CHAPTER 58
Hepatic fibrosis and cirrhosis

Don C. Rockey

Digestive Disease Research Center, Department of Internal Medicine, Medical University of South Carolina, Charleston, SC, USA,

This chapter provides additional images of interest in the area of liver fibrosis, and includes images spanning basic science to clinical concepts.

The cellular basis of hepatic fibrosis

One of the most important issues in the field relates to the cellular source of extracellular matrix (i.e., fibrosis) in the liver. These so‐called “effector” cells are important on a number of levels, but perhaps most importantly because they are a potential target for therapeutic intervention in the treatment of fibrosis.

A number of effector cells have been identified and have been advanced as critical to the fibrogenic response; these include (1) portal fibroblasts, (2) fibrocytes (derived from the bone marrow), (3) mesenchymal cells derived from hepatocytes through epithelial–mesenchymal transition (EMT), and (4) hepatic stellate cells (Figure 58.1). Considerable evidence suggests that portal fibroblasts contribute to fibrogenesis in the liver. Greater controversy exists as to the importance of bone marrow‐derived cells and cells arising from EMT in hepatic fibrogenesis. A number of studies have demonstrated through lineage tracing that hepatic stellate cells are the predominant effectors of fibrogenesis in the injured liver.

Portal fibroblasts

Portal fibroblasts (Figure 58.2) appear to be most prominent in liver diseases with portal‐based injury such as primary biliary cirrhosis, sclerosing cholangitis, or sarcoidosis. In experimental models, bile duct obstruction in particular leads to proliferation of portal fibroblasts.

Fibrocytes

The role of fibrocytes (Figure 58.3) in liver fibrogenesis is controversial. Available data clearly indicate that these CD45‐positive, collagen I‐producing cells migrate to the liver after injury, where they appear in small numbers. While they do not appear to be a major matrix‐producing cell in the liver, they appear to play a role in stimulation of stellate cell activation via inflammatory pathways.

Stellate cells

Stellate cells, residing in the sinusoidal space of Disse (physically located between the sinusoidal endothelial cell and the hepatocyte) (Figure 58.4), make up approximately 6% of the cells found in the liver (Figure 58.5). A remarkable feature of hepatic stellate cells is that they are rich in retinoid and lipids, and under normal circumstances appear to be responsible for significant storage of retinoid in the liver.

The rich retinoid and lipid content (Figures 58.8 and 58.9; see also Figure 58.6) of hepatic stellate cells is one of their most notable features. It helps identify them in cultures that have been developed from contemporary cell isolations. The lipid droplets in stellate cells readily take up oil red O. This diazo dye, used for staining of neutral triglycerides and lipids, can be seen in abundance in a perinuclear fashion in quiescent stellate cells (i.e., those soon after isolation). After stellate cells are grown in culture and become activated, the size of lipid droplets declines significantly (see Figures 58.6 and 58.9).

Photo depicts cellular sources of matrix in liver wound healing. — **Figure 58.1** Cellular sources of matrix in liver wound healing. Shown are characteristic cells that may transition to myofibroblast‐like matrix producing cells.

Source: Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 2008;88:125. Reproduced with permission of The American Physiological Society.

Photo depicts myofibroblasts and fibroblasts are present in the sclerosing portal fields, but not in the sinusoidal interstitium of human primary sclerosing cholangitis. — **Figure 58.2** Myofibroblasts and fibroblasts are present in the sclerosing portal fields, but not in the sinusoidal interstitium of human primary sclerosing cholangitis. Immunohistochemistry to detect smooth muscle α‐actin **(a)**, CD45 **(b).** and S100A4 **(c)**; **(d)** depicts picrosirius red staining, all on serial liver sections. Seen are smooth muscle α‐actin‐positive myofibroblasts **(a)** and infiltration of CD45‐positive **(b)** and S100A4‐positive **(c)** cells in the sclerosing areas around the injured bile ducts (black arrows). Smooth muscle α‐actin is also found in smooth muscle cells of the portal veins and the hepatic artery (white arrow). The picrosirius red staining **(d)** shows collagen around injured bile ducts.

Source: Strack I., Schulte S., Varnholt H., et al. β‐Adrenoceptor blockade in sclerosing cholangitis of Mdr2 knockout mice: antifibrotic effects in a model of nonsinusoidal fibrosis. Lab Invest 2011;91:252. Reproduced with permission of Nature Publishing Group.

Schematic illustration of fibrocytes from the bone marrow migrate to the liver. — **Figure 58.3** Fibrocytes from the bone marrow migrate to the liver. There, these cells transform into hepatic myofibroblasts. Although the role of mesenchymal stem cells in liver fibrosis is not well characterized due to the lack of specific markers and difficulties with their isolation, hematopoietic stem cells contribute to hepatic fibrocytes in response to liver injury. HSC, hepatic stellate cell.

Source: Kisseleva T., Brenner D.A. The phenotypic fate and functional role for bone marrow‐derived stem cells in liver fibrosis. J Hepatol 2013;56:965. Reproduced with permission of Elsevier.

Photo depicts hepatic stellate cells, residing in the sinusoidal space of Disse. — **Figure 58.4** Hepatic stellate cells, residing in the sinusoidal space of Disse. Backscattered scanning electron microscopic images of Golgi‐stained extensions of stellate cells that surround sinusoids in periportal (left) and centrolobular (right) zones of porcine liver. The former are thick and smooth, while the latter are bifurcated and more spiked in appearance.

Source: Wake K., Sato T. Cell Tissue Res 1993;273(2):227–237. Reproduced with permission of Springer Nature.

Photo depicts stellate cells in normal liver. — **Figure 58.5** Stellate cells in normal liver. Normal mouse liver was snap‐frozen and hepatic stellate cells were labeled with polyclonal antidesmin antibody, which was then detected with secondary antibody labeled with Alexa‐488. In **(a)**, smooth muscle cells are visualized in the portal tract (right upper portion of the figure) and in part of the central vein (left upper portion of the section. Stellate cells are seen labeled throughout the lobule. In **(b)**, labeled stellate cells are labeled throughout the liver lobule.

Source: Courtesy of Dr Songling Liu.

Table 58.1 Stellate cell characteristics.

	Quiescent	Activated
Physical properties
Size, shape, location	Small, close association with endothelium	Large, found in various locations, including fibrotic bands
Cytoplasm	Rich in retinoid, lipid droplets	Robust endoplasmic reticulum and Golgi, consistent with substantial protein synthesis, reduced retinoid content
Cytoskeleton	Intermediate filaments, especially desmin, present	Extensive intermediate filaments, robust actin cytoskeleton (including the smooth muscle isoform of actin), upregulation of focal adhesions
Functional properties
Fibrogenesis	Minimal	Extensive
Contractility	Minimal	Extensive, a result of robust smooth muscle protein machinery
Motility	Minimal	Extensive, a result of robust cytoskeletal machinery
Proliferation	Modest	Extensive
Other	Active retinoid metabolism	Reduced retinoid metabolism

Photo depicts quiescent and activated stellate cells. — **Figure 58.6** Quiescent and activated stellate cells. **(a)** Hepatic stellate cells isolated from normal rat livers, and grown for 1 day. They appear compact, with refractile, lipid droplets readily visible in a perinuclear fashion. **(b)** Cells from the same cell isolation, but grown in culture in medium containing serum for 5 days. These activated cells have lost significant amounts of their lipid droplets, have grown in size with the development of long cytoplasmic extensions, and have developed large nuclei.

Stellate cells are now accepted as the primary extracellular matrix (i.e., fibrosis)‐producing cell in the injured liver. They are found in much larger numbers proportionally than are the other putative fibrogenic cells as highlighted above. After essentially any form of liver injury, they become activated; in this process, they undergo a number of striking morphological changes (Table 58.1, Figures 58.6 and 58.7). These include the development of an extensive endoplasmic reticulum, the development of an intricate network of stress fibers, focal adhesions, and a remarkably robust actin cytoskeleton (see also below).

Stellate cells also have robust cytoskeletal features, especially after they become activated. They are enriched with a variety of intermediate filaments, including desmin, vimentin, and others (Figure 58.10). One of the classic features of stellate cell activation is the upregulation of the smooth muscle isoform of actin (Acta2), indicating that they represent liver‐specific myofibroblasts (Figure 58.11). The robust cytoskeleton typical of stellate cells further extends to other components; for example, cell‐matrix attachments such as focal adhesions are prominent. Stellate cells exhibit remarkable expression of vinculin (Figure 58.12), talin, paxillin, and focal adhesion kinase (FAK), to name a few.

Stellate cell activation is associated with a number of remarkable functional attributes (see Figure 58.7). Prominent among these functions is enhanced motility (Figure 58.13). Stellate cell motility is likely to be important as stellate cells move to certain parts of the liver (e.g., stellate cells likely home to areas of increased injury and inflammation and are often localized in areas of abundant ECM expression).

In addition to enhanced motility, activated stellate cells exhibit a remarkable contractile phenotype (Figure 58.14

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