of HSC cell lines from rodents and humans are abundantly used. Primary

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HSCs can be freshly isolated based on a density gradient centrifugation method [138140], and then cultivated in a mono-layer culture where HSCs would be activated over a 7–10 day culture period to acquire a myofibroblast-like phenotype [22]. Besides, a series of well-established cell lines are important for investigating the signaling pathway in HSC activation, such as human cell lines (LX-2, TWNT-4, GREF-X), rat 19

cell lines (HSC-T6, CFSC, PAV-1, T-HSC/Cl6, RGF), mouse cell lines (GRX, A640IS, JS1, Col-GFP) and rat portal myofibroblast cell lines (RGF-N2 and RGF) [141-145]. Monoculture systems are useful but inadequate to resemble the complicated progress in the development of fibrosis where HSCs interact with other “responded” cells. Co-culture systems gain more popularity for researching intercellular crosstalk of HSCs in subsequent studies. A multicellular in vitro system usually contains functional

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hepatocytes that could respond to external injury stimulation and HSCs that could be activated towards a myofibroblast phenotype. Human hepatoma cells, such as HepG2 [146] and HepaRG [147, 148], are the current options as sources of hepatocytes. HSC

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cell lines and primary HSCs from rodents or humans are available and represent the

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activated HSC phenotypes as well [149]. Hepatocyte-HSC co-culture system have been a potent system to study hepatocyte damage-dependent HSC activation. Giraudi and

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colleagues [29] demonstrated that cell-to-cell proximity between hepatocytes and HSCs

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is of importance to initiate the fibrotic process by a well-established co-culture system. To determine the role of immune cells in regulating HSC activation, researchers

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establish co-culture based on HSCs and KCs or BMDMs [73, 150]. By contrast, cocultures consisting of HSCs and ECs reflect the importance of ECs on regulating HSC

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activation [151]. Historically, in vitro co-cultures have been developed with the help of robust techniques including seeding between two layers of ECM compounds or by culturing in spheroids, micromolds of non-adherent materials, gravitational aggregation using hanging drop cultures, or the use of rotation of concave 96 well cell-repellent plates, making the systems more stable and controllable [22, 141]. 20

4.2 Ex vivo models Although co-cultures are good alternatives for liver fibrosis research and drug screening, they still exist with great differences to actual pathological mechanisms. In this sense, precision cut liver slices (PCLSs) are the best representation for the in vitro study of liver fibrosis, with all the cells including all cell-matrix interactions maintained in their original environment. PCLSs are fresh liver slices with a thickness of 100 to

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250 µm from rodents or humans, which can subsequently be cultured in cell culture

dishes, perfectly mimicking the multicellular characteristics of organs in vivo [152]. PCLSs have been extensively used to examine the potency of putative antifibrotic

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compounds [153-155]. Unfortunately, the limited viability of PCLSs has restricted their

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use to short-term study. In addition to PCLSs, liver organoids derived from stem cells or organ progenitors can also mimic the process in vivo and have been used to test anti-

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fibrotic compounds, which are important tools to transform the basic research to

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therapeutic applications [156, 157]. Leite and colleagues [158] developed a 3D microrganoids where functionality of both hepatocytes and HSCs can be maintained for

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at least 21 days, which optimizes the study of hepatocyte-dependent HSC activation and allows potential compounds testing.

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4.3. In vivo models

In vivo models are the most popular assessment models to study liver fibrosis for

researchers. A series of experiments in vivo are widely employed to mimic the complex pathology of liver fibrosis and hepatic cell-cell interactions. Traditionally, CCl4 toxicity model [159] and the common bile duct ligation model (CBDL) [160] are the “work21

horse” to study liver fibrosis in vivo , which can be used in multiple genetic backgrounds (transgenic, constitutive, or inducible knockout mice) and are easy to set up for both rats and mice [161]. Other rodent models, such as alcohol-induced alcoholic liver disease (ALD) [162, 163], diet-based models (MCD/HF diet-induced liver steatosis/NASH) [164], genetically modified models (Multidrug resistance-associated protein 2-deficient mice (Mdr2-/-)) [165], and hepatitis virus infection-based models

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[166], are available tools to study liver fibrosis induced by specific etiologies.

Considering the high prevalence of hepatitis virus infections worldwide, hepatitis virus infection-based models have been increasingly crucial. In fact, hepatitis virus infection

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induces liver fibrosis in humans but not in rodents. Nevertheless, humanized animal

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models with human immune and liver cells have allowed researchers to closely study liver fibrosis with HCV and long-term HBV infection in recent years, which are

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valuable tools to elucidate the contribution of immune system to HSC activation in

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humans [167, 168]. Chimeras with BM cells from other genetically modified mice are also powerful research models to study the specific proteins or signaling involved in

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the interaction between immune cells and HSCs [169]. For the significant roles of hepatocytes on activated HSCs, hepatocyte-specific gene knockout models have been

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widely used to explore the key molecular mechanisms existing in the process of HSC activation [48]. 5. Conclusion and perspective Liver fibrosis is a complex progress involving multiple events, in which HSC activation is publicly recognized as the central event. In responding to stimuli from the 22

intracellular and extracellular microenvironment, activated HSCs interact with other “responded” cells, triggering the wound-healing response. These “responded” cells play dual roles in HSC activation through diverse molecular mechanisms and cell signaling during different stages of fibrosis. Paracrine profibrogenic chemokines and molecular signals activate HSCs or promote their activation; inhibiting HSC activation and eliminating activated HSCs by reverting, transdifferentiating, or inducing HSC

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death and senescence. Deciphering the specific cellular mechanisms of cell-cell

interaction in HSC activation is essential to provide new insights into therapeutic interventions in liver fibrosis. Further investigation should focus on the key factors

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determining the dual regulation of HSC activation by these “responded” cells in order

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to discover precise intervention strategies for liver fibrosis. Furthermore, the intercellular crosstalk in regression of liver fibrosis is poorly understood. We still do

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not know what plays the leading role in regulating HSC activation in the process.

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Improved knowledge of the crucial process would develop more efficient strategies to promote fibrosis resolution and liver regeneration.

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Of equal importance, there is a lack of complete system building in the field of intercellular crosstalk to study the interaction between HSCs and other “responded”

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cells. Monolayer HSC culture is inadequate to unearth the complexity of HSC activation. As valuable alternatives, co-culture systems can resemble the multicellular interaction process in liver fibrosis and have been widely used in research labs. Nevertheless, we must be aware that the in vitro cell conditions may differ greatly from actual pathology in vivo . Most cells lose their characteristics in vitro, which may 23

change their function on HSC activation. In this regard, ex vivo models like PCLSs are needed and will be more popular in the upcoming years. Additionally, it has been confirmed that the latest liver-on-a-chip can recapitulate the hepatic microcirculatory architecture, allowing for more credible intercellular crosstalk research and drug

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screening in the future [170].

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Figure legends

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Fig. 1. Function and characteristic of activated HSCs.

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Activation of HSCs comprises two phases: initiation and perpetuation. In the initiation period, quieted HSCs response to injury stimuli and begin to transdifferentiate into

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proliferative and fibrogenic myofibroblast-like cells. When activated HSCs proliferate and secrete ECM proteins constantly, leading to accumulation of ECM and formation

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of scar tissue, HSCs are in the process of perpetuation, characterized by specific phenotypic changes including contractility, fibrogenesis, altered matrix degradation, chemotaxis and inflammatory signaling. If fibrosis begins to regress, activated HSCs may develop a potential third phase, resolution, where they may undergo apoptosis or become senescent, or revert to the quiescent phenotype. 47

Fig. 2. Intercellular crosstalk in the activation of HSCs. HSC activation is a dynamic process that mainly depends on the interaction with other “responded” cells, including hepatocytes, liver sinusoidal endothelial cells, hepatic macrophages, NK/NKT cells, biliary epithelial cells, hepatic progenitor cells, and platelets. Activation of HSCs is promoted (black arrows) or inhibited (red arrow) by

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paracrine chemokines and molecular signals from those cells. Pharmacological

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intervention to each candidate target is shown in the green box.

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Fig. 1. Function and characteristic of activated HSC

Fig. 2. Intercellular crosstalk in the activation of HSCs

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Jo

f Origin

Primary HSCs

Humans and rodent

LX-2, TWNT-4, GREF-X

Humans

HSC-T6, CFSC, PAV-1, T-HSC/Cl6, RGF

Rats

GRX, A640-IS, JS1, Col-GFP

Mice

e-

RGF-N2 and RGF

Pr

HSCs and hepatocytes

HSCs and KCs/BMDMs

Jo ur

Liver organoids Liver-on-a-chip

In vivo

HSCs and NKT cells

Close link with the in vivo situation

[138-140]

Ease of use; Supplied unlimitedly; Good reproducibility

[141-145]

Hepatocyte damage-dependent HSC activation

[146-148]

Roles of immune cells in the regulation of HSC activation

[73] [150]

Paracrine function of NKT cells on HSC activation

[100]

HSCs and ECs

Intercellular crosstalk between ECs and HSCs

[151]

HSCs and cholangiocytes

Effects of cholangiocytes on HSC activation

[107]

Fresh liver explants from rodents

Maintaining the complex cellular interactions that occur

and humans

in vivo; Limited human supply

Derived from stem cells or organ

Resembling an organ with self-organize through cell

progenitors

sorting

na l

Precision-cut liver slices Ex vivo

References

Rat portal myofibroblast

In vitro

Co-cultures

Characteristics

pr

Model

oo

Table 1 Experimental models of intercellular crosstalk systems

Containing cell culture equivalents of liver

[153-155] [156-158]

Recapitulate the hepatic microcirculatory architecture

[170]

CCl4 toxicity model

Rodent

High reproducibility; Showing the regression of fibrosis

[159]

Common bile duct ligation model

Rodent

Short time to develop; Close to human cholestatic injury

[160]

Humanized animal models

Mice

Similar to human viral infections

[167-168]

Chimeras with BM cells

Mice

Good model to study the specific proteins or signaling

[169]

Hepatocyte-specific gene knockout models

Mice

The role of hepatocyte in HSC activation

[40] [48]

50