The Matrisome, Inflammation, and Liver Disease

Abstract

Chronic fatty liver disease is common worldwide. This disease is a spectrum of disease states, ranging from simple steatosis (fat accumulation) to inflammation, and eventually to fibrosis and cirrhosis if untreated. The fibrotic stage of chronic liver disease is primarily characterized by robust accumulation of extracellular matrix (ECM) proteins (collagens) that ultimately impairs the function of the organ. The role of the ECM in early stages of chronic liver disease is less well-understood, but recent research has demonstrated that several changes in the hepatic ECM in prefibrotic liver disease are not only present but may also contribute to disease progression. The purpose of this review is to summarize the established and proposed changes to the hepatic ECM that may contribute to inflammation during earlier stages of disease development, and to discuss potential mechanisms by which these changes may mediate the progression of the disease.

The extracellular matrix (ECM) consists of a diverse range of components that work bidirectionally with surrounding cells to create a dynamic and responsive microenvironment that regulates cell and tissue homeostasis. The basic definition of the ECM comprises fibrillar proteins (e.g., collagens, glycoproteins, and proteoglycans). The definition has more recently been extended to include ECM affiliated proteins (e.g., collagen-related proteins), ECM regulator/modifier proteins (e.g., lysyl oxidases and proteases), and secreted factors that bind to the ECM (e.g., transforming growth factor-beta and other cytokines)¹; this broader definition has been coined the “matrisome.”² The ECM not only provides structure and support for the cells in a tissue but also acts as a reservoir for growth factors and cytokines and as a signaling mechanism by which cells can communicate with their environment and vice-versa.³

Perhaps the best-characterized function of the ECM is its role as a scaffold, providing support and structure to the surrounding tissue. There are two major components of structural ECM: the interstitial matrix and the basement membrane.⁴ Interstitial matrix proteins, including fibronectins, elastin, and fibrillar collagens, form support networks that provide the overall superstructure that shapes and encapsulates the organ.⁵ The basement membrane is a thin sheet of ECM that underlies epithelial and endothelial cells. Similar to the interstitial matrix, the basement membrane comprises collagens, glycoproteins, and proteoglycans that facilitate structure and growth of the cells. In most tissues, the basement membrane is continuous and dense and is a true barrier between the epithelial/endothelial cells and the adjacent parenchymal cell layer. In contrast, the hepatic basement membrane found in the Space of Disse between endothelial cells and hepatocytes is much less dense and is fenestrated.⁵ Although it possesses similar ECM as more strongly define basement membranes (e.g., collagen type IV and laminin),⁶ this region acts more as a structural filter and facilitates bidirectional exchange of proteins and xenobiotics between the sinusoidal blood and hepatocytes. Although it is clear that liver does not have a basal lamina, whether or not the ECM found in the space of Disse should be considered a basement membrane is a subject of a histological, rather than functional, debate.⁴

ECM (dys)Homeostasis

As mentioned above, the ECM responds dynamically to changes. Under normal conditions, these responses assist in maintaining organ homeostasis and appropriate responses to injury/stress. The orchestrated crosstalk between the coagulation cascade and the inflammatory response during subcutaneous wound healing is an excellent example of appropriate ECM changes in response to injury/stress.⁷ However, failure to properly regulate these responses can lead to qualitative and/or quantitative ECM changes that are maladaptive.⁸ For example, “aging” of the ECM with increased crosslinking is hypothesized to contribute to dysfunction in several organ systems, including the liver.^9–12 Key levels of ECM homeostasis, be it adaptive or maladaptive, include synthesis, proteolysis, and post-translational modifications.

ECM Synthesis

Under basal conditions, several hepatic cells contribute to the synthesis of ECM components, including hepatocytes, cholangiocytes, and sinusoidal endothelial cells.¹³ Kupffer cells do not normally synthesize fibrillar ECM, but they do produce several secreted factors (e.g., cytokines) that are associated with the ECM. The amount and content of ECM components produced by these cells change in response to injury or stress. Although it is unclear if hepatic stellate cells (HSC) generate significant ECM during normal tissue homeostasis, activated HSCs transdifferentiate into a myofibroblast-like phenotype and generate ECM.⁵ Furthermore, other myofibroblast-like cells have been identified, such as fibrocytes and periportal fibroblasts.^14–17 The contribution of extrahepatic sources to the hepatic ECM via de novo synthesis is unclear, but these compartments clearly contribute to ECM via other mechanisms of homeostasis.

Proteolysis

Protein families that degrade ECM include matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS), cathepsins, and plasmin.^18–22 The activity of these proteases is often balanced by protease inhibitors that directly inhibit their activity. For example, MMP activities are inhibited by tissue inhibitors of metalloproteinases (TIMPs), and contribute to collagen accumulation during hepatic fibrosis.²³ Similarly, macrophage-derived MMP-12 expression, activity and ratio to its inhibitor TIMP-1 regulates elastin turnover in liver injury and fibrosis.²⁴ This mechanism is proposed to be critical for the full reversibility of liver fibrosis.²⁵ Likewise, plasminogen activator inhibitors (e.g., PAI-1) inhibit the activity of the plasminogen activators and thereby contribute to the accumulation of fibrin ECM during hepatic injury.²² Proteases can also regulate the deposition of hepatic ECM by cleaving soluble precursors of the ECM proteins. The serine proteases of the coagulation cascade are a canonical example of an acute phase response that leads to the formation of a fibrin clot.²²

Post-Translational Modifications

Post-translational modification of ECM proteins regulates the formation of polymeric, helical structures, and cross-linked complexes associated with several fibrillary ECM proteins. For example, prolyl 4-hydroxylase targets terminal proline residues on collagen monomers to facilitate the formation of collagen oligomers and triple helices.²⁶ Recent studies indicate that lysyl oxidases and transglutaminases also contribute to ECM crosslinking.²⁷,²⁸ Although these events are important for stabilizing the proteins and preventing their degradation under normal conditions, their activation may contribute to excessive ECM accumulation in response to injury (e.g., fibrosis).²⁷ Furthermore, although fibrosis is potentially reversible if the causative insult is removed,²⁹ highly crosslinked ECM may be resistant to resolution.^30,31 Crosslinking of the ECM may be altered via nonezymatic means; for example, the formation of advanced glycation end products during diabetes is hypothesized to contribute to ECM crosslinking and increased matrix “aging.”³²

Inflammation as a Therapeutic Target for Chronic Liver Disease

The strategic location of the liver between the intestinal tract and the rest of the body makes it a critical physical and biochemical barrier against toxins/toxicants that enter the portal blood. However, as the main detoxifying organ in the body, the liver has a high likelihood of toxic injury.^33,34 It is, therefore, not surprising that the liver has tremendous regenerative capacity.^33,35 This capacity distinguishes it from other vital organs (e.g., the brain, heart, and lungs) that are far less able to replace functional tissue when damaged. Liver regeneration requires a tightly coordinated response to complement the regenerative process, so that the entire organ can be reconstituted. The complex and synchronized regenerative response in liver can be perturbed and thereby can impact normal tissue recovery from injury or damage.³⁶ When the cycle of injury and perturbed recovery from injury is repeated, damage can accumulate and initiate the process of chronic liver disease.³⁵

It is reported that ~30% of the US population has underlying liver disease.³⁷ There are numerous causative factors that drive liver disease, including extrinsic (e.g., diet, alcohol abuse, and viral infection) and intrinsic (e.g., genetic disorders and auto-immune diseases) factors.^38–42 No matter the etiology, chronic liver diseases share a well-documented, common natural history, which ranges initially from simple steatosis, to inflammation and necrosis (steatohepatitis), to fibrosis and cirrhosis.^43–46 Fibrosis may improve with removal of insult, but reversal of severe stages of fibrosis/cirrhosis is more limited.⁴⁷ Cirrhosis is often considered an end-stage liver disease and requires liver transplant. Even in the case of hepatitis C virus, where eradication of viral infection is nearly 100% with direct-acting antivirals, reportedly 30 to 60% of cirrhotic livers do not recover histologically after achieving a sustained virologic response (SVR).^48,49 Moreover, vascular changes (e.g., portal hypertension) and other sequelae of severe/decompensated cirrhosis do not appear to as readily reverse even after SVR.⁵⁰ Globally, over 1 million people die from complications of cirrhosis each year, and an estimated 1 million more people die from related diseases (e.g., hepatocellular carcinoma).⁵¹ Despite a clear understanding of disease progression, there is no universally accepted therapy available to halt or reverse this process in humans.⁵²

Given the poor prognosis of treating late-stage liver disease, much of the current research focuses on identifying at-risk individuals and preventing the progression of the disease during earlier phases, especially inflammation. Inflammation plays a central role in chronic liver diseases, and comprises components of both the innate and adaptive immune responses.^53,54 The net result is a chronic, low-grade inflammatory condition, in which innate immune cells are activated, and surveillance and tolerance by adaptive immune cells are dysregulated.^55–59 It is this vicious cycle of cell damage/death and inflammation, when it overwhelms the repair/recover responses of the liver, which leads to the chronicity of liver diseases.

The ECM in Liver Diseases-Thinking Beyond Collagen and Before Fibrosis

Beyond Collagen

The importance of the ECM in liver disease is well established. However, research on the hepatic ECM in the context of liver disease has been largely “collagenocentric,” or primarily focused on the role of the collagen matrix. This is not necessarily surprising, given the robust collagen ECM deposition found in fibrosis and cirrhosis and the ease of visualizing this matrix with histochemical stains. However, it is well-known that there are multiple ECMs that change qualitatively and quantitatively during hepatic fibrosis.^60,61 As mentioned above, the concept of the ECM as a microenvironment has been recently expanded to include nonfibrillar “matrisome” proteins that colocalize in and influence the ECM.¹ This dynamic subset of over 1000 matrisome proteins varies with organ and disease state.¹ It is therefore clear that changes in the hepatic ECM/matrisome during fibrosis are much more diverse than simply collagen.

Before Fibrosis

The study of the role of the ECM in liver disease has also been “fibrosocentric,” that is, focused on the dramatic ECM changes during the fibrosis stage of disease. Again, this is not necessarily a surprise, given that chronic liver disease is often asymptomatic,⁶² and the patient’s first presentation is often with already established late-stage disease. However, ECM changes are not solely relegated to fibrosis. As mentioned above, in some areas of research (e.g., subcutaneous wound healing), the changes to the ECM in response to acute injury have been well-understood.⁷

Work by this group and others has shown that the acute phase response in the liver in response to damage involves several of the ECM proteins found in subcutaneous wound healing, such as fibrin, osteopontin, and fibronectin.^63–65 We recently demonstrated that the hepatic matrisome changes robustly to acute injury (e.g., acute lipopolysaccharide), even under conditions in which the ECM appears histologically unchanged.⁴⁵ These subhistologic transitional changes to the matrisome appear to resolve after acute injury (⁶⁶,⁶⁷; ►Fig. 1); with chronic injury, the transitional matrisome is replaced by collagenous scarring in the liver, which is again in-line with subcutaneous wound healing.⁷ An improvement in referral practices and noninvasive tests has increased the rate of early detection of asymptomatic chronic liver diseases.⁶⁸ This paradigm change opens up the opportunity for mechanism-based therapies to halt disease progression during earlier (i.e., prefibrotic) phases of the disease progression.

Fig. 1.

Transitional remodeling of the hepatic extracellular matrix (ECM). Acute injury causes formation of a transitional or provisional ECM. This transitional ECM may contribute to injury and inflammation. The transitional ECM often resolves after removal of the insult and may contribute to the recovery from that insult. With continued injury, the transitional matrix may progress to a fibrotic matrix, which can also resolve. Resolution of both injuries release degraded proteins into the blood that may serve as alarmins for inflammatory injury, as a well as a potential source of biomarkers for recovery from liver disease. HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; MOF, multiple organ failure; PAI-1, plasminogen activator inhibitor-1; SIRS, systemic inflammatory response syndrome; TIMP, tissue inhibitors of metalloproteinase.

The Hepatic Matrisome and the Control of Inflammation

The hepatic matrisome’s dynamism during inflammation represents a potential therapeutic target for liver disease. This review will explore some of these functions in the context of inflammation (►Fig. 2): adhesion, structure, presentation and storage, and sensing.

Fig. 2.

Functions of the hepatic extracellular matrix (ECM) that contribute to the roles it plays in inflammation. The ECM plays multiple roles that contribute to the regulation of inflammation and injury in the liver. These roles are related to adhesion, structure, presentation of signaling molecules and/or receptors, storage of signaling molecules, and sensing. See Section “The Hepatic Matrisome and the Control of Inflammation” for details. Figure modified with permission from Rozario and DeSimone.⁸⁶

Adhesion

Hepatic inflammation after injury involves the recruitment of immune cells, including natural killer cells, natural killer T cells, dendritic cells, neutrophils, eosinophils, and monocytes.^69,70 Immune cells are known to interact with many ECM proteins, including fibronectin, collagens, laminin, tenascin, and hyaluronic acid through a variety of receptors, including integrins, and surface glycoproteins(e.g., CD54, CD44, and CD26).⁷¹ These interactions have important implications in liver disease; for example, inhibition of fibronectin binding to T-cells is part of the anti-inflammatory mechanism of pentoxifylline.⁷²

Interaction between leukocytes and the ECM is critical for the process of leukocyte adhesion and transmigration to sites of inflammation/injury.⁷³ Leukocyte surfaces contain ECM receptors/adhesion molecules that direct their migration through interaction with the ECM.⁷¹ Regulation of these receptors is important for the rapid change between adhesive and nonadhesive states of immune cells.⁷¹ Initial leukocyte capture and rolling in the microvasculature may be mediated by selectins (CD62),^73,74 although selectin-independent mechanisms have also been observed in the liver.^75,76 Leukocyte activation is mediated by chemokines as described below, and arrest is mediated by the binding of leukocyte integrins to endothelial cell adhesion molecules (e.g., CD54 and CD106).⁷³ Integrin-dependent leukocyte adhesion involves the β₁- and β₂-integrin.⁷⁴ Alternatively, integrin-independent adhesion involves CD44 and vascular adhesion protein-1.⁷⁴ Strengthening of adhesion is mediated by integrin outside-in signaling, and transmigration involves adhesion molecules (e.g., platelet and endothelial cell adhesion molecule 1 [PECAM1], Junctional adhesion molecule-A [JAM-A]) as well as barrier degradation by proteases.⁷³ The interaction between the ECM and cell infiltration is bidirectional; as leukocytes integrate structural and biochemical cues from the ECM, they in turn release matrix-degrading proteases,⁷⁷ which alter the extracellular composition and allow for easier cell migration.

The ECM not only adheres immune cells but also binds chemokines, creating a haptotactic gradient that directs immune cells to focal targets.⁷⁸ After liver injury, parenchymal cells and resident leukocytes secrete chemokines.⁶⁹ Chemokines bind to the glycosaminoglycan (GAG)/heparin sulfate components of basement membrane.⁷⁸ An example of this is the chemokine (C-X-C motif) ligand 16 (CXCL16)-promoted recruitment and retention of CXCR6⁺ T cells in the liver, likely through conformational activation of β₁ integrins and binding to vascular cell adhesion molecule 1 (VCAM-1).⁷⁹ Chemokines also play an important role in the activation step of leukocyte adhesion.⁷³ Indeed, it has been demonstrated that chemokines CCL2, CCL4, and CCL5 with mutations at their GAG binding sites are unable to recruit cells in vivo.⁸⁰ ECM proteins themselves can also be chemotactic. For example, osteopontin has been shown to be chemotactic for natural killer T cells, neutrophils, and macrophages.⁸¹ Additionally, fibronectin secreted by T cells potently triggers macrophage agglutination and mediates monocyte and neutrophil translocation through the ECM.⁸² Overall, the ECM is key in the adhesion and direction of immune cells.

Structure

The ECM plays an important direct structural role, partially through definition of tissue boundaries and zonation.⁸³ Furthermore, the ECM indirectly characterizes tissue morphology.⁸⁴ For example, during branching morphogenesis, the process by which the liver and other organs develop, heparin sulfates on the cell surface and ECM regulate growth factor-epithelial cell interactions.^85,86 In the mature liver, the ECM varies within the hepatic lobule and may help define zonation.^87,88 The ECM defines properties permissive and/or instructive to inflammation. For example, although basement membrane may physically impede leukocyte transmigration, changes in this ECM in response to injury may also drive homing of recruited/repopulated cells within the liver.⁸⁹

In addition to defining boundaries, the ECM also provides integrity and elasticity to the liver. The ECM is thereby key in maintaining or restoring normal physiology during or following insult. Remodeling of the hepatic ECM in response to acute injury can alter the super- and ultra-structure of the ECM. Acute toxic liver injury causes notable alterations in the ECM structural components (e.g., collagens I, IV, V, fibronectin, and elastin) and nonstructural proteins (e.g., olfactomedin-4 and thrombospondin-4).⁹⁰ Through altering these components of the ECM, injury affects the normal elasticity provided by the ECM.⁹⁰ It is known that inflammation impacts liver stiffness measurements (e.g., transient elastography), but is viewed as a “false positive” signal.^49,91 However, inflammation has been shown to directly increase ECM stiffness in other organs, such as the lungs and the vasculature.^92–95 It is, therefore, likely that the increase in liver stiffness measurements caused by inflammation is at least in part, a true signal. Indeed, three-dimensional magnetic resonance elastography, which measures shear stiffness, damping ratio, and magnetic resonance imaging proton density fat fraction, is an emerging technology to noninvasively differentiate inflammation from fibrosis.⁹⁶ As will be discussed later (see next section), ECM stiffness can also influence inflammation.

Invasive cells can also degrade the ECM during disease and inflammation. For example, leukocytes secrete MMP-9, which mediates their transmigration during liver injury.⁹⁷ This change in the ECM alters the interactions between the ECM and its receptors (e.g., integrins). Degradation can also expose self-antigens (e.g., collagen V) that can be used to promote infiltration of inflammatory cells.⁹⁸ Moreover, even in cases where there is a net increase in ECM, overall ECM turnover is also increased.⁹⁹ ECM turnover can also release proinflammatory ECM peptide fragments that may also serve as an alarmin/chemoattractant to recruit inflammatory cells to the site of injury (^100,101; ►Fig. 1).

Storage, Presentation, and Sensing

The ECM also serves as a reservoir of signaling molecules, including growth factors during development and angiogenesis, as well as cytokines and chemokines during inflammation and disease. These interactions may serve to present or restrict access of ligands to receptors, modulate the spatial distribution of growth factors or create chemotactic gradients, or sequester a signaling molecule for later release.⁸⁶ Damaged tissue can rapidly release mediators that attract components of the inflammation/wound healing response. The ECM sequesters and stores these molecules, predominantly via interactions with GAG, which protect them from normal proteolytic cleavage.¹⁰² However, these linkages can be rapidly cleaved and released by proteases (e.g., MMPs and ADAMs) induced by injury and/or inflammatory cytokines.^102–105 The localized release of these mediators also creates a gradient that acts as a “homing signal” to the origin of the injury.^78,105,106

The ECM is also a dynamic signaling moiety that allows the environment to interact with the cell and the cell to interact with the environment. One family of receptors that mediate these interactions is the integrins. Integrins are heterodimeric proteins composed of α and β subunits, with at least 24 different combinations having been identified in mammalian cells.¹⁰⁷ Integrins transfer information from the ECM to the cell, allowing rapid and dynamic responses to changes in the extracellular environment. Integrins play a myriad of roles within the body, including proliferation/angiogenesis, maintenance of differentiation, as well as inflammation and apoptosis.^108,109 Integrins are found on almost all cell-types in the liver, and dysregulated integrin signaling has been demonstrated to be involved in hepatic fibrogenesis in a wide variety of liver diseases, as well as inflammatory liver injury.¹¹⁰ There are also several nonintegrin receptors involved in signaling between the ECM and the cell. For example, CD44, a type I transmembrane glycoprotein with over 20 different isoforms, has been demonstrated to be involved in liver disease and inflammation.^111,112 The canonical CD44 ligand is hyaluronic acid. Interactions between this ECM GAG and CD44 are known to facilitate migration of leukocytes to inflamed tissue, as well as the progression of inflammatory injury.¹¹³ Alternatively, CD44 has been implicated in the resolution of injury by facilitating the migration of hematopoietic stem cells to the injured liver.¹¹⁴

Interaction of cells with the surrounding ECM can also impact downstream signaling of both proinflammatory and restorative (e.g., growth factor) signals. This control can be at the level of receptor affinity or downstream signaling. These receptors are laterally organized on the plasma membrane in lipid/lipoprotein-rich regions (lipid rafts); this close proximity facilitates ligand binding, receptor dimerization, and cooperative downstream signaling.¹¹⁵ It has been recently suggested that ECM proteins contribute to this lateral organization.¹¹⁶ Signal integration between ECM-binding receptors (e.g., integrins) and extracellular signaling molecules is also know to vary based on ECM stratum. This has best been described for growth factor signaling and categorized as concomitant signaling, collaborative activation, direct activation, amplification, and negative regulation.^117,118 Chronic inflammation has been shown to impair normal growth factor signaling, in part by altering the phenotype of the ECM.¹¹⁹ Moreover, ECM interactions have been shown to impact toll-like receptor and tumor necrosis factor alpha signaling to alter both effect and magnitude of the extracellular signal.¹²⁰

In addition to directly and indirectly impacting downstream signaling cascades, integrin/ECM interactions foster linkage to the intracellular cytoskeleton.^121,122 This interaction leads to the clustering of integrins into focal adhesions and plays key roles in influencing normal development, growth, cellular maintenance, and overall organ homeostasis.^121–123 Dysregulation of adhesion signaling via ECM/integrin interactions are also key steps in cancer progression.^124,125 This integration via integrins of the ECM and intracellular cytoskeleton contributes to mechanosensing, which likely influences the impact of ECM rigidity on the inflammatory response (see above¹²³).

Inflammation and ECM remodeling can become a feed-forward cycle.¹⁰⁴ The ECM facilitates immune cell migration and differentiation, while immune cells trigger new ECM deposition and proteolytic remodeling. Proteases subsequently cleave ECM producing proinflammatory degradation products. These processes can be adaptive, but aberrant ECM and bioactive degradation products perpetuate inflammation in a maladaptive response, such as in chronic hepatic inflammation.¹²⁶ Given the key role of the ECM in mediating inflammation, it is not surprising that this compartment also plays key roles in the resolution of inflammation (i.e., catabasis^127–129).

Conclusion

In conclusion, the ECM is complex and responds dynamically to internal/external stressors. Changes to the ECM in response to acute and/or inflammatory injury are well understood in some contexts. However, in the context of chronic liver diseases, most of the attention has been paid on the ECM changes associated with the end-stage of collagenic fibrosis. Although fibrosis is an important target that is relevant to clinical liver disease, it is also arguably the least sensitive to external interventions.¹³⁰ In contrast, improvements in detection methods make the concept of blunting/reversing earlier stages of disease progression viable. In this context, inflammation may be a key therapeutic target. The response of the matrisome to stress is a critical component of the inflammatory response. Although changes to the ECM during hepatic inflammation are partially understood, it is an emerging area of interest. There is an opportunity to cross-fertilize our understanding from other fields in which the ECM and inflammation are more well described.^131–133 These other fields may also be a source of laterally translated therapies that can be applied to chronic liver disease.¹³⁴