Unravelling the reversion mechanisms of activated hepatic stellate cell properties by extracellular vesicles from mesenchymal stem cells

Abstract

Hepatic fibrosis is a pathological process characterized by an imbalance between the deposition and degradation of extracellular matrix components. This process is initiated by chronic liver injuries resulting from viral infections, alcoholic liver disease, non-alcoholic fatty liver disease, and autoimmune-mediated hepatic damage. If left untreated, hepatic fibrosis can progress to life-threatening conditions such as cirrhosis and hepatocellular carcinoma. Central to the development of fibrosis is the transdifferentiation of quiescent hepatic stellate cells (HSCs) into proliferative and fibrogenic myofibroblast-like activated HSCs (aHSCs), which play a crucial role in extracellular matrix accumulation and fibrotic tissue formation. Beyond resmetirom, a recently Food and Drug Administration-approved medication for liver fibrosis and nonalcoholic steatohepatitis, there are currently no other established pharmacological treatments available to slow down the progression of these conditions. Moreover, activation of HSCs and formation of hepatic fibrosis have been considered irreversible. Recent studies reported transforming growth factor beta as one of the key regulators of HSCs activation and pathogenesis of hepatic fibrosis. It has been also reported that the features of aHSCs can be reversed to those of quiescent HSCs by modulating transforming growth factor beta mediated pathways. The potential of extracellular vesicles (EVs) as cell free therapeutics to treat hepatic fibrosis has been suggested earlier. However, detailed knowledge of the mechanisms involved in the alleviation of hepatic fibrosis using EVs from mesenchymal stem cells is still lacking. Hence, this review aims to describe the pathogenesis of hepatic fibrosis from the cellular and molecular point of views and shed light on the potential of EVs from mesenchymal stem cells in reversing the properties of aHSCs to their quiescent state.

Key Words: Exosomes; MicroRNAs; Myofibroblast; Nuclear factor kappa B; Transforming growth factor-beta

Core Tip: Liver fibrosis and cirrhosis are global health challenges resulting from persistent liver injury, hepatocyte damage, inflammation, and hepatic stellate cell activation. These processes dysregulate key signaling pathways such as transforming growth factor beta/small mother against decapentaplegic, Wnt/β-catenin, nuclear factor-kappa B, and mitogen-activated protein kinase, leading to excessive extracellular matrix deposition. Importantly, once the underlying cause is addressed, liver’s natural ability to regenerate and regress fibrosis is restored. Innovative approaches such as mesenchymal stem cell-derived extracellular vesicles and microRNA-based therapies are gaining attention due to their potential to deliver targeted anti-fibrotic molecules, regulate gene expression, and reverse liver fibrosis. Ultimately, these strategies could lessen the burden of chronic liver diseases globally.

INTRODUCTION

The liver is involved in performing crucial functions such as detoxification, metabolic regulation, and immunological support[1,2]. However, chronic liver diseases, particularly cirrhosis and hepatic fibrosis, are major global health concerns. According to the Centers for Disease Control and Prevention, in the United States, liver diseases cause around 40000 deaths annually[3]. Prevalent causes encompass viral hepatitis (B and C), alcoholic liver disease, and non-alcoholic fatty liver disease, the latter impacting up to 25% of Americans and frequently progressing to non-alcoholic steatohepatitis (NASH) and fibrosis[4].

High rates of morbidity and mortality are experienced by patients with chronic liver diseases who develop liver fibrosis and cirrhosis, which significantly burdens society financially[5]. It affects over 800 million people annually and has a mortality rate of about 2 million deaths equivalent to 4% of global mortality worldwide[6]. Cirrhosis-related problems account for one million deaths, whereas viral hepatitis and hepatocellular cancer contribute to the remaining one million deaths[7,8].

Liver fibrosis is a slow-developing, often clinically unmanifested condition where continuous liver injury leads to stiffening and loss of function, potentially progressing to cirrhosis or liver cancer[9-12]. It can be caused by various factors including alcohol, metabolic issues, viral infections, toxins, autoimmune diseases, and prolonged use of certain medications[11,13-15]. A key factor in the pathophysiology of liver fibrosis is the activation of hepatic stellate cells (HSCs). Usually quiescent HSCs (qHSCs) activate in response to liver damage, transforming into myofibroblast-like cells that overproduce extracellular matrix (ECM), leading to scar tissue formation[16]. This activation is driven by signals [like reactive oxygen species (ROS)] from damaged liver cells[17-20] and inflammatory cytokines from immune cells[21,22].

At present, liver transplantation remains the most proven effective intervention for managing advanced liver diseases[23-26]. The understanding and treatment of fibrosis are undergoing a significant shift. Previously, fibrosis was considered irreversible with no specific treatments. However, new research is overturning these long-held beliefs. A prime example of this evolving therapeutic landscape is the 2024 Food and Drug Administration approval of resmetirom, the first drug specifically developed for liver fibrosis and NASH. This approval highlights a major change in how fibrosis is viewed and managed therapeutically[27-29]. The potential of phytochemicals and other drugs in partially reversing the condition of fibrosis have been identified by a number of etiological and preclinical studies[30-35]. Although many mediators and routes implicated in hepatic fibrogenesis have been identified, a clear therapeutic drug to treat liver fibrosis is still elusive. Current therapeutic drugs frequently have poor selectivity, limited solubility, and low liver accumulation, which can result in non-target organ absorption and insufficient therapeutic doses in vivo. For example, poor water solubility, low absorption, and imprecise targeting hinder the useful chemicals curcumin and silymarin[36-38].

Recent studies have shown that extracellular vesicles (EVs) produced from mesenchymal stem cells (MSCs) have the potential to control HSC activity, reducing liver fibrosis and promoting tissue healing. For instance, MSC-derived EVs have been shown to inhibit the Wnt/β-catenin signaling cascade in HSCs, resulting in reduced activation and fibrosis in rat models of liver injury[39]. Furthermore, it has been revealed that EVs produced from human liver stem cells reduce the activated phenotype of HSCs; miR-146a-5p is a key mediator of this anti-fibrotic impact[40]. MSC-derived EVs have also been shown to reduce the expression of pro-fibrotic markers in liver spheroids and activated HSCs (aHSCs) that are driven by transforming growth factor beta 1 (TGF-β1), highlighting their potential as a therapy strategy for hepatic fibrosis[40,41]. Hence, this review will concentrate on the activation of HSCs, pathogenesis, and mechanisms of liver fibrosis, along with its clinical implications, by examining the cellular and molecular pathways of liver fibrosis resolution and the reversion mechanisms of aHSCs using EVs from MSCs.

CELLULAR PATHOPHYSIOLOGY OF LIVER FIBROSIS AND CIRRHOSIS

The liver maintains a perfect balance between building up and breaking down its ECM, a vital support structure. The ECM, a complex network of macromolecules, regulates numerous physiological processes via various signaling pathways. These processes include cell division, adhesion, migration, proliferation, growth, metabolism, injury healing, and tissue remodeling[42,43]. Chronic liver disease disrupts equilibrium, leading to more production than breakdown[44]. Liver fibrosis is characterized by continuous ECM deposition[45], harming the liver’s structure[5] and impairing normal activity[12]. Active interaction between immune, non-parenchymal, and hepatocytes causes the intricate process[35,46,47].

ROS, generated by various stimuli such as toxins, viruses, cholestasis, hypoxia, and insulin resistance, contribute to hepatocyte damage, apoptosis, steatosis, and infiltration of immune cells, especially Kupffer cells (KCs)[48,49]. Simultaneously, sinusoidal endothelial cells undergo capillarization, characterized by the loss of fenestrae, which alters sinusoidal structure[50]. This process is initiated by chronic hepatocyte injury, leading to the release of pro-fibrotic cytokines and growth factors-including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, TGF-β, and platelet-derived growth factor (PDGF) through interactions with natural killer cells and KCs. Primary responder cells, such as HSCs, become activated, resulting in excessive ECM deposition[51,52]. Although HSCs are the central mediators in liver fibrosis, interactions among various liver cell types including hepatocytes, liver sinusoidal endothelial cells (LSECs), and inflammatory cells are also involved in the fibrotic process[10]. The process of liver fibrosis, and the main functions of different cells and cytokines is briefly described below.

The key mediators of liver fibrosis: Hepatocytes

The liver consists of parenchymal cells and non-parenchymal cells. Parenchymal cells, account for 80% of the liver’s volume and 60% of its total cell count[10]. Hepatocytes are crucial for detoxification, energy metabolism, and lipid, protein, and bile secretion[53]. Because of its nature, liver to be affected by the xenobiotic drugs and toxins, which lead to the hepatocyte death. Dead hepatocytes release damage-associated molecular patterns (DAMPs), which encircle KCs and HSCs, leading to inflammation and fibrosis[54,55].

One key DAMP, high-mobility group box-1 (HMGB1), can directly activate HSCs, especially in conditions like hepatitis B[56]. Besides, damaged hepatocytes can also release exosomes carrying signaling molecules and microRNAs (miRNAs) particularly miR-27a that could further boost HSC activation, suppress aHSC autophagy and eventually could accelerate liver scarring in metabolic-associated fatty liver disease[57]. In brief, healthy hepatocytes keep the liver running smoothly, but their damage and death are critical triggers in the complex process of liver inflammation and scarring.

Inflammation and macrophages in liver fibrosis

KCs, the liver’s resident macrophages, are a vital part of the innate immune system and are essential for maintaining liver homeostasis[58,59]. KCs exhibit a dual role in liver fibrosis[60]. During fibrosis progression, KCs exacerbate fibrosis by recruiting more inflammatory cells to the injured liver, increasing the production of pro-inflammatory cytokines and chemokines, and activating HSCs to produce excessive fibrotic tissue. In contrast, during fibrosis regression, macrophages change their function, aiding recovery by producing matrix-degrading enzymes like matrix metalloproteinase (MMP)-9 and MMP-2 to dismantle ECM. This resolution phase also entails lowering the production of inflammatory and fibrogenic mediators[61].

In case of persistent liver injury inflammation is triggered. Damaged cells release ROS and other signaling molecules activate immune cells, specifically lymphocytes and macrophages[62]. As a consequence, activated KCs rapidly secrete a variety of inflammatory chemicals, namely IL-1β, TNF-α, chemokine (C-C motif) ligand 2, and chemokine (C-C motif) ligand 5[5,10,63]. Furthermore, KCs activation can increase nuclear factor kappa-B (NF-κB) in HSCs, which further promotes the release of pro-inflammatory cytokines. The release of these substances worsens liver fibrosis and directly activates HSCs, the primary collagen-producing cells[64].

Sinusoidal endothelial cells in the liver

Non-parenchymal cells called LSECs make up the sinusoidal wall and serve as the contact between hepatocytes and HSCs on one side and the blood and KCs on the other. In a healthy liver, LSECs are considered “guardians of hepatic homeostasis” due to their vasodilatory, anti-inflammatory, antithrombotic, anti-angiogenic, antifibrotic, and regeneration-promoting properties[65]. HSCs are kept in a resting condition by endothelium-derived nitric oxide, which is mostly produced by LSECs. When liver damage occurs, LSECs become capillarized, which can increase the synthesis of vasoconstrictors (endothelin 1, thromboxane A2, and angiotensin II) and decrease the production of vasodilators (like nitric oxide, cyclooxygenase, and prostaglandin I2). This imbalance causes inflammation and liver fibrosis in addition to changing the LSEC phenotype and activating HSCs[66-68]. Additionally, LSECs can activate signaling pathways like Wnt/β-catenin or release signaling molecules like TGF-β and PDGF, which in turn stimulate HSCs[35].

HSCs: From quiescence to fibrosis

HSCs are found in the liver’s subendothelial region. In a healthy liver, they remain quiescent. Their normal functions include storing fat and metabolizing vitamin A, working closely with surrounding hepatocytes and sinusoidal endothelial cells[69,70]. qHSCs play a crucial role in maintaining the ECM homeostasis by secreting moderate amounts of ECM proteins like collagen type III, collagen type IV, and laminin[35,71]. They also produce MMPs, such as MMP-1, which help break down ECM. Tissue inhibitors of MMP (TIMP)-1 and TIMP-2, two TIMPs, are also released by qHSCs. TIMP-1 is particularly important as it inhibits MMPs and helps prevent ECM breakdown, ensuring a healthy balance between ECM production and degradation[72-75]. This precise interplay between MMPs and TIMPs is vital for liver matrix regeneration and maintaining healthy liver architecture[76].

One crucial stage in hepatic fibrogenesis is the activation of qHSCs[77]. This activation is triggered by various factors, such as inflammatory stimuli, fibrogenic cytokines like TGF-β, ROS, activated KCs, platelets, and products from injured hepatocytes. Upon activation, aHSCs not only secrete large amounts of ECM (primarily collagen I and III) but also release cytokines like TGF-β that sustain their own activation, creating a vicious cycle[78]. Besides, a significant increase in the expression of TIMP-1, further hindering ECM breakdown[78]. This continuous activity leads to the excessive deposition of mature collagen fibers in the Disse space, causing liver fibrosis[79].

MOLECULAR PATHWAYS INVOLVED IN LIVER FIBROSIS AND CIRRHOSIS

Among several pathways that are involved in the liver fibrosis TGF-β/small mother against decapentaplegic (SMAD), Wnt/β-catenin, NF-κB pathway, mitogen-activated protein kinase (MAPK) pathways are considered the most important. The roles of these pathways are described in brief below.

TGF-β/SMAD pathway in liver fibrosis and cirrhosis

The TGF-β/SMAD pathway plays a central and critical role in the development of liver fibrosis and cirrhosis, primarily through its influence on HSC activation and ECM production. TGF-β is a potent fibrogenic cytokine released by injured hepatocytes, inflammatory cells, and KCs during liver injury. TGF-β binds to TGF-β receptors (type I and II) on HSCs, initiating intracellular signaling cascades. Upon TGF-β binding, type I TGF-β receptor phosphorylates receptor-regulated SMADs (SMAD2 and SMAD3). Phosphorylated SMAD2/3 forms a complex with SMAD4, then translocate into the nucleus. Inside the nucleus, the SMAD complex regulates the transcription of target genes involved in fibrosis[80,81]. Upregulated genes encode ECM components, especially collagen types I and III. Stimulates alpha-smooth muscle actin (α-SMA) expression, marking the transdifferentiation of qHSCs into aHSC[82]. Enhances production of TIMPs that eventually reduce ECM degradation. Elevated TGF-β levels sustain HSC activation and fibrogenesis[83].

Wnt/β-catenin pathway in liver fibrosis and HSC activation

The Wnt/β-catenin signaling pathway plays an important role in liver fibrogenesis and the activation of HSCs during the progression to cirrhosis. Glycoproteins called Wnt proteins are secreted and bind to cell surface Frizzled receptors and co-receptors (like lipoprotein receptor-related protein 5/6. In the absence of Wnt signaling, a destruction complex that contains glycogen synthase kinase-3β and other elements targets β-catenin for degradation. When Wnt binds to its receptors (such as Frizzled and lipoprotein receptor-related protein 5/6), this destruction complex is inhibited, preventing β-catenin degradation. As a result, β-catenin stabilizes and accumulates in the cytoplasm. The accumulated β-catenin then translocates into the nucleus, where it interacts with T-cell factor/Lymphoid enhancer factor transcription factors to regulate the expression of target genes. These genes are involved in critical cellular processes such as proliferation, survival, and differentiation[84,85].

NF-κB pathway in pathogenicity of liver cirrhosis

The NF-κB signaling pathway is reported in controlling inflammation, fibrosis, and even the development of hepatocellular carcinoma[86]. NF-κB primarily influences liver scarring through three main cell types: (1) By regulating hepatocyte damage, which acts as the primary trigger for fibrogenic responses; (2) By controlling inflammatory signals generated by macrophages and other immune cells within the liver; and (3) By affecting the fibrogenic activity of HSCs. NF-κB is expressed in nearly all cell types and controls a wide array of genes involved in innate and adaptive immunity, cell growth, differentiation, survival, and apoptosis[87]. Two main pathways activate NF-κB: The canonical pathway involving RELA (p65) and the non-canonical pathway involving RELB. Activation of NF-κB results in the transcription of hundreds of genes containing κB binding sites, most of which regulate inflammation, immune responses, and cell survival[88]. Abnormal or excessive NF-κB activation is observed in many chronic liver diseases, including alcoholic steatohepatitis, NASH, and viral infections, highlighting its vital role in maintaining liver homeostasis, immune cell interactions, and tissue repair processes. More specifically, NF-κB signaling upregulates the expression of activation markers, such as α-SMA and fibrogenic genes like collagen types I and III. NF-κB drives the expression of pro-inflammatory cytokines (e.g., IL-6, monocyte chemotactic protein-1) and chemokines, which recruit immune cells to the liver, sustain inflammatory milieu and further promote HSC activation and fibrosis progression[21,22]. NF-κB promotes HSC survival by inducing anti-apoptotic genes, thereby prolonging their activated state. This contributes to persistent fibrogenic activity in chronic liver injury.

MAPK pathway in pathogenicity of liver cirrhosis

The MAPK pathway influences processes like inflammation, cell death, and fibrosis, with specific MAPK members like p38 and c-Jun N-terminal kinase (JNK) implicated in the progression of liver injury and fibrosis[89]. MAPK cascades are signaling pathways that carry external inputs to the nucleus. Each of the three primary MAPK cascades-which have been thoroughly described-converges on extracellular signal-regulated kinases (ERKs), JNKs, and p38 MAPKs and is made up of three classes of serine/threonine kinases: MPKKkinase, MAPK, and MAPK kinase[90,91]. p38 MAPK is activated during liver injury and participates in inflammatory responses, potentially contributing to liver damage and fibrosis. Inhibition of p38 MAPK may be beneficial in treating liver cirrhosis. JNK consists of three isoforms-JNK1, JNK2, and JNK3-encoded by distinct genes. JNK1 and JNK2 are expressed in nearly all tissues, including the liver, while JNK3 is primarily found in neuronal tissue[92]. JNK plays versatile roles in regulating cellular processes such as proliferation, differentiation, and apoptosis, primarily through phosphorylation and modification of downstream targets. Its activity is stimulus- and cell-type dependent. ERK1/2 are part of the MAPK pathway that governs cell proliferation, differentiation, and development. They are activated by signaling[93]. In the liver, ERK1/2 promotes autophagy in hepatocytes, which can help reduce liver steatosis.

Notch signaling in liver fibrosis

The Notch signaling pathway is highly conserved and plays essential roles in liver development, biliary cell differentiation, and fibrosis[94]. In fibrotic liver tissue, abnormal activation of Notch encourages the ductular reaction and the proliferation of reactive cholangiocytes, which release pro-fibrotic cytokines like TGF-β1 and PDGF that, in turn, activate HSCs[95,96]. Moreover, Notch promotes epithelial-to-mesenchymal transition-like changes in cholangiocytes and increases the expression of fibrogenic genes such as α-SMA and collagen type I. Experimental studies have shown that blocking Notch signaling with γ-secretase inhibitors can reduce fibrosis, highlighting its potential as a therapeutic target[97,98].

Hedgehog signaling in liver fibrosis

The Hedgehog signaling pathway remains inactive in the healthy adult liver but becomes reactivated in response to liver injury. When Hedgehog ligands-Sonic Hedgehog, Indian Hedgehog, or Desert Hedgehog-bind to the Patched receptor, they relieve the suppression of smoothened, leading to activation of GLI transcription factors (GLI1-3). This activation promotes the survival, proliferation, and activation of HSCs, and also attracts immune cells such as macrophages and natural killer T cells, which contribute to inflammation and fibrosis. Hedgehog signaling supports the maintenance of myofibroblast-like cells and stimulates the expression of pro-fibrogenic genes, resulting in increased ECM production. In models of cholestasis and NASH, activation of the Hedgehog pathway has been directly linked to the severity of fibrosis. Pharmacological inhibition of this pathway has been shown to reduce collagen accumulation and attenuate HSC activation in vivo[99-101].

Hippo signaling in liver fibrosis

The Hippo signaling pathway is essential for essential for controlling tissue regeneration, cell division, and organ growth[102]. The downstream effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are dephosphorylated and go into the nucleus when Hippo signaling is interfered with. There, they interact with YAP-transcriptional enhancer factor domain family member transcription factors. Genes like connective tissue growth factor, CYR61, and amphiregulin that are involved in cell development and fibrogenesis are expressed more frequently as a result of this interaction. YAP is elevated in hepatocytes, cholangiocytes, and HSCs in chronic liver damage, which causes enhanced proliferation and a fibrogenic phenotype. The crucial role of YAP in liver fibrogenesis is highlighted by the fact that blocking YAP activity significantly lowers ECM deposition, while experimentally activating YAP in hepatocytes or cholangiocytes causes rapid fibrosis in murine models[103-106].

Cross-talk among signaling pathways in liver fibrosis

Liver fibrosis is orchestrated by a complex interplay among several signaling pathways that regulate HSC activation, inflammation, and ECM remodeling. These pathways include the TGF-β/SMAD axis, Wnt/β-catenin signaling, the NF-κB inflammatory cascade, MAPK signaling modules, and PDGF-mediated proliferative signals. Importantly, these pathways exhibit extensive cross-talk, creating amplifying feedback loops that sustain fibrogenesis[5,60,83,107-113].

TGF-β/SMAD signaling, a central driver of fibrosis, not only induces ECM components such as type I and III collagen and α-SMA via SMAD2/3 activation, but also interfaces with other pathways[114-116]. Beyond its direct effects, TGF-β interacts with other signaling pathways to amplify fibrogenic responses. It enhances Wnt/β-catenin signaling by downregulating antagonists like Dickkopf-1, leading to stabilization and accumulation of β-catenin. The stabilized β-catenin then cooperates with SMAD proteins at promoters of pro-fibrotic genes, further increasing their transcription[117-119].

Additionally, TGF-β can indirectly activate NF-κB signaling via TGF-β-activated kinase 1, resulting in the upregulation of pro-fibrotic cytokines such as IL-6 and monocyte chemotactic protein-1, as well as increased TGF-β expression itself[120]. Pro-inflammatory cytokines like TNF-α and IL-1β, released by KCs and damaged hepatocytes, stimulate NF-κB and MAPK pathways (including ERK and JNK), which in turn modulate transcription factors to enhance SMAD signaling and promote fibrogenesis[121]. Furthermore, NF-κB and TGF-β increase TIMP expression, limiting MMP-mediated ECM degradation and promoting net ECM accumulation[122,123]. This intricate network underscores the interconnected nature of fibrogenic signaling pathways in liver injury[121].

The MAPK pathway plays a significant role in liver fibrosis through both direct and indirect mechanisms. JNK, a member of the MAPK family, can phosphorylate SMAD3 at linker regions, altering its DNA-binding affinity and transcriptional activity, thereby influencing fibrogenic gene expression. Meanwhile, ERK promotes proliferation and survival of aHSC[124,125]. Additionally, MAPKs are activated by PDGF, a potent mitogen for HSCs, which works synergistically with TGF-β to sustain aHSCs proliferation and fibrogenesis[126]. Furthermore, DAMPs, such as HMGB1, initiate fibrogenic signaling by binding to pattern recognition receptors like Toll-like receptor 4. This engagement leads to rapid activation of NF-κB and MAPK pathways, which amplify cytokine production and reinforce the fibrotic response, creating a feedback loop that accelerates liver fibrosis progression[127].

The Notch, Hippo, and Hedgehog pathways form a critical regulatory axis in fibrogenesis, particularly influencing biliary proliferation, ductular reaction, and stromal activation[128-130]. The transcriptional co-activators YAP/TAZ, effectors of the Hippo pathway, promote expression of Notch ligands (e.g., Jagged1), enhancing Notch signaling in cholangiocytes and aHSCs. Conversely, Notch activation supports YAP nuclear localization, reinforcing profibrotic transcriptional programs. Additionally, YAP/TAZ interact with GLI transcription factors of the Hedgehog pathway, while Hedgehog signaling reciprocally upregulates YAP expression. This reciprocal feed-forward loop drives HSC activation, progenitor cell expansion, and ECM production[95,130-134].

ECM remodeling is controlled by the balance between MMPs and TIMPs. TGF-β, Notch, and YAP/TAZ signaling promote TIMP1/2 expression, limiting ECM degradation and favoring matrix accumulation. In contrast, early activation of MAPKs and inflammatory cytokines transiently promotes MMP expression to facilitate cell migration. This dynamic regulation ensures both tissue remodeling and matrix deposition are coordinated during fibrogenesis[135,136].

Pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, along with DAMPs like HMGB1, serve as upstream initiators of fibrosis by activating Toll-like receptor, NF-κB, Janus kinase (JAK)/signal transducer and activator of the transcription, and MAPK pathways. These signals stimulate the release of TGF-β, PDGF, and Wnt ligands, indirectly modulating Hippo, Notch, and Hedgehog activity[120,121,137]. Additionally, ROS generated during chronic injury further enhance TGF-β expression and ECM gene transcription, establishing a vicious cycle of injury and fibrogenesis[138-140]. These synergistic and feedback interactions create a robust, self-perpetuating fibrotic program. Therefore, therapeutic strategies that target multiple key nodes within this integrated network rather than isolated pathways may prove more effective in halting or reversing liver fibrosis associated with chronic liver diseases.

ROLE OF EVS IN LIVER FIBROSIS

Cell-based therapies, particularly those involving MSCs, have emerged as promising alternatives for treating chronic liver diseases. MSCs are multipotent stem cells known for their tissue repair and immune regulation capabilities. They exert anti-fibrotic effects through various mechanisms, including modulating hepatic immune responses, secreting trophic cytokines to reduce hepatocyte apoptosis, exhibiting antioxidant properties, inhibiting HSC proliferation, and regulating TIMP-1 expression[89,141]. MSCs can be sourced from various tissues, such as bone marrow, liver, and umbilical cord. While MSC transplantation shows promise in improving liver function in conditions like alcoholic cirrhosis, a potential limitation is their capacity to differentiate into myofibroblasts, which could inadvertently contribute to fibrosis[142-145].

Recently, attention has shifted to EVs, particularly exosomes derived from MSCs, as a novel therapeutic strategy for liver fibrosis. miRNAs are small non-coding RNAs that regulate key cellular processes such as division, differentiation, and cell death. Over 1000 human miRNAs have been identified, with estimates suggesting they control about one-third of gene expression[146]. The human genome comprises approximately thirty collagen genes that generate different types of collagen fibrils vital for cellular functions. Clinical trials are being conducted to test the safety and effectiveness of MSC-EVs for a variety of disorders[147]. MSC-derived exosomes carry a similar therapeutic payload as their parent MSCs and can significantly reduce HSC activation[148,149]. They offer advantages over MSCs, such as smaller size, reduced complexity, lower immunogenicity, and no risk of tumor development. These characteristics make exosomes promising natural nanovectors for therapeutic agents in hepatic fibrosis treatment[150,151].

A key mechanism by which MSCs and their EVs exert their anti-fibrotic effects is through the transfer of miRNAs[146]. They play a crucial role in regulating gene expression in nearly all cellular functions and are implicated in various liver conditions, including fibrosis[40]. During liver fibrosis, miRNAs can be either up- or down-regulated. Specific miRNAs, such as miR-125b from MSC-derived exosomes, can prevent HSC activation by suppressing smoothened expression[152]. Similarly, miR-122 from adipose tissue-derived MSCs can inhibit HSC activation and proliferation[153,154].

Furthermore, miRNAs are involved in regulating collagen accumulation and the TGF-β/SMAD signaling pathway, both central to liver fibrosis. miRNAs like let-7 g, miR-29b, and miR-29c directly target collagen expression, while miR-29a suppresses collagen type 1 alpha 1 (COL1A1) expression and promotes apoptosis[155-157]. miRNAs such as miR-30e, miR-133b, and miR-19b have been shown to control collagen production and inhibit fibrotic pathways by targeting key components like prolyl 4-hydroxylase 2 (P4HA2) and TGF-β signaling[158-160]. Moreover, a range of miRNAs, including miR-15a, miR-15b, miR-16, miR-29a, miR-125b, miR-126, miR-146a, miR-195, miR-199a, and miR-200, have been identified as having roles in reversing fibrosis by regulating genes involved in HSC activation and the TGF-β pathway[161,162]. miR-21, miR-29a, miR-146a, miR-125b, miR-126, miR-195, and miR-199a are frequently identified in MSC-derived microvesicles across different tissue sources[163,164]. These miRNAs contribute to the regenerative, anti-inflammatory, and pro-angiogenic effects of MSCs and their derived EVs.

POSSIBLE WAYS OF REGRESSION OF LIVER FIBROSIS

The following succinctly describes the potential role of different cells and molecules in reversing the pathogenicity of liver fibrosis. An increasing number of clinical studies indicate that fibrosis can partially regress once the underlying cause is eliminated, challenging earlier beliefs that the condition was irreversible[165-167]. Unlike organs such as the kidney and lung, the liver possesses a greater capacity for regeneration, making the regression of fibrosis and restoration of normal tissue structure more likely. These characteristics may simplify the development of effective therapies for liver fibrosis[9,167].

It is required to find more effective medicines to postpone fibrosis progression or encourage regression of liver fibrosis, which can assist prevent the advancement of cirrhosis or cancer[70,165]. Understanding the mechanisms underlying liver fibrosis regression can help identify new treatment targets. The main ways to achieve liver fibrosis regression[167] are to eliminate the factors causing liver damage, reduce the number of aHSCs[168], inhibit the signal pathways linked to liver fibrosis[169], and promote the degradation of the ECM[170].

Lowering the aHSC count

The intricate mechanisms underlying liver fibrosis involve changes in cell populations, cytokines, and their molecular pathways[171]. Notably, activation of HSCs is a hallmark feature of liver fibrosis[171,172]. Upon removal of chronic liver injury, fibrosis tends to regress, and aHSCs either decrease in number or disappear[173]. Additionally, both experimental and clinical studies have demonstrated that apoptosis contributes to the elimination of aHSCs[174], or while inactivation into a quiescent-like state[175,176], or senescence[177] may also facilitate fibrosis regression. Due to their pivotal role in fibrosis development, aHSCs are considered a primary target for therapeutic intervention aimed at disease regression[178]. Therefore, strategies that reduce the number of aHSCs such as inhibiting their proliferation or activation[177-179], promoting apoptosis or autophagy[171,172,180], and deactivating the aHSCs[175,176] may serve as effective approaches to reverse liver fibrosis[181].

Apoptosis, a form of programmed cell death, aids in regulating the HSCs’ equilibrium between growth and death[167]. The condition may worsen as a result of HSCs’ abnormal activation and proliferation during liver fibrosis progression, which may hasten the death of other healthy liver cells[54,182]. Clinical studies revealed that, regardless of HSC activation or proliferation, the quantity of apoptotic HSCs declined as the fibrosis stage increased[183]. Research on animal models of liver damage caused by various techniques has demonstrated a strong correlation between the regression of fibrosis and the reduction of HSCs through apoptosis induction, indicating that HSC apoptosis is also a key factor in the regression of liver fibrosis[174,183]. Thus, increasing the rate at which aHSCs die and decreasing the overall quantity of aHSCs can reverse liver fibrosis[184,185]. The researchers have shown that liver fibrosis treatment could rely on endoplasmic reticulum stress (ERS)-mediated apoptosis[186]. Additionally, in the HSC line HSC-T6, the calpain-2/Ca²⁺-dependent ERS pathway can be used by the ERS inducer tunicamycin to induce HSC apoptosis[186].

The liver is the primary site of expression for the metabolic enzyme alcohol dehydrogenase (ADH), and ADH-I is the traditional liver ADH among these family members[187]. According to research, the human fibrotic liver exhibits substantially greater ADHI activity than the normal liver. ADH-I may enhance the proliferation, motility, adhesion, and invasion of HSCs[188]. Therefore, ADH-I might be a target for future hepatic fibrosis medications.

aHSC inhibition and reversal

Anti-fibrotic medications target HSCs because of their significant role in liver fibrosis[52]. Focusing on aHSC deactivation has become a novel and effective approach to liver fibrosis treatment[176,189]. Fibrosis experimental models consistently show that fibrosis can reverse when aHSCs are eliminated by apoptosis or other mechanisms[190,191]. Recent research has demonstrated that transcriptional reprogramming, including ectopic expression of GATA-binding protein 4 (GATA4), forkhead transcription factor 3, hepatocyte nuclear factor 1 homeobox A, and hepatocyte nuclear factor 4 homeobox A in vivo, can convert aHSCs into qHSCs[192].

It has been shown that abnormal ECM component development and HSC activation in the embryonic liver occur when the zinc finger transcription factor GATA4 in HSCs is deactivated[189]. Reactivation of GATA4 has been found to reverse liver fibrosis by transforming aHSCs into a qHSCs[82]. Therefore, GATA4 might be a suitable target for liver fibrosis resolution. In another study, the activation of HSCs has been linked to peroxisome proliferator-activated receptor-γ (PPAR-γ)[175]. Significantly lower levels of PPAR-γ are observed during HSC activation[193], and a shift to qHSCs could be brought by PPAR-γ overexpression in aHSCs[194,195]. Transcription factor 21 has been found to be a deactivation factor of aHSCs[196]. Both in vitro and in vivo experiments, overexpression of transcription factor 21 in aHSCs reduced the expression of genes linked to fibrosis, moderately reverse the cells into a quiescent state, and eventually helped to decrease and improved liver function[196].

Reducing of scar evolution

Excess ECM removal is one of the main goals in treating liver fibrosis. Collagen is the ECM protein that is most common in liver fibrosis. A small interfering RNA to the procollagen α1(I) gene has now been shown to significantly reduces the synthesis of collagen 1 fibrils in animals, and in fibrosis models[197]. This could be another modality to treat liver fibrosis.

Inhibiting the signal pathways associated with liver fibrosis

The entire process of liver fibrosis involves a number of molecular mechanisms, signaling channels, and cytokines. The disease can be reversed by interfering with these substances and pathways[178,198]. Many signals are crucial to the progression of liver fibrosis, including TGF-β[199], Wnt/β-catenin[200], Notch[128], Hedgehog[201], Hippo[185,202], and inflammasome signaling pathways[203]. From initial liver damage to cirrhosis and fibrosis, TGF-β, the most potent fibrogenic cytokine, influences each stage of the disease’s progression[9,204]. TGF-β is increased during liver fibrosis and primarily acts to stimulate HSCs. Additionally, it can directly stimulate the creation of interstitial fibrillar collagens and increase the synthesis of TIMPs[205]. Consequently, inhibiting TGF-β or interfering with its downstream signaling pathways offers an effective approach to prevent liver fibrosis[206]. Strategies such as targeting specific TGF-β isoforms or blocking TGF-β receptor activation have been explored extensively to disrupt TGF-β-mediated signaling and suppress fibrosis progression[205]. Numerous cytokines, including IL, can activate the JAK signaling pathway, which is crucial to the pathophysiology of hepatic fibrosis. According to studies, using the JAK2 receptor antagonist TG101348 in animal models can lessen hepatic fibrosis[207-209]. The Notch signaling system may be one of the targets for reversing hepatic fibrosis because it can also be used to treat liver fibrosis[10]. Beyond the pathways previously mentioned, liver fibrosis reversal can also be achieved by promoting ECM degradation, boosting immune cell activity and their signaling pathways, employing stem cell transplantation, and applying other innovative approaches. However, further clinical trials are necessary to establish effective methods for reversing liver fibrosis.

REVERSING LIVER FIBROSIS USING MIRNA

A number of cytokines control HSC activation and aHSCs are closely linked to liver fibrosis. miRNAs regulate gene expression post-transcriptionally by binding to target mRNAs, leading to their degradation or translational repression. Given their ability to modulate key pathways involved in liver pathology, miRNAs are closely linked to various liver conditions, including liver fibrosis, making them promising therapeutic targets. During liver fibrosis, the expression levels of specific miRNAs can be both up- and down-regulated. Anti-miRNA oligonucleotides and miRNA masking can reverse up-regulated miRNA[207-209]. In contrast, some studies have observed that downregulated miRNAs may actually play a role in inhibiting liver fibrosis[210-212]. According to reports, miRNAs have antifibrogenic qualities and have the ability to regulate the production of genes including COL1A1, ACTA2, TGF-β receptor type-1, and TIMP1 that are involved in the activation of HSCs[79,176,189]. The role of different miRNAs in regulating pathways involved in liver fibrosis has been extensively studied, revealing their crucial influence on the progression or inhibition of fibrogenic processes. Below are an overview of key miRNAs and their functions.

miRNAs in down-regulating the genes involved in collagen accumulation

Many miRNAs influence the production and regulation of collagen, and miR-21 playing major role in controlling the expression of collagen type 1 in particular cell types[213]. miRNAs such as let-7 g, miR-29b, and miR-29c directly target the expression of various collagens by binding to their 3’ untranslated regions[157]. Collagen synthesis induced by TGF-β is activated in HSCs, which play a role in liver fibrosis. Due to its anti-fibrotic effects, miR-29a directly suppresses COL1A1 expression and promotes apoptosis[155,156]. The integrity of the collagen triple helix depends on the hydroxylation of proline residues on collagen, which is mediated by collagen prolyl 4 hydroxylase. Collagen prolyl 4 hydroxylase is an α2β2 tetramer made up of three isoenzymes comprising various catalytic subunits (encoded by the genes P4HA1, P4HA2, and P4HA3) and an a-subunit (the gene P4HB)[214]. P4HA2 promotes collagen deposition in the liver both in vitro and in vivo, which accelerates the development of liver fibrosis and liver cancer. miR-30e, targeting P4HA2 mRNA, controls collagen production[158]. In addition, miR-133b and miR-19b have been shown to inhibit fibrotic pathways by targeting TGF-β signaling components, thereby reducing collagen accumulation[159,160].

miRNAs in down-regulating the genes involved in TGF-β/SMAD signaling pathway

In numerous conserved biological signaling cascades, miRNAs play a role in post-transcriptional gene regulation[215,216]. A component of these signaling cascades is the TGF. miRNAs regulate the TGF-β signaling pathway. miRNAs are involved in many biological processes including regulation of apoptosis, development, signal transduction, cell proliferation, and immune defense[217]. Almost every biological activity and cell type, including those in the liver, is targeted and regulated by miRNAs, which also affect gene expression in almost every cellular function. Multiple research studies have demonstrated that alterations in intracellular miRNAs such as miR-155, miR-132, miR-21, miR-26a, and miR-217 are linked to various liver disorders[218-220]. Specifically, miR-155 has been shown to be elevated in KCs following alcohol consumption, with TNF identified as a target of miR-155 that promotes liver inflammation[218]. Additionally, TGF-β1 exerts its biological functions through activation of both SMAD-dependent and SMAD-independent signaling pathways. It is well established that TGF-β primarily mediates its effects by activating downstream mediators SMAD2 and SMAD3, while its activity is negatively regulated by the inhibitory SMAD7[221]. In contrast, miR-15a, miR-15b, miR-16, miR-21, miR-29a, miR-122, miR-125b, miR-126, miR-146a, miR-150-5p, miR-195, miR-199a, miR-200, miR-223, and miR-486-5p have the role in reversing the fibrosis (Table 1). For instance, miR-200 family known to target zinc-finger E-box-binding homeobox 1 and zinc-finger E-box-binding homeobox 2, which are downstream of TGF-β signaling and involved in epithelial-to-mesenchymal transition. They can also directly target components of the TGF-β pathway to inhibit fibrosis[161]. miR-29 family, besides regulating collagen genes, miR-29 can target TGF-β receptor type-2 and SMAD3, reducing TGF-β signaling and fibrotic responses[162].

Table 1 Role of anti-fibrotic microRNAs in liver fibrosis by modulating specific pathway(s).

miRNA	Target protein/pathway	Mechanism	Ref.
miR-29a/b/c	TIMP, SMAD, TGF-β, PDGF, Wnt, MMP, HMGB-1, TLR-4	Directly targets collagen genes, reduces ECM production, increases MMPs via TIMP suppression, inhibits Wnt signaling	[12,155,156,162]
miR-146a	NF-κB, IL-6, TNF-α, IL-1β, MCP-1, TGF-β, Wnt	Targets IRAK1, TRAF6 to inhibit NF-κB pathway; reduces pro-inflammatory cytokines, inhibits HSC activation	[40,222]
miR-125b	TGF-β, SMAD, Wnt	Targets SIRT7, suppresses TGF-β/SMAD and Wnt pathways, regulate Hedgehog signaling	[152]
miR-122	Collagen I/III, TIMP, α-SMA	Downregulated in fibrosis; restoration reduces collagen and HSC activation	[153,154]
let-7 g	TGF-β, SMAD	Inhibits TGF-β signaling, reduces ECM production, inhibit migration of HCC	[157]
miR-30a/e	TGF-β, α-SMA	Targets Beclin-1 and autophagy pathways, suppresses HSC activation	[158]
miR-133b	TGF-β	Directly targets TGF-β, inhibits HSC activation	[159]
miR-19b	TGFβR2	May inhibit TGF-β pathway via receptor targeting, inhibit fibrogenesis	[160]
miR-26a	Notch, TGF-β	Inhibits HSC proliferation, collagen production, EMT	[223]
miR-15a/b, miR-16	Bcl-2, TGF-β/SMAD, Notch, Wnt	Inhibit HSC proliferation and collagen expression	[224]
miR-126	p5, PI3K-AKT	Maintains endothelial integrity; fibrosis role less defined; regulates HSCs cycle and maintains HSCs	[225]
miR-199a/199a-3p	Collagen I/III, caveolin-2, GSK, MAPK	Improve chemo-sensitivity through mTOR pathway	[151,163]
miR-200	EMT, TGF-β, SMAD	Inhibits EMT and TGF-β induced fibrogenesis through SMAD pathway	[161]
miR-223	NF-κB, PDGF, MCP-1, HMGB1	Inhibits inflammasome activation, reduces inflammation and fibrosis	[226-228]
miR-150-5p/miR-150	TGF-β, c-Myb	Suppresses HSC activation	[229]
miR-486-5p	Hedgehog signaling	Suppresses HSC activation and fibrosis	[148]

miRNA: MicroRNA; TIMP: Tissue inhibitors of matrix metalloproteinase; SMAD: Small mother against decapentaplegic; TGF-β: Transforming growth factor-β; PDGF: Platelet derived growth factor; MMP: Matrix metalloproteinase; HMGB-1: High-mobility group box-1; TLR-4: Toll-like receptor 4; ECM: Extracellular matrix; NF-κB: Nuclear factor-kappa B; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β; MCP-1: Monocyte chemotactic protein-1; IRAK1: Interleukin-1 receptor associated kinase 1; TRAF6: Tumor necrosis factor receptor associated factor 6; HSC: Hepatic stellate cell; SIRT7: Sirtuin7; α-SMA: Alpha-smooth muscle actin; HCC: Hepatocellular carcinoma; TGFβR2: Transforming growth factor-beta receptor 2; EMT: Epithelial-mesenchymal transition; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; GSK: Glycogen synthase kinase; MAPK: Mitogen-activated protein kinase; mTOR: Mammalian target of rapamycin.

CONCLUSION

Liver fibrosis and cirrhosis represent significant global health challenges characterized by complex cellular and molecular mechanisms, notably the activation of HSCs and dysregulation of key signaling pathways such as TGF-β/SMAD, Wnt/β-catenin, NF-κB, and MAPK. The progression of these conditions results from persistent liver injury, involving hepatocyte damage, inflammatory responses, and alterations in liver cell interactions that promote excessive ECM deposition. Importantly, emerging research highlights the liver’s inherent capacity for regeneration and fibrosis regression once the underlying cause is addressed. Therapeutic strategies targeting HSC activation, promoting apoptosis, inhibiting pro-fibrotic cytokines, and modulating signaling pathways show promise. Innovative approaches such as MSC-derived EVs and miRNA-based therapies are gaining attention due to their potential to deliver targeted anti-fibrotic molecules, regulate gene expression, and reverse fibrosis effectively. While current treatments are limited, these advances suggest a hopeful future for more effective, targeted therapies capable of halting or reversing liver fibrosis, ultimately reducing the burden of chronic liver diseases worldwide.

ACKNOWLEDGEMENTS

The authors would like to thank Md Sanower Hossain, Md Mahfuzul Islam, Md Hasan Ali, Munira Mohtasim, Naureen Sharmi, and Mahamuda Afroze for their support and reviewing this manuscript.