Extracellular Matrix
Alex Y. Hui, Scott L . Friedman
6
6.1
Introduction
The hepatic extracellular matrix (ECM) is a complex network of macromolecules that not only provides cells with an extracellular scaffold but also plays an important role in the regulation of cellular activities (Fig. 6.1) [50]. In normal liver, the ECM comprises less than 3% of the relative area on a tissue section and approximately 0.5% of the wet weight [13, 21]. In addition to Glisson’s capsule, ECM is found main- ly in the portal tracts and the central veins. Small amounts of ECM, the perisinusoidal matrix, are also found in the space of Disse. The sinusoids are lined by fenestrated endothelial cells, which lack an underlying basement membrane (BM). This facili- tates the flow of plasma between sinusoidal lumen and the hepatocytes, and vice versa. The strategic position of the perisinusoidal matrix at the inter- face between blood and the epithelial components of the liver explains why quantitative or qualitative change of ECM may significantly influence hepatic function [3].
Generation of ECM, or fibrogenesis, occurs in re- sponse to different chronic injuries to the liver. This is a wound-healing response, which is reversible by ECM degradation upon elimination of the primary insult. Transition of a normal to a fibrotic liver in- volves both quantitative and compositional changes in ECM [16]. Intense research over the past 15 years has established that hepatic stellate cells (HSC) play a central role in the process (Fig. 6.2; see Part 1, Chap. 3). Other cell types including hepatocytes and hepatic sinusoidal cells have at most a modest contribution to the production of ECM (see Part 1, Chaps. 1 and 5).
6.2
Components of the ECM in Liver
The components of ECM in liver include collagens, non-collagenous glycoproteins, glycosaminogly- cans, proteoglycans, matrix-bound growth factors (Fig. 6.3) and matricellular proteins. In normal liv- er, the dense, interstitial ECM is largely confined to the capsule, around large vessels and in the portal triad. The perisinusoidal matrix, on the other hand, is composed of both an interstitial and a BM-like low-density ECM.
6.2.1
The Collagen Scaffold
More than 20 genetically distinct collagens have been
identified and are grouped into two main molecular
classes: the relatively homogeneous group of fibril-
forming collagens (collagens I, II, III, V and XI) and
the rather heterogeneous group of non-fibrillar col-
lagens. The fibril-forming collagens, which consist
of a triple helix of approximately 300 nm in length
and 1.5 nm in diameter, self-assemble into fibrils
in the extracellular space through the cleavage of
terminal procollagen peptides by C-propeptidase
and N-propeptidase. Types I, III and V are the main
components in the dense interstitial ECM in portal
tract and central vein wall of normal liver. Among
the non-fibrillar collagens, types IV, VI, VIII, XIV,
XIX, XV and XVIII are found in the liver with dif-
ferent locations and functions. Both fibril-forming
and non-fibrillar collagens are found in the perisi-
nusoidal matrix. These include fibrillar types I, III
and V, microfibrillar collagen VI, BM collagens IV
and XVIII, and FACIT (fibril-associated collagens
with interrupted triple helices) collagen [9, 50].
6.2.2
Proteoglycans
Proteoglycans belong to a distinct subset of non-collagenous glycoproteins that contain gly- cosaminoglycan (GAG) side chains. They interact with other ECM molecules via specific GAG-bind- ing domains in these molecules. By virtue of such properties, they regulate matrix architecture and spatial arrangement of structural polymers. They bind cytokines and growth factors and thus control their availability and biological activities [9].
Proteoglycans identified in liver so far include aggrecan, fibromodulin, decorin, biglycan, perle- can, betaglycan, glypicans and syndecan-1, -2, -3 and -4. Aggrecan belongs to the family of proteogly- cans characterized by an N-terminal globular do- main that interacts with hyaluronan, and a C-termi-
nal selectin domain. It is found in the interstitium with structural function.
Fibromodulin, decorin and biglycan are char- acterized by a protein core composed of leucine- rich repeats. These provide a horseshoe-like struc- ture, which favors protein–protein interaction. In fact, these small proteoglycans bind transforming growth factor- β1 (TGF-β1), a potent fibrogenic cy- tokine to HSC. In normal liver tissue, biglycan and decorin are detected in the space of Disse, while in liver of patients with chronic hepatitis, they are also found in fibrotic areas [26].
Betaglycan, syndecans and glypicans are mem- brane-anchored heparin sulfate proteoglycans. Be- taglycan is the type III TGF- β receptor, while syn- decans may function as co-receptors of cytokines [15, 49]. They are transmembrane proteins with an amino-terminal extracellular domain, a single transmembrane domain and a short cytoplasmic
Fig. 6.1. Molecules of the hepatic extracellular matrix. The he- patic ECM consists of collagens, non-collagenous glycoproteins, elastin, glycosaminoglycans and proteoglycans. ECM-bound molecules include fibrin, plasmin, urokinase plasminogen acti- vator (upa), plasminogen activator inhibitor (PAI)-1, tissue trans- glutaminase, lysyl oxidase, growth factors/cytokines, metallo- proteinases (MMPs), and tissue inhibitors of metalloproteinase (TIMP)-3. In addition, transmembrane proteoglycans, which may serve as cell surface receptors, are cleaved by proteases, becom- ing ECM-bound, including CTGF connective tissue growth fac-
tor, a/bFGF acidic/basic fibroblast growth factor, GM-CSF granu- locyte macrophage colony-stimulating factor, IFN interferon, IL interleukin, HGF hepatocyte growth factor, KGF keratinocyte growth factor, OSM oncostatin M, PDGF platelet-derived growth factor, SPARC secreted protein acidic and rich in cysteine (syn- onymous with osteonectin or BM-40), TGF transforming growth factor, TSP thrombospondin, VEGF vascular endothelial growth factor, BM basement membrane, S sulphate. (Reproduced from reference 50, with permission from the author and publisher)
tail. Glypicans are integral membrane proteogly- cans that are anchored to the membrane via glycosyl phosphatidylinositol. Overexpression of glypican-3 is seen in hepatocellular carcinoma, which lends evidence to the suggestion that glypican-3 is mainly involved in growth-regulatory functions [63].
6.2.3 Laminin
Laminin is a non-collagenous glycoprotein and, together with perlecan, nidogen and collagen IV, is
one of the main components of the basement mem- brane [9]. It is composed of three disulphide-linked chains ( α, β and γ) with a characteristic cross shape.
A number of homologs of these chains have been discovered – five α chains, three β chains and three γ chains. Not all possible combinations of the three chains are used and so far 12 distinct laminin iso- forms have been identified. Among them, at least four may be found in human liver [9, 36]. Laminin is important not only in its structural role in the BM but also in its range of effects on cellular activities, namely cell adhesion, cell migration and cell differ-
Fig. 6.2. Changes in ECM and stellate cells during hepatic fibro- sis. A In normal sinusoids, stellate cells, located between sinu- soidal endothelium and hepatocytes, are quiescent and contain vitamin A droplets. Their foot processes encircle the sinusoid.
Microvilli depicted on hepatocytes indicate normal differentiat- ed function. The sinusoid endothelial cell fenestrae facilitate the flow of solutes between sinusoidal lumen and the hepatocytes.
Fig. 6.3. Binding of growth factors by ECM. The ECM can localize and store growth factors (see also Fig. 6.1), sequestering them for release by con- trolled proteolysis. (Reproduced from reference 50, with permission from the author and publisher)
B In chronic liver injury, a fibrillar matrix accumulates in the sub- endothelial space, produced primarily by activated stellate cells.
This matrix leads to loss of hepatocyte microvilli and reduced sinusoidal porosity. (Reprinted from Friedman SL, Arthur MJP.
Reversing hepatic fibrosis. Science and Medicine 2002;8(4):194–
205, with permission)
entiation. It mediates the cell–matrix interaction via binding to the integrin receptors [9, 11].
6.2.4 Fibronectin
Fibronectin (FN) is a multifunctional glycoprotein and plays crucial roles in various cellular functions.
It is a major component of normal and fibrotic he- patic ECM. Fibronectin molecules found in ECM are insoluble (cellular fibronectin, cFN) while plasma fibronectin, pFN, is soluble [50].
Fibronectin has a domain structure, consist- ing of three internally homologous repeats, termed types I, II and III [39]. The repeats are assembled into different functional domains that bind to vari- ous ligands such as collagen, heparin, fibrin and integrin. FN mRNA is post-transcriptionally modi- fied by alternative splicing at three variable regions of type III homology, EIIIA, EIIIB and EIIICS. Two mRNA isoforms are generated by either inclusion or exclusion of EIIIA and EIIIB, respectively. The II- ICS region has three subdomains (CS1, CS5 and the portion between these) and five isoforms may arise by exon subdivisions within this region. Alternative spliced variants have different biological properties.
For example, only cFN has EIIIA and EIIIB regions.
In vivo, FN forms fibrils stabilized by intermolecu- lar disulfide bridges. The polymerization process is driven by cell surface receptors, especially by in- tegrin α5β1.
Hepatocytes are the major source of pFN while cFN is produced by hepatocytes, activated HSC and sinusoidal endothelial cells [32, 58]. In rats, none of the FN isoforms is present in quiescent HSC from normal liver. In response to injury, sinusoidal en- dothelial cells express EIIIA fibronectin. This is a critical early event as the EIIIA segment is biologi- cally active, resulting in activation of HSC. The ac- tivated HSC in turn synthesize an EIIIA-containing FN themselves [32].
6.2.5
Matricellular Proteins
Matricellular proteins are a group of matrix proteins that modulate cell–matrix interaction and cell func- tion but do not contribute directly to the formation of structural elements [8]. Members of this group of distinct molecules include SPARC (secreted protein acidic and rich in cysteine, also termed osteonec- tin), thrombospondins (TSP1 and TSP2), osteopon- tin, tenascin-C, tenascin-X and CCN (CYR61, CTGF [connective tissue growth factor], Nov) family of
proteins. Studies in various cell types demonstrat- ed that these molecules are capable of sequestering growth factors (e.g. SPARC and PDGF [platelet-de- rived growth factor]), binding ions (e.g., osteonectin and Ca
2+), inhibiting (TSP1) or clearing proteases (TSP2) and activating cytokines (e.g. TSP2 and TGF- β1). They regulate cell adhesion, migration, chemo- taxis, proliferation and apoptosis. Furthermore, complex effects are also exerted on ECM synthesis and collagen assembly.
In liver, expression of SPARC is upregulated dur- ing fibrogenesis and hepatocarcinogenesis [34, 42].
HSC are the main source of SPARC as shown both in vivo and in vitro [7, 18]. Tenascin and osteopontin have both been found in normal and fibrotic livers and hepatocellular carcinoma (HCC) [33, 44, 56, 59].
Furthermore, osteopontin overexpression is associ- ated with larger tumours, high-grade and late-stage HCC, intrahepatic metastases and higher early re- currence rate [44, 60].
6.3
Changes in ECM
from Normal to Fibrotic Liver
Hepatic fibrosis is associated with a significant change in both the quantity and composition of the ECM. Total collagen content increases by three- to tenfold [48]. The peri-sinusoidal low-density ECM is gradually replaced by a high-density ECM with accu- mulation of fibrillar collagens (types I and III) and an electron-dense BM [24]. There is also an increase in glycoproteins, proteoglycans, glycosaminoglycans and a shift from BM-type proteoglycans (heparan sulfate) to interstitial-type (dermatan sulfates and chondroitin sulfates) [23]. Changes in ECM are as- sociated with disappearance of endothelial fenestra- tions, a process termed “sinusoid capillarization”.
In vitro studies showed that interstitial matrix in- duces loss of fenestrations whereas physiologically derived BM maintains them [40].
Complex interactions exist between the cel-
lular components of liver and the ECM [16]. With
the modification of the ECM microenvironment,
cellular functions and phenotypes are inevitably
affected. This is evidenced by the loss of microvilli
in hepatocytes in fibrotic liver and compromised
synthetic activity of hepatocytes when deprived of
BM matrix [6]. Meanwhile, the high-density matrix
activates HSC, which further perpetuates the proc-
ess of fibrogenesis [17]. On the other hand, quies-
cent HSC cultured on BM-like matrix derived from
Englebreth-Holm-Sarcoma remain non-prolifera-
tive and non-fibrogenic [17]. A recent study further
showed that BM-like matrix (Matrigel) can induce quiescence of activated HSC, suggesting that resto- ration of normal ECM in liver might downregulate fibrogenesis by restoring HSC to a quiescent state as well [19].
Specific components of the ECM have been iden- tified to regulate cellular activities. One example is the EIIIA segment of fibronectin as described above. Another molecule that has been intensely researched is microfibrillar collagen VI, the expres- sion of which is upregulated in liver fibrosis. Colla- gen VI stimulates DNA synthesis and inhibits apop- totic cell death in HSC in vitro [2, 50].
6.4
Pathways of Cell–Matrix Interaction
6.4.1
The Integrin Family
The ECM interacts with cells via matrix receptors on the cell membrane; the best-characterized of these being the integrin family of heterodimeric trans- membrane receptors [14, 55]. Integrin receptors are composed of α and β subunits, and at least 18 α and 8 β subunits are currently known. The various combi- nations result in over 20 functional integrin dimers with different specificities. Their globular head do- main binds to ligands, which include components of the ECM and cell adhesion molecules. Most integrin ligands contain an Arg-Gly-Asp (RGD) tripeptide sequence, which is necessary but insufficient for sig- naling. Intracellularly, integrins are connected via associated proteins to the actin cytoskeleton.
The integrins are important not only in their adhesive function but also in their roles in modu- lating signal transduction pathways downstream of other receptors. Modulation of signaling pathways takes place via a number of mechanisms. Cell ad- hesion may result in change in shape and tension of the cell and nucleus via the cytoskeleton, which in turn influences gene expression [35, 37]. Mean- while, integrins may affect signal transduction via parallel activation of pathways that synergize at the level of phosphorylation of proteins [12, 47]. Signals generated by other receptors are also enhanced due to clustering of ECM-bound integrins in the plane of membrane. The activated integrins recruit signal- ing molecules that form complexes called focal ad- hesion complexes [22]. Examples of such molecules are caveolin, paxillin and tyrosine kinases such as fyn and focal adhesion kinase (FAK). Further down- stream, the complexes are associated with other ki-
nases and adaptor molecules. Clustering of these proteins results in amplification of the signal trans- duction. Lastly, integrin may cluster and transacti- vate signaling pathways involving receptor tyrosine kinases. An example of such functional cooperation is that between the PDGF pathway and integrin sig- naling as demonstrated by clustering of ligand-ac- tivated PDGF- β receptors in areas corresponding to focal adhesion complexes [10]. A complex web of crosstalk thus exists between the integrin pathways and signaling mechanisms of the growth factor re- ceptors.
The normal adult human hepatocytes express low levels of three integrin dimers: a collagen and laminin receptor, α1β1; a fibronectin receptor, α5β1;
and a tenascin receptor, α9β1. On the other hand, re- ceptors α1β1, α2β1, α5β1 and α6β4 have been iden- tified in cultured HSC [11, 45]. In experimental liver fibrosis, there is upregulation of laminin-binding integrins α6β1 and α2β1, and fibronectin-binding α5β1 [11, 46, 62]. Functionally, integrin antagonism by soluble integrin recognition sequence pentapep- tide GRGDS in rat HSC disturbs actin stress fiber formation and tyrosine phosphorylation of FAK caused by adhesion to ECM. The pentapeptide also induces p53 expression and apoptosis in the same cell type [30, 31].
Recent studies have also elucidated the roles of integrin in hepatocarcinogenesis. In HepG2 cells, stable transfection of β1 integrin modulates respon- siveness to hepatocyte growth factor (HGF) with resultant increased proliferation [61]. On the other hand, osteopontin binding to α5β3 integrin aug- ments in vitro adhesion of HepG2 to hyaluronate and may contribute to the mechanism by which os- teopontin enhances metastatic behavior in hepato- cellular cancer cells [20].
6.4.2
Discoidin Domain Receptor-2
Another matrix receptor recently found to have a
potential role in liver fibrosis is discoidin domain
receptor-2 (DDR2) [43]. DDR2 is a tyrosine kinase
receptor that responds to ECM ligands but not to
soluble peptide factors. It is activated primarily by
collagen type I, and to a lesser extent by collagen
types II, III and V. DDR2 is induced during HSC ac-
tivation, and the phosphorylated receptor mediates
growth stimulation and matrixmetalloproteinase-2
(MMP-2; see 6.5 below) production in response to
type I collagen; thus DDR2 can stimulate degra-
dation of normal liver ECM via MMP-2, while it is
further upregulated by accumulating interstitial
collagen, thereby establishing a positive feedback
loop. The importance of DDR2 in liver fibrosis is substantiated by a recent study showing its presence at elevated levels in the small bile ducts of patients suffering from primary biliary cirrhosis [38].
6.4.3
Growth Factors in ECM
In addition to its structural role and direct inter- action with cells, ECM also regulates cell function indirectly via modulation of the availability and activity of growth factors including PDGF, TGF- β (see Part II, Chap. 11), CTGF, vascular endothelial growth factor, HGF and so on [54]. Proteoglycans such as decorin, biglycan, fibromodulin and gly- cosaminoglycans are the main ECM components that bind growth factors and cytokine. They can in- teract with growth factors either via their core pro- teins or via their glycosaminoglucan side chains.
For example, decorin and biglycan bind TGF- β by their protein cores but interact with HGF through the heparan sulfate [25, 41]. Other ECM components such as fibronectin and laminin bind tumor necro- sis factor- α (TNF-α), while collagen binds PDGF, HGF and interleukin-2 (IL-2) [1, 51–53]. Binding of survival factors by interstitial matrix may prevent apoptosis of hepatocytes in liver that have acquired DNA damage, thereby perpetuating the expansion of cells with mutations and genomic instability. This observation may explain why cancer is more likely in livers that are cirrhotic, particularly in patients with hepatitis C. As well as protecting such factors from proteolysis, the ECM controls their release through the actions of proteases and their inhibitors, result- ing in further modulation of their activities.
6.5
Matrixmetalloproteinase and its Inhibitors Since the ECM components are highly stabilized and cross-linked molecules, they can only be broken down by a specific family of enzymes, the matrix- metalloproteinases (MMP). The activity of MMP is balanced by a group of proteins, the tissue inhibitors of the MMP family (TIMP) [4]. The integral activity of the MMP and TIMP determines the rate of ECM degradation. The MMP family comprises 25 differ- ent calcium- and zinc-dependent enzymes divided into five broad categories: interstitial collagenases, gelatinases, stromelysins, membrane-type MMP (MT-MMP) and metalloelastase. This functional classification is somewhat arbitrary as there is over- lap in activities among categories.
HSC are the main source of MMP in the liver.
In early primary culture, HSC express MMP-3 (a stromelysin) and MMP-2 but not TIMP [27, 29, 57].
Although this matrix-degrading phenotype could be a result of the isolation process, it might also re- flect the early phase of “pathological” matrix degra- dation in vivo after injury, during which the normal subendothelial matrix and BM are damaged. With prolonged culture of HSC, MMP-1/MMP-13 are downregulated while expression of TIMP-1, TIMP-2, MMP-2 and MMP-14 is enhanced [4]. In vivo stud- ies in rodent and human specimens concurred with such findings by demonstrating increased TIMP expression associated with fibrosis [5, 27]. Accord- ing to one proposed model, increased MMP-2 and MMP-14 production by HSC causes degradation of the pericellular matrix. This results in an altered HSC–matrix interaction and further HSC activa- tion [4]. Meanwhile, as the expression of TIMP is increased, degradation of newly synthesized colla- gen is inhibited, with consequent accumulation of ECM.
Resolution of fibrosis, on the other hand, is as- sociated with degradation of fibrillar ECM and res- toration of normal hepatic architecture. In carbon tetrachloride-treated rats, liver fibrosis regresses after cessation of treatment. The process is associ- ated with marked reduction in TIMP activity and an approximately fivefold increase in hepatic colla- genase activity [28]. MMP-1/MMP-13 derived either from HSC or Kupffer cells, together with MMP-14 and MMP-2 contribute to the fibrolysis, leading to restoration of normal histology.
Development of HCC in a fibrotic liver is asso- ciated with increased expression of MMP-2, TIMPs and MMP-14. ECM components including collagen 1, collagen IV and laminin are also overexpressed.
Interestingly, non-encapsulated HCC, which is as- sociated with poorer prognosis, has the highest expression of these molecules. This may imply an enhanced aggressiveness in such tumors with im- pairment of capsule formation.
6.6 Conclusion
The hepatic ECM can no longer be seen as an inert
structural element of the liver. Dynamic changes in
its composition and quantity take place in response
to external stimuli. This plasticity and responsive-
ness serve important physiologic functions, as
typified by the wound-healing response of fibro-
genesis. HSC are the predominant cells responsible
for producing the components of ECM as well as
the enzymes that break down the ECM. Injurious stimuli directly or indirectly through hepatocytes or Kupffer cells activate HSC via various signaling pathways, resulting in the synthesis of these compo- nents. Yet interaction between ECM and their sur- rounding cells is bidirectional. The biological activ- ities contained in the ECM components, in addition to the growth factors sequestered, regulate cellular functions in a multitude of ways. Further research and better understanding of this complex web of interactions and its molecular basis may eventu- ally facilitate the development of new therapies for chronic liver diseases and carcinoma.
Selected Reading
Schuppan D et al. Matrix as a modulator of hepatic fibrogenesis.
Semin Liver Dis 2001;21:351–372. (A detailed and up-to-date review of different matrix components and their possible bi- ological functions in liver. It also contains a series of figures depicting different structures of matrix proteins.)
Bedossa P, Paradis V. Liver extracellular matrix in health and dis- ease. J Pathol 2003;200:504–515. (A succinct overview of composition of extracellular matrix in liver, and cellular and molecular basis of liver fibrogenesis.)
Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis 2001;21:373–
384. (An excellent review on the role and interaction of met- alloproteinases and their inhibitors in liver fibrosis providing important insight on possible mechanisms of fibrolysis.) Friedman SL. Mechanisms of Disease: mechanisms of hepatic
fibrosis and therapeutic implications. Nat Clin Pract Gastro- enterol Hepatol. 2004;1:98–105. (A comprehensive and up- to-date series on the mechanism, natural history, diagnostic and therapeutic approches of liver fibrosis.)
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