Liver fibrogenesis: Mechanisms and clinical relevance

Liver fibrosis represents a scarring response to either acute or chronic liver injury. After acute liver injury, parenchymal cells regenerate to successfully preserve hepatocellular mass and function. This acute process is associated with an inflammatory and fibrogenic response but with limited deposition of extracellular matrix (ECM). In contrast, prolonged liver injury leads to sustained production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines. These events culminate in the accumulation of ECM, forming septa that coalesce into broad bands of scar tissue encircling nodules of hepatocytes and leading to altered microvascular structure ^, ( Fig. 7.1 ). This late stage of fibrosis, termed cirrhosis , ultimately impairs liver function and leads to portal hypertension and its complications, including ascites, encephalopathy, and hepatocellular carcinoma, or primary liver cancer (see Chapters 74 and 79 ).

Typically, progression of fibrosis to cirrhosis evolves for decades before clinical events ensue, but disease may progress more rapidly after repeated episodes of severe acute alcoholic hepatitis and subfulminant hepatitis (especially because of drug toxicity). In addition, there have been reports of rapidly progressive acute hepatitis C virus (HCV) with fibrosis in men coinfected with human immunodeficiency virus (HIV), a syndrome that has become much rarer, with good control of HIV using highly active antiretroviral therapies.

Genetic and environmental factors also influence the course of liver diseases. For example, in HCV infection, polymorphisms in a number of candidate genes involving the inflammatory (e.g., Toll-like receptor 4 [TLR4]) or the immune responses may influence the progression of liver fibrosis in humans. Although genetic polymorphisms in these and other pathways have been linked to progression risk in HCV, they have become far less relevant with the development of direct-acting antiviral drugs that cure the infection in more than 95% of patients, regardless of the disease stage or risk factors. Unfortunately, the development of a similar genetic risk score for nonalcoholic fatty liver disease (NAFLD) has been elusive, possibly because the disease is more heterogeneous. Nonetheless, genetic determinants have been identified that influence the risk and severity of NAFLD (see Chapter 69 ).

The main etiologies of liver fibrosis in Western countries are chronic HCV and hepatitis B virus (HBV) infection, alcohol abuse, and nonalcoholic steatohepatitis (NASH; see Chapters 68 and 69 ). As a generalized tissue response to chronic injury, fibrosis also occurs in many other organs (heart, lung, kidneys) and typically represents the result of an ongoing inflammation. Remarkably, as many as 45% of all deaths are related to some kind of fibrosis, which underscores the importance of this response and explains the growing interest in this field of research. For decades, fibrosis was considered an irreversible disease that progresses to cirrhosis with a greater risk for hepatocellular carcinoma and with development of liver failure. This meant that the only potential treatment for liver fibrosis was liver transplantation once cirrhosis was present.

Research during the past 35 years has yielded increasing insight into the cellular and molecular mechanisms of this disease, uncovering an orchestrated pathophysiology, identifying the hepatic stellate cell (HSC) as the central cell type in fibrogenesis, and, most importantly, revealing the potential reversibility of the disease and the hope for effective antifibrotic drugs.

Molecular and cellular mechanisms of fibrosis

The anatomic arrangement of the parenchymal and nonparenchymal cells of the liver contributes to its unique role as an immune organ and helps explain how the liver responds to an insult. The liver is composed primarily of epithelial cells (hepatocytes and cholangiocytes), as well as resident nonparenchymal cells that include resident hepatic macrophages (Kupffer cells), sinusoidal endothelium, and HSCs. In addition to Kupffer cells, several specialized immune cells have been characterized, including dendritic cells, natural killer (NK) cells, and natural killer T (NKT) cells, which reveal that the liver represents a key organ in the regulation of innate immunity ^, (see Chapter 10 ).

The liver capsule extends as septae into the liver, delineating hepatic lobules that form the structural units of the liver. The lobule forms a hexagonal structure with portal triads (including branches of the hepatic portal vein, the hepatic artery, and the bile duct) localized in the periphery of the lobule and with a portal vein branch in the center (see Fig. 7.1 ; see Chapter 5 ). Hepatocyte plates radiate outward from the central vein and are separated from each other by sinusoids. The latter form the connecting element between the branches of the hepatic portal veins and hepatic arteries with the central vein. Kupffer cells, NK cells, NKT cells, and dendritic cells, all of which are important components of the innate immune system, reside in the hepatic sinusoids. The subendothelial space between the sinusoidal endothelium and hepatocytes is also termed the space of Disse . Thus the HSCs, which lie in the space of Disse, have direct contact with endothelial cells and hepatocytes. Sinusoidal endothelial cells are highly fenestrated, which allows for unimpeded flow of plasma from sinusoidal blood into the space of Disse. Through this arrangement, hepatocytes and HSCs are exposed directly to plasma derived largely from venous blood draining the intestine.

Common triggers of hepatic fibrogenesis

Ongoing insult to the liver will lead to an increased inflammatory state with activation of HSCs, which ultimately tilts the profibrotic and antifibrotic balance toward fibrosis. Viral infection, reactive oxygen species (ROS), endoplasmic reticulum stress with protein misfolding, damage associated molecular patterns (DAMPs), pathogen-associated molecular patterns, and bile acids are among the most common stress signals for the liver ( Fig. 7.2 ). Additionally, free cholesterol promotes fibrogenesis by indirect activation of HSCs, which may be relevant to the pathogenesis of NAFLD (see Chapter 69 ).

FIGURE 7.2, Pathways of cellular injury and fibrosis.

In alcoholic liver disease, ethanol decreases gut motility, increases epithelial permeability, and promotes overgrowth of gram-negative bacteria. Consequently, lipopolysaccharide (LPS) concentration is elevated in portal blood, through the TLR4 signaling complex, to generate ROS via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Oxidants then upregulate nuclear factor kappa B (NF-κB) in Kupffer cells, which leads to increased tumor necrosis factor-α (TNF-α) production (see Fig. 7.2 ). In turn, TNF-α induces neutrophil infiltration and stimulates mitochondrial oxidant production in hepatocytes, which are then sensitized to undergo apoptosis. Furthermore, ROS and acetaldehyde, the main degradation product of alcohol, both activate HSCs and stimulate inflammatory signals. Interestingly, many of the same gut defects in alcoholic liver disease are now also implicated in NASH, with additional focus on the nature of the microbiome as well as the integrity of the gut mucosa as determinants of this disease. ^,

Bile acids are hepatotoxic agents and typically target hepatocytes but may also injure biliary epithelium. In addition to their potential role in provoking damage, bile acids are also ligands for nuclear receptors, in particular the farnesoid X receptor (FXR), which drives an entire cellular program that can alter hepatocellular metabolism and bile secretion and composition. Remarkably, the therapeutic benefit of vertical sleeve gastrectomy has also been ascribed to FXR signaling in an animal model, raising the possibility that intestinal FXR alone may be sufficient to drive weight loss and improve metabolic parameters in NASH. ^,

Oxidant stress, mediated by ROS, is a common mediator of injury in many liver diseases in which damaged hepatocytes become apoptotic or necrotic, thereby releasing ROS and NADPH oxidase, which both activate HSCs. Injured hepatocytes also release inflammatory cytokines and soluble factors that activate Kupffer cells and stimulate the recruitment of activated T cells. This inflammatory milieu further stimulates the activation of resident HSCs.

NAFLD is increasingly prevalent because of increased rates of childhood and adult obesity in the United States and Western Europe (see Chapter 69 ). In fact, the percentage of liver transplantations performed for this indication is rapidly rising and is overtaking viral hepatitis not only because of curative antiviral therapy for HCV but also because of the rising prevalence of metabolic syndrome, which predisposes to NAFLD. NAFLD can progress to NASH, with consequent fibrosis and cirrhosis. Although a hierarchy of disease causality is still lacking, there are multiple convergent defects that clearly drive disease progression and fibrosis in NASH, including insulin resistance, oxidant stress, altered adipokine balance, lipotoxicity, effects of the microbiome, and enhanced inflammation. ^, ^, ^,

Hepatic stellate cell activation: Hepatic myofibroblasts

The HSC is a central regulator of the liver’s fibrotic and repair responses ( Fig. 7.3 ). In a healthy liver, the HSC is a quiescent cell type that contains cytoplasmic retinoid droplets, representing the major storage site for vitamin A in the body, and expresses the markers desmin and glial fibrillary acidic protein. During liver injury, HSCs undergo activation in response to a range of inflammatory and injury signals produced by damaged hepatocytes and biliary cells, by changes in the composition of the ECM, by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin, and by fibrogenic cytokines that include transforming growth factor-β (TGF-β1), connective tissue growth factor (CTGF), angiotensin II, and leptin. Recent studies using single cell RNA sequencing have uncovered significant heterogeneity among stellate cells in both normal and injured liver in both man and mouse, reinforcing earlier studies that documented stellate cell heterogeneity based on the types of intracellular filaments they express as assessed by immunohistochemistry.

FIGURE 7.3, Functions, features, and phenotypes of hepatic stellate cells in normal and diseased liver.

Activation of HSCs is accompanied by loss of retinoid droplets and accumulation of α-smooth muscle actin, a myogenic filament that confers increased cellular contractility. Activated HSCs are characteristically positive for α–smooth muscle actin and desmin and are called hepatic myofibroblasts (MFB), a cell type that is also characteristic of wound healing in a range of tissues, including the skin, kidney, lung, bone marrow, and pancreas. ^, The relative importance of each fibrogenic cell type in liver fibrogenesis may depend on the origin of the liver injury. Fate-tracing studies using genetically engineered reporter mice implicate stellate cells as the dominant source of MFBs in parenchymal liver disease ; however, a contribution from biliary portal fibroblasts is important in cholestatic liver disease. ^,

HSC activation can be divided conceptually into two phases. First there is initiation , with early changes in gene expression and phenotype, resulting from paracrine stimulation, primarily because of changes in surrounding ECM, as well as exposure to lipid peroxides and products of damaged hepatocytes. Next there is perpetuation , which results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis. Within the nucleus, a growing number of transcription factors regulate HSC activation, including peroxisome proliferator–activated receptors (PPARs), retinoid receptors, liver X receptor, REV-ERBα, NF-κB, FXR, GATA4, vitamin D receptor, JunD, Kruppel-like factor 6, and FOXF1. ^, A number of general and cell type–specific membrane receptors and signaling pathways also control HSC biology, including receptor tyrosine kinases, chemokine receptors, and integrins. ^, Not only is HSC activation under transcriptional control, but a growing range of epigenetic changes further regulates this HSC transdifferentiation into myofibroblasts.

As previously noted, portal fibroblasts and bone marrow–derived MFBs have also been identified as collagen-producing cells in the injured liver, although their overall contribution is minor. Earlier studies implicated epithelial-mesenchymal transition as a source of fibrogenic cells, but more recent findings strongly refute its importance in liver.

Functions of hepatic myofibroblasts

Hepatic MFBs have functions that are distinct from their quiescent cells of origin. They are profibrogenic and promitotic, they have a chemotactic and vasoregulatory role, and they control the degradation of ECM. They also have important immune and phagocytic functions. The regulation of ECM accumulation and degradation by HSCs is reviewed in the next section.

Fibrogenesis

The major profibrogenic signal in liver is the cytokine TGF-β1. TGF-β1 is secreted mainly by MFBs but also by platelets cells and liver macrophages. It functions by activating the type II TGF-β receptor, which recruits the type I TGF-β receptor. SMAD2 and SMAD3 then associate with the TGF-β1 receptor, are phosphorylated, and recruit SMAD4. This tri-heteromeric complex then translocates to the nucleus, where it activates profibrogenic transcription factors. TGF-β also activates the mitogen-activated protein kinase (MAPK) p38 pathway, which stimulates additional SMAD-independent collagen type 1 synthesis and, in contrast to the SMAD-dependent collagen type 1 synthesis, also leads to a post-transcriptionally regulated stabilization of the collagen type 1 messenger RNA (mRNA). Local activation of TGF-β1 at the cell surface by integrins has led to the prospect of antagonizing integrins as an antifibrotic therapy. In addition to TGF-β1, CTGF and Hedgehog signaling have also been implicated as important fibrogenic mediators in liver injury and repair. ^,

Proliferation

The predominant stimulus to MFB proliferation is the mitogen platelet-derived growth factor (PDGF) in addition to other mitogens, including epidermal growth factor, VEGF, and fibroblast growth factor. All pathways downstream of the β-PDGF receptor, the key receptor isoform in HSCs, promote proliferation. First, c-Jun N-terminal kinase is stimulated through MAPK; second, PDGF receptor stimulates the RAS/RAF complex, followed by mitogen-induced extracellular kinase and extracellular signal-regulated kinase engagement; and third, the PI3K pathway is activated, leading to AKT (protein kinase B) activation and phosphorylation of the 70S6 kinase.

Immunoregulation

The liver is a microenvironment of diminished immunogenicity, which is necessary to cope with the high exposure of antigens from the portal vein ^, (see Chapter 10 ). This feature also accounts for the tolerance of liver transplantation across ABO barriers and may contribute to the chronic nature of HBV or HCV, in which the virus persists despite the development of an immune response. Upon entry of the antigen to the sinusoid, classic antigen-presenting cells (Kupffer cells, dendritic cells) are first encountered. Subsequently, HSCs in the space of Disse may contact antigens. Indeed, HSCs display a wide range of immunoregulatory functions and are an essential part of the liver’s immune response. ^,

Hepatic MFBs produce a range of proinflammatory and antiinflammatory cytokines (see Chapter 10 ) and recruit lymphocytes through secretion of chemokines (monocyte chemoattractant protein-1, interleukin-8 [IL-8], C-C chemokine 21 [CCL21], regulated on activation, normal T-cell expressed and secreted [RANTES], C-C chemokine receptor 5 [CCR5]), thus amplifying the inflammatory response. Nevertheless, upon activation, they exert a profound immunosuppressive activity by inducing T-cell apoptosis. In the setting of liver transplantation, MFB can induce T-cell apoptosis via programmed death ligand-1 and may foster local immunotolerance of the liver. In liver fibrosis, MFBs may further regulate the contribution of lymphocytes to the course of hepatic fibrosis by ingesting disease-associated lymphocytes or by activating in response to engulfment of apoptotic bodies.

The interaction between HSCs and immune cells is bidirectional. T cells activate HSCs by interferon-γ (IFN-γ), which upregulates both stimulatory (CD80, CD86, CD54) and inhibitory (B7-H1) surface molecules and enhances both inflammatory and suppressive cytokines. The inhibitory molecules, however, are thought to override the stimulatory counterparts, resulting in immunosuppression. Lymphocytes can also mediate hepatic fibrosis by activating HSCs. CD8-positive T lymphocytes are more fibrogenic toward stellate cells than CD4 T lymphocytes. This may explain, in part, why patients co-infected with untreated HIV and HCV have accelerated fibrosis because their CD4:CD8 cell ratios are reduced. Of the CD4-positive T lymphocytes, previously called T-helper cells, the humoral immunity mediated by T-helper 2 cells (Th2) is profibrogenic in liver injury, whereas the cell-mediated immunity by the Th1 cells via IFN-γ, TNF-α, and IL-2 is antifibrogenic.

HSCs can also function as antigen-presenting cells. They can interact with bacterial LPS directly via TLR4, which amplifies their activation. TLR4 signaling leads to downregulation of a TGF-β pseudoreceptor, BMP (bone morphogenic protein), and activin membrane-bound inhibitor, which thereby amplifies fibrogenic activity of MFBs. Signaling through TLR4 may be elicited not only by exogenous ligands, including LPS, but also by endogenous ligands, including high-mobility group box 1 protein. The discovery of endogenous ligands for TLR4 has been part of a larger recognition that many cells, including HSCs, possess an intracellular complex known as the inflammasome , which transduces signals arising from cellular damage. The inflammasome is especially pertinent to understanding the pathogenesis of inflammation and fibrosis in NAFLD and NASH.

Vasoregulation

MFBs play an important role in the regulation of sinusoidal blood flow and may contribute to portal hypertension that is characteristic of advanced liver disease (see Chapter 74 ). The release of endothelin-1 (ET-1) can stimulate their contraction through the endothelin type A (ETA) receptor, thereby promoting tissue contraction, increasing portal resistance, and generating portal hypertension. On the other hand, MFBs and endothelial cells also secrete nitric oxide (NO), which is the physiologic antagonist of ET-1.

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