Studying the influence of liver cells on stem cells. Endothelial cells, Kupffer cells and Ito About the treatment of organ pathology

Intercellular communication might be realized by paracrine secretion and direct cell-to-cell contacts. It is known that hepatic perisinusoidal cells (HPC) establish regional stem cells niche and determine their differentiation. At the same time, HPC remains poorly characterized on molecular and cellular level.

The aim of the project was to study interactions between rat hepatic perisinusoidal cells and various stem cells such as mononuclear cell fraction of human umbilical cord blood (UCB-MC) and rat bone-marrow derived multipotential mesenchymal stromal cells (BM-MMSC).

Materials and methods. Rat BM-MSC and HPC, human UCB-MC cells were derived using standard techniques. To study HPC paracrine regulation we co-cultured UCB-MC or BM-MMSC cells with HPC using Boyden chambers and conditioned HPC cells media. Differentially labeled cells were co-cultured and their interactions were observed by phase-contrast fluorescent microscopy and immunocytochemistry.

Results. During the first week of cultivation there was autofluorescence of vitamin A because of fat-storing ability of PHC. BM-MMSC demonstrated high viability in all co-cultural models. After 2 day incubation in conditioned media co-culture of BM-MMSC with HPC we observed changes in morphology of MMSC - they decreased in size and their sprouts became shorter. The expression of α-Smooth Muscle Actin and desmin was similar to myofibroblast - an intermediate form of Ito cells culture in vitro. These changes could be due to paracrine stimulation by HPC. The most profound effect of HPC on UCB-MC cells was observed in contact co-culture, thereby it is important for UCB-MC cells to create direct cell-to-cell contacts for maintaining their viability. We did not observe any cell fusion between HPC /UCB and HPC /BM-MMSC cells in co-cultures. In our further experiments we plan to study growth factors produced by HPC for hepatic differentiation of stem cells.

Introduction.

Of particular interest among the diversity of liver cells are liver perisinusoidal cells (Ito cells). Thanks to the secretion of growth factors and components of the intercellular matrix, they create a microenvironment of hepatocytes, and a number of scientific studies have shown the ability of liver stellate cells to form a microenvironment for progenitor cells (including hematopoietic ones) and influence their differentiation into hepatocytes. Cell-to-cell interactions of these cell populations may occur through paracrine secretion of growth factors or direct cell-to-cell contacts, but the molecular and cellular basis of these processes remain poorly understood.

Purpose of the study.

Study of interaction mechanisms Ito cells with hematopoietic (HSC) and mesenchymal (MMSC) stem cells under in vitro conditions.

Materials and methods.

Rat liver Ito cells were isolated by two different enzymatic methods. At the same time, stromal MMSCs were obtained from rat bone marrow. The mononuclear fraction of hematopoietic stem cells was isolated from human umbilical cord blood. The paracrine influences of Ito cells were studied by culturing MMSCs and HSCs in the medium in which Ito cells grew, and by co-cultivating cells separated by a semipermeable membrane. The influence of intercellular contacts was studied during co-culture of cells. For better visualization, each population was labeled with an individual fluorescent tag. Cell morphology was assessed by phase contrast and fluorescence microscopy. Phenotypic characteristics of cultured cells were studied using immunocytochemical analysis.

Results.

Within a week after isolating perisinusoidal cells, we noted their ability to autofluorescent due to their fat-accumulating ability. Next, the cells entered an intermediate phase of their growth and acquired a stellate shape. At the initial stages of co-cultivation of Ito cells with rat bone marrow MMSCs, the viability of MMSCs was maintained in all cultivation options. On the second day, when MMSCs were cultivated in the culture medium of Ito cells, a change in the morphology of MMSCs occurred - they decreased in size, and their processes shortened. The expression of alpha-smooth muscle actin and desmin in MMSCs increased, indicating their phenotypic similarity to myofibroblasts, an intermediate growth stage of activated Ito cells in vitro. Our data indicate the influence of paracrine factors secreted by Ito cells on the properties of MMSCs in culture.

Based on co-cultivation of hematopoietic stem cells with Ito cells, it was shown that hematopoietic stem cells retain viability only during contact co-cultivation with Ito cells. According to the fluorescent analysis of mixed cultures, the phenomenon of fusion of cells of different populations was not detected.

Conclusions. To maintain the viability of hematopoietic stem cells, the presence of direct intercellular contacts with Ito cells is a decisive factor. Paracrine regulation was observed only when MMSCs were cultured in the nutrient medium in which Ito cells grew. It is planned to study the influence of specific factors produced by Ito cells on the differentiation of HSCs and MMSCs in cell culture in the following studies.

Shafigullina A.K., Trondin A.A., Shaikhutdinova A.R., Kaligin M.S., Gazizov I.M., Rizvanov A.A., Gumerova A.A., Kiyasov A.P.
State Educational Institution of Higher Professional Education "Kazan State Medical University of the Federal Agency for Health and Social Development"

1

An ultrastructural, immunohistochemical and morphometric analysis of the population of liver stellate cells was carried out in the dynamics of the development of fibrosis and cirrhosis of infectious viral origin. Fibrogenic activation of hepatic stellate cells was revealed, which is characterized by a reduction of lipid droplets and synchronous expression of fibroblast-like characteristics - a positive immunohistochemical reaction to smooth muscle α-actin, hyperplasia of the granular cytoplasmic reticulum and pericellular formation of numerous collagen fibrils. It has been shown that, despite the progressive decrease in the numerical density of lipid-containing stellate cells during the development of fibrosis, the need to maintain the function of retinoid deposition remains: in liver cirrhosis, lipid-containing stellate cells were found in fibrous septa and inside the lobules. It was concluded that hepatic stellate cells are a polymorphic heterogeneous population with a wide range of functional activity.

fibrogenesis

liver stellate cells

ultrastructure

immunohistochemistry

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10. Senoo H. Structure and function of hepatic stellate cells // Med. Electron. Microsc. – 2004. – Vol. 37. – P. 3–15.

Liver stellate cells (lipocytes, Ito cells, fat-accumulating liver cells) are localized in the spaces of Disse between hepatocytes and the endothelial lining of the sinusoids and play a leading role in the regulation of retinoid homeostasis, depositing up to 80% of vitamin A. Disse's space is the area of ​​greatest functional responsibility, providing transsinusoidal exchange. It has been demonstrated in experimental models and in cell culture that hepatic stellate cells differentiate into large cytoplasmic lipid droplets containing vitamin A; this phenotype is interpreted as "resting".

Increasing importance is being attached to the role of stellate cells in the development of liver fibrosis and cirrhosis. Upon receiving fibrogenic stimuli, "quiescent" stellate cells "transdifferentiate" to a myofibroblast-like phenotype and begin to produce collagen, proteoglycans, and other components of the extracellular matrix. Fibrosis at the level of the central veins, sinusoids or portal vessels limits the normal hemodynamics of the liver, which leads to a reduction in metabolically effective parenchyma, subsequently to portal hypertension and portosystemic shunting. The accumulation of connective tissue in the spaces of Disse disrupts normal metabolic traffic between the blood and hepatocytes, interfering with the clearance of circulating macromolecules, altering cell-cell interactions, and leading to liver cell dysfunction.

There are conflicting opinions regarding whether activated stellate cells are capable of reverting to a quiescent phenotype. Evidence has been obtained that fibrogenic hepatic stellate cells can partially neutralize the activation process, for example, when exposed to retinoids or when interacting with components of the extracellular matrix, including fibrillar collagen type I or components of the basement membrane. The solution to this issue lies at the heart of the problem of fibrosis reversibility and the development of therapeutic approaches to the treatment of liver cirrhosis.

Purpose of the study- conduct a comprehensive study of the structural and functional characteristics of liver stellate cells in the dynamics of fibrotic changes in a model of chronic HCV infection.

Material and research methods

A comprehensive light-optical, electron microscopic and morphometric study of liver biopsy samples from chronic HCV infection at various stages of fibrotic changes was carried out (100 samples divided into 4 equal groups according to the severity of fibrosis). It is important to note that lipid-containing stellate cells are best visualized on semithin sections, while fibrogenic stellate cells are best visualized only on ultrathin sections or by immunohistochemical imaging.

Liver samples were fixed in a 4% paraformaldehyde solution cooled to 4 °C, prepared in Millonig's phosphate buffer (pH 7.2-7.4); Paraffin sections were stained with hematoxylin and eosin in combination with the Perls reaction, according to Van Gieson with additional staining of elastic fibers with Weigert's resorcinol fuchsin, and the CHIC reaction was performed. Semi-thin sections were stained with Schiff's reagent and Azure II. The study was carried out using a Leica DM 4000B universal microscope (Germany). Microphotographs were taken using a Leica DFC 320 digital camera and Leica QWin computer program. Ultrathin sections, contrasted with uranyl acetate and lead citrate, were examined in a JEM 1010 electron microscope at an accelerating voltage of 80 kW.

The stage of liver fibrosis was determined on a 4-point scale, ranging from portal fibrosis (stage I) to cirrhosis with the formation of porto-central vascularized septa and nodular transformation of the parenchyma. Liver stellate cells and other matrix-producing cellular elements were identified in the dynamics of fibrosis development by the expression of smooth muscle α-actin.

Smooth muscle α-actin expression in liver matrix-producing cells was tested using a two-step indirect immunoperoxidase method with a streptavidin-biotin negative control product imaging system. Mouse monoclonal antibodies to smooth muscle α-actin (NovoCastra Lab. Ltd, UK) at a dilution of 1:25 were used as primary antibodies; as secondary antibodies - universal biotinylated antibodies. The products of the immunohistochemical reaction were visualized using diaminobenzidine, then the sections were counterstained with Mayer's hematoxylin. The numerical density of lipid-containing stellate cells was assessed on semi-thin sections per unit field of view equal to 38,000 μm2. When statistically processing the data, the Student's test was used; differences in the compared parameters were considered significant if the probability of error P was less than 0.05.

Research results and discussion

With minimal fibrous changes in the liver of patients with chronic hepatitis C, as a rule, a fairly large number of stellate cells are found, which are clearly visible only in semi-thin and ultra-thin sections and are differentiated in the spaces of Disse by the presence of large lipid droplets in the cytoplasm. The transformation of stellate cells from “resting” cells containing retinoids to fibrogenic ones is accompanied by a gradual decrease in the number of lipid droplets. In this regard, the true number of stellate cells can be determined using a comprehensive electron microscopic and immunohistochemical study.

At the initial stages of fibrosis (0, I) in chronic hepatitis C, when studying semi-thin sections, the population of liver stellate cells was distinguished by pronounced polymorphism - the size, shape, number of lipid droplets and their tinctorial properties varied sharply: differences in the osmiophilicity of lipid-containing material in different cells. The numerical density of liver stellate cells, visualized in preparations by the presence of cytoplasmic lipid droplets, was 5.01 ± 0.18 per unit field of view.

Features of the ultrastructure of stellate cells are associated with the heterogeneity of the electron density of lipid droplets not only within one cell, but also between different lipocytes: against the background of an electron-transparent lipid substrate, a more osmiophilic marginal rim stood out; In addition, the nuclei were sharply polymorphic, and the length of the cytoplasmic processes varied. Among the ultrastructural features of lipid-containing stellate cells, along with the presence of lipid droplets, one can note a very small amount of cytoplasmic matrix, poor in membrane organelles, including mitochondria, and therefore, apparently, this phenotype of lipocytes is called “resting” or “passive” .

At stages of fibrosis II and III, the ultrastructure of most stellate cells acquired the so-called mixed, or transitional, phenotype - the simultaneous presence of morphological characteristics of both lipid-containing and fibroblast-like cells. In such lipocytes, the nuclei had deep invaginations of the nucleolemma, a larger nucleolus, and an increased volume of cytoplasm that retained lipid droplets. At the same time, the number of mitochondria, free ribosomes, polysomes and tubules of the granular cytoplasmic reticulum sharply increased. As a rule, there was membrane contact between lipid droplets and mitochondria, indicating the “utilization” of lipids. In many cells, lipid droplets are degraded by the formation of autophagosomes, which are then eliminated by exocytosis. In some cases, proliferation of stellate cells of mixed phenotype was noted.

Matrix-producing stellate cells, most numerous at the stage of liver cirrhosis, were characterized by a complete absence of lipid granules, a fibroblast-like form, a developed protein-synthesizing compartment, and the formation of contractile fibrillar structures in the cytoplasm; Numerous bundles of collagen fibrils with specific transverse striations were localized pericellularly in the spaces of Disse.

In general, with the progression of chronic hepatitis C, accompanied by intralobular perisinusoidal fibrogenesis, there were morphological signs of activation of liver stellate cells, their transformation from the so-called “passive” ones, accumulating vitamin A, into fibrogenic and proliferating cells.

At the stage of transformation into liver cirrhosis, there was a significant decrease in the numerical density of lipid-containing stellate cells, indicating their fibrogenic transformation. However, with established liver cirrhosis, in isolated cases there were areas of the liver parenchyma with perisinusoidal lipid-containing stellate cells. In addition, in one sample, numerous lipocytes were found in the periportal fibrous tissue, which likely indicates the important role of stellate cells in the metabolism of retinoids in the body, even at the stage of cirrhosis of the organ. In addition, stellate cells appear to have a number of other functions, they are also found in extrahepatic organs such as the pancreas, lungs, kidneys and intestines, and it is believed that hepatic and extrahepatic stellate cells form the disseminated stellate cell system of the body , similar to the APUD system. For example, despite the association of fibrogenic stellate cells with liver cirrhosis, their activation may play a beneficial role in cases of acute injury because it results in the formation of an appropriate stromal circuit for parenchymal cell regeneration.

The severity of perihepatocellular fibrosis in chronic HCV infection, according to morphometric analysis, had a significant inverse correlation with the numerical density of lipid-containing stellate cells - at stage III fibrosis and in cirrhosis of the organ it was 0.20 ± 0.03 per unit of visual field, which is significant less (p< 0,05), чем на стадиях фиброза 0 - I (5,01 ± 0,18) и II (2,02 ± 0,04).

We tested the fibrogenic activity of matrix-producing liver cells using an immunohistochemical study of the expression of smooth muscle alpha actin. Products of the immunohistochemical reaction of varying intensity were found in the cytoplasm of activated stellate cells localized inside the hepatic lobules. Particularly significant expression of smooth muscle α-actin was observed in the cytoplasm of fibroblasts and myofibroblasts of the portal zones, vascular smooth muscle cells and myofibroblasts around the central veins.

Most data on the cellular mechanisms of fibrogenesis come from studies performed on hepatic stellate cells, but it is clear that various matrix-producing cells (each with a distinct location, immunohistochemical and ultrastructural phenotype) contribute to the development of liver fibrosis. They include fibroblasts and myofibroblasts of the portal tracts, vascular smooth muscle cells and myofibroblasts around the central veins, which are activated in conditions of chronic liver damage.

Conclusion

The role of liver stellate cells in the development of organ fibrosis in chronic hepatitis C has been demonstrated. As fibrosis progresses, the numerical density of lipid-containing stellate cells significantly decreases, while part of the population retains the so-called “resting” phenotype to carry out metabolic function. “Myofibroblast-like” liver stellate cells in a state of fibrogenic activation are characterized by the following structural and functional features: a decrease in the number and subsequent disappearance of lipid droplets, hyperplasia of the granular cytoplasmic reticulum and mitochondria, focal proliferation, immunohistochemical expression of fibroblast-like characteristics, including smooth muscle α-actin, and the formation pericellular collagen fibrils in the spaces of Disse.

Thus, hepatic stellate cells are not a static, but a dynamic population that is directly involved in the remodeling of the intralobular perihepatocellular matrix.

Reviewers:

Vavilin V.A., Doctor of Medical Sciences, Professor, Head. Laboratory of Drug Metabolism, Research Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk;

Kliver E.E., Doctor of Medical Sciences, leading researcher at the Laboratory of Pathomorphology and Electron Microscopy, Novosibirsk Research Institute of Circulatory Pathology named after Academician E.N. Meshalkin Ministry of Health and Social Development of the Russian Federation, Novosibirsk.

The work was received by the editor on August 15, 2011.

Bibliographic link

Postnikova O.A., Nepomnyashchikh D.L., Aidagulova S.V., Vinogradova E.V., Kapustina V.I., Nokhrina Zh.V. STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF LIVER STELLATE CELLS IN THE DYNAMICS OF FIBROSIS // Fundamental Research. – 2011. – No. 10-2. – P. 359-362;
URL: http://fundamental-research.ru/ru/article/view?id=28817 (access date: 01/30/2020). We bring to your attention magazines published by the publishing house "Academy of Natural Sciences"

Stellate cells

Above, a schematic representation of an Itoh cell (HSC) adjacent to nearby hepatocytes (PCs), below hepatic sinusoidal epithelial cells (ECs). S - liver sinusoid; KC - Kupffer cell. Bottom left - Ito cells in culture under a light microscope. Bottom right - Electron microscopy reveals numerous fat vacuoles (L) of Itoh cells (HSCs) that store retinoids.

Ito cells(synonyms: liver stellate cell, fat storage cell, lipocyte, English Hepatic Stellate Cell, HSC, Cell of Ito, Ito cell ) - pericytes contained in the perisinusoidal space of the hepatic lobule, capable of functioning in two different states - calm And activated. Activated Ito cells play a major role in fibrogenesis - the formation of scar tissue in liver damage.

In an intact liver, stellate cells are found in calm state. In this state, the cells have several projections covering a sinusoidal capillary. Another distinctive feature of cells is the presence of vitamin A (retinoid) reserves in their cytoplasm in the form of fat droplets. Quiet Ito cells make up 5-8% of all liver cells.

Ito cell outgrowths are divided into two types: perisinusoidal(subendothelial) and interhepatocellular. The first emerge from the cell body and extend along the surface of the sinusoidal capillary, covering it with thin finger-like branches. The perisinusoidal projections are covered with short villi and have characteristic long microshoots that extend even further along the surface of the capillary endothelial tube. Interhepatocellular projections, having overcome the plate of hepatocytes and reaching the adjacent sinusoid, are divided into several perisinusoidal projections. Thus, on average, an Ito cell covers slightly more than two adjacent sinusoids.

When the liver is damaged, Ito cells become activated state. The activated phenotype is characterized by proliferation, chemotaxis, contractility, loss of retinoid stores, and the formation of myofibroblast-like cells. Activated hepatic stellate cells also show increased levels of novel genes such as α-SMA, chemokines, and cytokines. Activation indicates the onset of the early stage of fibrogenesis and precedes the increased production of ECM proteins. The final stage of liver healing is characterized by increased apoptosis of activated Ito cells, as a result of which their number is sharply reduced.

Gold chloride staining is used to visualize Ito cells under microscopy. It has also been established that a reliable marker for differentiating these cells from other myofibroblasts is their expression of the Reelin protein.

Story

Links

• Young-O Queon, Zachary D. Goodman, Jules L. Dienstag, Eugene R. Schiff, Nathaniel A. Brown, Elmar Burkhardt, Robert Schoonhoven, David A. Brenner, Michael W. Fried (2001) Reduced fibrogenesis: an immunohistochemical study of paired biopsies liver cells after lamivudine therapy in patients with chronic hepatitis B. Journal of Haepothology 35; 749-755. - translation of an article in the journal “Infections and Antimicrobial Therapy”, Volume 04/N 3/2002, on the Consilium-Medicum website.
• Popper H: Distribution of vitamin A in tissue as revealed by fluorescence microscopy. Physiol Rev 1944, 24:205-224.

Notes

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Above, a schematic representation of an Itoh cell (HSC) adjacent to nearby hepatocytes (PCs), below hepatic sinusoidal epithelial cells (ECs). S sinusoids of the liver; KC Kupffer cell. Bottom left Ito cells in culture under a light microscope... Wikipedia

NERVE CELLS- NERVE CELLS, the main elements of nervous tissue. Discovered by N. K. Ehrenberg and first described by him in 1833. More detailed data about N. to. with an indication of their shape and the existence of an axial-cylindrical process, as well as ... ... Great Medical Encyclopedia

Large neurons of the cerebellar cortex (See Cerebellum) (M), the axons of which extend beyond its boundaries; described in 1837 by Ya. E. Purkin. Through P. k. the command influences of the M cortex on the motor centers subordinate to it (M nuclei and vestibular nuclei) are realized. U... ... Great Soviet Encyclopedia

Or Gephyrei class of the subphylum Vermiformes or Vermidea, a type of worms or Vermes. Animals belonging to this class are exclusively marine forms that live in the mud and sand of warm and cold seas. The class of stellate Ch. was established by Quatrphage... ...

Not to be confused with neutron. Pyramidal cells of neurons in the mouse cerebral cortex A neuron (nerve cell) is a structurally functional unit of the nervous system. This cell has a complex structure, highly specialized and in structure... ... Wikipedia

This name applies both to some pigment cells and to parts of cells (both animal and plant) containing pigment. X. are more often found in plants (see the previous article by N. Gaidukov), but they are also described in protozoa... Encyclopedic Dictionary F.A. Brockhaus and I.A. Ephron

- (cellulae flammeae), cells with a bundle of cilia and a long process, closing the proximal part of the protonephridium tubule. Center, part "P. k., having numerous. stellate processes, passes into the cavity, a tuft of long cilia descends into the cavity... ...

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FLAME CELLS (cellulae flammeae), cells with a bundle of cilia and a long process, closing the proximal part of the protonephridium tubule. Center. part of the P. to., having numerous. stellate processes, passes into the cavity, a bundle descends into the cavity... ... Biological encyclopedic dictionary

- (S. Golgi) stellate neurons of the granular layer of the cerebellar cortex ... Large medical dictionary

Genes & Cells: Volume V, No. 1, 2010, pp.: 33-40

Authors

Gumerova A., Kiyasov A.P.

Regenerative medicine is one of the most rapidly developing and promising areas of medicine, which is based on a fundamentally new approach to restoring a damaged organ by stimulating and (or) using stem (progenitor) cells to accelerate the regeneration. To implement this approach, it is necessary to know what stem cells are, and, in particular, regional stem cells, what their phenotype and potency are. For a number of tissues and organs, such as the epidermis and skeletal muscle, stem cells have already been identified and their niches described. However, the liver, an organ whose regenerative abilities have been known since ancient times, has not yet revealed its main secret - the secret of the stem cell. In this review, based on our own and literature data, we discuss the hypothesis that perisinusoidal stellate cells can lay claim to the role of liver stem cells.

Perisinusoidal liver cells (Ito cells, stellate cells, lipocytes, fat-storing cells, vitamin A-storing cells) are one of the most mysterious cell types of the liver. The history of studying these cells goes back more than 130 years, and there are still many more questions regarding their phenotype and functions than answers. The cells were described in 1876 by Kupffer, whom he named stellate cells and classified as macrophages. Later, true sedentary macrophages of the liver received the name Kupffer.

It is generally accepted that Ito cells are located in the space of Disse in direct contact with hepatocytes, accumulate vitamin A and are capable of producing macromolecules of intercellular substance, and also, having contractile activity, regulate blood flow in sinusoidal capillaries like pericytes. The gold standard for identifying Ito cells in animals is to identify the cytoskeletal intermediate filament protein characteristic of muscle tissue, desmin. Other fairly common markers of these cells are markers of neuronal differentiation - glial fibrillary acid protein (GFAP) and nestin.

For many years, Ito cells were considered only from the standpoint of their participation in the development of fibrosis and cirrhosis of the liver. This is due to the fact that when the liver is damaged, activation of these cells always occurs, which consists of increased expression of desmin, proliferation and transdifferentiation into myofibroblasts-like cell transformation expressing --smooth muscle actin (--SMA) and synthesizing significant amounts of intercellular substance, in particular type I collagen. It is the activity of such activated Ito cells that leads, according to many researchers, to the development of liver fibrosis and cirrhosis.

On the other hand, facts are gradually accumulating that allow us to look at Ito cells from completely unexpected positions, namely, as the most important component of the microenvironment for the development of hepatocytes, cholangiocytes and blood cells during the hepatic stage of hematopoiesis, and, moreover, as possible stem cells ( progenitor cells of the liver. The purpose of this review is to analyze modern data and views on the nature and functional significance of these cells with an assessment of their possible membership in the population of liver stem (progenitor) cells.

Ito cells are the most important participant in the restoration of parenchyma during liver regeneration due to the macromolecules of the intercellular matrix they produce and its remodeling, as well as the production of growth factors. The first doubts about the truth of the established theory, which considers Ito cells exclusively as the main culprits of liver fibrosis, arose when it was found that these cells produce a significant number of morphogenic cytokines. Among them, a significant group consists of cytokines, which are potential mitogens for hepatocytes.

The most important in this group is hepatocyte growth factor - hepatocyte mitogen, necessary for proliferation, survival and cell motility (it is also known as scatter factor. A defect in this growth factor and (or) its receptor C-met in mice leads to liver hypoplasia and destruction of its parenchyma as a result of suppression of hepatoblast proliferation, increased apoptosis and insufficient cell adhesion.

In addition to hepatocyte growth factor, Ito cells produce stem cell factor. This was shown in a model of liver regeneration after partial hepatectomy and exposure to 2-acetoaminofluorene. It has also been established that Ito cells secrete transforming growth factor and epidermal growth factor, which play an important role both in the proliferation of hepatocytes during regeneration and stimulate mitosis of the Ito cells themselves. The proliferation of hepatocytes is also triggered by the mesenchymal morphogenic protein epimorphin expressed by Ito cells, which appears in them after partial hepatectomy, and pleiotrophin.

In addition to paracrine mechanisms of interaction between hepatocytes and Ito cells, direct intercellular contacts of these cells with hepatocytes also play a certain role. The importance of intercellular contacts between Ito cells and epithelial progenitor cells was demonstrated in vitro when mixed culture proved more effective in differentiating the latter into albumin-producing hepatocytes than culture of membrane-separated cells, where they could only exchange soluble factors through cultural medium. Isolated from the fetal liver of mice on day 13.5. gestation, mesenchymal cells with the Thy-1+/С049!±/vimentin+/desmin+/ --GMA+ phenotype, after establishing direct intercellular contacts, stimulated the differentiation of a population of primitive hepatic endodermal cells - into hepatocytes (containing glycogen, expressing m-RNA tyrosine aminotransferases and tryptophan oxygen -names). The population of Thy-1+/desmin+ mesenchymal cells did not express markers of hepatocytes, endothelium and Kupffer cells, and, apparently, were represented specifically by Ito cells. High densities of desmin-positive Ito cells and their arrangement in close contact with differentiating hepatocytes have been noted in vivo in rat and human prenatal liver. Thus, all these facts allow us to conclude that this cell type is the most important component of the microenvironment, necessary for the normal development of hepatocytes in ontogenesis and their restoration in the process of reparative regeneration.

In recent years, data have been obtained indicating a significant influence of Ito cells on the differentiation of hematopoietic stem cells. Thus, Ito cells produce erythropoietin and neurotrophin, which influence the differentiation of not only liver epithelial cells, but also hematopoietic stem cells. A study of fetal hematopoiesis in rats and humans showed that it is these cells that constitute the microenvironment of hematopoietic islands in the liver. Ito cells express vascular cell adhesion molecule-1 (VCAM-1), a key molecule for maintaining the adhesion of hematopoietic progenitors to bone marrow stromal cells. In addition, they also express stromal derived factor-1 - (SDF-1 -) - a potential chemoattractant for hematopoietic stem cells, stimulating their migration to the site of hematopoiesis due to interaction with the specific receptor Cystein-X- Cystein receptor 4 (CXR4), as well as the homeobox protein Hlx, if defective, both the development of the liver itself and hepatic hematopoiesis are disrupted. Most likely, it is the expression of VCAM-1 and SDF-1a on fetal Ito cells that is the trigger for the attraction of hematopoietic progenitor cells to the fetal liver for further differentiation. Retinoids accumulated by Ito cells are also an important morphogenesis factor for hematopoietic cells and epithelia. One cannot fail to mention the influence of Ito cells on mesenchymal stem cells. Ito cells, isolated from rat liver and fully activated, modulate the differentiation of mesenchymal stem cells (multipotent mesenchymal stromal cells) of the bone marrow into hepatocyte-like cells (accumulating glycogen and expressing thetase and phosphoenolpyruvate carboxykinase) after 2 weeks. co-cultivation.

Thus, the accumulated scientific evidence allows us to conclude that Ito cells are one of the most important cell types necessary for the development and regeneration of the liver. It is these cells that create the microenvironment both for fetal hepatic hematopoiesis and for the differentiation of hepatocytes during prenatal development, as well as for the differentiation of epithelial and mesenchymal progenitor cells into hepatocytes in vitro. Currently, these data are beyond doubt and are accepted by all liver researchers. What then served as the starting point for the emergence of the hypothesis put forward in the title of the article?

First of all, its appearance was facilitated by the identification in the liver of cells expressing both epithelial markers of hepatocytes and mesenchymal markers of Ito cells. The first work in this area was carried out in the study of prenatal histo- and organogenesis of the mammalian liver. It is the development process that is the key event, the study of which makes it possible to trace in natural conditions the dynamics of the primary formation of the definitive phenotype of various cell types of an organ using specific markers. Currently, the range of such markers is quite wide. In works devoted to the study of this issue, various markers of mesenchymal and epithelial cells, individual cell populations of the liver, and stem (including hematopoietic) cells were used.

In the studies conducted, it was found that desmin-positive Ito cells of rat fetuses transiently on days 14-15. gestations express epithelial markers characteristic of hepatoblasts, such as cytokeratins 8 and 18. On the other hand, hepatoblasts at the same time of development express the Ito cell marker desmin. This is what allowed us to make the assumption that in the liver during intrauterine development there are cells with a transitional phenotype expressing both mesenchymal and epithelial markers, and, therefore, to consider the possibility of the development of Ito cells and hepatocytes from one source and (or) to consider these cells as one and the same cell type at different stages of development. Further studies of histogenesis conducted on human embryonic liver material showed that at 4-8 weeks. intrauterine development of human liver, Ito cells expressed cytokeratins 18 and 19, which was confirmed by double immunohistochemical staining, and weak positive staining for desmin was noted in hepatoblasts.

However, in a study published in 2000, the authors were unable to detect the expression of desmin in hepatoblasts in the liver of mouse fetuses, and E-cadherin and cytokeratins in Ito cells. The authors obtained positive staining for cytokeratins in Ito cells only in a small proportion of cases, which they associated with nonspecific cross-reaction of primary antibodies. The choice of these antibodies is somewhat puzzling - antibodies to chicken desmin and bovine cytokeratins 8 and 18 were used in the work.

In addition to desmin and cytokeratins, a common marker for Ito cells and fetal hepatoblasts of mice and rats is another mesenchymal marker - the vascular cell adhesion molecule VCAM-1. VCAM-1 is a unique surface marker that distinguishes Ito cells from myofibroblasts in the adult rat liver and is also present on several other liver cells of mesenchymal origin, such as endothelial cells or myogenic cells.

Another evidence in favor of the hypothesis under consideration is the possibility of mesenchymal-epithelial transdifferentiation (conversion) of Ito cells isolated from the liver of adult rats. It should be noted that the literature mainly discusses epithelial-mesenchymal rather than mesenchymal-epithelial transdifferentiation, although both directions are recognized as possible, and often the term “epithelial-mesenchymal transdifferentiation” itself is used to refer to transdifferentiation in either direction. Having analyzed the expression profile of m-RNA and corresponding proteins in Ito cells isolated from the liver of adult rats after exposure to carbon tetrachloride (CTC), the authors found both mesenchymal and epithelial markers in them. Among the mesenchymal markers, nestin, --GMA, and matrix metalloproteinase-2 (MMP-2) were identified, and among the epithelial ones, muscle pyruvate kinase (MPK), characteristic of oval cells, cytokeratin 19, α-FP, E-cadherin, as well as the transcription factor Hepatocyte nuclear factor 4- (HNF-4-), specific for cells that are destined to become hepatocytes. It was also found that in the primary culture of human epithelial hepatic progenitor cells, m-RNA expression of Ito cell markers occurs - nestin, GFAP - epithelial progenitors co-express both epithelial and mesenchymal markers. The possibility of mesenchymal-epithelial transdifferentiation is confirmed by the appearance in Ito cells of Integrin-linked kinase (ILK), an enzyme necessary for such transdifferentiation.

Mesenchymal-epithelial transdifferentiation was also revealed in our in vitro experiments, where an original approach was taken to cultivate a pure population of Ito cells isolated from rat liver until a dense monolayer of cells was formed. After this, the cells stopped expressing desmin and other mesenchymal markers, acquired the morphology of epithelial cells and began to express markers characteristic of hepatocytes, in particular cytokeratins 8 and 18. Similar results were obtained during organotypic cultivation of rat fetal liver.

Within the last year, two papers have been published that consider Ito cells as a subtype of oval cells, or as their derivatives. Oval cells are small oval-shaped cells with a narrow rim of cytoplasm that appear in the liver in some models of toxic liver injury and are currently considered to be bi-potent progenitor cells capable of differentiating into both hepatocytes and cholangiocytes. Based on the fact that the genes that are expressed by isolated Ito cells coincide with the genes expressed by oval cells, and under certain culturing conditions of Ito cells, hepatocytes and bile duct cells appear, the authors tested the hypothesis according to which Ito cells are a type of oval cells capable of generate hepatocytes to regenerate damaged liver. Transgenic GFAP-Cre/GFP (Green fluorescent protein) mice were fed a methionine-choline-deficient/ethionine-enriched diet to activate Ito cells and oval cells. Quiescent Ito cells had a GFAP+ phenotype. After activation of Ito cells by injury or culture, their GFAP expression decreased and they began to express markers of oval and mesenchymal cells. The oval cells disappeared as GFP+ hepatocytes appeared, beginning to express albumin and eventually replacing large areas of liver parenchyma. Based on their findings, the authors hypothesized that Ito cells are a subtype of oval cells that differentiate into hepatocytes through a “mesenchymal” phase.

In experiments performed on the same model of activation of oval cells, when the latter were isolated from the liver of rats, it was found that oval cells in vitro express not only the traditional markers 0V-6, BD-1/BD-2 and M2RK and markers trees of the extracellular matrix, including collagens, matrix metalloproteinases and tissue inhibitors of metalloproteinases - marker features of Ito cells. After exposure of cells to TGF-pl, in addition to suppression of growth and morphological changes, an increase in the expression of these genes, as well as desmin and GFAP genes, the appearance of expression of the transcription factor Snail, responsible for epithelial-mesenchymal transdifferentiation, and cessation of the expression of E-cadherin were noted, which indicates the possibility of “reverse” transdifferentiation of oval cells into Ito cells.

Since oval cells are traditionally considered as bipotent precursors of both hepatocytes and cholangiocytes, attempts have been made to establish the possibility of the existence of transitional forms between the epithelial cells of the intrahepatic bile ducts and Ito cells. Thus, it was shown that in normal and damaged liver small structures of the ductal type were stained positively for the Ito cell marker - GMA, however, in the photographs presented in the article, which reflect the results of immunofluorescent staining, it is possible to determine what these - GMA+ ductal structures - bile ducts or blood vessels - are not possible. However, other results have been published indicating the expression of Ito cell markers in cholangiocytes. In the already mentioned work by L. Yang, expression of the Ito cell marker GFAP by bile duct cells was shown. The cytoskeletal intermediate filament protein synemin, present in normal liver in Ito cells and vascular cells, appeared in the duct cells involved during the development of the ductular reaction; it was also expressed in cholangiocarcinoma cells. Thus, if there is quite a lot of various evidence regarding the possibility of mutual transdifferentiation of Ito cells and hepatocytes, then with cholangiocytes such observations are still sporadic and not always unambiguous.

To summarize, we can say that the patterns of expression of mesenchymal and epithelial markers both during histo- and organogenesis of the liver, and in a variety of experimental conditions, both in vivo and in vitro, indicate the possibility of both mesenchymal-epithelial and epithelial-mesenchymal small transitions between Ito cells/oval cells/hepatocytes, and therefore allow Ito cells to be considered as one of the sources of hepatocyte development. The above facts undoubtedly indicate an inextricable connection between these cell types, and also indicate significant phenotypic plasticity of Ito cells. The phenomenal plasticity of these cells is also evidenced by their expression of a number of neural proteins, such as the already mentioned GFAP, nestin, neurotrophins and their receptors, the neural cell adhesion molecule (N-CAM), synaptophysin, and neural growth factor. factor, NGF), brain-derived neurotrophic factor (BDNF), on the basis of which a number of authors discuss the possibility of the development of Ito cells from the neural crest. However, over the last decade, another version has attracted enormous attention from researchers - namely, the possibility of the development of hepatocytes and Ito cells from hematopoietic and mesenchymal stem cells.

The first work in which this possibility was proven was published by V.E. Petersen et al., who showed that hepatocytes are capable of developing from a hematopoietic stem cell. Subsequently, this fact was repeatedly confirmed in the works of other scientists, and a little later the possibility of differentiation into hepatocytes was shown for mesenchymal stem cells. How this happens - by fusion of donor cells with recipient liver cells, or by their transdifferentiation - is still not clear. However, we also found that human cord blood hematopoietic stem cells, when transplanted into the spleen of partial hepatectomy rats, colonize the liver and are able to differentiate into hepatocytes and liver sinusoid cells, as evidenced by the presence of human cell markers in these cell types. In addition, we were the first to show that preliminary genetic modification of umbilical cord blood cells does not have a significant effect on their distribution and differentiation capabilities in the recipient’s liver after transplantation. As for the possibility of the development of hepatocytes from hematopoietic stem cells during prenatal histogenesis, although this possibility cannot be completely excluded, it nevertheless seems unlikely, since the morphology, localization and phenotype of these cells differ significantly from similar indicators for liver cells. Apparently, if such a path exists, it does not play a significant role in the formation of epithelial and sinusoidal cells during ontogenesis. The results of recent studies, conducted both in vivo and in vitro, have cast doubt on the established theory of the development of hepatocytes only from the endodermal epithelium of the foregut, and therefore the assumption naturally arose that the regional stem cell of the liver may be located among its mesenchymal cells. Could such cells be Ito cells?

Considering the unique properties of these cells, their phenomenal plasticity and the existence of cells with a transitional phenotype from Ito cells to hepatocytes, we assume that these cells are the main candidates for this role. Additional arguments in favor of this possibility are that these cells, like hepatocytes, can be formed from hematopoietic stem cells, and they are the only sinusoidal cells of the liver that are capable of expressing markers of stem (progenitor) cells.

In 2004, it was determined that Ito cells can also develop from a hematopoietic stem cell. After transplantation of bone marrow cells from GFP mice, GFP+ cells expressing the Ito cell marker GFAP appeared in the liver of recipient mice, and processes of these cells penetrated between hepatocytes. If the recipient's liver was damaged by CCU, the transplanted cells also expressed Ito blast-like cells. When the fraction of non-parenchymal cells was isolated from the liver of recipient mice, GFP+ cells with lipid droplets accounted for 33.4+2.3% of the isolated cells; they expressed desmin and GFAP, and after 7 days. cultivation

On the other hand, transplantation of bone marrow cells leads to the formation of not only Ito cells, but also the type I collagen gene, on the basis of which it was concluded that such transplantation contributes to the development of fibrosis. However, there are also works that have demonstrated a decrease in liver fibrosis due to the migration of transplanted cells into fibrous septa and the production of matrix metalloproteinase-9 (Matrix Metalloproteinase-9, MMP-9) by these cells, which is one of the most important characteristics of Ito cells. Our preliminary data also showed a decrease in the number of myofibroblasts and a decrease in the level of fibrosis after autotransplantation of a fraction of peripheral blood mononuclear cells in patients with chronic hepatitis with severe liver fibrosis. In addition, as a result of hematopoietic stem cell transplantation, other cell types capable of producing extracellular matrix may appear in the recipient's liver. Thus, in liver injury induced by bile duct ligation, the transplanted cells are differentiated fibrocytes expressing collagen, and only when cultured in the presence of TGF-pl they are differentiated myofibroblasts, potentially promoting fibrosis. Thus, the authors associated the danger of liver fibrosis after bone marrow cell transplantation not with Ito cells, but with a “unique population of fibrocytes.” Due to the inconsistency of the data obtained, a discussion arose on another issue - whether Ito cells, which appeared as a result of differentiation of transplanted hematopoietic stem cells, will contribute to the development of fibrosis, or will they ensure complete regeneration of liver tissue and reduction of fibrosis. In recent years, it has become obvious (including from the data above) that the origin of myofibroblasts in the liver can be different - from Ito cells, from portal tract fibroblasts, and even from hepatocytes. It has also been established that myofibroblasts of different origins differ in a number of properties. Thus, activated Ito cells differ from portal tract myofibroblasts in vitamin content, contractile activity, response to cytokines, especially TGF-p, and the ability to undergo spontaneous apoptosis. Additionally, these cell populations are distinct and may express the vascular cell adhesion molecule VCAM-1, which is present on Ito cells and absent on myofibroblasts. It should also be noted that in addition to the production of proteins of the intercellular matrix, activated Ito cells also produce matrix metalloproteinases, which destroy this matrix. Thus, the role of Ito cells, including those formed from hematopoietic stem cells, in the development of fibrosis is far from being as clear as previously thought. Apparently, they do not so much promote fibrosis as remodel the extracellular matrix during liver repair after damage, thus providing a connective tissue framework for the regeneration of liver parenchymal cells.

normal and damaged rat livers. Rat Ito cells also express another marker of stem (progenitor) cells - CD133, and demonstrate the properties of progenitor cells, capable, depending on conditions, of differentiating in various - 2) with the addition of cytokines that facilitate differentiation into endothelial cells, form branched tubular structures with induction of marker expression endothelial cells - endothelial NO synthase and vascular endothelial cadherin; 3) when using cytokines that promote the differentiation of stem cells into hepatocytes - into round cells expressing hepatocyte markers - FP and albumin. Rat Ito cells also express 0ct4, a characteristic of pluripotent stem cells. Interestingly, only a portion of the Ito cell population could be isolated by magnetic sorter using anti-CD133 antibodies, but after standard (pronase/collagenase) isolation, all plastic-adherent cells expressed CD133 and 0kt4. Another marker for progenitor cells, Bcl-2, is expressed by desmin+ cells during prenatal development of the human liver.

Thus, various researchers have shown the possibility of Ito cells expressing certain markers of stem (progenitor) cells. Moreover, an article was recently published in which the hypothesis was first put forward that the Disse space, formed by basement membrane proteins, endothelial cells and hepatocytes, in which Ito cells are located, may constitute a microenvironment for the latter, acting as a “niche” of stem cells. cells. This is evidenced by several features characteristic of the table cell niche and identified in the components of the microenvironment of Ito cells. Thus, cells located in close proximity to the stem cell must produce soluble factors, as well as carry out direct interactions that keep the stem cell in an undifferentiated state and trap it in a niche, often located on the basement membrane. Indeed, endothelial cells of liver sinusoidal capillaries synthesize soluble SDF-1, which binds specifically to the Ito cell receptor CXR4 and stimulates the migration of these cells in vitro. This interaction plays a key role in the migration of hematopoietic stem cells to their final niche in the bone marrow during ontogenesis and their permanent residence there, as well as in their mobilization into the peripheral blood. It is logical to assume that such an interaction can play a similar role in the liver, keeping Ito cells in the Disse space. During the early stages of liver regeneration, increased expression of SDF-1 may also contribute to the recruitment of additional stem cell compartments in the body. The innervation of niche cells must involve the sympathetic nervous system, which is involved in regulating the recruitment of hematopoietic stem cells. Noradrenergic signals from the sympathetic nervous system play a critical role in GCSF (Granulocyte colony-stimulating factorl-induced mobilization of hematopoietic stem cells from the bone marrow. The location of nerve endings in close proximity to Ito cells has been confirmed in several studies. It has also been found that in response to sympathetic stimulation Ito cells secrete prostaglandins F2a and D, which activate glycogenolysis in nearby parenchymal cells. These facts suggest that the sympathetic nervous system may have an influence on the Ito cell niche. Another function of the stem cell niche is the maintenance of a "slow" cell cycle and an undifferentiated state of stem cells cells. The maintenance of the undifferentiated state of Ito cells in vitro is facilitated by parenchymal liver cells - when culturing these two populations of cells separated by a membrane, Ito cells retain the expression of stem cell markers CD133 and 0kt4, whereas in the absence of hepatocytes, Ito cells acquire the myofibroblast phenotype and lose stem cell markers. Thus, the expression of stem cell markers is clearly a hallmark of quiescent Ito cells. It has also been established that the influence of parenchymal cells on Ito cells may be based on the interaction of the paracrine factors Wnt and Jag1 synthesized by hepatocytes with the corresponding receptors (Myc, Notchl) on the surface of Ito cells. Wnt/b-catenin and Notch signaling pathways support the ability of stem cells to self-renew through slow symmetrical division without subsequent differentiation. Another important component of the niche is the basement membrane proteins, laminin and collagen IV, which maintain the quiescent state of Ito cells and suppress their differentiation. A similar situation occurs in muscle fibers and convoluted seminiferous tubules, where satellite cells (muscle stem cells) and undifferentiated spermatogonia are in close contact with the basement membrane of the muscle fiber or “spermatogenic epithelium,” respectively. It is obvious that the interaction of stem cells with extracellular matrix proteins suppresses the initiation of their final differentiation. The data obtained thus make it possible to consider Ito cells as stem cells, the niche for which can be the Disse space.

Our data on the stem potential of Ito cells and the possibility of the formation of hepatocytes from these cells were confirmed in experiments studying liver regeneration in vivo using models of partial hepatectomy and toxic liver damage by lead nitrate. It is traditionally believed that in these models of liver regeneration there is no activation of the stem compartment and there are no oval cells. We were able to establish, however, that in both cases one can observe not just the activation of Ito cells, but also the expression in them of another stem cell marker, namely, the receptor for the stem cell factor C-kit. Since C-kit expression was also observed in single hepatocytes (in them it was less intense), mainly located in contact with C-kit-positive Ito cells, it can be assumed that these hepatocytes differentiated from C-kit+ Ito cells. It is obvious that this cell type not only creates the conditions for restoration of the hepatocyte population, but also itself occupies the niche of regional stem cells of the liver.

Thus, it has now been established that Ito cells express at least five stem cell markers under various developmental, regenerative, and culture conditions. All the data accumulated to date suggest that Ito cells can act as regional stem cells of the liver, being one of the sources of the development of hepatocytes (and possibly cholangiocytes), and are also the most important component of the microenvironment for liver morphogenesis and hepatic hematopoiesis. However, it is apparently somewhat premature to draw definitive conclusions about whether these cells belong to the population of liver stem (progenitor) cells. However, there is an obvious need for new research in this direction, which, if successful, will open up prospects for the development of effective methods for treating liver diseases based on stem cell transplantation.

For quotation: Kurysheva M.A. Liver fibrosis: past, present and future // Breast cancer. 2010. No. 28. S. 1713

Liver fibrosis is a local or diffuse increase in the amount of connective tissue, extracellular matrix (collagen fibrous tissue in the perisinusoidal space) and the main path of progression of chronic diffuse liver diseases. In the early stages of fibrosis there are no clinical manifestations, and only histological examination of the biopsy specimen reveals excessive accumulation of connective tissue. Subsequently, fibrosis leads to the formation of regenerate nodes, vascular anastomoses - the formation of liver cirrhosis. Non-cirrhotic liver fibrosis is rare and is not considered in this work.

The processes of fibrosis in the liver have been studied for many years (Table 1), but only after the discovery of the role of stellate cells in fibrosis processes were new opportunities for antifibrotic therapy obtained.

Pathogenesis of liver fibrosis
Sinusoidal cells - endothelial, Kupffer cells, stellate cells (Ito cell, stellate cell, retinoid-storing cell, lipocyte), together with the area of ​​hepatocytes facing the lumen of the sinusoids, form a functional unit. In addition to cells, in the sinusoid area there is an extracellular matrix (ECM), visible only in liver diseases. All cells that form sinusoids can participate in the formation of the ECM. Normally, there is a balance between fibrogenesis factors and antifibrotic factors. The main role in fibrosis is played by Ito cells, which produce profibrotic and antifibrotic factors. Antifibrotic factors include matrix metalloproteases (MMPs), which are involved in the destruction of ECM proteins (collagenases, gelatinases, stromolysins). MMP activity is suppressed by tissue inhibitors of matrix metalloproteases (TIMPs), which are also produced by Ito cells.
When the liver is damaged, biologically active substances are released that activate macrophages and sinusoid endothelium, releasing IL-1, TNFα, nitric oxide, endothelin, acting on Ito cells. When activated, stellate cells produce platelet-activating factor PDGF and transforming growth factor TGFβ 1. Under the influence of TGFβ 1, Ito cells begin to activate themselves and migrate to areas of inflammation. There is a change in the phenotype of Ito cells - they transform into myofibroblasts, which continue to produce TGFβ 1, and begin to produce ECM. An imbalance between fibrotic and antifibrotic factors leads to a 3-10-fold increase in ECM components and a change in its composition (the predominance of collagen types I and III). Redistribution of the matrix into the space of Disse, its expansion, capillarization of sinusoids is accompanied by a disturbance in the exchange between hepatocytes and blood, shunting of the blood due to the development of false lobules and the development of liver cirrhosis. If the action of inflammatory mediators ceases, Ito cells again begin to produce profibrotic substances and a decrease in ECM components in the space of Disse occurs. Thus, fibrosis in the early stages of development is a reversible process.
The pathogenesis of liver fibrosis in chronic viral hepatitis is associated with the induction of inflammatory cell activity by infected hepatocytes, which leads to stimulation of Ito cells. In alcoholic liver disease, acetaldehyde and oxygen free radicals activate Ito cells. In addition, ethanol promotes the growth of gram-negative microflora in the intestine, increasing the level of lipopolysaccharides in the portal blood and activating Kupffer cells that produce TNFα, acting on Ito cells. The pathogenesis of liver fibrosis in nonalcoholic fatty liver disease is associated with hyperglycemia and insulin resistance, leading to increased levels of free fatty acids and hepatic steatosis, and free radicals and proinflammatory cytokines lead to apoptosis of hepatocytes and activation of inflammatory cells with the progression of liver fibrosis. In primary biliary cirrhosis, biliary cells secrete fibrogenic mediators that activate Ito cells, triggering fibrogenesis.

Reversibility of liver fibrosis
For a long time, liver fibrosis was considered an irreversible pathological condition. However, 50 years ago, cases of the reverse development of fibrosis were described after effective therapy for hemochromatosis and Wilson-Konovalov disease, and subsequently data were repeatedly published on the reverse development of fibrosis in autoimmune hepatitis as a result of immunosuppressive therapy, secondary biliary cirrhosis after surgical decompression of the biliary tract, non-alcoholic steatohepatitis with a decrease in body weight, alcoholic hepatitis during abstinence.
Reversibility of fibrosis was observed with long-term abstinence from alcohol intake, when after 4-6 weeks a decrease in the content of type IV collagen, laminin and hyaluronic acid was detected in the walls of the sinusoids during biopsy and in the blood serum - regression of the process of “sinusoid capillarization” occurred. Changes reflecting the function of Ito cells were also noted - an increase in the level of MMP-2 and a decrease in the level of its inhibitor TIMMP-2. At certain time intervals, a decrease in the number of actin myofibrils was observed in the walls of the sinusoids, which indicates a drop in the activity of Ito stellate cells and their switching from the synthesis of the extracellular matrix to its degradation.
At the same time, only with the introduction of antiviral therapy into clinical practice, the concept of liver fibrosis, as a dynamic process with the possibility of both progression and regression, was recognized as a scientifically proven fact.
Progress has led to a clear understanding that liver fibrosis is reversible and to realistic expectations that effective antifibrotic therapy will significantly change the management of patients with liver disease and provide a favorable prognosis even in those with established cirrhosis.
Diagnosis of liver fibrosis
The gold standard for diagnosing liver fibrosis is a biopsy with histological examination. Histological assessment is carried out according to Desmet scales (1984) as modified by Serov; JSHAK or METAVIR scale. Depending on the location and prevalence, the following forms of liver fibrosis are distinguished: venular and perivenular (in the center of the lobules and the walls of the central veins - characteristic of chronic alcoholic hepatitis); pericellular (around hepatocytes in chronic viral and alcoholic hepatitis); septal (concentric growth of fibrous tissue around the bile canaliculi - with viral hepatitis); portal and periportal (for viral, alcoholic, autoimmune hepatitis); periductal fibrosis (around the bile canaliculi in sclerosing cholangitis); mixed (different forms of fibrosis are presented).
Due to the invasiveness, the rather large error of histological examination associated with “mistakes in the needle” during puncture biopsy of the liver, and differences in the interpretation of results, for the early diagnosis of pathological processes, great attention is currently paid to non-invasive methods for diagnosing fibrosis. These include bioprognostic laboratory tests; liver elastometry and MR elastography; Ultrasound, CT, MRI of the liver, Doppler ultrasound of the vessels of the liver and spleen with calculation of indices of fibrosis and portal hypertension.
Markers of fibrosis are divided into direct (biomarkers), reflecting ECM metabolism, and indirect, indicating liver failure. Direct markers include carboxy-terminal peptide of type I procollagen, amino-terminal peptide of type III procollagen, TIMP-1, 2, type IV collagen, hyaluronic acid, laminin, MMP-2. The determination of these substances is used in clinical studies.
For clinical practice, various calculated prognostic indices have been proposed to assess the severity of liver fibrosis using indirect markers: APRI, ELF, FIB-4, FibroFast, FibroIndex, FibroMeter, FPI, Forns, GUCI, Hepascore, HALT-C, MDA, PGA, PGAA.
To assess the severity of liver fibrosis, the Fibro-test and Acti-test systems are used, considering them as an alternative to biopsy. The fibro-test includes 5 biochemical indicators: alpha 2-macroglobulin (activates Ito cells), haptoglobin (reflects stimulation of liver cells by interleukins), apolipoprotein A1, gamma-glutamyl transpeptidase, total bilirubin. Acti-test (viral necroinflammatory activity is assessed) in addition to the listed components includes alanine aminotransferase - ALT. FibroMax is a combination of five non-invasive tests: FibroTest and ActiTest, Steato-Test (diagnoses liver steatosis), NeshTest (diagnoses non-alcoholic steatohepatitis), AshTest (diagnoses severe alcoholic steatohepatitis). FibroMax detects alpha 2-macroglobulin, haptoglobin, apolipoprotein A1, gamma-glutamyl transpeptidase, total bilirubin, ALT, AST, glucose, triglycerides, cholesterol. Based on the data obtained, taking into account the age and gender of the patient, the stage of fibrosis and the level of hepatitis activity are calculated. The use of tests is limited by signs of cholestasis, which negatively affect the diagnostic value of the tests, and the high cost of the study.
The operation of the device, based on ultrasound elastography of the liver by passing waves (vibrations) through the liver and capturing them with a sensor, allows one to assess the degree of fibrosis in the liver in the early stages. The device is of little information for obesity and ascites.
Magnetic resonance elastography is a direct method for determining liver density, allowing the determination of F0 in comparison with healthy volunteers, which has not yet been demonstrated using other methods for assessing fibrosis.
In the future, it is possible to determine the presence and rate of fibrosis progression depending on the etiological factor. Solving these problems makes it possible to diagnose the early stages of fibrosis and, therefore, effectively treat it.

Treatment
Antifibrotic therapy is inextricably linked with the etiological and pathogenetic treatment of chronic hepatitis (Table 2). In most cases, drugs to eliminate the etiological factors of hepatitis are also antifibrotic agents. An antifibrotic effect was detected in antiviral drugs, pentoxifylline, phosphatidylcholine, glucocorticosteroids, nitric oxide donors, vitamin E, endothelin receptor antagonists, angiotensin receptor antagonists, angiotensin-converting enzyme inhibitors, silymarin. A search is underway for drugs that inhibit fibrogenesis for use in situations where the effect on the causative factor is difficult: antioxidants (betaine, probucol, N-acetylcysteine), hepatoprotectors (silymarin, UDCA, S-adenosylmethionine, essential phospholipids), reducing the activity of tumor necrosis factor (pentoxifylline , adiponectin, infliximab).
A search is underway for drugs with targeted antifibrotic effects:
- elimination of the damaging agent (interleukin 10, TNF inhibitors - anti-inflammatory effect; antioxidants - suppression of fibrotic processes in response to oxidative stress);
- suppression of profibrotic activity of stellate cells (interferons, hepatocyte growth factor, PPARγ agonists);
- maintaining active antifibrotic activity of stellate cells (TGFβ 1 antagonists - reduce matrix synthesis and increase its breakdown; PDGF antagonists, nitric oxide, ACE inhibitors - suppress the proliferation of Ito cells);
- influence on the secretion of collagens by stellate cells of the liver (ACE inhibitors, polyhydroxylase inhibitors, interferon γ - reduce fibrosis; endothelin receptor antagonists - reduce fibrosis and portal hypertension);
- effect on apoptosis of Ito cells (hylotoxin, NGF - neuronal growth factor - stimulate apoptosis);
- increased breakdown of the collagen matrix (metalloproteinases, tissue inhibitor MMP antagonists; TGFβ 1 antagonists - reduce TIMP activity and increase MMP activity; relaxin - reduce TIMP activity and increase MMP activity).
The use of the drug silymarin (Legalon) for antifibrotic purposes seems promising. Silymarin is the official name of a group of four flavonolignan isomers (silibinin, isosilibinin, silicristin and silydianin), isolated from extracts of the fruits of milk thistle (Cardui mariae fructus) and included in Legalon 70 and 140 (silymarin dose).
During clinical studies, it was revealed that, along with anti-inflammatory, antioxidant, antitoxic, hypolipidemic and anticarcinogenic effects, silymarin has a pronounced antifibrotic effect. This is due to effects on transforming growth factor β and gene expression in Ito cells, as well as increased free radical clearance and direct inhibition of collagen synthesis.
The relationship between the pharmacodynamics of silymarin/silibinin and the clinical effect of Legalon® is given in Table 3. The indicated mechanisms of action determine the therapeutic value of Legalon® in diffuse liver diseases. Numerous studies have shown the high effectiveness of Legalon® with long-term use in suppressing the inflammatory-necrotic reaction in the liver, inhibiting the development of fibrosis and reducing the risk of malignant transformation of hepatocytes in liver cirrhosis.
In a model of alcoholic liver fibrosis in monkeys, a morphological study of the liver and a study of serum markers of fibrosis revealed that animals treated with silymarin had significantly less fibrosis progression and less often developed liver cirrhosis.
The effect of Legalon on liver fibrosis was studied in 792 patients with chronic liver diseases, including cirrhosis. The P-III-NP indicator was chosen as a marker of fibrogenesis. The observation period averaged 107 days. With an initially elevated level of P-III-NP, after 3 months of treatment with Legalon, the level of P-III-NP decreased to normal.
The results of 5 international placebo-controlled studies (600 patients participated) showed that the 4-year survival rate of patients with alcoholic cirrhosis while taking Legalon was statistically significantly higher compared to the group of patients receiving placebo. When analyzing subgroups, it was revealed that treatment with Legalon was effective in alcoholic cirrhosis, regardless of its severity and stage of cirrhosis, and in the subgroup with Chaid-Pugh stage A cirrhosis, regardless of its etiology. In the subgroup of patients with alcoholic cirrhosis due to viral hepatitis, no deaths were recorded during the observation period, while in the placebo group there were 4 deaths from decompensation of cirrhosis.
Fibrosis is currently called the cornerstone of chronic liver pathology. It is this that causes the formation of liver cirrhosis, so early diagnosis and treatment of fibrosis is extremely relevant at the present time and is a task for future scientific research.

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