Stellate Cells in Health and Disease

Stellate Cells in Health and Disease is a comprehensive reference providing the most up-to-date knowledge and perspectives on the function of stellate cells affecting the liver and other organs.

The text presents comprehensive coverage of their already established role in hepatic fibrosis along with the newer emerging evidence for stellate cell participation in the liver cell (hepatocyte) survival and regeneration, hepatic immunobiology, transplant tolerance, and liver cancer.

Chapters describe both animal and human research and the relevance of findings from animal research to human pathophysiology, and also contain sections on future directions which will be of special interest to basic and clinical researchers working on liver fibrosis, hepatic biology, and pathobiology.

• Presents coverage of the mechanisms of liver fibrosis with stellate cells as a target for therapy.
• Shows stellate cells as a major participant in hepatic immunobiology, including transplantation immunology.
• Key illustrations show the phenotypical changes in stellate cells in situ and tissue culture, their interactions with other cell types, signaling pathways and demonstrate the functions and roles of stellate cell in pathological processes.

• Language: English
• Publisher: Academic Press
• Release date: Apr 10, 2015
• ISBN: 9780128005446

Book preview

56:532–543.

Preface

Chandrashekhar Gandhi and Massimo Pinzani

One and a half centuries ago von Kupffer identified an interesting group of cells that he initially called perivascular cell4/7/20154/7/2015s of the connective tissue. These were subsequently identified as fat-storing cells or stellate cells. The field of stellate cells has expanded exponentially in the last four decades, primarily due to increasing evidence that they are the major cell type to cause liver fibrosis. Thus, hepatic stellate cells have become synonymous with liver fibrosis and targeting them is a realistic approach to treating fibrosis and cirrhosis of the liver.

Overwhelming interest in stellate cells research is evident with nearly 10,000 research articles including nearly 1,000 reviews (and several book chapters) published to date. From about 100 research articles before the 1970s, the number of publications quadrupled in the next decade and has consistently doubled in each of the next three decades. Another 2,000 articles have been published since 2011. With initial interest as a major cell type of storage and metabolism of retinoids (including vitamin A), and subsequently for its function in hepatic fibrosis, stellate cells continued to guard its secrets in regard to its role in other facets of hepatic pathophysiology. Emerging evidence in the last 15 years has revealed its role in liver cell (hepatocyte) survival and regeneration, hepatic inflammation, and immunobiology including transplantation tolerance and hepatocellular carcinoma.

All 16 chapters of this book have been contributed by leading experts in their respective fields, and cover the entire spectrum of stellate cell biology known to date. Thus they provide an up-to-date comprehensive treatise and perspectives of the established and putative functions of stellate cells in various pathophysiological conditions that affect the structure and functions of the liver. An independent chapter has also been devoted to characteristics of stellate cells in pancreatic pathobiology. This book is expected to serve as a reference to researchers in the field (cell and molecular biologists, hepatologists, gastroenterologists, and liver surgeons) as well as those who have an interest in hepatic biology and pathobiology. It is also intended to be used for graduate courses and for training residents and fellows in Gastroenterology and Hepatology.

Chapter 1

History and Early Work

Massimo Pinzani¹ and Chandrashekhar R. Gandhi²,     ¹UCL Institute for Liver and Digestive Health, Division of Medicine, University College London, Royal Free Hospital, London, UK; ²Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Department of Surgery, University of Cincinnati and Cincinnati VA Medical Center, Cincinnati, OH, USA

The history of the discovery of stellate cells is quite intriguing. In 1876, von Kupffer identified stellate cells as cells of the connective tissue, but later named them phagocytic special endothelial cells of the liver. Stellate cells were properly identified by Ito in 1952, and characterized by Wake two decades later in 1972 as the major storage site of retinoids and vitamin A homeostasis. However, the findings in early and mid-1970s that stellate cells could be the precursors of the cells responsible for liver fibrosis moved the field exponentially to discover mechanisms of their transformation and fibrogenic activity. Important discoveries made since have made inhibition of stellate cell pro-fibrogenic activity or induction of their apoptosis a therapeutic reality. These and other aspects of stellate cell biology are discussed in the subsequent chapters. In this chapter, we provide an historical account of the discovery of stellate cells and the early work done up until the late 1980s that formed a strong platform for the research that followed.

Keywords

Hepatic stellate cell

Ito cell

fat-storing cell

Kupffer cells

retinoids

fibrosis

vitamin A

1.1 Discovery of Hepatic Stellate Cells

The discovery of hepatic stellate cells (HSCs) is credited to a German anatomist Karl Wilhelm von Kupffer (Figure 1.1) who, while studying the liver’s nervous system, observed star-shaped, gold, chloride-stained cells within the hepatic lobules (Figures 1.2 and 1.3). Because these cells were localized around blood capillaries within the liver (now known as sinusoids), von Kupffer described them as perivasculäre bindgewebszellen (perivascular cells of the connective tissue) in an 1876 letter to his colleague Waldeyer [3]. While continuing his investigation of these "perivasculäre bindgewebszellen, von Kupffer observed uptake of intravenously injected India ink or sheep red blood cells by the rabbit liver cells stained with gold chloride and he changed his earlier opinion, concluding that they were phagocytes and also called them specialized endothelial cells of the sinusoids [4]. In a paper published in 1912, Pearce and Austin from the University of Pennsylvania, USA [5] referred to the liver cells that phagocytosed red blood cells in splenectomized dogs as stellate endothelial cells or Kupfer’s (instead of Kupffer’s) cells. Such a multitude of nomenclature continued for several more decades. In 1952, Toshio Ito of Keiko University, Japan, found that the cells in the perisinusoidal space contained abundant fat droplets (which were originally observed by von Kupffer), and were distinct from the phagocytosing cells in the sinusoids (now known as Kupffer cells). He called them fat-storing cells [6]. Almost two decades passed before Kenjiro Wake at Osaka University, Japan, using gold chloride and silver impregnation technique and vitamin A autofluorescence, confirmed that von Kupffer and Ito had described the same cells and further observed that they contained well-developed rough endoplasmic reticulum [7]. This observation in 1971 suggested the strong ability of stellate cells to synthesize proteins. In a subsequent study 3 years later, Wake reported the presence of cytosolic lipid droplets in stellate cells that were differentiated into two types: Type I droplets were electron dense, variable in size (up to 2 µm in diameter), membrane-bound, and appeared to be derived from multivesicular bodies, whereas Type II droplets were larger and uniform in size [8]. Type I lipid droplets were found to accumulate in the cells of animals with hypervitaminosis A in the intermediate and central zones of the liver lobule [1,8]. The number of stellate cells containing lipid droplets was estimated to be up to 75% both in rat and human [1,9]. The lipid droplets in isolated stellate cells contained high concentrations of both retinol and retinyl palmitate [10]. The observation of vitamin A stores (lipid droplets) in the 1980s led investigators to refer to stellate cells variously as fat-storing cells, lipocytes, perisinusoidal cells, and Ito cells (after Toshio Ito). Due to potential confusion arising from such different nomenclature, a consensus was reached in 1996 to call this cell type as HSC [11]. It is important to appreciate the efforts of von Kupffer in distinguishing these cells with the very basic techniques available at that time. Thus it was only apt that hepatic resident macrophages were named Kupffer cells" in recognition of von Kupffer’s original contribution to the field.

Figure 1.1 Karl von Kupffer.

Figure 1.2 Reproduction of von Kupffer’s drawing of the HSCs (Sterzellen) in dog liver. From Ref. [1] (Courtesy of Prof. Kenjiro Wake.)

Figure 1.3 Stellate cells in the porcine liver. The long cytoplasmic processes encompass sinusoids at regular intervals. Golgi’s silver method.×840. From Ref. [2] (Courtesy of Prof. Kenjiro Wake.)

1.2 HSCs and Vitamin A Homeostasis

Wake’s work in the 1970s established that HSCs were the major storage site of vitamin A (nearly 80% of the body’s retinoids is stored in these cells). This was confirmed in 1982 by Knook and co-workers of the Institute of Gerontology, the Netherlands. They found that HSCs isolated from the livers of 12-month-old rats contained much higher levels of retinoids (32 µg retinol and 685 µg retinyl palmitate per mg cellular protein) as compared with the whole liver homogenate (1 µg retinol and 15 µg retinyl palmitate per mg protein) [12]. Further analysis revealed that HSCs contained 300-fold greater amount of retinyl esters than hepatocytes [13]. Approximately 42% retinyl ester, 28% triglycerides, 13% cholesterol, and 4% phospholipids were estimated to constitute the lipid droplets in HSCs [14]. In another study, lipid droplets isolated from HSCs were found to be much larger (up to 8 µm diameter) than those isolated from parenchymal cells (up to 2.5 µm diameter), but both contained similar concentration of neutral lipids [15]. Whereas retinyl- and cholesteryl esters comprised 65% and triglycerides 20% of the lipid droplet fractions of HSCs, lipid droplet fractions of the parenchymal cells comprised 62% triglycerides and up to 30% cholesteryl esters [15]. These findings in the 1980s opened up a major area of investigation to determine the role of HSCs in vitamin A homeostasis, which, as we know now, is critical in maintaining the liver’s physiologic function [16]. The loss of retinoids by HSCs is paralleled by their transformation into a highly proliferative, contractile, and fibrogenic myofibroblastic phenotype that is responsible for liver fibrosis, cirrhosis, portal hypertension, and also implicated in playing a role in hepatocellular carcinoma (see Chapters 4, 6, 8, and 9).

A large amount of work on vitamin A homeostasis by HSCs has been performed by Blomhoff at the University of Oslo, Norway. In the intestine, retinol is esterified and retinyl esters are packaged in chylomicrons for delivery to the liver via the lymph. Hepatocytes are the primary cell type to endocytose retinyl esters, which are transferred to adjacent HSCs upon binding to specific retinol binding proteins [17]. However, HSCs were shown to possess an ability to directly take up retinol and its esters (e.g., retinyl acetate), as demonstrated by in vitro experiments [18,19]. Intracellular retinol-binding protein (CRBP), whose concentration in HSCs is almost 20 times greater than in hepatocytes, regulates the uptake, storage as well as the mobilization of retinoids to meet the peripheral demand [20,21]. In addition to CRBP, HSCs also contain other proteins related to retinoid metabolism such as cellular retinoic acid-binding protein, retinol palmitate hydrolase, bile salt-dependent and -independent retinol ester hydrolase, and acyl coenzyme A:retinol acyltransferase [22,23]. Furthermore, HSCs have been shown to express nuclear retinoid receptors including retinoic acid receptor (RAR) α, β, and γ [23,24], and retinoid X receptor (RXR) α and β [25]. Thus HSC is not just a cell type that simply stores and releases retinoids, but it also regulates their physiological and pathophysiological metabolism.

1.3 Morphological Characteristics of HSCs

An elaborate electron microscopic examination of the bat liver performed by Tanuma and Ito in 1978 described the special arrangement of the three main nonparenchymal cell types, namely sinusoidal endothelial cells, Kupffer cells, and HSCs [26]. Accordingly, the cytoplasmic extensions of sinusoidal endothelial cells consist of continuous thicker parts (cytoplasmic processes) and discontinuous thinner parts (sieve plates) containing fenestrations that allow a free exchange of molecules between the blood flowing through the sinusoids and hepatocytes. Figure 1.4 (transmission electron micrograph of rat liver section) shows some of the characteristics of the interactions of HSC with other hepatic cells. The endothelium is single layered and devoid of basal lamina, with junctions between closely apposed margins of the cytosolic processes. Kupffer cells adhering to the sinusoidal walls protrude into the sinusoidal lumen and contain many microvillous pseudopods. HSCs localized in the space of Disse contain fat droplets, large Golgi complex and well developed rough endoplasmic reticulum filled with electron dense material. In normal human liver also HSCs located in the space of Disse were seen making direct physical contacts with hepatocytes and endothelial cells, and their nucleus-to-nucleus distance was estimated to be about 40 µm [27] (Figure 1.5).

Figure 1.4 Transmission electron micrographs of rat liver. (Left) An HSC containing lipid droplets (L) is seen in close association with a Kupffer cell (KC), and making physical contact with the microvilli of a hepatocyte (H). S, sinusoid; SD, space of Disse. (Right) A sinusoidal cell (SEC) lining the sinusoid. Endothelial fenestrations (arrows) are clearly visible. The images were kindly provided by Dr. Donna Stolz, Center for Biologic Imaging, University of Pittsburgh.

Figure 1.5 A mouse liver section stained for GFAP, a marker of HSC. With long cytosolic processes, one HSC can physically contact 1–2 hepatocytes as well as sinusoidal endothelial and Kupffer cells (40×magnification).

Zonal distribution studies of human liver performed in 1981 by Giampieri et al. showed that the number of HSCs was higher in the centrilobular than in the periportal region [28]. HSCs in the periportal zone 1 were found to be relatively small with short cytosolic processes and contained smaller lipid droplets, those in zone 2 displayed longer branching processes and abundant vitamin A stores in the lipid droplets, and those in centrilobular zone 3 demonstrated dendritic appearance and progressively lower vitamin A content [27,29]. However, administration of excess vitamin A to rats caused an increase in the number and size of vitamin A-lipid droplets in HSCs located in the central zone, indicating that the intralobular heterogeneity may reflect differences in the metabolic handling of vitamin A. Such heterogeneity was also apparent with diffuse immunostaining for desmin (a marker for HSCs) in zone 1, intense staining in zone 2, and progressively diminishing staining moving toward the centrilobular vein [27,29]. HSCs were found to extend cytoplasmic processes ramifying beneath the endothelial lining, occasionally surrounding the sinusoids almost completely. These processes of HSCs seemed to reinforce the endothelial lining, potentially providing a regulation of the sinusoidal tone. In this regard, the subendothelial processes of HSCs were found to be equipped with massive actin-like filaments [1]. In fact, subsequent work demonstrated contractile properties of HSCs [30,31] and their role in contractile component of portal hypertension, which is almost always present in advanced cirrhosis (see Chapter 8). With their number (up to 10% of the total number of liver cells) [12,28] and ability to form physical association with all of the hepatic cell types, it became apparent that HSCs form a unique cell population organized in a three-dimensional network with the potential to closely regulate several important functions of the liver.

1.4 HSCs and Liver Fibrosis

Although the focus of research in the 1970s was to understand the mechanisms of uptake, storage, composition, and release of retinoids by HSCs, evidence started accumulating that these cells could be a major source of extracellular matrix deposited in the liver during fibrogenesis [32–35]. McGee and Patrick (Royal Infirmary, Glasgow) in 1972 [32,33] observed massive hepatocellular necrosis that preceded fibrogenesis in rats injected with carbon tetrachloride, and suggested that HSCs were responsible for the latter event. Subsequently in 1977, Kent (University of Chicago, Illinois, USA), Popper (Mount Sinai School of Medicine, New York, USA) and co-workers demonstrated accumulation of transitional cells with morphological features of HSCs (lipocytes) and fibroblasts along the fibrous septa during carbon tetrachloride-induced liver injury in rats that also received subcutaneous injections of vitamin A [34]. Because collagen type III deposits were associated with these transitional cells, the authors postulated that they originated from HSCs. The relevance of these findings to humans became apparent later in a 1984 study with nonhuman primates. The hypothesis that HSCs were the precursors of the transitional cells that caused hepatic fibrosis was drawn based on the cellular depletion of lipid droplets and hypertrophy of endoplasmic reticulum observed in experiments performed in baboons maintained on a diet containing alcohol [35]. Investigation of HSCs as the primary fibrogenic cells during wound healing, hepatic fibrosis, and cirrhosis became, and continues to be, a major area of research described in Chapter 4. We now know that inflammatory mediators and reactive oxygen species produced by infiltrating blood cells (e.g., neutrophils and monocytes) as well as Kupffer cells and apoptotic bodies derived from dying hepatocytes are major stimuli for the transformation of HSCs into fibrogenic phenotype. It is interesting to note that McGee and Patrick [32,33] postulated a link between hepatocellular necrosis and fibrosis as early as 1972, although this reality remained obscure until the late 1980s.

1.5 Isolation and Culture of HSCs

Until the early 1980s, knowledge of the functions of HSCs was limited. Based on the ultrastructural characteristics, they resembled fibroblasts and there were strong indications for an active role of these cells in hepatic fibrosis [1,32–35]. Thus, a major breakthrough to determine precisely the functions of HSCs required isolation and culture of highly purified cells. In retrospect, this was critical because contaminating Kupffer cells, endothelial cells, or hepatocytes can influence the properties of HSCs. In the 1980s, attempts were made to isolate HSCs based on their relatively lower density (high lipid content) than other hepatic cells. In 1980, Otto and Veech at the National Institute on Alcohol Abuse and Alcoholism [10] reported isolation of HSCs from the livers of rats treated with vitamin A to increase the number and size of lipid droplets. The livers were digested with pronase, and total nonparenchymal cell fraction was separated by differential centrifugation; this was followed by purification of HSCs by centrifugation in a linear metrizamide gradient [10]. Based on the quickly fading green autofluorescence at 330 nm due to vitamin A, the procedure yielded a fourfold enrichment of HSCs in the cell fraction with gradient density of 1.043 g/mL. Shortly afterwards, Knook et al. [12] employed similar metrizamide density gradient centrifugation technique to isolate HSCs from rats without prior vitamin A treatment, and purified them from other nonparenchymal cells by centrifugal elutriation. The yield of HSCs from the livers of old animals (12 months of age) was much superior as compared with younger animals (3 months of age). Most investigators now use old rats and mice (retired breeders) for isolation of HSCs. However, even with the centrifugal elutriation procedure [12], the purity of HSCs was estimated to be only about 70–75%, the major contaminants being lymphocytes and endothelial cells. In 1985, Friedman et al. (Liver Center, University of California, San Francisco) achieved>95% pure HSC preparation from rat liver [36]. The investigators digested the livers of rats pre-treated with retinyl acetate with pronase and collagenase; following separation of nonparenchymal cells by low-speed centrifugation, they used stractan (arabinogalactan) gradient (6%, 8%, 12%, and 20%) and obtained highly purified HSC fraction between the medium and 6% stractan [36]. These procedures or modifications thereof have become established techniques and are now routinely used by researchers to isolate HSCs from various species including human. Details of various procedures of HSC isolation and purification are addressed in Chapter 2.

1.6 Activation and Transdifferentiation of HSCs

In their follow-up investigation, Knook’s group demonstrated successful culture of HSCs that divided, and could be established as cell lines for at least two passages [37]. The cells contained vimentin (indicating fibroblastic or myogenic origin), actin filaments, collagen type I and IV, and laminin in first passage and only collagen type I in the fourth passage. Because prolonged culture of HSCs was associated with gradual disappearance of vitamin A and phenotypic transition to more fibroblastic phenotype, the authors concluded that they might be the cell type involved in liver fibrosis, a view proposed much earlier (1972) by McGee and Patrick [32,33]. This was later confirmed by Friedman et al. using highly purified HSCs; they further demonstrated that the fibrogenic potential of HSCs, as assessed by collagen synthesis, was far superior to that of hepatocytes and endothelial cells [36]. It was further demonstrated in 1987 that highly purified desmin-positive rat HSCs lost retinoids progressively upon repeated sub-culture as they transdifferentiated into the myofibroblast-like phenotype [38]. In vivo experiments with carbon tetrachloride-treated rats also showed significant reduction in the hepatic vitamin A content in association with increased fibrosis, providing additional evidence that the loss of vitamin A might be a prerequisite for the transition of HSCs into the fibrogenic phenotype [39]. In fact, administration of vitamin A to rats was found to suppress carbon tetrachloride-induced fibrosis [39]. This issue (loss of retinoids and change of HSC phenotype) is addressed in detail in Chapter 4. Even in lower vertebrates, such as lamprey, evidence for the role of HSCs in hepatic fibrosis during biliary atresia has been presented [40].

In situ, HSCs have a small cell body from which cytosolic processes radiate and make physical contacts with surrounding cells (Figure 1.5). Upon isolation and culture, HSCs appear spherical and stain strongly for lipid droplets with oil red O (Figure 1.6A), but start flattening and generating cytosolic processes shortly in culture in association with the loss of lipid droplets (Figure 1.6B). Prolonged culture or passaging induces increase in size and myofibroblastic appearance with very low or absence of oil red O staining (Figure 1.6C and D). The process of the loss of retinoids and transformation of HSCs into myofibroblasts is known as activation. Such spontaneous activation in culture indicated that HSCs produced factors that initiated and perpetuated their activation and proliferation, and provided an excellent model to discover extracellular factors that induce or accelerate the process of activation/transdifferentiation and the underlying intracellular signaling mechanisms. For culture-induced activation, it is essential to incubate HSCs in a medium supplemented by 5–10% serum. The cells at different stages of activation can be used to characterize their functions and relevance to the stages of liver injury, bearing in mind that HSCs are a highly heterogeneous population. It should also be considered that the findings derived from experiments with HSCs from laboratory animals that are maintained in controlled pathogen-free environments may not necessarily be similar to the cells obtained from humans because humans are subjected to variable environmental conditions/stresses, food-derived antigens, drugs, xenobiotics, and microbial products that impact liver cell characteristics significantly.

Figure 1.6 Rat stellate cells at indicated times in culture after isolation. The cells were stained with Oil Red O to show lipid stores. Note that with time in culture, HSCs progressively lose their lipid content and become flattened, gaining a myofibroblast-like phenotype.

Because HSCs isolated from normal liver rapidly begin to change their phenotype, it was necessary to develop a method to maintain them in the quiescent state to investigate their physiological characteristics and functions. Friedman et al. [41] found that HSCs retained their quiescent physiologic phenotype when cultured on basement membrane-like matrix (Matrigel™) derived from Engelbreth-Holm-Swarm murine tumor. These cells produced 70% less collagen (consisting only of type III collagen) than those cultured on plastic, which differentiated into myofibroblasts and produced large amounts of type I collagen. Although these findings provided key information on the relationship between HSCs and the surrounding three-dimensional extracellular matrix microenvironment in the process of HSC activation, this method has not been extensively employed in routine laboratory practice because of the inherent non-standardized content of growth factors and cytokines of Matrigel™ and its barrier effect toward exogenously added experimental agonists.

The search for exogenous factors that induce activation of HSCs directed attention to Kupffer cells, which were already known to produce reactive oxygen species and a variety of cytokines and growth mediators [42]. Indeed, medium conditioned by Kupffer cells induced proliferation of HSCs and increased their collagen production [43]. The mediator in Kupffer cell medium that caused proliferation was identified to be platelet-derived growth factor (PDGF) [43,44], which is perhaps the most potent mitogen for HSCs. Whereas PDGF induced proliferation of HSCs, TGFβ1, which is also produced by Kupffer cells, was identified to be a very potent cytokine to induce collagen production by HSCs [44].

1.7 Markers for HSCs

Until the mid-1980s, identification and quantification of HSCs largely depended on microscopic examination and ultrastructural analysis in combination with vitamin A autofluorescence [12,45]. Because a significant number of HSCs might not contain vitamin A [27,29], it was essential to identify specific markers. Knook et al. demonstrated that HSCs expressed vimentin [37], a type III intermediate filament protein that is expressed in mesenchymal cells. However, Kupffer cells as well as endothelial cells also express vimentin [38]. Although Yokoi et al. found desmin, a smooth muscle protein consisting of intermediate filaments, to be a reliable marker for rat HSCs [40], it was later determined that there was significant variability in desmin expression by HSCs within the liver lobule [27,29]. Rat HSCs were also reported to express glial fibrillary acidic protein (GFAP), the major component of intermediate filament in astrocytes [46]. This indicated an interesting possibility that HSCs may have neural origin during embryonic development. The accumulation of HSCs around necrotic area and their potential role in fibrosis was postulated in the 1970s [32–34], and was confirmed by Burt et al. in 1986 using anti-desmin antibody to identify HSCs in areas of hepatocellular necrosis caused by a single injection of carbon tetrachloride [45]. It is important to mention that human quiescent HSCs may not express desmin or GFAP [47]. Thus lipid deposits, vitamin A autofluorescence, and electron microscopy remain the primary mode of their identification. However, in the normal human liver some HSCs may express α-smooth muscle actin (α-sma), and regardless of the species all HSCs express α-sma upon activation.

As mentioned earlier, HSCs differentiate into proliferative and fibrogenic phenotype during liver injury and in culture. In addition to the loss of retinoids, the activation process is associated with the expression of α-sma, which is a highly reliable marker of HSC activation in vivo and in vitro (Figure 1.7). It has become a standard practice to assess the purity of freshly isolated HSCs by vitamin A autofluorescence and immunostaining for GFAP and desmin (HSC) in conjunction with markers for Kupffer cells, endothelial cells, and epithelial cells (see Chapter