Perisinusoidal Stellate Cells Research Articles

Ductular reaction or hepatic reparative complex: immunohistochemical features in liver cirrhosis in patients with chronic hepatitis

Until recently, a discussion about the mechanisms of development and the biological role of the ductular reaction, which develops in patients with chronic liver diseases continues among hepatologists and pathomorphologists. Purpose of the study. To characterize the pathomorphological features and significance of the ductular response in liver cirrhosis in patients with chronic non-alcoholic, alcoholic and viral hepatitis in hepatobioptats with the use of immunohistochemical (IHC) techniques. Material and methods of investigation. Histological, histochemical and IHC study of the ductular liver reaction in liver biopsies of 52 patients aged 24 to 66 years with cirrhosis of the liver on the background of non-alcoholic steatohepatitis (13 patients), and alcoholic steatohepatitis (13 patients) and on the background of chronic viral hepatitis C (10 patients, 26–47 years), as well as those suffering from severe biliostasis (8 patients) and focal nodular liver hyperplasia (8 patients). Results. The ductular reaction can be detected in the active phase with maximum manifestations in patients with liver cirrhosis on the background of chronic hepatitis, it may have an average or weak degree of severity; in a significant number of patients, the effects of the ductular reaction of the liver are revealed. Cellular chains and groups of cells with the immunophenotype of the progenitor cells of the liver appear in the active phase of the ductular reaction at the periphery of the hepatic lobules in the projection of the Goering canals, in the fibrotically altered portal tracts, in the subcapsular zone of the liver and in the thickened fibrosis septa (c-kit CD117+, CD34+, CD56+ CK7-, CK19-, Hepar-) without presence of figures of mitosis or increased level of expression of Кі-67 in them. In small ductules localized in the projection of the Goering canals, single cells with the expression of c-kit CD 117+, CD44 Std./HCAM+, CD34+, CD56+, expressing the markers of biliary (CK7+, CK19+) and hepatocyte (Hepar+, α-fetoprotein+) differentiation are revealed. Cellular chains and ductules in the intermediate zones of the hepatic lobules are represented by cells of biphasic differentiation: biliary (CK7+, CK19+), hepatocyte (Hepar+) differentiation and intermediate hepatobiliary CK7+ cells. Hepatocyte differentiation of progenitor cells in lobules of the liver is facilitated by local expression of laminin by perisinusoidal stellate cells. New pseudolobes arise in the active phase of the ductular reaction in the lobes with perisinusoidal pericellular fibrosis from disorderly located large hepatocytes without linear perisinusoid spaces and centrolobular veins, with the presence of “intermediate” SC7+ hepatocytes on the periphery. One-two-row chains and ductules from SC7+ and CK19+ cells dominate in the fibro-altered portal tracts, septa and the subcapsular zone of the liver among the collagen fibers of I, III, IV type and the processes of α-SMA+ myofibroblasts, and small short chains from Hepar+ and α- fetoprotein-cells. When the severity of micronodular liver cirrhosis increases, when the area of hepatic lobules and pseudotypes becomes equal to or less than the area of the surrounding fibrosis, there is a parallel increase in the number of Ki-67+ cells in portal-lobular ductules and chains, as well as the number of Ki-67+ hepatocytes in hepatic lobules and pseudolobes. The long-term consequences of the ductular reaction in liver cirrhosis reflect the presence of the different number of bile-free ductules with CK7 + epithelium in the fibrous tissue of portal tracts, septa and under the liver capsule. In the lobules of the liver with perisinusoidal pericellular fibrosis, fuzzy outlines of hepatocyte hyperplasia without ordered sinusoids and liver-celled beams are found. Conclusions. The ductular reaction in liver cirrhosis in patients with chronic non-alcohol, alcoholic and viral hepatitis is the process of activation and evolution of a complex liver repair complex aimed at replenishing the deficit of hepatocytes and biliary structures. The reparative process, activating by liver cirrhosis on the background of the disturbed portal-lobular intercellular matrix and the progressive deficit of hepatocytes, does not provide the reconstruction of full-fledged liver structures, it is discordant towards the creation of new, functionally low-value hepatocellular pseudolobes and the accumulation of biliary terminals in fields of portal-septal fibrosis.

星細胞(ビタミンA貯蔵細胞)の細胞生物学-レチノイド代謝と細胞外マトリックスの機能の視点から

星細胞(ビタミンA貯蔵細胞)の細胞生物学-レチノイド代謝と細胞外マトリックスの機能の視点から

Pathomorphology of liver fibrosis in trepanobioptates of patients with steatohepatitis: main types, sources of development, features of progression

Until recently, among the pathologists, hepatologists and gastroenterologists, a discussion continues on the morphogenesis and gradation of liver fibrosis in non-alcoholic patients (NASH) and alcoholic steatohepatitis (ASH).Purpose of the study. Studying the main types and sources of liver fibrosis in patients with nonalcoholic and alcoholic steatohepatitis, justifying the gradation of its severity, taking into account the quantitative dynamics of fibrogenic producer cells, the relative area of fibrosis and the deposition of type I, III and IV collagen in the liver.Material and methods. Histological and histochemical examination of liver fibrosis was performed in 198 patients with NASH of 18-79 years and in 79 patients with ASH of 47-63 years. Immunohistochemical study with measurement of the area of expression of activated αSMA + perisinusoidal stellate cells and αSMA + portal myofibroblasts with F1 (mild), F2 (moderate), F3 (severe) fibrosis and F4 fibrosis / cirrhosis was performed in 80 trepanobioptates of patients with NASH (20 cases in each group), electron microscopic examination of the liver – in 10 deceased patients suffering from NASH.Results. In patients with NASH and ASH, there are two major types of liver fibrosis progress: the perisinusoidal pericellular and portal-Z3 perisinusoidal fibrosis, the development of which is the new generation of αSMA + star cells and αSMA + portal myofibroblasts of the fibrogenic immunophenotype, with co-expression of fascin and vimentin and absence of desmine expression. As the perisinusoidal pericellular fibrosis progresses from the mild F1 degree, to the severe F3 fibrosis and to the extremely severe F4 fibrosis / cirrhosis of the pericellular type, the area of the Masson-positive extracellular molecular-fibrous matrix (H = 88,70 р = 0,05 ) and the area of αSMA + collagen-producing stellate cells of perisinusoidal-pericellular localization (H = 45,12, p = 0,05). According to the data of photometric morphometry, with increasing severity of portal-Z3 perisinusoidal hepatic fibrosis from F1 to F4, the area of the Masson-positive extracellular molecular-fibrous matrix (N = 76,56 p = 0,05) in areas fibrofacitiously increases, as well as the area of activated αSMA + collagen-producing portal myofibroblasts and perisinusoid stellate cells of Z3 zones of hepatic lobules (H = 43,18, p = 0,05). Activation and increase in the number of CD-68+ macrophages in the centers of intensive hepatocyte steatosis, as well as the appearance of S100 (A4)+ macrophages that activate stellate cells, suggests that the phenomenon of pericellularity of liver fibrosis in NASH and ASH is due to exosomal macrophage involvement in zone of balloon steatonecrosis and necroptosis of hepatocytes, as well as local exosomal-cytokine activation of perisinusoid stellate cells of fibrogenic type.Conclusion: As the progression of fibrosis from F1 to F4 degree in the liver the area of activated αSMA + fibroblasts, and the area of deposition of collagen I, III and IV type perisinusoidal pericellular or portal-Z3 perisinusoidal localization significantly increase.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Translating an Understanding of the Pathogenesis of Hepatic Fibrosis to Novel Therapies

The response to injury is one of wound healing and fibrogenesis, which ultimately leads to fibrosis. The fibrogenic response to injury is a generalized one across virtually all organ systems. In the liver, the injury response, typically occurring over a prolonged period of time, leads to cirrhosis (although it should be pointed out that not all patients with liver injury develop cirrhosis). The fact that many different diseases result in cirrhosis suggests a common pathogenesis. The study of hepatic fibrogenesis over the past 2 decades has been remarkably active, leading to a considerable understanding of this process. It clearly has been shown that the hepatic stellate cell is a central component in the fibrogenic process. It also has been recognized that other effector cells are important in the fibrogenic process, including resident fibroblasts, bone marrow-derived cells, fibrocytes, and even perhaps cells derived from epithelial cells (ie, through epithelial to mesenchymal transition). A key aspect of the biology of fibrogenesis is that the fibrogenic process is dynamic; thus, even advanced fibrosis (or cirrhosis) is reversible. Together, an understanding of the cellular basis for liver fibrogenesis, along with multiple aspects of the basic pathogenesis of fibrosis, have highlighted many exciting potential therapeutic opportunities. Thus, although the most effective antifibrotic therapy is simply treatment of the underlying disease, in situations in which this is not possible, specific antifibrotic therapy is likely not only to become feasible, but will soon become a reality. This review highlights the mechanisms underlying fibrogenesis that may be translated into future antifibrotic therapies and to review the current state of clinical development.

Immunohistochemical Identification of Alpha-Smooth Muscle Actin Positive Myofibroblasts in Toxic Hepatitis

The crucial role played by the myofibroblast in wound healing and pathological organ remodeling is well established. Hepatic myofibroblasts constitute a heterogeneous population of pro-fibrogenic cells found in chronically injured livers. Liver fibrogenesis is sustained by populations of highly proliferative, pro-fibrogenic, pro-angiogenic and contractile myofibroblasts that, originate by a process of activation involving perisinusoidal hepatic stellate cells, portal fibroblasts, bone marrowderived mesenchimal stem cells as well as, in certain conditions, by a process of epithelial to mesenchymal transition involving hepatocytes and cholangiocytes. (Parola 2008). In this study we used 60 male Wistar rats with an average weight of 200g, who received chronic carbon tetrachloride treatment in dose of 1,2 ml/kg, two times a week, for 12 weeks. Liver samples were taken after euthanasia at 4, 8 and 12 weeks after the start of the experiment. The formalin-fixed hepatic samples were embedded in paraffin, sectioned at 4 μm, stained by Haematoxilin-Eosine and Masson's trichrome methods. Immunohistochemistry was performed, using a Rabbit polyclonal antibody to alpha smooth muscle Actin (Abcam, Cambridge, ab5694). Alpha smooth muscle actin (SMA) positive myofibroblast were localized in the expanded connective tissue around central veins, within fibrous septa, at the interface between fibrous septa and parenchyma and in the parenchyma around sinusoids (activated hepatic stellate cells).

Bringing retinoid metabolism into the 21st century

The requirement of vitamin A (retinoids) for normal growth has been recognized for almost a century (1, 2). It plays an important role in a number of physiological processes including vision, cell differentiation, reproduction, and immunity. Vitamin A is a fat-soluble organic compound that cannot be synthesized endogenously by humans and thus, it is an essential nutrient. The main sources of vitamin A in the diet are provitamin A carotenoids from vegetables and retinyl esters from animal tissues [mainly the liver (3)]. All of the retinyl esters are enzymatically converted to retinol in the intestinal lumen before absorption by intestinal enterocytes (see Fig. 1). Subsequently, retinol is esterified with long-chain fatty acids to form retinyl esters, which are then incorporated into chylomicrons. Fig. 1. Retinoid transport in the body. See first three paragraphs for explanation. RE, retinol ester; ROH, retinol; TG, triglycerides; CE, cholesterol ester; LPL, lipoprotein lipase; RBP, retinol binding protein. Chylomicrons are secreted into intestinal lymph and then move into the blood. Triglycerides in chylomicrons are extrahepatically hydrolyzed by LPL and fatty acids are taken up by muscle and adipose tissues (4). Cholesteryl esters and retinyl esters in chylomicron-remnants are simultaneously taken up by the liver parenchymal cells [hepatocytes (5)] through the remnant receptor, of which the molecular structure is still under active discussion (6, 7). Intracellularly, both cholesteryl esters and retinyl esters are hydrolyzed either at the plasma membrane or in early endosomes (5). The majority of the formed cholesterol appears in the bile, mainly as bile acids (5), whereas the retinol is associated with retinol-binding protein 4 for secretion from the cells. Most of the retinol that is derived from chylomicron retinyl esters is transferred from hepatocytes to hepatic perisinusoidal stellate cells [fat-storing cells, Ito cells (3)]. In animals, about 50 to 80% of the total body Vitamin A is stored in liver stellate cells as retinyl esters (3). The outline of the mechanism of absorption, transport, and cellular fate of retinyl esters has been intensively studied in the 1980s and 1990s of the 20th century with mostly rats as experimental animals. In this issue, Schreiber et al. (8) revitalize this field by an attempt to characterize the enzyme(s) responsible for the hydrolysis of retinoids in the liver. It is no surprise that from the 47 references in this paper, there are no citations of publications in the last 4 years and only four citations to relevant publications in the 21st century (see Fig. 2). Actually, only the group of Blaner et al. (9, 10) from Columbia University brought transgenic mouse studies into this field. Historically, the Department of Medicine at the College of Physicians and Surgeons, Columbia University, headed by DeWitt S. Goodman, played a major role in establishing the pathway of vitamin A metabolism (11). This work was often accomplished in collaboration with outside laboratories (12–15), including such illustrious coworkers as O. Stein, Y. Stein, N. Fidge, K. Norum, and D. Steinberg, and developed classic technologies like thin-layer chromatography of sterols and steroids (16). Fig. 2. Number of citations per year in the article of Schreiber et al. (8) In work reported in this issue, the group of Rudi Zechner (8) report important new information on the mechanisms by which retinyl esters are hydrolyzed in the liver. They cloned 12 separate carboxyl esterases into a mammalian expression vector and expressed these recombinant His-tagged proteins in COS-7 cells. Using retinyl palmitate emulsified with cholate as substrate, they showed that esterase 22 was the most likely candidate responsible for physiological hydrolytic activity. It appears that esterase 22 specifically hydrolyzes retinyl esters and that esterase 22 overexpression attenuates accumulation of retinol esters in COS-7 cells. Esterase 22 can form a complex with β-glucuronidase and the β-glucuronidase exhibits hydrolyzing activity toward retinoyl β-glucuronide, a naturally occurring metabolite of vitamin A. These data make it likely that esterase 22 and β-glucuronidase act in concert in mobilizing retinol from retinol esters and retinoyl β-glucuronide. Both of these enzymes colocalize in the endoplasmic reticulum in hepatocytes. Although the present publication does not provide definitive proof for the exclusive role of esterase 22 and β-glucuronidase in hydrolyzing retinoids in mouse liver, it does bring up-to-date 21st century technology into the field of retinoid metabolism. Recently, the group of Zechner has provided definitive proof for the role of adipose triglyceride lipase in lipolysis and energy metabolism (17, 18). Haemmerle et al. (17) used genetic inactivation of adipose triglyceride lipase to prove the crucial and rate-limiting role of this enzyme in the catabolism of cellular fat depots and energy homeostasis. It can be anticipated that a similar approach will be used to provide more definitive data on the role of esterase 22 and β-glucuronidase in retinoid metabolism and the present manuscript thus forms an important first step in defining this pathway.

Vitamin D and the digestive system

Target tissues of in vivo receptor binding and deposition of 1,25(OH)2 vitamin D3 and its oxygen analog OCT are reviewed in rats, mice, hamsters and zebra finch, identified with high-resolution microscopic autoradiography. Throughout the digestive system numerous sites with nuclear receptor binding of 3H-1,25(OH)2 vitamin D3 and 3H-OCT exist: in the oral region, epithelial cells of the oral cavity, tongue and gingiva, teeth odontoblast and ameloblast precursor pulp and stratum intermedium cells; in the parotid, submandibular and sublingual salivary glands, epithelial cells of striated ducts and granular convoluted tubules, intercalated ducts and acinar cells, as well as myoepithelial cells; in the stomach, neck mucous cells of gastric glands, endocrine cells of the antrum, and muscle cells of the pyloric sphincter; in the small and large intestine, absorptive and crypt epithelial cells; in the pancreas, predominantly islet B-cells. Perisinusoidal stellate (Ito) cells in the liver concentrate and retain variable amounts of radiolabeled compound in regions of their cytoplasm after administration of 3H-I,25(OH)2 vitamin D3 and 3H-25(OH) vitamin D3, probably sites of specific storage, similar to vitamin A. Submucosa in stomach and intestine also retain variable amounts of radiolabel, however unspecific with all compounds studied. In pilot studies with 3H-25(OH)2 vitamin D3 and 3H-24,25(OH)2 vitamin D3, no nuclear concentration was detectable. The reviewed data for vitamin D and its oxygen analogue OCT indicate genomic effects on multiple target tissues of the digestive system that involve cell proliferation and differentiation, endo- and exocrine secretion, digestion and absorption for maintaining optimal functions, with potentials for health prophylaxis and therapies.

Uptake Behavior of Embryonic Chick Liver Cells

The capacity of selective uptake by liver cells, focusing particularly on the parenchymal and perisinusoidal stellate cells during chick liver development (8-18 days of incubation), was ultrastructurally examined after injection of 240-nm-diameter lecithin (phosphatidylcholine) -coated or noncoated beads into the extraembryonic circulation. Cytoplasmic projections of both cells as well as extrasinusoidal macrophages reached into the sinusoid-like vascular spaces. The primitive perisinusoidal stellate cells were identified by immunocytochemistry as being rich in desmin-positive cytoplasmic intermediate filaments. The cells demonstrated selective uptake of noncoated beads by means of their cytoplasmic projections. These findings were significant in the early period of incubation, indicating that the phagocytic activity is a characteristic and transient phenomenon of developmental differentiation. Large numbers of coated and a few noncoated beads penetrated into the perivascular spaces. The parenchymal cells incorporated only the coated beads that passed through the endothelial lining, suggesting that these cells express selective but limited phagocytic capacity against large "foreign" substances even long before their maturation. The cell projections were not engaged in uptake function. Extrasinusoidal macrophages, Kupffer cells, and intraluminal primitive macrophages all took up both beads; however, lecithin coating of the beads clearly suppressed their uptake function. These data suggest that the uptake function of large "foreign" substances appears to be intrinsic to liver cells and lecithin coating would be useful for delivering large substances to parenchymal cells.

Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

The expression of glial fibrillary acidic protein (GFAP) by perivascular cells of many mammalian organs suggests an as yet unknown function of this intermediate filament protein in the maintenance of homeostasis and vascular permeability at the blood-tissue interface. Although a similar situation may exist at the air-tissue interface, the cellular distribution of GFAP in skin tissue has never been demonstrated. To approach this issue, we have employed immunofluorescence and Western blotting techniques to detect GFAP in skin sections of young and adult humans, normal rodents, and two types of mutant mice, as well as in rat lung sections, and in cultured human keratinocytes and fibroblasts. Colocalization with antigens known to be associated with GFAP in other tissues was also tested. Epidermal and hair follicle keratinocytes and dermal fibroblasts showed distinct staining for GFAP as well as colocalization with alpha-actin, metallothionein, and antigens of the class-II major histocompatibility complex (MHC II). GFAP was also identified in rat alveolar fibroblasts which, in common with keratinocytes, form part of the air-tissue interface. GFAP was upregulated together with MHC II in nude mice but was barely detectable in the skin of non-obese diabetic severe combined immunodeficiency mice, suggesting a possible involvement in antigen-presenting functions. The intriguing distribution of a common set of antigens both in certain cells of the integumentary system and at the blood-tissue interfaces of internal organs suggests the involvement of these proteins in universal mechanisms controlling tissue homeostasis and protection.

Pregnane X Receptor Activators Inhibit Human Hepatic Stellate Cell Transdifferentiation In Vitro

The activated pregnane X receptor is antifibrogenic in rodent chronic liver injury in vivo models. The aim of this study was to determine the effects of human pregnane X receptor activators on human hepatic stellate cell transdifferentiation to a profibrogenic phenotype in vitro. Hepatic stellate cells were isolated from resected human liver and cultured under conditions in which they trans-differentiate into profibrogenic myofibroblasts. The pregnane X receptor was expressed in primary cultures at the level of messenger RNA and protein and was activated by the ligand rifampicin as judged by increases in binding of proteins to the pregnane X receptor ER6 DNA response element and by increases in ER6-dependent reporter gene expression. Short-term treatment of hepatic stellate cells with rifampicin inhibited the expression of selected fibrosis-related genes (transforming growth factor beta1, alpha-smooth muscle actin), proliferation-related genes, and WNT signaling-associated genes. There was also an increase in interleukin-6 secretion and an inhibition in DNA synthesis. Long-term treatment with rifampicin over several weeks reduced the proliferation and transdifferentiation of hepatic stellate cells. Small interfering RNA knockdown of the pregnane X receptor in a hepatic stellate cell line reduced the binding of proteins to the ER6 DNA response element and abrogated pregnane X receptor activator-dependent changes in transforming growth factor beta1 expression, interleukin-6 secretion, and proliferation. The pregnane X receptor is transcriptionally functional in human hepatic stellate cells and activators inhibit transdifferentiation and proliferation. The pregnane X receptor may therefore be an effective target for antifibrotic therapy.

Vitamin A Toxicity from over the Counter Health Supplements

Introduction: The growing popularity and availability of over the counter (OTC) health products is of concern. In United States, about 25% of adults ingest vitamin A containing supplements and 5% take supplements of vitamin A alone. We report a case of cirrhosis associated with habitual daily ingestion of OTC dietary supplement of vitamin A. Case: A 59-year-old white man was referred from primary care physician for massive ascites of six-months duration. He was abstinent from alcohol since 15 years and had been taking variety of unsupervised OTC multivitamin tablets containing an aggregate of 160,000 IU/d vitamin A for two years (Recommended Daily Amount is 5000 IU/d for adults). Laboratory workup was significant for total bilirubin of 0.5 mg/dL, ALP of 154 IU/L, ALT of 40 IU/L, AST of 45 IU/L, albumin of 2.7 g/dL, INR of 1.1, serum ascitic albumin gradient of 1.5, vitamin A level of 21 micrograms/L (normal 30–95 mcg/L) with normal serum levels of ceruloplasmin, ferritin, transferrin, alpha 1 antitrypsin and negative hepatitis and autoimmune serology. Echocardiogram showed ejection fraction of 55% with normal right ventricular systolic pressure. Ultrasonogram revealed a mildly shrunken liver. Grade I esophageal varices were present on esophagogastroduodenoscopy. Liver biopsy showed chronic hepatitis grade II stage II with hypertrophied stellate cells and perisinusoidal fibrosis consistent with vitamin A toxicity. Vitamin supplements were discontinued. At two year follow-up patient had minimal ascites with improved liver functions. Discussion: Symptoms may occur with too much or too little of vitamin A. Vitamin A hepatotoxicity is rare; ranging from elevated liver enzymes to cirrhosis and may be observed in the absence of extra hepatic signs of vitamin A intoxication or increase in serum vitamin A concentration like in our case. Hyperplasia and hypertrophy of perisinusoidal stellate cells and perisinusoidal fibrosis on liver biopsy is consistent with hepatic storage of excess vitamin A. Concomitant alcohol use further potentiates the intrinsic hepatotoxicity of vitamin A. This case highlights the collateral damage caused by OTC vitamin supplements and indicates the need for regulation of medically unsupervised use of such products. As a consequence of increasing use of OTC vitamin A supplements, physicians need to provide information regarding relative safety of such products and elicit history of vitamin A supplements use in evaluation of liver dysfunction.

Role of CD44 in Epithelial Wound Repair: MIGRATION OF RAT HEPATIC STELLATE CELLS UTILIZES HYALURONIC ACID AND CD44v6

The hyaluronic acid receptor, CD44, exists as multiple splice variants that appear to have a role in migration of tumor cells. The role of this receptor and its variants in normal wound repair is poorly understood. A central feature of wound repair in the liver is activation and migration of perisinusoidal stellate cells. We have examined CD44 expression by stellate cells from normal or injured rat liver, finding that it increases with injury and involves a distinct set of CD44 splice variants. Among the latter, variants containing the v6 exon (CD44v6) are strikingly increased. Analysis of migration of primary cells on transwell filter inserts reveals that only cells isolated from injured liver are migratory. Also, they move more rapidly on hyaluronic acid than on collagen I or collagen IV. A polyclonal antibody to recombinant CD44v6 blocks migration by 50%, whereas antibody to CD44v4 has no effect. The inhibition is specific for cells migrating on hyaluronic acid and is reversed by synthetic peptide representing the N terminus of the v6 protein. In conclusion, activated stellate cells use CD44v6 and hyaluronic acid for migration. Given the evidence that migration is required for progression of injury with scar formation, blockers of CD44v6 expression or function are candidates for preventing the deleterious effects of chronic fibrosis.

Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: Involvement of perisinusoidal stellate cells

To analyze the relationship between perisinusoidal stellate cell (PSC) activation and the dietary fat quantity and composition in the treatment of hepatic steatosis. Using an experimental rat model of steatosis based on the intake of a hyperlipidic diet (14% fat as olive oil or sunflower oil, HL-O and HL-S, respectively), we analyzed the liver's capability of recovery after the treatment with a normal-lipidic diet (5% fat as olive oil or sunflower oil, NL-O and NL-S, respectively) by immunocytochemical and Western blot analysis of glial fibrillary acidic protein (GFAP) expression in PSCs, collagen quantification and serum aminotransferase determination. The fatty infiltration in the steatotic livers decreased after the treatment with both NL diets, indicating liver recovery. This decrease was accompanied with a lower collagen deposition and aminotransferase level as well as changes in the PSC population that increased the GFAP expression. The above-mentioned effects were more pronounced in animals fed on NL-O based diet. Treatment with a balanced diet enriched in olive oil contributes to the liver recovery from a steatotic process. The PSC phenotype is a marker of this hepatic-recovery model.

Melatonin Prevents Oxidative Stress and Hepatocyte Cell Death Induced by Experimental Cholestasis

The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500 μg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine aminotransferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of α-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p<0.0001) and a reduction in GSH, GPx, catalase and SOD content (p<0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p<0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.

Hepatic blood flow regulation by stellate cells in normal and injured liver.

Hepatic stellate cells have received considerable attention as key components of the fibrogenic response to injury. Beyond this feature, they also have been implicated as regulators of sinusoidal vascular tone, and in disease states, in the pathogenesis of intrahepatic portal hypertension. The basis for this latter concept is derived from the following: (a) stellate cells are situated in a perisinusoidal orientation within the sinusoid, optimized for sinusoidal constriction; (b) a series of studies performed over the past decade have demonstrated that perisinusoidal stellate cells exhibit a remarkable capacity for cellular contraction, a characteristic that is most prominent after liver injury and stellate cell activation; and (c) in vivo microscopy studies have revealed that stellate cells can mediate sinusoidal constriction. Available evidence indicates that liver injury leads to a vascular disorder in which endothelin-1 is overproduced by stellate cells and endothelial cell-derived nitric oxide production is reduced. These abnormalities, in the context of exaggerated stellate cell contractility after liver injury, set up a paradigm in which stellate cells contribute to the increased intrahepatic resistance typical of portal hypertension. Furthermore, because stellate cell contractility and the mediators that control this function are dynamic processes, strategies that target exaggerated contractility provide an opportunity for novel therapeutics in intrahepatic portal hypertension.

Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension.

Reduced production of nitric oxide (NO) in the cirrhotic liver results from a defect in hepatic endothelial cell nitric oxide synthase (ecNOS) and appears to contribute to the high intrahepatic resistance and portal hypertension typical of cirrhosis. Therefore, we postulated that targeting a heterologous NOS isoform to sinusoidal endothelial cells or other perisinusoidal cells, such as hepatic stellate cells, would counter the defect in NO production and reduce resistance to blood flow. Recombinant adenovirus (Ad) carrying the neuronal NOS gene (nNOS) targeted liver sinusoidal endothelial cells, stellate cells, and hepatocytes more efficiently than the corresponding cells in cirrhotic livers, but transduction rates were substantial even in cirrhotic animals. Expression of nNOS in each liver cell type, whether from normal or injured liver, caused increased NO production and inhibited endothelin-1-induced contractility of perisinusoidal stellate cells. Finally, in 2 different in vivo models of cirrhosis and portal hypertension, transduction of livers with recombinant Ad.nNOS significantly reduced intrahepatic resistance and portal pressure. The data highlight the feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of a novel therapy for portal hypertension caused by cirrhosis.

Synaptophysin: A Novel Marker for Human and Rat Hepatic Stellate Cells

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.

Cells with morphological and immunohistochemical features of hepatic stellate cells (Ito cells) form an extralittoral (extrasinusoidal) compartment in the cirrhotic rat liver.

Systematic studies on hepatic stellate cells and myofibroblasts have so far mainly focused on cells located in the perisinusoidal space of Disse, the so-called littoral compartment. Here, these cells play a key role for intralobular fibrogenesis and sinusoidal capillarization. However, advanced hepatic fibrosis and cirrhosis are characterized by portal tract fibrosis and septal fibrosis, thus involving cells outside the perisinusoidal space. To study the question as to whether hepatic stellate cells occur and are expanded in an extralittoral (extrasinusoidal) compartment in cirrhogenesis, we systematically analyzed the distribution and density of desminreactive stellate cells in a rat model of hepatic fibrosis. Fibrosis and remodeling of the liver were induced by bile duct ligation, and stellate cells were identified by single and double immunohistochemistry. We can show that desmin-reactive cells are reproducibly detectable in extralittoral compartments of the normal and fibrotic rat liver. Periductular extralittoral stellate cells are significantly more frequent in cirrhosis, indicating that extralittoral stellate cells expand in concert with proliferating ductules. The findings suggest that ductular proliferation thought to represent a pacemaker of hepatic remodeling is accompanied by a population of cells exhibiting the same phenotype as perisinusoidal stellate cells.

Retinoids may increase fibrotic potential of TGF-β: Crosstalk between two multi-functional effectors

HepatologyVolume 26, Issue 4 p. 1067-1068 EditorialFree Access Retinoids may increase fibrotic potential of TGF-β: Crosstalk between two multi-functional effectors R Blomhoff, R BlomhoffSearch for more papers by this author R Blomhoff, R BlomhoffSearch for more papers by this author First published: 30 December 2003 https://doi.org/10.1002/hep.510260439Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int Rev Cytol 1980; 66: 303– 353. 2 Blomhoff R, Wake K. Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis. FASEB J 1991; 5: 271– 277. 3 Friedman SL. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med 1993; 328: 1828– 1835. 4 Davis BH, Kramer RT, Davidson NO. Retinoic acid modulates rat Ito cell proliferation, collagen, and transforming growth factor beta production. J Clin Invest 1990; 86: 2062– 2070. 5 Davis BH, Vucic A. The effect of retinol on Ito cell proliferation in vitro. Hepatology 1988; 8: 788– 793. 6 Sato T, Kato R, Tyson CA. Regulation of differentiated phenotype of rat hepatic lipocytes by retinoids in primary culture. Exp Cell Res 1995; 217: 72– 83. 7 Senoo H, Wake K. Suppression of experimental hepatic fibrosis by administration of vitamin A. Lab Invest 1985; 52: 182– 194. 8 Seifer WF, Bosma A, Hendriks HF, van Leeuwen RE, van Thil-de Ruiter GC, Seifert-Bock I, Knock DL, et al. Beta-carotene (provitamin A) decreases the severity of CCl4-induced hepatic inflammation and fibrosis in rats. Liver 1995; 15: 1– 8. 9 Seifer WF, Bosma A, Brouwer A, Hendriks HF, Roholl PJ, van Leeuwen RE, Ruiter GC van Thil-de, et al. Vitamin A deficiency potentiates carbon tetrachloride-induced liver fibrosis in rats. Hepatology 1994; 19: 193– 201. 10 Okuno M, Moriwaki H, Imai S, Muto Y, Kawada N, Suzuki Y, Kojima S. Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-β in liver stellate cells. Hepatology 1997; 26: 918– 926. 11 Blomhoff R. Vitamin A in Health and Disease. New York: Marcel Dekker, Inc., 1994: 1– 677. 12 Blomhoff R, Green MH, Berg T, Norum KR. Transport and storage of vitamin A. Science 1990; 250: 399– 404. 13 Blomhoff R, Helgerud P, Rasmussen M, Berg T, Norum KR. In vivo uptake of chylomicron retinyl ester by rat liver: evidence for retinol transfer from parenchymal to nonparenchymal cells. Proc Natl Acad Sci U S A 1982; 79: 7326– 7330. 14 Blomhoff R, Rasmussen M, Nilsson A, Norum KR, Berg T, Blaner WS, Kato M, et al. Hepatic retinol metabolism, distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells. J Biol Chem 1985; 260: 13560– 13565. 15 Blaner WS, van Bennekum AM, Brouwer A, Hendriks HF. Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions. FEBS Lett 1990; 274: 89– 92. 16 Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Kastner P, Mark M, et al. The nuclear receptor superfamily: the second decade. Cell 1995; 83: 835– 839. 17 Ohata M, Lin M, Satre M, Tsukamoto H. Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis. Am J Physiol 1997; 272: G589– G596. 18 Takahashi N, Bretiman TR. Retinoylation of proteins in mammalian cells, pp. 257– 273. In: R Blomhoff, ed. Vitamin A in Health and Disease. New York: Marcel Dekker, Inc., 1994: 257–273. 19 Koga H, Fujita I, Miyazaki S. Effects of all-trans retinoic acid on super-oxide generation in intact neutrophils and a cell-free system. Br J Haematol 1997; 97: 300– 305. 20 Buck J, Derguini F, Levy E, Nakanishi K, Hmmerling U. Intracellular signalling by 14-hydroxy-4, 14-retro-retinol. Science 1991; 254: 1654– 1656. 21 Achkar CC, Derguini F, Blumberg B. 4-oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors. Proc Natl Acad Sci USA 1996; 93: 4879– 4884. Citing Literature Volume26, Issue4October 1997Pages 1067-1068 ReferencesRelatedInformation