Hepatic Fibrosis: Mechanisms and Targets

By Pablo Muriel

Hepatic Fibrosis: Mechanisms and Targets is a complete volume of liver extracellular matrix biology, including molecular signaling pathways, cells and factors that modulate fibrogenesis and fibrosis. The book uses an integrated approach toward the molecular and cellular mechanisms involved in the synthesis and degradation of hepatic fibrotic tissue, emphasizing the possible molecular targets to fight fibrosis. This important reference describes, in detail and didactically, the cellular and molecular events that are conducive to fibrosis that leads to cirrhosis, hepatocellular carcinoma and death. The provided information allows readers to understand the molecular mechanisms of hepatic fibrogenesis to accelerate the development of new therapies.

• Presents progression from inflammation to fibrosis, with a special focus on the molecular mechanisms involved
• Didactically explains the participation of cells, cytokines and factors in profibrogenic pathways
• Illuminates the causative participation of free radicals in liver fibrogenesis
• Explains the role of gut dysbiosis in chronic liver diseases leading to fibrosis
• Provides experimental models to study liver fibrosis and describes available, noninvasive monitoring methods

• Language: English
• Publisher: Academic Press
• Release date: Apr 23, 2022
• ISBN: 9780323952897

Pablo Muriel

Dr. Muriel PhD has research experience in the pathophysiology and pharmacology of liver diseases including necrosis, fibrosis and cholestasis; the role of cytokines in liver diseases; and oxidative stress and its relation to liver disease. He has published over 100 articles (original and reviews) in addition to 7 book chapters in the area of the liver and has also directed the thesis on dozens of postgraduate students in the same area over the last 30 years. The majority of his work has been in the Medicine and Pharmacology areas. He shows strong and growing citation counts from 2010-2017.

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Chapter 1: The healthy and diseased extracellular matrix of the liver

Abstract

This chapter describes the composition of the extracellular matrix (ECM) of the liver under normal conditions and the quantitative and qualitative alterations observed under chronic hepatic damage conditions. This chapter also describes the molecular mechanisms of ECM turnover.

Keywords

Chronic hepatic damage; Collagen; ECM turnover; Extracellular matrix; Liver

Introduction

The extracellular matrix (ECM) is essential for the normal function of any organ in the body and is composed of two classes of macromolecules, namely, proteoglycans and fibrous proteins. The ECM is constantly remodeled to maintain tissue homeostasis. Altered or old components of the ECM are degraded and replaced by newly synthesized molecules. Remodeling of the ECM is a finely regulated process that requires crosstalk between the components of the ECM and the embedded cells. When the tissue is injured, proinflammatory and profibrogenic processes occur, leading to the imbalance in ECM turnover, resulting in qualitative and quantitative modifications of the ECM components and thereby fibrosis. ¹ , ²

The extracellular matrix

The extracellular matrix is composed of two main types of molecules

The interstitial matrix and the basement membrane are the main components of the ECM that perform specific roles, depending on their location in the tissue parenchyma. ³ The interstitial matrix is principally formed by fibrillar type I, III, and V collagens; proteoglycans; and fibronectin and constitutes most of the ECM in organisms. ⁴ The interstitial matrix, particularly type I, III, V, and VI collagens, is mainly produced by fibroblasts. ⁵ The other component of the ECM is the basement membrane, which possesses a variety of functions and consists of different molecular structures that allow quick dissemination of specific small molecules and a stratified scaffold for epithelial and endothelial cells. This basement membrane is formed by type IV collagen (a very important network collagen produced by epithelial and endothelial cells), laminins, and proteoglycans with collagen types XV, XVIII, and XIX and heparan sulfate. ⁶ Notably, the basement membrane is a reservoir of proinflammatory, profibrogenic, and angiogenic cytokines and growth factors that regulate processes such as cell migration, tissue regeneration, and the wound healing response ⁶ (Fig. 1.1).

Figure 1.1  The extracellular matrix (ECM) is formed by the interstitial matrix and basement membrane proteins.The main components of the interstitial matrix are collagen types I, III, V, and VI; fibronectin; and proteoglycans, which are mainly produced by fibroblasts and activated HSCs. The principal function of the interstitial matrix is to provide structure to the hepatic parenchyma; however, under chronic liver injury, interstitial matrix is produced in excess, leading to fibrosis. Interstitial matrix components are mainly localized in the portal tract stroma and the perisinusoidal space and form fibrotic septa under fibrosis conditions. The basement membrane includes collagens IV, XV, XVIII, and XIX; laminins; and proteoglycans. The main function of the basement membrane is the support of hepatocytes, allowing the free diffusion of molecules between blood and liver cells, permitting the polarized and differentiated functions of hepatic cells. Basement membrane proteins are synthesized by quiescent HSCs, hepatocytes, and endothelial cells. Basement membrane components are mainly situated along the sinusoids, bile ducts, and vessels of the portal tract.

The importance of collagens for the extracellular matrix

Twenty-eight different types of collagen that are encoded by 42 different genes have been described. ⁷ Collagens share a triple helix structure that is highly stable and is formed by three chains, which possess a repetitive amino acid sequence (glycine-proline-hydroxyproline). Apart from the collagens that form fibrils, other collagen types possess interruptions of the glycine-proline-hydroxyproline triple-helical region that permit them to form flexible networks and supramolecular organization structures. ⁴ , ⁷ The collagens are divided into six classes: fibrillar collagens, fibril-associated collagens with interrupted triple helices (FACITs), multiplexins, network-forming collagens, transmembrane collagens, and others. ⁷ The organization and composition of the collagen chains define their function.

The extracellular matrix signals

In addition to providing structural support and protection to cells, the ECM is critical for the development of various processes that regulate organ function and the cell response to diseases. ⁵ , ⁷ , ⁸ Several collagen-derived proteolytic fragments produced by the activity of specific proteases trigger a plethora of signaling molecular pathways. Matrix metalloproteinase (MMP) activity on type IV collagen produces fragments of arrestin, tetrastatin, canstatin, pentastatin, and tumstatin that block endothelial cell proliferation, angiogenesis, and tumor growth. ⁴ , ⁹–¹³

Healthy liver collagens

Most of the collagens present in a disease-free liver are fibrillar types I, III, and V, which are mainly found in the interstitial matrix and synthesized by fibroblasts, and their function consists of providing structure to the hepatic parenchyma. Chronic liver injury induces fibrosis, and then collagens undergo changes in their quantity, proportion, and distribution. Collagen IV, which is produced by quiescent hepatic stellate cells (HSCs), hepatocytes, and endothelial cells, ¹⁴ is present in the normal liver and supports hepatocytes, permitting the diffusion of compounds between the blood and hepatic cells in the space of Disse. Type IV collagen and nonfibrillar proteins, such as laminins and entactin/nidogen, form a low-density basement membrane-like matrix within vessels, bile ducts and sinusoids, which allows the polarized and differentiated functions of hepatic cells. ⁸ , ¹⁵ Collagen type XVIII, mainly produced by hepatocytes, and collagen type XV, which is a basement membrane collagen, can be found in the portal tract. ¹⁶ , ¹⁷ In a healthy liver, fibrillar collagens are frequent components of the portal tract stroma, and minor quantities can be observed in the perisinusoidal space and increase in the fibrotic septa during chronic liver damage (Fig. 1.1).

Collagens

All organs in the body express collagens, and they are the most abundant molecules in connective tissues. Collagens can form supramolecular aggregates by interacting with other proteins of the ECM. Collagens contain nontriple-helical domains and triple-helical domains that serve as building blocks. According to their molecular structure and supramolecular assembly, collagens can be divided into six classes: fibril-forming collagens, fibril-associated collagens with interrupted triple helices, network-forming collagens, membrane-anchored collagens, interconnecting collagens, microfibrils, and short collagens. ⁷

Fibroblasts produce collagen types I, III, and V, which are important in the interstitial matrix, providing the characteristic structure, and collagen type IV and other networking collagens, which are produced by epithelial cells, are mainly found in the basement membrane, underlying epithelial or endothelial cells, and provide specialized function and polarization. ¹⁸–²⁰ Collagens are diverse in distribution, structure, and function in the ECM. ⁴ Notably, collagen type IV is essential for the processes of tissue repair and allows the survival and function of polarized cells, such as hepatocytes in the liver parenchyma, whereas collagen types I and III are important for providing support and structure during wound healing in response to chronic injury. ²¹

The synthesis and degradation of collagens

Several cellular chaperones and extracellular helper molecules oversee collagen synthesis. In the endoplasmic reticulum, the heat shock proteins HSP 90 and 47 bind to procollagen, ²² and the chaperone HSP 47 binds to procollagen during type I collagen synthesis. ²³ Moreover, the secreted protein acidic and rich in cysteine protein participates in collagen fibril formation in the extracellular space. ²⁴ Collagen is synthesized in the form of procollagen and needs to be activated by a maturation process in which propeptides are cleaved by specific proteases. N-Proteinases are in charge of cleaving the N-propeptide. The N-propeptide of the pro-α1(V) chain is cleaved by bone morphogenetic protein-1. ²⁵ Young animals possess a better collagen turnover capacity than older animals ¹⁹ and have a similar pattern to that reported for humans. ²⁶ , ²⁷ This point should be considered when studies are performed in young animals to avoid direct extrapolation with cirrhotic patients who are usually not very young, thus exhibiting a limited regenerative capacity.

Collagens as signaling molecules

The ECM is not only a scaffold to support and protect embedded cells but also plays a role in various important physiological and pathophysiological processes. ⁴ Moreover, ECM degradation produces fragments that acquire a paracrine or endocrine function, acting as cell signaling molecules. MMP-2 and MMP-9 act on the collagen α1(I) chain of collagen type I, producing a matricryptin that has angiogenic activity and promotes the deposition of ECM in the cardiac tissue of patients with myocardial infarction. ²⁸ Tumstatin, tetrastatin, pentastatin, arresten, and hexastatin are produced by MMP activity on collagen type IV, which possesses antiangiogenic, antitumor, and antiproliferative activity. ⁹–¹³ Endotrophin is a C-terminal fragment of the α3(VI) chain of collagen type IV that upregulates transforming growth factor β1 (TGF-β1) and promotes angiogenesis, inflammation, and fibrosis, mainly in adipose tissue. ²⁹ Vastatin, which is a C-terminal fragment of collagen type VIII, induces apoptosis and proliferation of endothelial cells. ³⁰ Restin, which reduces the migration of endothelial cells, is the noncollagenous C-terminal fragment of collagen type XV. ³⁰–³² Endostatin, which is derived from hepatocytes, ³³ is the noncollagenous C-terminus of collagen type XVIII; endostatin interacts with several receptors ³³ , ³⁴ and binds to heparin sulfate proteoglycans in the ECM, competing with proangiogenic heparin-binding and endothelial growth factors. ³⁴ , ³⁵ Notably, because of its antiangiogenic properties, endostatin may block cancer growth ³⁶ and, through controlling the cell phenotype, can ameliorate fibrosis of the liver and other organs ³⁷ , ³⁸ (Fig. 1.2).

Figure 1.2  The proteolytic activity of metalloproteinases (MMPs) on the extracellular matrix (ECM) produces several signaling molecules.This figure shows some examples of the signaling molecules produced by the enzymatic activity of MMPs on some components of the ECM that may modulate inflammation, fibrosis, cell phenotype, and organ function.

Collagen and other components of the ECM may induce inflammation of several organs through TLR activation, and collagen-derived fragments may act as chemokines; moreover, the ECM may control cell phenotype and organ function ⁴ (Fig. 1.2).

The wound healing response

Epithelial and endothelial cells respond to damage by a clotting process that stops bleeding by binding von Willebrand factor to the ECM, inducing reepithelialization and reendothelialization of the damaged tissue. von Willebrand factor binding to the ECM recruits platelets to the injured parenchyma, which initiates the secondary clotting process, leading to a crosslinked fibrin clot. After activation, platelets produce and release platelet-derived growth factor (PDGF) and TGF-β1 to induce angiogenesis of endothelial cells and ECM deposition from fibroblasts or HSCs to produce the desired wound-healing effect. Then, neutrophils, monocytes, T cells, and other inflammatory cells reach the site of damage to remove damaged ECM and cell debris, generating fragments that have important signaling roles, leading to either remission of fibrosis, characterized by controlled ECM deposition and degradation, or to fibrogenesis, producing fibrosis. ⁴ In the chronic injury scenario, inflammation of the tissue triggers the activation of Th2 cells and M2 macrophages (see Chapter 2 of this volume), and in an attempt at continuous repair, uncontrolled ECM deposition produces large fibrotic areas within the affected organ. On the one hand, MMPs degrade ECM proteins; on the other hand, activated fibroblasts produce exacerbated amounts of ECM, and the net result is scarring of the affected tissue. As these processes take place, multiple fragments of the ECM are produced and released, exhibiting signaling functions and providing biomarkers of fibrosis. ¹ , ²¹ , ³⁹ , ⁴⁰

Cells that produce extracellular matrix components

Fibroblasts are the main producers of interstitial collagens. Upon activation, fibroblasts, myofibroblasts, and HSCs are responsible for exacerbated ECM deposition in organ fibrosis, mainly collagen types I and III and fibronectin. ⁴¹–⁴⁴ Additionally, fibroblasts produce connective tissue, which is the ECM with its embedded cellular components, and are responsible for the maintenance and degradation of the ECM by the production and secretion of MMPs and MMP inhibitors. ⁴⁵ The most abundant collagen is type I (80%), followed by type III collagen (15%) and type V collagen (5%). ⁴⁶ Collagen types VI, XII, and XIV constitute only a minor fraction; however, they are important modulators of thick collagen fibrils, regulating the interaction with cells and other ECM components. ⁴⁷ Several ECM proteins of the basement membrane, including type IV and XVIII collagens, are not produced by fibroblasts but by hepatocytes and proliferating bile duct epithelial cells. ⁴⁸–⁵¹

Liver fibrosis is more than the exacerbated deposition of extracellular matrix proteins

At the beginning of the fibrogenic process, fibrin and fibronectin form a provisional matrix suitable for the formation of type I and III fibrillar networks. ⁵²–⁵⁴ In turn, the progression of fibrosis is characterized by an increase in the type I/III collagen ratio and a decrease in the basement membrane and FACIT collagens ⁵⁵ that induce the ECM to become less flexible and denser. However, in most chronic hepatic disorders, there is also a significant increase in basement membrane components, including type IV collagen. ⁴ , ³⁹ , ⁵⁶ , ⁵⁷ Therefore, hepatic fibrosis is more than just the simple accumulation of scar tissue but is characterized by a modification in the composition of the various components that constitute the ECM. Moreover, collagens in advanced fibrosis are deposited in places in the hepatic parenchyma that do not correspond to the normal physiological location. Established cirrhosis exhibits up to a 10-fold increase in fibrillar collagens and an up to a sixfold increase in basement membrane components of the ECM, leading to an increase in liver stiffness. ⁵⁸ , ⁵⁹ Importantly, in advanced cirrhosis, the basement membrane types IV and XVIII collagen content increase in the space of Disse, provoking capillarization of the hepatic sinusoids and thus disrupting the exchange of gases and nutrients between hepatocytes and the blood. ⁶⁰ , ⁶¹

The basement membrane matrix serves as a substrate for hepatocytes, and interstitial collagens are required to fill up the gaps. ⁶² ECM degradation normally precedes HSC activation in liver fibrosis. ⁶² The early fibrotic stage of the liver is characterized by small collagen fibers placed within the hepatocytes in a pattern known as chicken wire fibrosis with delicate reticular fibers in the perisinusoidal space.

Different liver diseases exhibit a characteristic pattern of fibrosis development. Biliary fibrosis has a characteristic pattern, consisting of myofibroblasts, leading to portal-to-portal septum formation. ⁶³ Chronic viral hepatitis induces the activation of inflammatory cells such as Kupffer cells and infiltrated macrophages and other proinflammatory cells, leading to the formation of portal-to-central septa. ⁶³ Metabolic liver diseases, as well as alcohol-induced liver injury, possess a chicken wire fibrotic pattern appearance where deposition of ECM proteins is found around hepatocytes and sinusoids. ⁶⁴ Depending on the location of tissue injury and the profibrogenic cells and molecular pathways involved, different patterns of fibrosis develop. ⁴ , ⁶⁵

Metalloproteinases in liver fibrosis

Twenty-five MMPs have been reported to degrade several proteins of the ECM. ⁶⁶ , ⁶⁷ The proteolytic activity of MMPs modulates several physiological processes of the liver by producing and releasing several signaling molecules that regulate the behavior of a variety of cell types. ⁶⁸–⁷¹

Because MMPs are produced in a latent inactive form, they require activation. ⁷² Moreover, there are at least four MMP inhibitors, named tissue inhibitors of metalloproteinases (TIMPs). ⁷³ Proinflammatory and profibrogenic factors and cytokines such as PDGF, interferon (IFN) α, interleukin (IL)-1α, tumor necrosis factor (TNF)-α, IL-6, IL-8, and TGF-β1 ⁷⁴–⁷⁸ regulate the expression and activity of MMPs, which in turn modulate the activation of HSCs and thus ECM production. ⁷⁹ , ⁸⁰ Therefore, MMP activity is tightly modulated and involves epigenetic and transcriptional regulation, modulation by miRNAs, and translational and posttranslational modifications, influencing their release, substrate specificity, activators, and catabolism. ⁸¹–⁸⁷ It has been reported that MMP-12 deficiency increases ECM degradation of MMPs, which reduces IL-13-dependent fibrosis. ⁸⁸ Importantly, inflammation may destroy tissue by activating MMP cascades. ⁸⁹ Efficient crosstalk exists between MMPs and TIMPs in a normal liver that leads to moderate ECM turnover, preserving vascular rearrangement and liver homeostasis, where MMP-2 and MMP-9 play a fundamental role by proteolytically modulating the basal activity of cytokines and growth factors. ⁹⁰ , ⁹¹ The illumination of the interplay between these processes may pave the way for a rational approach aimed at alleviating the deleterious properties of MMPs in hepatic disorders while maintaining their normal functions ⁷¹ , ⁸⁴ (Fig. 1.3).

Figure 1.3  Matrix metalloproteinases (MMPs) play a fundamental role in fibrosis.Activated hepatic stellate cells (HSCs) are the main executors of fibrogenesis. Several factors and cytokines, including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), transforming growth factor-α (TNF-α), several proinflammatory interleukins (ILs) and IFN-α, prime HSCs to produce ECM proteins, leading to fibrosis. HSCs synthesize several MMPs and their inhibitors, namely, tissue inhibitors of metalloproteinases (TIMPs), to produce regulated turnover of ECM. MMPs play a dual role in activating or inhibiting HSCs, depending on the product formed by a given MMP's proteolytic activity on a specific component of the ECM. The ECM may positively or negatively regulate the fibrogenic activity of HSCs. The figure shows several possible antifibrotic targets.

The role of metalloproteinases in acute hepatic damage

Hepatic damage may affect liver functions such as glycogen storage, metabolism, clearance of pathogens, and toxins. ⁹² Therefore, to preserve these vital functions, the liver has an extraordinary ability to regenerate due to the capacity of mature hepatocytes and cholangiocytes to divide and repopulate the organ. ⁹³–⁹⁵ Several agents, such as toxins and hepatotropic viruses, may induce apoptosis and necrosis of hepatocytes. ⁹⁶ Then, killed hepatocytes release free radicals, cytokines, growth factors, hormones, and apoptotic bodies that may activate HSCs, thus triggering the fibrogenic response. ⁹⁷ In addition to ECM, ⁹⁸ HSCs release TIMPs, TGF-β, IL-6, and vascular endothelial growth factor (VEGF), ⁶⁷ which stimulate and recruit T cells, macrophages, and endothelial cells of the sinusoid to dedifferentiate toward a capillarized phenotype, losing endothelial fenestration. ⁹⁹ , ¹⁰⁰ TIMP-1 is abundantly secreted by activated HSCs, leading to the inhibition of MMP activity; consequently, ECM proteins are not degraded, and fibrosis ensues. Therefore, it seems that proteolytic degradation of the normal ECM occurs at the beginning of hepatic fibrogenesis. ¹⁰¹ Indeed, several MMPs are only basally expressed under normal conditions but are upregulated after liver damage, thereby degrading ECM and modulating cellular functions. ¹⁰² Notably, the breakdown of ECM proteins by MMP activity regulates HSC activation, proliferation, migration, and apoptosis. ¹⁰³ Upregulation of MMP-2, ¹⁰³ , ¹⁰⁴ MMP-3, ¹⁰²–¹⁰⁵ MMP-9, ⁷⁴ , ¹⁰³–¹⁰⁹ MMP-10, ⁷⁴ MMP-13, ¹⁰⁵ and MMP-14 ¹¹⁰ has been demonstrated after hepatic injury before fibrogenesis in rodent and in vitro models; this large body of evidence supports the importance of MMPs in triggering fibrosis. It has been hypothesized that some MMPs, which are induced in response to liver damage, modify healthy ECM, thus inducing hepatocyte necrosis, ⁹⁰ , ¹¹¹ which in turn triggers the transdifferentiation of HSCs and alters the phenotype of sinusoidal endothelial cells and hepatocytes. ¹¹²–¹¹⁴ Accordingly, pharmacological or genetic inhibition of MMP-2, MMP-3, MMP-8, and MMP-9 can prevent acute liver injury. ¹⁰⁴ , ¹¹⁵ HSCs, in addition to being the main ECM-producing cells, are the major source of MMPs in acute liver injury in the early stage of activation. ⁷⁴ Additionally, it has been reported that active MMP-2 and MMP-14 induce the activation, migration, and proliferation of HSCs in vitro. ¹¹⁶–¹¹⁹ Moreover, MMP-8 proteolytic activity induces the activation of HSCs in vitro, suggesting a role in the development of hepatic fibrosis. ¹²⁰ Apoptosis of activated HSCs can be regulated by MMP-9 activity, ¹²¹ and mice lacking MMP-9 exhibit a decreased fibrotic response, ¹²² indicating the important role of this MMP in the regulation of the fibrotic process. However, in chronic liver damage-induced fibrosis, it has been demonstrated that the expression of MMP decreases and TIMP increases, leading to the net accumulation of ECM in the hepatic parenchyma. ⁷⁴ MMP-9 breaks down basement membranes ¹²³ and disrupts cell adhesion molecules in damaged tissue. ¹²⁴ Furthermore, ischemia-reperfusion injury was minimized in MMP-9-deficient animals compared with control animals ¹⁰⁸