**3. Possible mechanisms of collagen network formation**

As described in Section 2.1, it has been believed that collagen-network formation depends on the fibronectin matrix in culture [12, 13]. Indeed, the prominent expression of fibronectin is observed during adult tissue repair [11, 109]. Another line of evidence shows that TGF-β plays a central role as a profibrogenic cytokine in the accumulation of ECMs, including fibronectin. Therefore, it would be possible that TGF-β-induced ECM accumulation is dependent on fibronectin. However, to date, fibronectin/TGF-β interdependence in the fibrogenic response to tissue damage has not yet been addressed. Furthermore, it remains to be elucidated how ECM remodeling by myofibroblasts results in changes in mechanical tension and supports the activation of pathogenic signaling pathways during the development of chronic fibrotic diseases. We hypothesized that the removal of fibronectin or TGF-β signaling *in vivo* could prevent extensive ECM network formation following tissue damage. To define the functional identity of fibronectin and TGF-β signaling in adult tissue remodeling, we recently established two animal models lacking fibronectin (both isoforms) or TRII, respectively, in adult liver [14, 62]. Our new findings suggest fibronectin-/TGF-β-independent mechanisms are involved in the development of liver fibrosis.

#### **3.1. Fibronectin-dependent assembly**

#### *3.1.1. Fibronectin matrix assembly*

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

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

**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

as a drug target for liver fibrosis.

ECM stiffness via LOX activation.

Fibronectin matrix assembly consists of multistep process (**Figure 1**) [110–113]. Importantly, fibronectin assembly is cell dependent; binding of fibronectin to cell surface and cellular contractility are required. In the first step, fibronectin binds to cell surface, and integrin plays an important role. Integrins α5β1 and αvβ3 are characterized as fibronectin receptors [114]. Lines of evidence show that α5β1 is a primary receptor in fibronectin matrix assembly, whereas αvβ3 dominates the formation of focal contacts [115–117]. However, the binding of fibronectin to the cell is not sufficient for fibronectin assembly. A critical step of this assembly is considered to be the cell-driven exposure of cryptic site for self-association in fibronectin. Although one mechanism for exposing the cryptic site could be conformational changes induced by fibro‐ nectin binding to integrin, cellular contractility is necessary for fibronectin fibrillogenesis [88, 111, 118]. Indeed, loss of cellular contractility by RhoA inhibitor prevents fibronectin matrix formation [119]. Furthermore, recent study demonstrates that β1 cytoplasmic domain modu‐ lates fibronectin assembly via recruitment of cytoplasmic adaptor protein talin, which links integrin to the actin cytoskeleton [120, 121]. Thus, integrin-mediated association of fibronectin with cytoskeleton is important for fibronectin assembly.

**Figure 1.** Proposed mechanism of fibronectin-dependent collagen assembly. (A) Type I collagen assembly with fibro‐ nectin. (B) Fibronectin assembly. ①Fibronectin binding to activated integrin; ②conformational changes of fibronectin cryptic site by cellular contraction; ③exposure of fibronectin cryptic site, resulting in acquiring ability to associate with other fibronectin molecules; and ④formation and extension of fibronectin fibril assembly. (C) Fibronectin structure. See details in the text.

#### *3.1.2. Collagen assembly with fibronectin matrix*

Fibronectin contains collagen-binding domain [122] and directly binds to collagens. *In vitro* studies have shown an extensive codistribution of fibronectin and Col I/III (**Figure 1**) [12, 123]. A very recent study using fibroblasts demonstrates that type I collagen fibrils preferen‐ tially colocalize with more-relaxed fibronectin fibrils in the ECM *in vitro* [124]. Fibronectin-null embryonic fibroblastic cells cannot organize collagen fibril networks *in vitro*, but they can form collagen networks when fibronectin is exogenously added [123]. Interestingly, collagenbinding integrins α2β1 and α11β1 are not required for collagen polymerization in fibronectinnull embryonic fibroblastic cells when cultured with exogenous fibronectin [12]. Furthermore, integrin β1-null line GD25 cells, which express fibronectin and its receptor αvβ3, contract collagen gel more forcefully than integrin α2- and β1-transfected GD25 cells that contract collagen gel via α2β1 but not αvβ3 [125]. Other studies show that fibronectin polymerization stimulates collagen gel contraction [126] and that the disruption of fibronectin–collagen association inhibits this contraction [13]. These findings demonstrate that fibronectin matrix is required for collagen assembly and enhances ECM contraction of cells.

### *3.1.3. Initial incorporation of latent TGF-β complex*

to be the cell-driven exposure of cryptic site for self-association in fibronectin. Although one mechanism for exposing the cryptic site could be conformational changes induced by fibro‐ nectin binding to integrin, cellular contractility is necessary for fibronectin fibrillogenesis [88, 111, 118]. Indeed, loss of cellular contractility by RhoA inhibitor prevents fibronectin matrix formation [119]. Furthermore, recent study demonstrates that β1 cytoplasmic domain modu‐ lates fibronectin assembly via recruitment of cytoplasmic adaptor protein talin, which links integrin to the actin cytoskeleton [120, 121]. Thus, integrin-mediated association of fibronectin

**Figure 1.** Proposed mechanism of fibronectin-dependent collagen assembly. (A) Type I collagen assembly with fibro‐ nectin. (B) Fibronectin assembly. ①Fibronectin binding to activated integrin; ②conformational changes of fibronectin cryptic site by cellular contraction; ③exposure of fibronectin cryptic site, resulting in acquiring ability to associate with other fibronectin molecules; and ④formation and extension of fibronectin fibril assembly. (C) Fibronectin structure.

Fibronectin contains collagen-binding domain [122] and directly binds to collagens. *In vitro* studies have shown an extensive codistribution of fibronectin and Col I/III (**Figure 1**) [12,

with cytoskeleton is important for fibronectin assembly.

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

See details in the text.

*3.1.2. Collagen assembly with fibronectin matrix*

Growing evidence suggests a mechanism by which fibronectin plays a role in the initial incorporation of latent TGF-β complex into ECM [18, 33]. LTBP-1 associates with not only fibrillin-1 but also fibronectin [33, 36]. Fibronectin-null fibroblasts fail to incorporate LTBP into ECM [33], and minimally activate latent TGF-β [127]. Furthermore, cells lacking fibronectin receptor integrin α5β1 show defective activity of latent TGF-β by αvβ6 [127]. These findings suggest that fibronectin regulates latent TGF-β activation via deposition of latent TGF-β into the matrix.

#### **3.2. Fibronectin-independent collagen assembly**

As described above, based on *in vitro* findings, it has been postulated that collagen network organization and assembly depends on the fibronectin matrix in culture [12, 13]. However, the contribution of fibronectin to these processes remains to be defined *in vivo*. We therefore investigated whether fibronectin is a suitable molecular target for ameliorating the fibrogenic response to liver injury. Since mice with complete inactivation of fibronectin gene die at an early embryonic stage [10], we generated conditional fibronectin-floxed and liver-specific adult fibronectin-null mice (lacking both plasma and cellular isoforms of fibronectin) using *Cre-loxP* technology [11, 14], investigated their phenotypes, and have demonstrated fibronec‐ tin-independent mechanisms for collagen network formation following liver injury.

#### *3.2.1. TGF-β and Col V-mediated collagen assembly in fibronectin-null liver*

The adult mouse model lacking fibronectin shows no abnormalities in anatomical and histological analyses of the liver and hepatic biochemical markers under standard laboratory conditions. Unexpectedly, the lack of fibronectin did not interfere with the reconstruction of collagen fibril organization in response to both acute liver and chronic liver injuries up to 8 weeks induced by CCl4 [14]. Fibronectin-null livers show significant increased HSC activation with elevated Smad signaling following injury. To determine whether TGF-β is involved in Col III/I collagen network formation in the absence of fibronectin, we further assessed which factors that regulate activated HSC phenotypes were involved in collagen fibrillogenesis. We have identified TGF-β1-induced Col V as a novel and essential element for Col I/III fibrogenesis in hepatic stellate cells (**Figure 2**). Thus, our study provides compelling evidence that collagen fibrillogenesis in response to adult tissue/organ damage is mediated by both fibronectin and type V collagen.

**Figure 2.** Proposed mechanism of fibronectin-independent collagen assembly mediated by tissue growth factor beta (TGF-β) signaling and type V collagen (Col V). (A) Type I collagen assembly with Col V. (B) Pro-α1(V) collagen struc‐ ture. PARP, proline- and arginine-rich domain; VAR, variable domain. (C) Local TGF-β activation. See details in the text.

Interestingly, type V collagen–mediated Col III/I fibril assembly following liver injury seems to be specific for adult HSCs because TGF-β1 supports neither Col V nor Col III/I fibril assembly in fibronectin-null embryonic fibroblasts *in vitro* [14]. Furthermore, fibronectin-null livers show substantial depositions of LTBP-1, -3, and -4 with fibrillar structures in the ECM following both acute and chronic liver injuries [14, 65], whereas fibronectin-null embryonic fibroblasts fail to incorporate LTBP-1 into ECM *in vitro* [33]. Therefore, further studies for mechanisms underlying Col V-mediated collagen network formation remain to be elucidated, i.e., the phenotypic differences among myofibroblasts, and the contribution to adult tissue/ organ remodeling following injury.

#### *3.2.2. Elevated collagen matrix stiffness in advanced stage of liver fibrosis/cirrhosis in fibronectin-null liver*

We showed that collagen network organization can be formed even without fibronectin in response to liver injury [14]. However, it remains unknown whether the initial deposition of fibronectin could contribute to the turning point from normal healing to chronic fibrotic disorders. Furthermore, it remains to be elucidated how ECM remodeling by myofibroblasts affects mechanical tension and supports the activation of pathogenic signaling during the development of chronic fibrotic diseases. We therefore have further investigated whether fibronectin could be a suitable target for ameliorating fibrosis during advanced stages of chronic liver injury [65]. Fibronectin-null livers have exhibited constitutively elevated local TGF-β activity, induced more myofibroblast phenotypes, and accumulated highly disorgan‐ ized/diffused collagenous ECM networks during chronic liver fibrogenesis induced by CCl4. The deposition and network formation of Col V are also significantly increased. Consequently, fibronectin-null livers have led to more extensive liver cirrhosis, which is accompanied by significant increased liver matrix stiffness and deteriorated hepatic functions. Mechanistically, fibronectin-null livers have shown elevated LOX expressions, and a significant amount of active LOX is released in fibronectin-null hepatic stellate cells in response to TGF-β1. Further‐ more, treatment of fibronectin in fibronectin-null stellate cells recovers collagen fibril stiffness to wild-type levels [65].

We propose that there are the functional links between fibronectin-mediated control of TGFβ bioavailability and collagen fibril stiffness regulated by LOX. All these novel findings strongly suggest that locally activated TGF-β signaling and Col V are essential elements for collagen fibrogenesis without fibronectin in adult tissue remodeling. Although the contribu‐ tion of Col V-nucleated Col I fibrogenesis in adult tissues is largely undetermined, our finding raises the hypothesis that the accumulation of Col V-mediated ECMs during persistent chronic damage could influence a formation of disorganized ECM architecture.

### **3.3. TGF-β-independent collagen assembly**

## *3.3.1. CTGF-mediated collagen assembly*

have identified TGF-β1-induced Col V as a novel and essential element for Col I/III fibrogenesis in hepatic stellate cells (**Figure 2**). Thus, our study provides compelling evidence that collagen fibrillogenesis in response to adult tissue/organ damage is mediated by both fibronectin and

**Figure 2.** Proposed mechanism of fibronectin-independent collagen assembly mediated by tissue growth factor beta (TGF-β) signaling and type V collagen (Col V). (A) Type I collagen assembly with Col V. (B) Pro-α1(V) collagen struc‐ ture. PARP, proline- and arginine-rich domain; VAR, variable domain. (C) Local TGF-β activation. See details in the

Interestingly, type V collagen–mediated Col III/I fibril assembly following liver injury seems to be specific for adult HSCs because TGF-β1 supports neither Col V nor Col III/I fibril assembly in fibronectin-null embryonic fibroblasts *in vitro* [14]. Furthermore, fibronectin-null livers show substantial depositions of LTBP-1, -3, and -4 with fibrillar structures in the ECM following both acute and chronic liver injuries [14, 65], whereas fibronectin-null embryonic fibroblasts fail to incorporate LTBP-1 into ECM *in vitro* [33]. Therefore, further studies for mechanisms underlying Col V-mediated collagen network formation remain to be elucidated, i.e., the phenotypic differences among myofibroblasts, and the contribution to adult tissue/

*3.2.2. Elevated collagen matrix stiffness in advanced stage of liver fibrosis/cirrhosis in fibronectin-null*

We showed that collagen network organization can be formed even without fibronectin in response to liver injury [14]. However, it remains unknown whether the initial deposition of

type V collagen.

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

text.

*liver*

organ remodeling following injury.

TGF-β signaling is the dominant pathway of ECM productions in HSCs [58]. To address whether the elimination of this signaling is sufficient to prevent liver fibrosis, we have generated adult liver-specific TRII-null mice (TGFβIIR[flox/flox]/Mx-Cre+) [62]. Actually, this mutant liver exhibits a significant decrease of ECM deposition and α-SMA expression in CCl4-induced chronic liver injury. However, elimination of TRII does not completely prevent the collagen accumulation in chronic liver injury, and TRII-null livers still remain ~46.4% fibrosis compared to wild type. Furthermore, we have found that matricellular protein CTGF/ CCN2 expression is significantly upregulated (1.94-fold compared to wild type) in mutant livers following chronic liver injury. Therefore, these findings clearly indicate that TGF-βindependent mechanisms play an alternative role in developing liver fibrosis. Since CTGF/ CCN2 synergizes with the action of TGF-β, CTGF/CCN2 is considered to act as a TGF-β downstream modulator [128]. Accompanied by the elevated TGF-β activity, CTGF/CCN2 expression is upregulated in several fibrotic tissues, including kidney, lung, heart, liver, pancreas, bowel, and skin [128, 129]. A recent study has demonstrated that overexpression of CTGF/CCN2 in fibroblasts alone is sufficient to cause spontaneous multiorgan fibrosis *in vivo* and that this signal pathway does not involve canonical TGF-β-Smad signaling *in vitro*. However, the liver does not show significant fibrosis [130]. We have found that TRII-null livers in chronic injury show elevated expression of CTGF/CCN2 in spite of a lack of TGF-β signaling, indicating CTGF/CCN2 as a potent mediator in liver fibrosis [62]. We propose two hits, induction of CTGF/CCN2 and adult tissue/organ injury, for the progression of liver fibrosis. In fibrotic livers, CTGF/CCN2 is known to be synthesized in a diversity of cells such as hepatocytes, myofibroblasts (activated HSCs), endothelial cells, proliferating bile duct epithelial cells, and inflammatory cells [128]. Thus, it remains to be elucidated to identify cellular contribution and mechanisms underlying CTGF/CCN2 production in the progression of tissue/organ fibrosis.
