**2.1. Fibronectin**

receptor; LAP, latency associated protein; LLC, large latent complex; LOX, lysyl oxidase; LTBP, latent TGF-β-binding protein; MMP, matrix metalloproteinase; OB, obliterative bronchiolitis; SLRPs, small leucine-rich proteoglycans/proteins; TGF-β, transforming growthfactor-β; TSP-1, Thrombospondin-1; TRI, TGF-β type I receptor; TRII, TGF-β type II receptor; TMLC, mink lung

Cells in virtually all tissues are in contact with organized complexes of structural molecules collectively called the extracellular matrix (ECM). ECM induces a variety of signals that regulate the behavior of cells, such as differentiation, adhesion, and migration, and also fundamental physiological processes such as embryonic development and tissue regeneration and remodeling [1]. As a consequence, tissues or organs keep their normal architecture and homeostasis. Aberrations in signal transduction from the ECM cause chronic degenerative and

Considering the adult tissue/organ remodeling following injury, an important unresolved question is how newly deposited ECM contributes to the critical turning point from normal to abnormal healing. Wound healing is a crucial response to maintain tissue/organ structure and integrity after tissue damage, and also tissue/organ homeostasis [2]. Fibrosis is a part of the wound-healing response that maintains organ structure and integrity following tissue damage. However, excessive fibrosis contributes to a number of diseases. Indeed, fibrosis is the common pathological end result of many chronic inflammatory diseases. Fibrosis is an abnormal extension of the wound-healing process that follows tissue damage, characterized by the excessive accumulation of collagenous ECMs. The hallmark of fibrosis is excessive accumulation of mainly type I collagen (Col I) containing ECMs, and therefore involves both wound-healing and fibrotic processes. Fibrosis is recognized as a major cause of morbidity and mortality in most chronic diseases and chronic graft rejection, and also influences tumor invasion and metastasis. Importantly, a critical event in all fibrotic diseases is the activation of

myofibroblast, which are the key mediators of fibrotic tissue remodeling [3].

The clarification of regulatory mechanisms underlying excessive accumulation of ECMs in parenchymal organs such as livers during the development of chronic fibrotic diseases is a critical issue. However, currently, the main barrier to designing novel antifibrotic strategies is due to our insufficient understanding of the mechanisms responsible for ECM-network formations following tissue/organ injury. This gap in knowledge translates to lack of experi‐ mental models of repair *in vivo*, including gaps in the understanding of the identity and molecular control of factors and cells participating in the repair processes following injury. In this chapter, we will focus on the present knowledge of the mechanisms of the collagennetwork organization following tissue/organ damage and pathological processes of chronic

cell line; PAI-1, plasminogen activator inhibitor-1.

236 Composition and Function of the Extracellular Matrix in the Human Body

**1. Introduction**

fibrotic disorders.

fibrotic diseases.

Fibronectin, a dimeric glycoprotein, exists in two isoforms: a soluble isoform in plasma (plasma-type fibronectin, produced solely by hepatocytes) and an insoluble isoform in tissue ECM (cellular-type fibronectin, produced by a variety of cells). Both isoforms are generated from a single gene by alternative splicing [4]. Although considerable *in vitro* functional studies have indicated that fibronectin isoforms play key roles in cell differentiation, proliferation, migration, and survival [5, 6], knowledge of the functional identity of each fibronectin isoform in adult tissue remodeling remains loosely defined due to the complexity and the lack of the systems. Indeed, a prominent expression of fibronectin is often observed during adult tissue repair [7]. In response to adult tissue damage, the initial "provisional matrix" formation between plasma-type fibronectin and fibrinogen stabilizes wounded area, which acts as a nidus for subsequent collagen fibrillogenesis [7, 8]. Little insight into the pathophysiological roles of fibronectin has emerged from studies of genetic changes in humans. There are no documented cases of fibronectin-null patients, the nearest condition being familial glomeru‐ lonephritis in which there are mutations in the type III modules of fibronectin [9]. While complete fibronectin-null mice show an embryonic lethal phenotype [10], experimental evidence has documented that skin wounds heal normally in mice lacking plasma-type fibronectin [11]; hence, an absolute requirement for fibronectin in response to adult tissue damage has been speculative.

Based on *in vitro* findings, it has been postulated that collagen network formation depends on the fibronectin matrix [12, 13]. It was, therefore, hypothesized that the removal of fibronectin from the *in vivo* system could abolish extensive ECM network formation following tissue damage. To define the functional identity of fibronectin in adult tissue remodeling, we recently established a null condition for both fibronectin isoforms in adult liver (fibronectin(fl/fl)/Mx-Cre+). We have demonstrated an unexpected finding that the lack of fibronectin does not interfere with reconstruction and resolution of collagen fibril organization after the initial stages of liver injury. We have discovered a fibronectin-independent mechanism of collagen fibrillogenesis and identified transforming growth factor beta (TGF-β) signaling and type V collagen (Col V) as essential elements for collagen fibrillogenesis in response to liver injury [14] (further discussed in Section 3).

#### **2.2. Transforming growth factor-β (TGF-β)**

Transforming growth factor (TGF)-β is a profibrogenic master cytokine responsible for promoting differentiation of tissue-resident fibroblasts into myofibroblasts, upregulation of ECM production including fibronectin, and downregulation of ECM degradation [15–18]. TGF-β is secreted as a biologically inactive (latent) form, and importantly, the active TGF-β levels do not often correlate with mRNA and protein levels [19]. Indeed, the activation of latent TGF-β occurs independently of transcription [20], and the bioassay to measure active TGF-β levels has been developed using a mink lung cell line (TMLC) stably transfected with a plasminogen activator inhibitor-1 (PAI-1) promoter fused to luciferase [21].

TGF-β is secreted in a biologically inactive (latent) form in a complex (large latent complex [LLC]) with TGF-β latency associated protein (LAP) [22] and latent TGF-β binding proteins (LTBPs) [23]. Prior to the secretion, TGF-β is synthesized as a precursor and forms dimerized complex through disulfide bonds intracellularly. The dimer precursor is proteolytically cleaved by endopeptidase furin to generate LAP and mature TGF-β peptide from the Nterminal and C-terminal portions, respectively [24, 25]. Interestingly, LAP still associates with mature TGF-β noncovalently, termed as small latent complex. *In vitro* studies demonstrate that LAP is required for the secretion of TGF-β from cells [26, 27]. Furthermore, LAP shields the receptor binding epitope of mature TGF-β, indicating that LAP plays an inhibitory role in binding to its receptors [18]. Small latent complex is further associated with a secreted large glycoprotein LTBP via disulfide bonds. These trimolecular complexes (TGF-β, LAP, and LTBP) termed as LLC are formed intracellularly, then secreted and incorporated into ECM. An *in vitro* observation reveals that LLC and free LTBPs are secreted rapidly from cells as early as 30 min after synthesis, whereas small latent complex is secreted slowly [28]. Another study using TGF-β expressing CHO cells shows that only 50% of recombinant TGF-β precursor is secreted at 6 h after radio labeling with [35S] cysteine and [35S] methionine, and the level of its secretion becomes plateau at 20 h [29]. Secreted LLCs deposit in ECM via LTBPs. LTBPs are extracellular multidomain glycoproteins and share homology with fibrillins, which are the major constituents of connective tissue microfibrillar structure [25, 30–32]. Four different isoforms (LBTP 1-4) have been identified, and each isoform includes four 8-cystein domains and numerous epidermal growth factor (EGF)-like motifs [32]. Three isoforms, LTBP-1, 3, and 4, are known to associate with LAP via the third 8-cystein domain, whereas LTBP-2 does not bind to latent TGF-β [32]. In addition, LTBPs interact with extracellular proteins such as fibrillin-1, fibronectin, heparin, and myostatin [32–36]. Indeed, LTBP-1 colocalizes with both fibrillin-1 and fibronectin *in vitro* [33, 36]. Thus, LTBP-1 plays a central role in secreting and anchoring latent TGF-β into ECM (see Section 3).

In response to injury, the conformation of LLC changes and/or TGF-β is released from LAP, resulting that active TGF-β is exposed to its receptor binding site [37]. Indeed, elevated TGFβ bioavailability is frequently observed in chronic fibrotic diseases, and the inhibition of local TGF-β activation can protect against the progression of fibrosis in several adult chronic fibrotic diseases [38–41]. There are several mechanisms of local TGF-β activation, which mediates αvβ6 and αvβ8 integrins, and thrombospondin-1 (TSP-1). Integrin αvβ6 can directly activate latent TGF-β, which depends on an interaction with RGD amino acid sequence of LAP [25]. In response to injury, integrin αvβ6 induces a conformational change (deformation) of LAP via the interaction between αvβ6 and the cytoskeleton [25, 42, 43]. Consequently, such a defor‐ mation makes it possible to release active TGF-β from LAP and then cause mature TGF-β to interact with TGF-β type II receptor (TRII) [25, 42, 43]. Interestingly, this activation process is independent of any proteolysis [25]. In contrast, integrin αvβ8-mediated activation of TGF-β is shown to be dependent on membrane type 1-matrix metalloprotease (MT1-MMP, also known as MMP-14) [44]. Furthermore, this activation does not require β8-cytoplasmic domain [44]. These findings indicate that the mechanism of αvβ8-mediated TGF-β activation is clearly different from that of αvβ6. *In vitro* and *in vivo* studies demonstrate that TSP-1 can also activate latent TGF-β [45, 46]. TSP-1 is a matricellular protein prominently expressed in response to tissue damage and plays a role as a transient component of ECM during tissue repair [47]. TSP-1 directly interacts with the LAP [48], and the interaction is supposed to induce a confor‐ mational change of LAP, thereby presumably uncover TGF-β receptor binding site [49, 50]. In addition, MMP-2, 9, 13, bone morphogenetic protein (BMP)-1, and serine proteases (plasmin, thrombin, neutrophil elastase, and kallikrein) have been shown to play an important role in TGF-β activation at least *in vitro* [51, 52]. A very recent study reveals plasma kallikreindependent TGF-β activation in fibrotic liver in both animal models and patients [53].

TGF-β is secreted in a biologically inactive (latent) form in a complex (large latent complex [LLC]) with TGF-β latency associated protein (LAP) [22] and latent TGF-β binding proteins (LTBPs) [23]. Prior to the secretion, TGF-β is synthesized as a precursor and forms dimerized complex through disulfide bonds intracellularly. The dimer precursor is proteolytically cleaved by endopeptidase furin to generate LAP and mature TGF-β peptide from the Nterminal and C-terminal portions, respectively [24, 25]. Interestingly, LAP still associates with mature TGF-β noncovalently, termed as small latent complex. *In vitro* studies demonstrate that LAP is required for the secretion of TGF-β from cells [26, 27]. Furthermore, LAP shields the receptor binding epitope of mature TGF-β, indicating that LAP plays an inhibitory role in binding to its receptors [18]. Small latent complex is further associated with a secreted large glycoprotein LTBP via disulfide bonds. These trimolecular complexes (TGF-β, LAP, and LTBP) termed as LLC are formed intracellularly, then secreted and incorporated into ECM. An *in vitro* observation reveals that LLC and free LTBPs are secreted rapidly from cells as early as 30 min after synthesis, whereas small latent complex is secreted slowly [28]. Another study using TGF-β expressing CHO cells shows that only 50% of recombinant TGF-β precursor is secreted at 6 h after radio labeling with [35S] cysteine and [35S] methionine, and the level of its secretion becomes plateau at 20 h [29]. Secreted LLCs deposit in ECM via LTBPs. LTBPs are extracellular multidomain glycoproteins and share homology with fibrillins, which are the major constituents of connective tissue microfibrillar structure [25, 30–32]. Four different isoforms (LBTP 1-4) have been identified, and each isoform includes four 8-cystein domains and numerous epidermal growth factor (EGF)-like motifs [32]. Three isoforms, LTBP-1, 3, and 4, are known to associate with LAP via the third 8-cystein domain, whereas LTBP-2 does not bind to latent TGF-β [32]. In addition, LTBPs interact with extracellular proteins such as fibrillin-1, fibronectin, heparin, and myostatin [32–36]. Indeed, LTBP-1 colocalizes with both fibrillin-1 and fibronectin *in vitro* [33, 36]. Thus, LTBP-1 plays a central role in secreting and

In response to injury, the conformation of LLC changes and/or TGF-β is released from LAP, resulting that active TGF-β is exposed to its receptor binding site [37]. Indeed, elevated TGFβ bioavailability is frequently observed in chronic fibrotic diseases, and the inhibition of local TGF-β activation can protect against the progression of fibrosis in several adult chronic fibrotic diseases [38–41]. There are several mechanisms of local TGF-β activation, which mediates αvβ6 and αvβ8 integrins, and thrombospondin-1 (TSP-1). Integrin αvβ6 can directly activate latent TGF-β, which depends on an interaction with RGD amino acid sequence of LAP [25]. In response to injury, integrin αvβ6 induces a conformational change (deformation) of LAP via the interaction between αvβ6 and the cytoskeleton [25, 42, 43]. Consequently, such a defor‐ mation makes it possible to release active TGF-β from LAP and then cause mature TGF-β to interact with TGF-β type II receptor (TRII) [25, 42, 43]. Interestingly, this activation process is independent of any proteolysis [25]. In contrast, integrin αvβ8-mediated activation of TGF-β is shown to be dependent on membrane type 1-matrix metalloprotease (MT1-MMP, also known as MMP-14) [44]. Furthermore, this activation does not require β8-cytoplasmic domain [44]. These findings indicate that the mechanism of αvβ8-mediated TGF-β activation is clearly different from that of αvβ6. *In vitro* and *in vivo* studies demonstrate that TSP-1 can also activate latent TGF-β [45, 46]. TSP-1 is a matricellular protein prominently expressed in response to

anchoring latent TGF-β into ECM (see Section 3).

238 Composition and Function of the Extracellular Matrix in the Human Body

Smads are the central as direct downstream modulators in canonical TGF-β signaling [54–56]. Smads consist of three classes: regulatory/receptor-activated (Smad2 and Smad3), coactivating (Smad4), and inhibitory Smads (Smad6 and Smad7). TGF-β binding to TRII initiates the formation of the complex with the TGF-β type I receptor (TRI) and phosphorylation of TRI. Subsequently, they activate TRI phosphorylates of Smad2 and Smad3, and then they form complexes with Smad4. These complexes bind to specific motifs "Smad-binding element" with transcription factors/coactivator such as Ap-1, Sp1, and CBP/p300, and promote the gene expression [54, 57]. Lines of evidence suggest that TGF-β signaling plays a key role in regu‐ lating myofibroblast phenotypes and fibrosis in the heart, lungs, liver, kidneys, and skin [3, 54]. For example, TGF-β directly induces the transdifferentiation of fibroblasts into collagensecreting active myofibroblasts [3, 14, 58], and overexpression of TGF-β results in the induction of hepatocyte apoptosis and liver fibrosis [18, 59, 60]. In contrast, TGF-β1 knockout mice show remarkable (~80%) decrease of collagen accumulation in response to liver injury [58, 61]. Furthermore, Smad3-null mice show reduction in liver fibrosis with decreased myofibroblast activation and ECM production in response to liver injury, whereas the disruption of inhibitory Smad7 results in an enhancement of damage and fibrogenesis in chronic liver injury [7]. We have previously generated adult mice lacking TRII from livers (TGF-βIIR(fl/fl)/Mx-Cre+); because TRII is the exclusive type II receptor for all TGF-β ligands, lack of this receptor abolishes all TGF-mediated signaling in the liver. Knockout livers actually show significantly lower ECM deposition (~46% compared to controls) in carbon tetrachloride (CCl4)-induced chronic injury, which is accompanied by the decreased expression of myofibroblast marker alpha-smooth muscle actin (α-SMA). These findings indicate that TGF-β signaling is indeed a dominant pathway in the development of liver fibrosis [62]. However, elimination of TGF-β or TRII does not completely prevent the accumulation of Col I in chronic liver injury, and in particular, TRII knockout livers still remain ~46.4% fibrosis compared to wild type [61, 62]. Therefore, these findings clearly indicate the TGF-β-independent mechanism(s) in the development of liver fibrosis (see Section 3). Indeed, we have found that TRII-null livers significantly upregulated connective tissue growth factors (CTGF/CCN2) following chronic liver injury, suggesting that CTGF/CCN2 can be an alternative mediator in liver fibrosis.

As described above, the local activation of latent TGF-β is a critical step in TGF-β-mediated fibrosis [37, 42, 63, 64]. We have discovered that fibronectin-null livers show elevated local TGF-β bioavailability and upregulate Smad signaling in activated hepatic stellate cells (HSCs) following injury [14, 65]. This novel finding implies that fibronectin regulates the balance between active and inactive (latent) TGF-β, which in turn modulates ECM production and remodeling following injury, and consequently retains adult tissue/organ functions. We further describe this important topic in the following section.

#### **2.3. Type I/type V collagen**

Collagens are the most abundant scaffolding ECM in tissue/organ stroma and contribute significantly to tissue/organ integrity [66, 67]. The collagen superfamily is large and hetero‐ geneous, and there are at least 28 different collagen types in vertebrates [66, 68]. A collagen molecule consists of three polypeptide chains called α chains, displays a right-handed supercoil, and also has one or more triple helical regions with common sequence repeats, Gly-X-Y [69]. Collagens are divided into six subfamilies (or subgroups) based on their structure, function, and distribution [66, 67]. The collagens that we focus in this review are Col I and Col V and those belong to fibril-forming collagens. Fibril-forming collagens are synthesized as procollagen and three pro α chains fold a triple helical structure from its C-terminus to Nterminus [67]. To form ordered fibrils, both N- and C-propeptides should be cleaved by procollagen N-proteinases/ADAMTS-2, 3, 14, and procollagen C-proteinases/BMP-1/tolloid proteinases, respectively [68]. An exception to this is the case in Col α1(V) chain. Its Cprocollagen is cleaved by furin, and its N-terminal is cleaved by BMP-1 [70]. The peptides cleaved by proteinase are called "propeptides" and noncollagenous peptides remain after the enzyme cleavages called "telopeptides". The telopeptides of Col I contain intermolecular crosslinking sites for fibrillogenesis [71]. Fibril-forming collagens form the 64–67 nm regularly repeated striated fibrils [72]. When collagen I molecules form fibrils, there is a unique space in the collagen fibrils termed "gap zone". The gap zone is present between the N-terminus of one molecule and C-terminus of the next in the triple helix-formed collagen fibrils, and the gap zone is suggested to play a role in a variety of molecular interactions, including lysyl oxidase (LOX)-mediated collagen cross-linking [73, 74].

Collagens are essential for tissue-specific macromolecular structure and organizations in the ECM. Indeed, collagen-mediated ECM networks affect many important biological properties such as matrix/tissue stiffness and tissue/organ structure. Collagens participate in numerous physiological processes such as embryonic development, and tissue regeneration and remod‐ eling [75]. Collagen networks provide the biomechanical scaffold for cell attachment and trap of macromolecules, and regulate cell growth and proliferation, and also the shape and structural integrities of cells and tissues [1, 76]. While collagens exist outside of cell and are composed of ECM structures, cells always sense the alterations of their ECMs, produce new ECMs, and/or degrade their ECMs, and consequently, tissues/organs maintain their homeo‐ stasis. More importantly, collagens induce intracellular signaling pathways, and it is mediated by cell surface ECM receptor, integrins. Integrins are transmembrane αβ heterodimeric receptors that mediate organization of focal contacts, actin-containing cytoskeleton, and ECM. Integrins are a major family of cell-surface-adhesion receptors (composed of 18 α-subunits and 8 β-subunits) [77]. The ligation of integrins by adhesive ligands can induce intracellular signaling events ("outside-in" signaling) and intracellular signaling pathways can control binding avidity of integrins for extracellular ligands ("inside-out" signaling) [77]. Interesting‐ ly, there is a "cross-talk" between integrins and receptor tyrosine kinases in certain cell types [78–80].

remodeling following injury, and consequently retains adult tissue/organ functions. We

Collagens are the most abundant scaffolding ECM in tissue/organ stroma and contribute significantly to tissue/organ integrity [66, 67]. The collagen superfamily is large and hetero‐ geneous, and there are at least 28 different collagen types in vertebrates [66, 68]. A collagen molecule consists of three polypeptide chains called α chains, displays a right-handed supercoil, and also has one or more triple helical regions with common sequence repeats, Gly-X-Y [69]. Collagens are divided into six subfamilies (or subgroups) based on their structure, function, and distribution [66, 67]. The collagens that we focus in this review are Col I and Col V and those belong to fibril-forming collagens. Fibril-forming collagens are synthesized as procollagen and three pro α chains fold a triple helical structure from its C-terminus to Nterminus [67]. To form ordered fibrils, both N- and C-propeptides should be cleaved by procollagen N-proteinases/ADAMTS-2, 3, 14, and procollagen C-proteinases/BMP-1/tolloid proteinases, respectively [68]. An exception to this is the case in Col α1(V) chain. Its Cprocollagen is cleaved by furin, and its N-terminal is cleaved by BMP-1 [70]. The peptides cleaved by proteinase are called "propeptides" and noncollagenous peptides remain after the enzyme cleavages called "telopeptides". The telopeptides of Col I contain intermolecular crosslinking sites for fibrillogenesis [71]. Fibril-forming collagens form the 64–67 nm regularly repeated striated fibrils [72]. When collagen I molecules form fibrils, there is a unique space in the collagen fibrils termed "gap zone". The gap zone is present between the N-terminus of one molecule and C-terminus of the next in the triple helix-formed collagen fibrils, and the gap zone is suggested to play a role in a variety of molecular interactions, including lysyl oxidase

Collagens are essential for tissue-specific macromolecular structure and organizations in the ECM. Indeed, collagen-mediated ECM networks affect many important biological properties such as matrix/tissue stiffness and tissue/organ structure. Collagens participate in numerous physiological processes such as embryonic development, and tissue regeneration and remod‐ eling [75]. Collagen networks provide the biomechanical scaffold for cell attachment and trap of macromolecules, and regulate cell growth and proliferation, and also the shape and structural integrities of cells and tissues [1, 76]. While collagens exist outside of cell and are composed of ECM structures, cells always sense the alterations of their ECMs, produce new ECMs, and/or degrade their ECMs, and consequently, tissues/organs maintain their homeo‐ stasis. More importantly, collagens induce intracellular signaling pathways, and it is mediated by cell surface ECM receptor, integrins. Integrins are transmembrane αβ heterodimeric receptors that mediate organization of focal contacts, actin-containing cytoskeleton, and ECM. Integrins are a major family of cell-surface-adhesion receptors (composed of 18 α-subunits and 8 β-subunits) [77]. The ligation of integrins by adhesive ligands can induce intracellular signaling events ("outside-in" signaling) and intracellular signaling pathways can control binding avidity of integrins for extracellular ligands ("inside-out" signaling) [77]. Interesting‐

further describe this important topic in the following section.

240 Composition and Function of the Extracellular Matrix in the Human Body

(LOX)-mediated collagen cross-linking [73, 74].

**2.3. Type I/type V collagen**

Several unique features of collagens are spotlighted as targets of medical treatment or biomarkers for diagnosis. For examples, oral administration of Col V induces immunologic tolerance to lung allografts and downregulates lung allograft rejection [81]. Another study shows using a specific monoclonal antibody against Col V C-terminal propeptide that, in the liver fibrosis patients, Col V C-terminal propeptide levels released in the serum have a positive correlation with the total amount of collagens deposited in the fibrotic livers. This finding has a potential to be used as diagnostic and potentially prognostic markers in monitoring liver fibrosis [82].

Considering the mechanisms in collagen fibril network organization, two key molecules, Col V and Col XI, are involved in this process. Although both collagens are fibrillar collagens and minor components in tissues/organs, they play an important role in controlling fibril diameter of assembled collagen [83]. Col V [two α1(V) and one α2(V)] is known to form heterotypic fibrils with Col l [76]. Col V has a conserved multidomain structure, N-terminal domain [73]. Col V controls collagen fibril diameter through both triple helical [84] and N-terminal domains [83, 85]. There is evidence that Col V regulates the fibril diameter *in vitro* [73, 84, 86]: Col I alone formed a broad distribution of relatively large diameter fibrils, while Col V alone formed much thinner nonperiodic fibrils. Interestingly, fibrils formed from Col I in the presence of increasing amounts of Col V displayed a significant decrease in the mean fibril diameter. The variance of the fibril population is also decreased as the percentage of Col V increases. Despite of presence of Col I, deletion of *col 5a1* gene causes embryonic lethal in a mouse model due to a virtual lack of fibril formation in the mesenchyme [87]. The heterozygous mice (*col 5a1* [+/–]) are viable, but show the reduction of fibril number and collagen content in the skin compared to wildtype mice. Furthermore, the abnormal collagen fibrils are observed in the deep dermis of *col 5a1* (+/–) mice; the mutant fibrils display larger diameter and broad distribution, and the diameter is inconsistent along the fibril length. These findings show that col V is essential for Col I fibril assembly.

Col XI [α1(XI), α2(XI), α3(XI)] is known to form heterotypic fibrils with Col II. Col XI α-chains shares structural homology with Col V and appears to have a similar nucleating function [88]. The first half of the human α1(V) N-propeptide has 73% homology with the human α1(XI) chain [83]. The α1 (XI) chain loses its function by chondrodysplasia mutation [89]. Loss of function mutations in the α1 (XI) chain leads to the assembly of Col II and Col V fibrils with abnormally large diameters [73, 89]. This interesting observation clearly indicates that Col XI also have a role to regulate the fibril diameter as Col V. To date, very little evidence in Col XImediated fibrillogenesis has been identified, and more detailed studies in the functional role of Col XI remain to be elucidated.

Recently, Burlingham et al. have shown an attractive evidence that Col V is involved in immune response to adult human lung disease, obliterative bronchiolitis (OB), which is characterized by narrowing of bronchiole lumens due to the inflammation and fibrosis [90]. Col V is recognized as an antigen by monocyte and presented to CD4+ T cells (called Th-17), and Col V-specific responses are required both IL-17A produced by Th-17 and the monokines TNF-α and IL-1β, suggesting that IL-17A induced in response to Col V plays a role in fibrosis. Indeed, IL-17A plays a variety of significant roles in neutrophil recruitment, angiogenesis, inflammation, and autoimmune disease [91]. Very recently, Vittal et al. have shown a unique observation about the relationship between Col V and IL-17A [92]. IL-17A can induce epithe‐ lial–mesenchymal transition in rat lung epithelial T-antigen negative cells through upregula‐ tion of S100A4 and mesenchymal marker α-SMA, and downregulation of epithelial markers ZO-1 and E-cadherin. Mechanistically, IL-17A results in the downregulation of *Smad7*, and upregulation of *TGF-*β and Smad3 activation, clearly indicating that IL-17A is involved in TGFβ pathway. Col V is upregulated by TGF-β during osteogenesis [93], and we have demon‐ strated TGF-β-induced Col V-mediated *de novo* Col I and Col III network organization even in the absence of fibronectin [14]. Thus, these findings suggest that IL-17A can act as an upstream mediator to regulate the expression of Col V via TGF-β signaling-mediated pathway, and as a consequence, IL-17A could modulate Col V-mediated fibrogenesis. There is another obser‐ vation showing that IL-17A is involved in liver fibrosis. Mouse primary HSCs are shown to express IL-17A receptor (IL-17R). The treatment of HSC with recombinant IL-17A upregulates α-SMA, collagens, and TGF-β mRNA expression levels. Furthermore, the IL-17RA-null mouse model shows ~50% reduction of liver fibrosis induced by CCl4 with decreased levels liver damage and inflammations [94], suggesting that IL-17A plays a significant role in HSC transdifferentiation into active myofibroblasts during the development of liver fibrosis. Thus, further analysis of the regulatory mechanisms of Col V by IL-17A could open the new avenue as a drug target for liver fibrosis.

#### **2.4. Other modifying molecules such as lysyl oxidase and small leucine-rich proteoglycans**

Collagen contributes significantly to tissue/organ integrity, and collagen cross-linking stiffens the ECM [1]. A recent elegant study has demonstrated that collagen cross-linking leads to cancer progression by enhancing ECM receptor integrin signaling [95, 96]. However, the functional contribution of collagen cross-linking to noncancer pathogenesis remains largely unknown. The LOX family enzymes are copper-dependent amine oxidase and catalyze the post-translational modification of peptidyl lysine to the peptidyl aldehyde, α-aminoadipic-δsemialdehyde [97]. This chemical change enables the covalent cross-linking of collagen and elastin, resulting in insolubilizing and stabilizing ECM proteins. Collagen cross-linking stiffens the ECM and is accompanied by tissue/organ fibrosis that is mediated by several profibrogenic cytokines [95, 98]. Indeed, LOX inhibitor β-aminopropionitrile (BAPN) reduces organ stiffness following injury and TGF-β1-induced collagen fibril stiffness *in vitro* [65, 99]. Lines of study show that LOX localizes in ECM of several tissue such as skin, aorta, heart, lung, liver, and cartilage [97]. LOX is secreted as inactive proenzyme (proLOX) and then proteolytically cleaved to active enzyme. *In vitro* study shows the possibility that the activation of proLOX occurs on the cell surface in a complex with cellular fibronectin. Indeed, LOX colocalizes well with assembled fibronectin fibrils in cultured fibroblasts and normal human tissues [100]. Furthermore, fibronectin-null mouse embryonic fibroblasts exhibit drastic decrease of the proteolytic processing of proLOX [100], strongly suggesting that fibronectin matrix regulates ECM stiffness via LOX activation.

Small leucine-rich proteoglycans/proteins (SLRPs) such as decorin, biglycan, and fibromodu‐ lin are known to contribute significantly to collagen assembly. SLRPs consist of five classes, and almost all SLRPs bind collagen fibrils through their leucine-rich repeat domain [101–103]. Accumulating evidence using SLRP-null mouse models shows that SLRPs regulate the diameter and/or alter structure of collagen fibrils [101–103]. For example, decorin knockout mouse skin exhibits a broader range of collagen fibril diameters [104], and treatment of decorin with decorin-null skin fibroblasts results in the formation of more uniform collagen fibrils [105]. Fibromodulin-deficient tail tendons exhibit thinner collagen fibrils compared to wildtype controls [106, 107]. A dynamic modulus in biglycan-null tendons is significantly increased compared to wild-type tendons [108]. The elasticity of collagen fiber networks in cultured decorin-siRNA-transfected mouse NIH3T3 fibroblasts is declined during the incubation period, whereas it remains unchanged in untransfected cells. It is therefore likely that SLRPs could regulate the physiological properties of ECM (e.g., mechanical strength).
