Hepatic Fibrosis -- Role of Hepatic Stellate Cell Activation

Scott L. Friedman, MD

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In This Article

Stellate Cell Activation

As noted above, stellate cell activation is the central event in hepatic fibrosis. Activation consists of 2 major phases: (1) initiation (also called apreinflammatory stage) and (2) perpetuation (Figure 2).[6] Initiation refers to early paracrine-mediated changes in gene expression and phenotype that render the cells responsive to other cytokines and stimuli. Perpetuation then results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis.

Figure 2.

Phenotypic features of hepatic stellate cell activation during liver injury and resolution. Following liver injury, hepatic stellate cells undergo "activation" which connotes a transition from quiescent vitamin A-rich cells into proliferative, fibrogenic, and contractile myofibroblasts. The major phenotypic changes after activation include proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis, retinoid loss, and WBC chemoattraction. Key mediators underlying these effects are shown. The fate of activated stellate cells during resolution of liver injury is uncertain, but may include reversion to a quiescent phenotype and/or selective clearance by apoptosis. (Reprinted with permission from Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 2000;275:2247-2250).

Initiation of Stellate Cell Activation

The earliest changes in stellate cells are likely to result from paracrine stimulation by all neighboring cell types, including sinusoidal endothelium, Kupffer cells, hepatocytes, platelets, and leukocytes. Endothelial cells are also likely to participate in activation, both by production of cellular fibronectin and via conversion of transforming growth factor (TGF)-beta from the latent to active, profibrogenic form.

Kupffer cell infiltration and activation also play a prominent role in the process. The influx of Kupffer cells coincides with the appearance of stellate cell activation markers. Kupffer cells can stimulate matrix synthesis, cell proliferation, and release of retinoids by stellate cells through the actions of cytokines (especially, TGF-beta 1) and reactive oxygen intermediates/lipid peroxides. Proliferation has been attributed to Kupffer cell-derived TGF-alpha.[9,10] TGF-beta derived from Kupffer cells markedly stimulates stellate cell ECM synthesis.[9,11] Kupffer cells produce anti-inflammatory as well as proinflammatory cytokines, including interleukin (IL)-10.[12] Of interest, IL-10 downregulates fibrogenesis in cultured stellate cells by decreasing collagen synthesis and increasing production of collagenase.[13]

Another means by which Kupffer cells can influence stellate cells is through secretion of matrix metalloproteinase 9 (MMP-9; gelatinase B).[14] MMP-9 can activate latent TGF-beta, which in turn can stimulate stellate cell collagen synthesis.[15] Lastly, Kupffer cells are an important source of reactive oxygen species (ROS) in the liver. ROS, whether produced internally within stellate cells[16,17] or released into the extracellular environment,[18] are capable of enhancing stellate cell activation and collagen synthesis. Kupffer cells also produce nitric oxide (NO), which can counterbalance the stimulatory effects of ROS by reducing stellate cell proliferation and contractility.

Hepatocytes, as the most abundant cell type in liver, are a potent source of fibrogenic lipid peroxides in inflammatory liver diseases.

Platelets are a potent source of growth factors, and are present in the injured liver.[19] Potentially important platelet mediators include platelet-derived growth factor (PDGF), TGF-beta 1, and epidermal growth factor (EGF).

Leukocytes that are recruited to the liver during injury join with Kupffer cells in producing compounds that modulate stellate cell behavior. Neutrophils are an important source of ROS, which have a direct stimulatory effect on stellate cell collagen synthesis. The specific role of neutrophils as a stimulus to stellate cells was demonstrated in a coculture experiment in which cells activated by N-formyl-methionyl-leucyl-phenylalanine were plated in direct contact with stellate cells.[20] Activated neutrophils increased stellate cell collagen synthesis 3-fold over control levels. Superoxide was identified as the principal mediator of the neutrophil effect. Activated neutrophils also produced NO, which dampened the effect of superoxide on collagen expression but did not abrogate it completely. Lymphocytes, including CD4+ T-helper (Th) cells, reside in the liver[21,22] and represent a further potential source of cytokines. Th lymphocytes help orchestrate the host-response via cytokine production and can differentiate into Th1 and Th2 subsets, a classification that is based on the pattern of cytokines produced. In general, Th1 cells produce cytokines that promote cell-mediated immunity and include interferon (IFN)-gamma, tumor necrosis factor (TNF), and IL-2. Th2 cells produce IL-4, IL-5, IL-6, and IL-13, and promote humoral immunity. Th1 cytokines inhibit the development of Th2 cells and Th2 cytokines inhibit the development of Th1 cells. Thus, the host response to infection or injury frequently polarizes to either a Th1 or Th2 response, but not both.

Several experimental models offer evidence implicating Th cell-derived cytokines in determining the immune response. The polarization of the immune response is enhanced when chronic exposure to an agent occurs, such as with persistent infections or exposure to environmental toxins. Furthermore, polarization of the immune response to Th1 or Th2 cytokines is under genetic control, as demonstrated by divergent responses of different inbred strains of mice to experimental murine leishmaniasis.[23] Genetically resistant mice -- such as C57BL/6 mice -- exhibit an expansion of IFN-gamma-producing Th1 cells and control the infection, whereas susceptible strains (BALB/c mice) develop an IL-4-mediated Th2 response.[24,25]

More intriguing data have been obtained from studies comparing the effects of T lymphocytes on liver fibrosis. Overall, data suggest that Th2 lymphocytes favor fibrogenesis in liver injury over Th1 lymphocytes. The effects of CCl4 have been examined in mice with several different lymphocyte profiles, including T-cell depletion (severe combined immunodeficiency, SCID), Th1 predominance (C57/BL6), and Th2 predominance (BALB/c).[26] SCID mice from both C57/BL6 and BALB/c backgrounds develop liver fibrosis after treatment with CCl4 for 4 weeks. The degree of fibrosis is modified significantly, however, in immunocompetent mice from both strains. Immunocompetent C57/BL6 mice, whose lymphocyte cytokine profile includes IFN-gamma, actually exhibit less fibrosis than SCID mice from the same background. Indeed, when C57/BL6 mice with targeted disruption of IFN-gamma are treated with CCl4, fibrosis returns to the level seen in C57/BL6 SCID mice. However, immunocompetent BALB/c mice, whose lymphocyte cytokine profile includes the fibrogenic compounds IL-4 and TGF-beta, exhibit more fibrosis than BALB/c SCID mice. Among all of the studies examining immunoregulation of fibrosis, those demonstrating modulation by T lymphocytes appear the most convincing.

Perpetuation of Stellate Cell Activation

Perpetuation of stellate cell activation involves several discrete changes in cell behavior, as discussed below.

Proliferation. PDGF is the most potent stellate cell mitogen identified. Induction of PDGF receptors early in stellate cell activation increases responsiveness to this potent mitogen.[27] Downstream pathways of PDGF signaling have been carefully characterized in stellate cells

Chemotaxis. Stellate cells can migrate towards cytokine chemoattractants,[28,29] an action that is characteristic of wound-infiltrating mesenchymal cells in other tissues as well. Chemotaxis of stellate cells explains in part why stellate cells align within inflammatory septa in vivo.

Fibrogenesis. Increased matrix production is the most direct way that stellate cell activation generates hepatic fibrosis. The most potent stimulus to collagen I production is TGF-beta, which is derived from both paracrine and autocrine sources.

Lipid peroxidation products are emerging as important stimuli to ECM production; their effects may be amplified by loss of antioxidant capacity of stellate cells as they activate.[30] These important insights have provoked efforts to use antioxidants as therapy for hepatic fibrosis (see section on Therapy of Hepatic Fibrosis, below).

Contractility. Contractility of stellate cells may be a major determinant of early and late increases in portal resistance during liver fibrosis. Activated stellate cells impede portal blood flow by both constricting individual sinusoids and contracting the cirrhotic liver, because the collagenous bands typical of end-stage cirrhosis contain large numbers of activated stellate cells (see [31] for review). The major contractile stimulus towards stellate cells is endothelin-1. Receptors for the latter are expressed on both quiescent and activated stellate cells, but its subunit composition may vary.[32] Contractility of stellate cells in response to endothelin-1 has also been observed in vivo.[33] Other, less potent contractile stimuli have also been identified.[31] For example, Kupffer cells produce eicosanoids, including prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), and thromboxanes.[34,35] Eicosanoids modulate stellate cell contractility, with thromboxanes typically promoting contraction and PGE2 mediating relaxation.[36]

Locally produced vasodilator substances may counteract the constrictive effects of endothelin-1.[31] NO, which is also produced by stellate cells, is a well-characterized endogenous antagonist to endothelin.

Matrix degradation. Quantitative and qualitative changes in matrix protease activity play an important role in ECM remodeling accompanying fibrosing liver injury (see section on Extracellular Matrix Degradation, above).

Stellate cells express virtually all of the key components required for pathologic matrix degradation and therefore play a key role not only in matrix production, but also in matrix degradation. An enlarging family of matrix-metalloproteinases has been identified that are calcium-dependent enzymes which specifically degrade collagens and noncollagenous substrates (see [37] for review). Broadly, these enzymes fall into 5 categories based on substrate specificity: (1) interstitial collagenases (MMP-1, -8, -13); (2) gelatinases (MMP-2,-9) and fibroblast activation protein;[38] (3) stromelysins (MMP-3, -7, -10, 11); (4) membrane-type (MMP-14, 15, -16, -17, -24, -25); and (5) a metalloelastase (MMP-12). Inactive metalloproteinases can be activated through proteolytic cleavage by either membrane-type matrix metallproteinase 1 (MT1-MMP) or plasmin, and inhibited by binding to specific inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). Thus, net collagenase activity reflects the relative amounts of activated metalloproteinases and their inhibitors, especially TIMPs.

In liver, "pathologic" matrix degradation refers to the early disruption of the normal subendothelial matrix that occurs through the actions of at least 4 enzymes: MMP2 and MMP-9 degrade type IV collagen; membrane-type metalloproteinase-1 or -2 activates latent MMP2; and stromelysin-1 degrades proteoglycans and glycoproteins, and also activates latent collagenases. Stellate cells are a key source of MMP-2[39,40] as well as increases in the specific MMP inhibitor molecules, TIMP-1[41,42] and TIMP-2,[43] leading to a net decrease in protease activity, and therefore, more unopposed matrix accumulation. Moreover, an emerging role for TIMPs in regulating apoptosis suggests that their influence on liver homeostasis extends beyond that of direct effects on ECM turnover.

Retinoid loss. As stellate cells activate, they lose their characteristic perinuclear retinoid (vitamin A) droplets and acquire a more fibroblastic appearance. In culture, retinoid is stored as retinyl esters, whereas as stellate cells activate, the retinoid released outside the cell is retinol, suggesting that there is intracellular hydrolysis of esters prior to export.[8] However, it is generally unknown whether retinoid loss is required for stellate cells to activate and which retinoids may accelerate or prevent activation in vivo. Recently, peroxisome proliferator-activated receptors (PPAR), in particular PPAR gamma, have also been identified in stellate cells, and their expression increases with activation.[44,45] Ligands for this newly identified nuclear receptor family downregulate stellate cell activation.[45]

WBC chemoattractant and cytokine release. Increased production and/or activity of cytokines may be critical for both autocrine and paracrine perpetuation of stellate cell activation. Direct effects on stellate cell matrix production and contractility have been attributed to autocrine TGF-beta and endothelin-1, respectively.

Stellate cells can amplify the inflammatory response by inducing infiltration of mono- and polymorphonuclear leukocytes.

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