**4. Microenvironment-regulating DR expansion and differentiation**

The literature brings every day new evidence that the orchestrated interplay between proliferating hepatocytes or cholangiocytes, extracellular matrix-producing myofibroblasts, inflammatory cells (such as macrophages, neutrophils, or lymphocytes), and endothelial cells is pivotal in the regulation of DR expansion and differentiation. We will thus compare the microenvironment accompanying DR in the CDE versus DDC model.

#### **4.1. Extracellular matrix, collagen, and laminin**

Extracellular matrix and collagen deposition associates with DR. In the CDE model, a thin and loose web of collagen fibers is associated with invading DR cells, while collagens in DDC livers thicken the portal mesenchyme and abundant extracellular matrix accumulates in clots or thick concentric layers around the neo-formed pseudo-ductular structures (**Figure 2A**–**D**).

The localization of myofibroblasts, the cells chiefly involved in matrix synthesis and remodeling, adopted a pattern similar to that of the collagen deposition in both models, meaning that DR is at all times associated with myofibroblasts. In CDE livers, myofibroblasts chaperone the DR cells while they penetrate deep into the liver lobule (**Figure 2E**). Conversely, in the DDC model, myofibroblasts densely populate the portal mesenchyme and accumulate rather concentrically around DR (**Figure 2F**).

Laminin is a component of the basal membrane delineating the basal pole of cholangiocytes. Basement membrane is essential to establish the cholangiocytes polarity and to support a tubular structure with a lumen [39]. By contrast, hepatocytes do not lie on a basement membrane. In the CDE model, DR is anchored onto a laminin-rich basal membrane intermingled with collagen. This layer of laminin has been proposed to maintain the immature/biliary phenotype of DR cells and to provide a holding structure facilitating migration of DR into the lobular parenchyma in the CDE model [28, 40]. Moreover, decreased density of laminin and extracellular matrix in CDE livers is associated with enhanced hepatocytic differentiation of DR cells [28]. Indeed, during DR differentiation process, DR cells progressively lose contact with the laminin-rich basement. And when animals are reversed to a standard chow (supply of choline and cessation of ethionine administration) after CDE exposure, the injury reverses, DR, extracellular matrix, and laminin deposition progressively lessen, and concomitantly the number of DR-derived hepatocytes increases.

In DDC livers, laminin deposits as thin basal membrane outlining the DR in a pattern similar to that seen around normal bile ducts, with collagen stacked as separate sheets encircling newly formed DR.

#### **4.2. Inflammatory environment**

In response to liver injury, Kupffer cells, the hepatic macrophages, activate and participate to the recruitment of the inflammatory reaction. In CDE livers, enlarged and proliferative Kupffer cells are strongly associated with invading DR within the parenchyma while no portal inflammation is observed [41]. DDC-induced proliferation of the ducts is accompanied by a dense macrophage and neutrophil granulocytic infiltrate around small and larger bile ducts, further supporting that biliary structures are first concerned by the injurious and healing responses in this model [16].

biliary specification via the Notch pathway. While during hepatocyte regeneration, macrophage-derived canonical Wnt signaling maintains Numb within LPC and Notch signaling is

**Figure 2.** Comparison of the extracellular matrix deposition and the myofibroblast expansion between the CDE and DDC models. Liver sections obtained from mice receiving the CDE (A, C, E) or DDC diet (B, D, F) were stained with Sirius red to highlight fibrillary collagen (A-D), or αSMA and CK19 expression (E and F). In CDE livers, a collagen meshwork covers the whole parenchyma (A) with fibers elongating from the portal area into the lobule (C). In DDC livers, collagen fibers accumulate around the portal area to shape the portal mesenchyma (D), delimiting the boundaries of DR (F). At lower magnification, portal-portal bridging is observed (B). αSMA+ myofibroblasts show a distribution pattern similar as the collagen deposition. αSMA+ myofibroblasts infiltrate the lobule, chaperoning CK19+ DR cells in the CDE model (E), while in the DDC model, αSMA+ myofibroblasts rather accumulate concentrically around the DR structures (F).

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Experiments performed in lymphocyte-deficient mice fed on CDE suggest that natural killer cells and T-cells participate also to LPC expansion, presumably through their proinflammatory cytokine production [43]. Moreover, TNF-like weak inducer of apoptosis (TWEAK), produced

reduced, promoting hepatocyte specification [42].

Kupffer cells do not influence DR expansion but modulate its invasive behavior and its specification, through modulation of the density of extracellular matrix as well as via Notch and Wnt signaling pathways [41, 42]. Numb, a direct transcriptional target of Wnt and a negative regulator of Notch, is downregulated in LPC during biliary regeneration, promoting Relevance of the CDE and DDC Mouse Models to Study Ductular Reaction in Chronic Human... http://dx.doi.org/10.5772/intechopen.69533 151

**4.1. Extracellular matrix, collagen, and laminin**

150 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

centrically around DR (**Figure 2F**).

hepatocytes increases.

**4.2. Inflammatory environment**

responses in this model [16].

newly formed DR.

Extracellular matrix and collagen deposition associates with DR. In the CDE model, a thin and loose web of collagen fibers is associated with invading DR cells, while collagens in DDC livers thicken the portal mesenchyme and abundant extracellular matrix accumulates in clots or thick concentric layers around the neo-formed pseudo-ductular structures (**Figure 2A**–**D**). The localization of myofibroblasts, the cells chiefly involved in matrix synthesis and remodeling, adopted a pattern similar to that of the collagen deposition in both models, meaning that DR is at all times associated with myofibroblasts. In CDE livers, myofibroblasts chaperone the DR cells while they penetrate deep into the liver lobule (**Figure 2E**). Conversely, in the DDC model, myofibroblasts densely populate the portal mesenchyme and accumulate rather con-

Laminin is a component of the basal membrane delineating the basal pole of cholangiocytes. Basement membrane is essential to establish the cholangiocytes polarity and to support a tubular structure with a lumen [39]. By contrast, hepatocytes do not lie on a basement membrane. In the CDE model, DR is anchored onto a laminin-rich basal membrane intermingled with collagen. This layer of laminin has been proposed to maintain the immature/biliary phenotype of DR cells and to provide a holding structure facilitating migration of DR into the lobular parenchyma in the CDE model [28, 40]. Moreover, decreased density of laminin and extracellular matrix in CDE livers is associated with enhanced hepatocytic differentiation of DR cells [28]. Indeed, during DR differentiation process, DR cells progressively lose contact with the laminin-rich basement. And when animals are reversed to a standard chow (supply of choline and cessation of ethionine administration) after CDE exposure, the injury reverses, DR, extracellular matrix, and laminin deposition progressively lessen, and concomitantly the number of DR-derived

In DDC livers, laminin deposits as thin basal membrane outlining the DR in a pattern similar to that seen around normal bile ducts, with collagen stacked as separate sheets encircling

In response to liver injury, Kupffer cells, the hepatic macrophages, activate and participate to the recruitment of the inflammatory reaction. In CDE livers, enlarged and proliferative Kupffer cells are strongly associated with invading DR within the parenchyma while no portal inflammation is observed [41]. DDC-induced proliferation of the ducts is accompanied by a dense macrophage and neutrophil granulocytic infiltrate around small and larger bile ducts, further supporting that biliary structures are first concerned by the injurious and healing

Kupffer cells do not influence DR expansion but modulate its invasive behavior and its specification, through modulation of the density of extracellular matrix as well as via Notch and Wnt signaling pathways [41, 42]. Numb, a direct transcriptional target of Wnt and a negative regulator of Notch, is downregulated in LPC during biliary regeneration, promoting

**Figure 2.** Comparison of the extracellular matrix deposition and the myofibroblast expansion between the CDE and DDC models. Liver sections obtained from mice receiving the CDE (A, C, E) or DDC diet (B, D, F) were stained with Sirius red to highlight fibrillary collagen (A-D), or αSMA and CK19 expression (E and F). In CDE livers, a collagen meshwork covers the whole parenchyma (A) with fibers elongating from the portal area into the lobule (C). In DDC livers, collagen fibers accumulate around the portal area to shape the portal mesenchyma (D), delimiting the boundaries of DR (F). At lower magnification, portal-portal bridging is observed (B). αSMA+ myofibroblasts show a distribution pattern similar as the collagen deposition. αSMA+ myofibroblasts infiltrate the lobule, chaperoning CK19+ DR cells in the CDE model (E), while in the DDC model, αSMA+ myofibroblasts rather accumulate concentrically around the DR structures (F).

biliary specification via the Notch pathway. While during hepatocyte regeneration, macrophage-derived canonical Wnt signaling maintains Numb within LPC and Notch signaling is reduced, promoting hepatocyte specification [42].

Experiments performed in lymphocyte-deficient mice fed on CDE suggest that natural killer cells and T-cells participate also to LPC expansion, presumably through their proinflammatory cytokine production [43]. Moreover, TNF-like weak inducer of apoptosis (TWEAK), produced by T-cells and activating its receptor fibroblast growth factor-inducible 14 (Fn14), is suggested to be an exclusive LPC mitogen. After both CDE and DDC treatment on Fn14 knockout mice, a significant reduction of the LPC response is observed [31, 44].

to LPC fate, the observation of a phenotypic continuum between DR cells and hepatocytes in the livers of patients suffering from HCV supports differentiation of LPC toward hepatocytes [4, 50]. Besides hepatitis, the CDE diet also recapitulates features of the DR associated with lipid accumulation (steatosis) as in chronic alcoholic and nonalcoholic fatty liver diseases [11, 29, 42, 53].

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The DR pattern seen in DDC livers is more comparable to that of chronic fibrosing cholangitis such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) with DR proliferation restricted within the portal area and accompanied by concentric periportal fibrosis (**Figure 3E** and **F**). In these diseases as in DDC, the primary damage is directed toward cholangiocytes. Intrahepatic bile duct destruction and ductopenia seen in advanced PBC and the fibrous obliterative lesions of PSC do not occur in the DDC model, a phenomenon most likely related to

**Figure 3.** DR observed in human chronic hepatitis C infection and in primary sclerosing cholangitis. Liver stained with anticytokeratin 7 (CK7) and Masson-trichrome of an HCV case with mild inflammation (A and B), AIH with moderate inflammation (C and D), and PSC with cholangitis, edema, and portal fibrosis (E and F). HCV- and AIH-associated CK7+ DRs (A and C) have only a vague or no lumen and comprise and elongated cells with little cytoplasm extending in the periportal parenchyma and associated with dense collagen fibbers (B and D). While the CK7+ ductular proliferation seen

in PSC (E) is enclosed in portal mesenchyma and concentric periductular fibrosis occurs (F).

the specific immune component of PBC and PSC which is lacking in the DDC model.

#### **4.3. Nearby endothelial cells**

As described above, DR requires a typical niche provided by extracellular matrix-producing and inflammatory cells, which are located in the sinusoids closely adjacent to DR. Additionally, sinusoidal endothelial cells themselves could also have an important role in regulating DR. Signaling molecules specifically expressed within the endothelial compartment of the central vein have been shown to have a crucial role in liver zonation [45]. Moreover, in another model of liver injury, hepatocytes divide along the closest microvessel as order principle to restore liver architecture [46]. Either a signaling or a guiding role of endothelial cells on LPC response could be envisaged. However, so far, no experiments have been done to study endothelial regulation of DR in the CDE or DDC model.
