Could perisinusoidal cells be regional stem cells of the liver? Study of the influence of liver cells on stem cells Stellate cells

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.

Long years Ito cells were considered only from the standpoint of their participation in the development of liver fibrosis and cirrhosis. 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 cell-to-cell 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 normal development hepatocytes in ontogenesis and their restoration in the process of reparative regeneration.

IN last years Data were 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 bone marrow mesenchymal stem cells (multipotent mesenchymal stromal cells) 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 process of development that is the key event, the study of which allows us to trace 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 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, nerve growth factor (Neural growth 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 intercellular matrix during the process of liver restoration after damage, thus providing a connective tissue framework for the regeneration of parenchymal liver 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 different - 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 and permanent residence in their final niche in the bone marrow during ontogenesis, as well as in their mobilization in 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 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. One more an important component The niches are 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 conditions for the restoration of the hepatocyte population, but also occupies the niche of stem cells. regional cells 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.

In this case, these cells respond by multiplying to the influence of cytokines, growth factors and chemokines (pro-inflammatory cytokines) produced by the damaged liver. Chronic activation of stellate cells in response to replication-induced oxidative stress HBV virus and HCV, may contribute to fibrogenesis and increased proliferation of hepatocytes chronically infected with HBV and HCV.

Thus, stellate cells take part in the regulation of growth, differentiation and turnover of hepatocytes, which, together with the activation of MAP kinases, can lead to the development of liver cancer [Block, 2003].

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Studying the influence of liver Ito cells on stem cells

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.

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"

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Ito cage

calm And activated. Activated Ito cells

calm state

perisinusoidal(subendothelial) and interhepatocellular. The first leave 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.

activated state

Liver cells

The human liver is made up of cells, like any organic tissue. Nature has designed it in such a way that this organ performs the most important functions: it cleanses the body, produces bile, accumulates and deposits glycogen, synthesizes plasma proteins, manages metabolic processes, and participates in normalizing the amount of cholesterol and other components necessary for the functioning of the body.

To fulfill their purpose, liver cells must be healthy, have a stable structure, and each person must protect them from destruction.

About the structure and types of liver lobules

The cellular composition of the organ is characterized by diversity. Liver cells form lobules, and segments are made up of lobules. The structure of the organ is such that hepatocytes (the main liver cells) are located around the central vein, branch from it, connect with each other, forming sinusoids, that is, gaps filled with blood. Blood moves through them as if through capillaries. The liver is supplied with blood from the portal vein and artery located in the organ. The liver lobules produce bile and discharge it into the flow channels.

Other types of liver cells and their purpose

Endothelial - cells lining the sinusoids and containing fenestrae. The latter are intended to form a stepped barrier between the sinusoid and the Disse space.
The space of Disse itself is filled with stellate cells; they ensure the outflow of tissue fluid into the lymph vessels of the portal zones.
Kupffer cells are associated with the endothelium, they are attached to it, their function is to protect the liver when a generalized infection enters the body, or during injury.
Pit cells are killers of hepatocytes affected by the virus; in addition, they have cytotoxicity to tumor cells.

The human liver consists of 60% hepatocytes and 40% other types of cellular compounds. Hepatocytes have a polyhedron shape; there are at least 250 billion of them. Normal operation hepatocytes is due to the spectrum of components that are secreted by sinusoidal cells filling the sinusoidal compartment. That is, the Kupffer listed above, stellate and pit cells (intrahepatic lymphocytes).

The endothelial is a filter between the blood in the sinusoidal space and the plasma in the Disse space. This biological filter sorts out large compounds that are excessively rich in retinol and cholesterol and does not allow them to pass through, which is beneficial for the body. In addition, their function is to protect the liver (namely hepatocytes) from mechanical damage by blood cells.

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The process of interaction of organ elements

There is an interaction between all particles of the organ, which has a rather complex pattern. A healthy liver is characterized by the stability of cellular connections; in pathological processes, the extracellular matrix can be traced under a microscope.

Organ tissue undergoes changes under the influence of toxins, for example, alcohol, viral agents. They are as follows:

deposition in the organ of products formed due to metabolic disorders;
cell degeneration;
hepatocyte necrosis;
fibrosis of liver tissue;
inflammatory process of the liver;
cholestasis.

About the treatment of organ pathology

It is useful for each patient to know what the changes that the organ undergoes mean. Not all of them are catastrophic. For example, dystrophy can be mild or severe. Both of these processes are reversible. Currently, there are drugs that restore cells and entire segments of the liver.

Cholestasis can be cured even with folk remedies - decoctions and infusions. They help normalize the synthesis of bilirubin and eliminate disturbances in the outflow of bile into the duodenum.

With cirrhosis in initial stage treatment begins with a diet, then hepatoprotector therapy is prescribed. The most effective way to treat cirrhosis and fibrosis is stem cells, which are injected into the umbilical vein or intravenously; they restore hepatocytes damaged by various agents.

The main causes of liver cell death are alcohol abuse and drug exposure, including drugs and medications. Any toxin that enters the body is a liver destroyer. Therefore, you should give up bad habits so that you have a healthy liver.

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Education: Rostov State Medical University (RostSMU), Department of Gastroenterology and Endoscopy.

ENDOTHELIAL CELLS, KUPFER CELLS AND ITO

We will look at the structure of endothelial cells, Kupffer and Ito cells, using the example of two drawings.

The figure to the right of the text shows the sinusoidal capillaries (SC) of the liver - intralobular capillaries of the sinusoidal type, increasing from the entrance venules to the central vein. Hepatic sinusoidal capillaries form an anastomotic network between the hepatic plates. The lining of sinusoidal capillaries is formed by endothelial cells and Kupffer cells.

In the figure to the left of the text, the hepatic plate (LP) and the two sinusoidal capillaries (SCs) of the liver are cut vertically and horizontally to show the perisinusoidal Ito cells (Ito cells). The cut bile canaliculi (BC) are also marked in the figure.

ENDOTHELIAL CELLS

Endothelial cells (EC) are highly flattened squamous cells with an elongated small nucleus, poorly developed organelles and a large number of micropinocytotic vesicles. The cytomembrane is dotted with irregular openings (O) and fenestrae, often grouped into cribriform plates (RP). These holes allow blood plasma to pass through, but not blood cells, allowing it access to hepatocytes (D). Endothelial cells do not have a basement membrane and do not exhibit phagocytosis. They are connected to each other using small connecting complexes (not shown). Together with Kupffer cells, endothelial cells form the internal border of the space of Disse (PD); its outer border is formed by hepatocytes.

KUPFER CELLS

Kupffer cells (KCs) are large, non-persistent stellate cells within the hepatic sinusoidal capillaries, partly at their bifurcations.

Kupffer cell processes pass without any connecting devices between endothelial cells and often cross the lumen of the sinusoids. Kupffer cells contain an oval nucleus, many mitochondria, a well-developed Golgi complex, short cisterns of granular endoplasmic reticulum, many lysosomes (L), residual bodies and rare annular plates. Kupffer cells also include large phagolysosomes (PLs), which often contain obsolete red blood cells and foreign substances. Inclusions of hemosiderin or iron can also be detected, especially with supravital staining.

The surface of Kupffer cells displays variable, flattened cytoplasmic folds called lamellipodia (LP) - lamellar stalks - as well as processes called filopodia (F) and microvilli (MV) covered with glycocalyx. The plasmalemma forms vermiform bodies (VB) with a centrally located dense line. These structures may represent a condensed glycocalyx.

Kupffer cells are macrophages, very likely forming an independent genus of cells. They usually originate from other Kupffer cells due to mitotic division of the latter, but can also originate from the bone marrow. Some authors believe that they are activated endothelial cells.

Occasionally, an occasional autonomic nerve fiber (ANF) passes through the space of Disse. In some cases, the fibers have contact with hepatocytes. The edges of hepatocytes are delimited by interhepatocyte recesses (MU) dotted with microvilli.

ITO CELLS

These are stellate cells localized within the spaces of Disse (SD). Their nuclei are rich in condensed chromatin and are usually deformed by large lipid droplets (LDs). The latter are present not only in the perikaryon, but also in the processes of the cell and are visible from the outside as spherical protrusions. Organelles are poorly developed. Perisinusoidal cells show weak endocytotic activity but do not possess phagosomes. The cells have several long processes (O) that contact neighboring hepatocytes, but do not form connecting complexes.

The processes envelop the sinusoidal capillaries of the liver and in some cases pass through the hepatic plates, coming into contact with adjacent hepatic sinusoids. The processes are not constant, branched and thin; they can also be flattened. By accumulating groups of lipid droplets, they lengthen and take on the appearance of a bunch of grapes.

It is believed that perisinusoidal Ito cells are poorly differentiated mesenchymal cells that can be considered as hematopoietic stem cells, since they can transform into fat cells, active blood stem cells or fibroblasts.

Under normal conditions, Ito cells are involved in the accumulation of fat and vitamin A as well as in the production of intralobular reticular and collagen fibers (KB).

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Ito liver cells

Universal online popular science encyclopedia

LIVER

LIVER, the largest gland in the body of vertebrates. In humans, it makes up about 2.5% of body weight, on average 1.5 kg in adult men and 1.2 kg in women. The liver is located in the upper right part of the abdominal cavity; it is attached by ligaments to the diaphragm, abdominal wall, stomach and intestines and is covered with a thin fibrous membrane - Glisson's capsule. The liver is a soft but dense organ of a red-brown color and usually consists of four lobes: a large right lobe, a smaller left lobe and much smaller caudate and quadrate lobes that form the posterior lower surface of the liver.

Functions.

The liver is an essential organ for life with many different functions. One of the main ones is the formation and secretion of bile, a transparent liquid of orange or yellow color. Bile contains acids, salts, phospholipids (fats containing a phosphate group), cholesterol and pigments. Bile salts and free bile acids emulsify fats (i.e. break them into small droplets), making them easier to digest; convert fatty acids into water-soluble forms (which is necessary for the absorption of both the fatty acids themselves and fat-soluble vitamins A, D, E and K); have an antibacterial effect.

All nutrients absorbed into the blood from the digestive tract - products of the digestion of carbohydrates, proteins and fats, minerals and vitamins - pass through the liver and are processed there. At the same time, some amino acids (protein fragments) and some fats are converted into carbohydrates, so the liver is the largest “depot” of glycogen in the body. It synthesizes blood plasma proteins - globulins and albumin, and also undergoes amino acid conversion reactions (deamination and transamination). Deamination - the removal of nitrogen-containing amino groups from amino acids - allows the latter to be used, for example, for the synthesis of carbohydrates and fats. Transamination is the transfer of an amino group from an amino acid to a keto acid to form another amino acid ( cm. METABOLISM). The liver also synthesizes ketone bodies (products of fatty acid metabolism) and cholesterol.

The liver is involved in regulating glucose (sugar) levels in the blood. If this level increases, liver cells convert glucose into glycogen (a substance similar to starch) and store it. If the blood glucose level drops below normal, glycogen is broken down and glucose enters the bloodstream. In addition, the liver is capable of synthesizing glucose from other substances, such as amino acids; this process is called gluconeogenesis.

Another function of the liver is detoxification. Medicines and other potentially toxic compounds can be converted into a water-soluble form in liver cells, which allows them to be excreted in bile; they can also be destroyed or conjugate (combine) with other substances to form harmless products that are easily excreted from the body. Some substances are temporarily deposited in Kupffer cells (special cells that absorb foreign particles) or in other liver cells. Kupffer cells are particularly effective at removing and destroying bacteria and other foreign particles. Thanks to them, the liver plays an important role in the body's immune defense. Possessing a dense network of blood vessels, the liver also serves as a blood reservoir (it constantly contains about 0.5 liters of blood) and is involved in the regulation of blood volume and blood flow in the body.

In general, the liver performs more than 500 different functions, and its activity cannot yet be reproduced artificially. Removal of this organ inevitably leads to death within 1–5 days. However, the liver has a huge internal reserve; it has amazing ability recover from damage, so humans and other mammals can survive even after 70% of the liver tissue is removed.

Structure.

The complex structure of the liver is perfectly adapted to perform its unique functions. The lobes consist of small structural units - lobules. In the human liver there are about one hundred thousand of them, each 1.5–2 mm long and 1–1.2 mm wide. The lobule consists of liver cells - hepatocytes, located around the central vein. Hepatocytes are united into layers one cell thick - the so-called. liver plates. They diverge radially from the central vein, branch and connect with each other, forming a complex system of walls; the narrow gaps between them, filled with blood, are known as sinusoids. Sinusoids are equivalent to capillaries; passing one into another, they form a continuous labyrinth. The hepatic lobules are supplied with blood from the branches of the portal vein and hepatic artery, and the bile formed in the lobules enters the tubular system, from them into the bile ducts and is excreted from the liver.

The hepatic portal vein and hepatic artery provide the liver with an unusual, dual blood supply. Nutrient-rich blood from the capillaries of the stomach, intestines and several other organs is collected in the portal vein, which, instead of carrying blood to the heart like most other veins, carries it to the liver. In the liver lobules, the portal vein breaks up into a network of capillaries (sinusoids). The term “portal vein” indicates an unusual direction of blood transport from the capillaries of one organ to the capillaries of another (the kidneys and pituitary gland have a similar circulatory system).

The second source of blood supply to the liver, the hepatic artery, carries oxygenated blood from the heart to the outer surfaces of the lobules. The portal vein provides 75–80%, and the hepatic artery 20–25% of the total blood supply to the liver. In general, about 1500 ml of blood passes through the liver per minute, i.e. a quarter of cardiac output. Blood from both sources ultimately enters the sinusoids, where it mixes and flows to the central vein. From the central vein, the outflow of blood to the heart begins through the lobar veins into the hepatic vein (not to be confused with the portal vein of the liver).

Bile is secreted by liver cells into the smallest tubules between the cells - bile capillaries. It is collected through the internal system of tubules and ducts into the bile duct. Some bile goes directly into the common bile duct and is released into the small intestine, but most of it travels through the cystic duct back for storage in the gallbladder, a small, muscular-walled sac attached to the liver. When food enters the intestines, the gallbladder contracts and releases the contents into the common bile duct, which opens into the duodenum. The human liver produces about 600 ml of bile per day.

Portal triad and acini.

The branches of the portal vein, hepatic artery and bile duct are located nearby, at the outer border of the lobule and form the portal triad. At the periphery of each lobule there are several such portal triads.

The functional unit of the liver is the acinus. This is the part of tissue that surrounds the portal triad and includes lymphatic vessels, nerve fibers and adjacent sectors of two or more lobules. One acini contains about 20 liver cells located between the portal triad and the central vein of each lobule. In a two-dimensional image, a simple acinus looks like a group of vessels surrounded by adjacent sections of lobules, and in a three-dimensional image it looks like a berry (acinus - lat. berry) hanging on a stalk of blood and bile vessels. Acinus, the microvascular framework of which consists of the above circulatory and lymphatic vessels, sinusoids and nerves, is the microcirculatory unit of the liver.

Liver cells

(hepatocytes) have the shape of polyhedra, but they have three main functional surfaces: sinusoidal, facing the sinusoidal channel; tubular - involved in the formation of the wall of the bile capillary (it does not have its own wall); and intercellular - directly adjacent to neighboring liver cells.

Ito cage

Ito cells (synonyms: hepatic stellate cell, fat-storing 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 the sinusoidal capillary. Another distinctive feature cells is the presence in their cytoplasm of reserves of vitamin A (retinoid) in the form of fat drops. 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 leave 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, ICAM-1, 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

In 1876, Karl von Kupfer described cells he called "Sternzellen" (stellate cells). When stained with gold oxide, inclusions were visible in the cytoplasm of the cells. Mistakenly considering them to be fragments of red blood cells captured by phagocytosis, Kupfer in 1898 revised his views on the “stellate cell” as a separate type of cell and classified them as phagocytes. However, in subsequent years, descriptions of cells similar to Kupffer's “stellate cells” appeared regularly. They were given various names: interstitial cells, parasinusoid cells, lipocytes, pericytes. The role of these cells remained a mystery for 75 years, until Professor Toshio Ito discovered certain cells containing fat inclusions in the perisinusoidal space of the human liver. Ito called them "shibo-sesshu saibo" - fat-absorbing cells. Realizing that the inclusions were fat produced by cells from glycogen, he changed the name to “shibo-chozo saibo” - fat-storing cells. In 1971, Kenjiro Wake proved the identity of Kupffer's Sternzellen and Ito's fat-storing cells. Vake also found that these cells play an important role in storing vitamin A (prior to this it was believed that vitamin A was stored in Kupffer cells). Shortly thereafter, Kent and Popper demonstrated the close association of Ito cells with liver fibrosis. These discoveries began the process of studying Ito cells in detail.

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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). 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:.

Notes

Geerts A. (2001) History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis. 21(3):311-35. PMID
Wake, K. (1988) Liver perivascular cells revealed by gold- and silver-impregnation method and electron microscopy. In “Biopathology of the Liver. An Ultrastructural Approach" (Motta, P. M., ed) pp. 23-36, Kluwer Academic Publishers, Dordrecht, Netherlands
Stanciu A, Cotutiu C, Amalinei C. (2002) New data about ITO cells. Rev Med Chir Soc Med Nat Iasi. 107(2):235-9. PMID
John P. Iredale (2001) Hepatic Stellate Cell Behavior During Resolution of Liver Injury. Seminars in Liver Disease, 21(3):PMID- (English) on Medscape.
Kobold D, Grundmann A, Piscaglia F, Eisenbach C, Neubauer K, Steffgen J, Ramadori G, Knittel T. (2002) Expression of reelin in hepatic stellate cells and during hepatic tissue repair: a novel marker for the differentiation of HSC from other liver myofibroblasts. J Hepatol. 36(5):607-13. PMID
Adrian Reuben (2002) Hepatology. Volume 35, Issue 2, Pages 503-504 (English)
Suematsu M, Aiso S. (2001) Professor Toshio Ito: a clairvoyant in pericyte biology. Keio J Med. 50(2):66-71. PMID (English)
Querner F: Der mikroskopische Nachweis von Vitamin A im animalen Gewebe. Zur Kenntnis der paraplasmatischen Leberzellen-einschlüsse. Dritte Mitteilung. Klin Wschr 1935, 14:.

Excerpt characterizing Ito's Cell

Half an hour later, Kutuzov left for Tatarinova, and Bennigsen and his retinue, including Pierre, went along the line.

Bennigsen went down from Gorki high road to the bridge, which the officer from the mound pointed out to Pierre as the center of the position and on the bank of which lay rows of mown grass that smelled of hay. They drove across the bridge to the village of Borodino, from there they turned left and past a huge number of troops and cannons they drove out to a high mound on which the militia was digging. It was a redoubt that did not yet have a name, but later received the name Raevsky redoubt, or barrow battery.

Pierre did not pay much attention to this redoubt. He did not know that this place would be more memorable for him than all the places in the Borodino field. Then they drove through the ravine to Semenovsky, in which the soldiers were taking away the last logs of the huts and barns. Then, downhill and uphill, they drove forward through broken rye, knocked out like hail, along a road newly laid by artillery along the ridges of arable land to the flushes [a type of fortification. (Note by L.N. Tolstoy.) ], also still being dug at that time.

Bennigsen stopped at the flushes and began to look ahead at the Shevardinsky redoubt (which was ours only yesterday), on which several horsemen could be seen. The officers said that Napoleon or Murat was there. And everyone looked greedily at this bunch of horsemen. Pierre also looked there, trying to guess which of these barely visible people was Napoleon. Finally, the riders rode off the mound and disappeared.

Bennigsen turned to the general who approached him and began to explain the entire position of our troops. Pierre listened to Bennigsen's words, straining all his mental strength to understand the essence of the upcoming battle, but with grief he felt that mental capacity it was insufficient for this. He didn't understand anything. Bennigsen stopped talking, and noticing the figure of Pierre, who was listening, he suddenly said, turning to him:

– I think you’re not interested?

“Oh, on the contrary, it’s very interesting,” Pierre repeated, not entirely truthfully.

From the flush they drove even further to the left along a road winding through a dense, low birch forest. In the middle of it

forest, a brown hare with white legs jumped out onto the road in front of them and, frightened by the clatter of a large number of horses, he was so confused that he jumped along the road in front of them for a long time, exciting general attention and laughter, and only when several voices shouted at him, he rushed to the side and disappeared into the thicket. After driving about two miles through the forest, they came to a clearing where the troops of Tuchkov’s corps, which was supposed to protect the left flank, were stationed.

Here, on the extreme left flank, Bennigsen spoke a lot and passionately and made, as it seemed to Pierre, an important military order. There was a hill in front of Tuchkov’s troops. This hill was not occupied by troops. Bennigsen loudly criticized this mistake, saying that it was crazy to leave the height commanding the area unoccupied and place troops under it. Some generals expressed the same opinion. One in particular spoke with military fervor about the fact that they were put here for slaughter. Bennigsen ordered in his name to move the troops to the heights.

This order on the left flank made Pierre even more doubtful of his ability to understand military affairs. Listening to Bennigsen and the generals condemning the position of the troops under the mountain, Pierre fully understood them and shared their opinion; but precisely because of this, he could not understand how the one who placed them here under the mountain could make such an obvious and gross mistake.

Pierre did not know that these troops were not placed to defend the position, as Bennigsen thought, but were placed in a hidden place for an ambush, that is, in order to be unnoticed and suddenly attack the advancing enemy. Bennigsen did not know this and moved the troops forward for special reasons without telling the commander-in-chief about it.

On this clear August evening on the 25th, Prince Andrei lay leaning on his arm in a broken barn in the village of Knyazkova, on the edge of his regiment’s location. Through the hole in the broken wall, he looked at a strip of thirty-year-old birch trees with their lower branches cut off running along the fence, at an arable land with stacks of oats broken on it, and at bushes through which the smoke of fires—soldiers’ kitchens—could be seen.

No matter how cramped and no one needed and no matter how difficult his life now seemed to Prince Andrei, he, just like seven years ago at Austerlitz on the eve of the battle, felt agitated and irritated.

Orders for tomorrow's battle were given and received by him. There was nothing else he could do. But the simplest, clearest thoughts and therefore terrible thoughts did not leave him alone. He knew that tomorrow's battle was going to be the most terrible of all those in which he participated, and the possibility of death for the first time in his life, without any regard to everyday life, without consideration of how it would affect others, but only according to in relation to himself, to his soul, with vividness, almost with certainty, simply and horribly, it presented itself to him. And from the height of this idea, everything that had previously tormented and occupied him was suddenly illuminated by a cold white light, without shadows, without perspective, without distinction of outlines. His whole life seemed to him like a magic lantern, into which he looked for a long time through glass and under artificial lighting. Now he suddenly saw, without glass, in bright daylight, these poorly painted pictures. “Yes, yes, these are the false images that worried and delighted and tormented me,” he said to himself, turning over in his imagination the main pictures of his magic lantern of life, now looking at them in this cold white light of day - a clear thought of death. “Here they are, these crudely painted figures that seemed to be something beautiful and mysterious. Glory, public good, love for a woman, the fatherland itself - how great these pictures seemed to me, what deep meaning they seemed filled with! And all this is so simple, pale and rough in the cold white light of that morning, which I feel is rising for me. Three major sorrows of his life in particular occupied his attention. His love for a woman, the death of his father and the French invasion that captured half of Russia. "Love. This girl seemed to me full of mysterious powers. How I loved her! I made poetic plans about love, about happiness with it. Oh dear boy! – he said out loud angrily. - Of course! I believed in some kind of ideal love, which was supposed to remain faithful to me during the whole year of my absence! Like the tender dove of a fable, she was to wither away in separation from me. And all this is much simpler... All this is terribly simple, disgusting!

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"

The main source of endotoxin in the bodyis a gram-negative intestinal flora. Currently, there is no doubt that the liver is the main organ carrying out endotoxin clearance. Endotoxin is taken up primarily by cells kami Kupffer (KK), interacting with the membrane receptor CD 14. Can bind to the receptor itself lipopolysaccharide(LPS), and its complex with lipid A-binding protein com plasma. The interaction of LPS with liver macrophages triggers a cascade of reactions based on the production and release of reduction of cytokines and other biologically active mediators.

There are many publications on the role of macrophgov of the liver (LC) in the uptake and clearance of bacterial LPS, but the interaction of the endothelium with other mesenchymal cells, in particular, with perisinusoidal Ito cells have been practically not studied.

RESEARCH METHODOLOGY

White male rats weighing 200 g intraperitoneally in 1 ml of sterile saline solution introduced highly purified lyophilized LPS E. coli strain 0111 in doses of 0.5,2.5, 10, 25 and 50 mg/kg. At periods of 0.5, 1, 3, 6, 12, 24, 72 hours and 1 week, internal organs were removed under anesthesia and placed in buffered 10% formalin. The material was poured into paraffin blocks. Sections 5 µm thick were stained immunohistochemicalstreptavidin-biotin anti-desmin antibody method, α - smooth- muscle actin (A-GMA) and nuclear antigen well proliferating cells ( PCNA, " Dako"). Desmin was used as a marker perisinusoidalIto cells, A-GMA - as ve marker myofibroblasts, PCNA - proliferating cells. To detect endotoxin in liver cells, purified anti-Re-glycolipidantibodies (Institute of General and Clinical Pathology KDO, Moscow).

RESEARCH RESULTS

At a dosage of 25 mg/kg and higher, fatal shock was observed 6 hours after LPS administration. Acute exposure to LPS on liver tissue caused activation of Ito cells, which was manifested by an increase in their number. Number desmin positive cells increased from 6 hours after LPS injection and reached a maximum ma by 48-72 hours (Fig. 1, a, b).

Rice. 1. Sections of the roof's liver owls, processed LSAB -me- valuableantibodies to des mine(a, b) and α - smooth cervical actin (c), x400 (A, b), x200 (in).

a - before endotoxin administrationon, single desminpositiveIto cells in the periportal zone; b- 72 hoursafter administration of endotoxin on: numerous desminpositive Ito cells; V- 120 hours after administration of en dotoxin: α - smooth muscle active actin is present onlyto smooth muscle cells kah vessels.

In 1 week number desmin positive cells decreased, butwas higher than the control indicators. At In this case, in no case did we observe the appearance A-GMA-positive cells in the sinus give the liver. Internally positive control when stained with antibodies to A-GMA served to identify blood smooth muscle cellsvein vessels of the portal tracts containing A-GMA (Fig. 1, V). Therefore, despite the increase in the number of Ito cells, a one-time exposure to LPS does not lead to transformation ( transdifferentiation) them into myofibroblasts.

Rice. 2. Liver sectionsrats treated LSAB -labeled antibodies to PCNA. a - before the introduction of en dotoxin: singleproliferating genes pathocytes, x200; b - 72 hours after endotoxin administration: numerous proliferating hepatocytes, x400.

Increase in quantity desmin positive cells began within the portal zone. From 6 hours to 24 hours after LPS administration perisinusoidal cells were found only around the portal tracts, i.e. in the 1st zone of ACI noosa. At 48-72 hours, when poppy was observedmaximum quantity desmin positive glue current, they also appeared in other areas of the acinus; nevertheless, most of the Ito cells were still located periportally.

Perhaps this is due to the fact that periportallocated CCs are the first to capture endotoxin coming from the intestine through the portal vein or from the systemic circulation. Ak activated CCs produce a wide spectrum cytokines, which are thought to trigger the activation of Ito cells and transdifferentiation them into myofibroblasts. Obviously, this is why Ito cells located near activated liver macrophages (in the 1st zone of the acinus) are the first to react to the release of cytokines. However, we did not observe them in our study. transdifferentiation V myofibroblasts, and this suggests that cytokines secreted by CC and hepatocytes can serve as a factor supporting the process that has already begun transdifferentiation, but they are probably not able to trigger it with a single exposure of the liver to LPS.

An increase in the proliferative activity of cells was also observed mainly in the 1st zone of the acinus. This probably suggests that all (or almost all) processes aimed at out O- and paracrine regulation of intercellular interactions occur in the periportal zones. An increase in the number of proliferating cells was observed from 24 h after LPS administration; the number of positive cells increased up to 72 hours (maximum proliferative activity, Fig. 2, a, b). Both hepatocytes and sinusoid cells proliferated. However, staining on PCNA does not give the ability to identify the type of proliferation ruminating sinusoid cells. According to the literature, the action of endotoxin leads to increased depending on the amount of CC. They think it's about comes both from the proliferation of liver macrophages and from the migration of monocytes from other organs. Cytokines released by CKs can increase the proliferative capacity of Ito cells. Therefore, it is logical to assume that proliferating cells are represented perisinusoidal Ito cells. The increase in their number that we recorded is apparently necessary to increase the synthesis of growth factors and restore the intercellular matrix under conditions of damage. This may be one of the links in the compensatory-restorative reactions of the liver, since Ito cells are the main source of components of the intercellular matrix, stem cell factor and hepatocyte growth factor, which are involved in repair and differentiation formation of liver epithelial cells. Absent vie transformation of Ito cells into myofibroblasts indicates that one episode of endotoxin aggression is not enough for the development of liver fibrosis.

Thus, the acute effects of endotok syna causes an increase in the number desmin positive Ito cells, which is an indirect sign of liver damage. Quantity perisinusoidal cells increases, apparently as a result of their proliferation. A single episode of endotoxin aggression causes reverse my activation perisinusoidal Ito cells and does not lead to them transdifferentiation into myofibroblasts. In this regard, it can be assumed that in the mechanisms of activation and transdifferentiation Ito cells involve not only endotoxin and cytokines, but also some other factors of intercellular interactions.

LITERATURE

1. Mayansky D.N., Wisse E., Decker K. // New frontiers hepatology. Novosibirsk, 1992.

2. Salakhov I.M., Ipatov A.I., Konev Yu.V., Yakovlev M.Yu. // We will make progress, biol. 1998. T. 118, Issue. 1. pp. 33-49.

3. Yakovlev M.Yu. // Kazan . m units magazine 1988. No. 5. P. 353-358.

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Keywords

LIVER / STELLATE CELLS ITO/ MORPHOLOGY / CHARACTERISTIC / VITAMIN A / FIBROSIS / LIVER / HEPATIC STELLATE CELLS / MORPHOLOGY / CHARACTERISTIC / VITAMIN A / FIBROSIS

annotation scientific article on fundamental medicine, author of the scientific work - Tsyrkunov V.M., Andreev V.P., Kravchuk R.I., Kondratovich I.A.

Introduction. The role of Ito stellate cells (ISC) has been identified as one of the leading ones in the development of fibrosis in the liver, however, intravital visualization of the ISC structure is minimally used in clinical practice. Purpose of the work: to present the structural and functional characteristics of PCI based on the results of cytological identification of intravital liver biopsies. Materials and methods. Classical methods of light and electron microscopy of biopsy specimens and original techniques using ultrathin sections, fixation and staining were used. Results. Photo illustrations of light and electron microscopy of liver biopsies from patients with chronic hepatitis C shows the structural characteristics of HSCs at different stages (rest, activation) and in the process of transformation into myofibroblasts. Conclusions. Application of original methods of clinical morphological identification and assessment functional state ZCI will improve the quality of diagnosis and prognosis of liver fibrosis.

Text of scientific work on the topic “Clinical Liver Cytology: Ito stellate cells”

UDC 616.36-076.5

CLINICAL CYTOLOGY OF THE LIVER: ITO STELLATE CELLS

Tsyrkunov V. M. ( [email protected]), Andreev V. P. ( [email protected]), Kravchuk R. I. ( [email protected]), Kondratovich I. A. ( [email protected]) EE "Grodno State Medical University", Grodno, Belarus

Purpose of the work: to present the structural and functional characteristics of PCI based on the results of cytological identification of intravital liver biopsies.

Materials and methods. Classical methods of light and electron microscopy of biopsy specimens and original techniques using ultrathin sections, fixation and staining were used.

Results. Photo illustrations of light and electron microscopy of liver biopsies from patients with chronic hepatitis C show the structural characteristics of PCIs at different stages (rest, activation) and in the process of transformation into myofibroblasts.

Conclusions. The use of original methods for clinical morphological identification and assessment of the functional state of the liver will improve the quality of diagnosis and prognosis of liver fibrosis.

Key words: liver, Ito stellate cells, morphology, characteristics, vitamin A, fibrosis.

Introduction

An unfavorable outcome of most chronic diffuse liver lesions of various etiologies, including chronic hepatitis C (CHC), is liver fibrosis, in the development of which the main participants are activated fibroblasts, the main source of which are activated Ito stellate cells (Ito stellate cells).

Hepatic Stellate Cell, HSC, Cell of Ito, Ito cell. ZCIs were first described in 1876 by K. Kupffer and named by him stellate cells (“Stemzellen”). T. Ito, having discovered drops of fat in them, first designated them fat-absorbing (“shibo-sesshusaibo”), and then, having established that fat was produced by the cells themselves from glycogen, fat-storing cells (“shibo-chozosaibo”) . In 1971, K. Wake proved the identity of Kupfffer stellate cells and Ito fat-storing cells and that these cells “store” vitamin A.

About 80% of vitamin A in the body accumulates in the liver, and up to 80% of all liver retinoids are deposited in fat droplets of the liver. Retinol esters in the composition of chylomicrons enter hepatocytes, where they are converted into retinol, forming a complex of vitamin A with retinol binding protein (RBP), which is secreted into the perisinusoidal space, from where it is deposited by the cells.

The close connection between PCI and liver fibrosis established by K. Popper demonstrated their not static, but dynamic function - the ability to directly participate in the remodeling of the intralobular perihepatocellular matrix.

The main method of morphological examination of the liver, carried out to assess changes in intravital biopsies, is light microscopy, which in clinical practice makes it possible to determine the activity of the liver.

burning and the stage of chronicity. The disadvantage of the method is its low resolution, which does not allow one to evaluate the structural features of cells, intracellular organelles, inclusions, and functional characteristics. Intravital electron microscopic examination of ultrastructural changes in the liver makes it possible to supplement light microscopy data and increase their diagnostic value.

In this regard, the identification of liver HCIs, the study of their phenotype in the process of transdifferentiation, and the determination of the intensity of their proliferation are the most important contribution to predicting the outcomes of liver diseases, as well as to the pathomorphology and pathophysiology of fibrogenesis.

The goal is to present the structural and functional characteristics of PCI based on the results of cytological identification of intravital liver biopsies.

Materials and methods

An intravital liver biopsy was obtained by aspiration biopsy liver in patients with CHC (HCV RNA+), from whom written informed consent was obtained.

For light microscopy of semi-thin sections, liver biopsy samples of patients measuring 0.5^2 mm were fixed using a double fixation method: first, using the Sato Taizan method, then tissue samples were additionally fixed for 1 hour in 1% osmium fixative prepared with 0.1 M phosphate Sorensen buffer, pH 7.4. To better identify intracellular structures and interstitial substances on semi-thin sections, potassium dichromate (K2Cr2O7) or chromic anhydride crystals (1 mg/ml) were added to 1% osmium tetroxide. After dehydration of samples in a series alcohol solutions increasing concentration and acetone, they were placed in a prepolymerized mixture of butyl methacrylate and styrene and polymerized at 550C. Semi-thin sections (1 µm thick) were sequentially stained

azure II-basic fuchsin. Microphotographs were taken using a digital video camera (Leica FC 320, Germany).

Electron microscopic examination was carried out in liver biopsy samples measuring 0.5x1.0 mm, fixed with a 1% solution of osmium tetroxide in 0.1 M Milloniga buffer, pH 7.4, at +40C for 2 hours. After dehydration in ascending alcohols and acetone, the samples were embedded in Araldite. Semi-thin sections (400 nm) were prepared from the resulting blocks using a Leica EM VC7 ultramicrotome (Germany) and stained with methylene blue. The preparations were examined under a light microscope and a similar area was selected for further study of ultrastructural changes. Ultrathin sections (35 nm) were counterstained with 2% uranyl acetate in 50% methanol and lead citrate according to E. S. Reynolds. Electron microscopic preparations were studied in a JEM-1011 electron microscope (JEOL, Japan) at magnifications of 10,000-60,000 and an accelerating voltage of 80 kW. To obtain images, a complex consisting of an Olympus MegaViewIII digital camera (Germany) and iTEM image processing software (Olympus, Germany) was used.

Results and discussion

PCIs are located in the perisinusoidal space (Disse) in pockets between hepatocytes and endothelial cells, have long shoots, penetrating deeply between hepatocytes. Most publications devoted to this population of CCIs provide their schematic representation, which only allows one to indicate the “territorial” affiliation of CCIs in the liver and in relation to the surrounding “neighbors” (Figure 1).

PCIs have close contact with endothelial cells through components of the incomplete basement membrane and interstitial collagen fibers. Nerve endings penetrate between the PCI and parenchymal cells, which is why the space of Disse is defined as the space between the plates of parenchymal cells and

complex of HCI and endothelial cells.

It is believed that PCIs originate from poorly differentiated mesenchymal cells of the transverse septum of the developing liver. The experiment established that hematopoietic stem cells participate in the formation of HCI and that this process is not caused by cell fusion.

Sinusoidal cells (SCs), primarily HSCs, play a leading role in all types of liver regeneration. Fibrosing liver regeneration occurs as a result of inhibition of the stem functions of the liver and bone marrow stem cells. In the human liver, HSCs account for 5-15%, being one of 4 types of SCs that are of mesenchymal origin: Kupffer cells, endothelial cells, Pd cells. The SC pool also contains 20-25% leukocytes.

The cytoplasm of the HCI contains fatty inclusions with retinol, triglycerides, phospholipids, cholesterol, free fatty acids, α-actin and desmin. Gold chloride staining is used to visualize PCI. The experiment established that a marker of differentiation of HCI from other myofibroblasts is their expression of the Reelin protein.

HSCs exist in a quiet (“inactive HSC”), transient, and long-term activated states, each of which is characterized by gene expression and phenotype (α-MA, ICAM-1, chemokines and cytokines).

In an inactive state, HCIs have a round, slightly elongated or irregular shape, a large core and a clear visual feature - lipid inclusions (droplets) containing retinol (Figure 2).

The number of lipid droplets in the inactive HCI reaches 30 or more; they are close in size, adjacent to each other, pressing into the core and pushing it to the periphery (Figure 2). Small inclusions may be located between large drops. The color of the drops depends on the fixative and the color of the material. In one case they are light (Figure 2a), in the other they are dark green (Figure 2b).

Figure 1. - Scheme of the location of the PCI (stellatecell, perisinusoidal lipocyte) in the perisinusoidal space of Disse (space of Disse), Internet resource

Figure 2. - ZKI in an inactive state

a - round-shaped HCI with a high content of lipid droplets with a light color (white arrows), hepatocytes (Hz) with devastated cytoplasm (black arrow); b - HCI with dark-colored lipid droplets, in close contact with the macrophage (Mph); a-b - semi-thin sections. The color of azure II is basic magenta. Microphotographs. Increased 1000; c - ZCI with an abundance of lipid droplets (more than 30), having an irregular shape (magnitude 6,000); d-ultrastructural components of the ICI: l-lipid droplets, mitochondria (orange arrows), GRES (green arrows), Golgi complex (red arrow), uv. 15,000; v-d - electron diffraction patterns

With electron microscopy, a more osmiophilic marginal rim is formed against the background of a light lipid substrate (Figure 5a). In most “resting” HCIs, along with large lipid inclusions, there is a noticeably small amount of cytoplasmic matrix, poor in mitochondria (Mx) and granular endoplasmic reticulum (GRE). In this case, the compartments of a moderately developed Golgi complex are clearly visible in the form of a stack of 3-4 flattened cisterns with slightly widened ends (Figure 2d).

At certain conditions activating HSCs acquire a mixed or transitional phenotype, combining the morphological characteristics of both lipid-containing and fibroblast-like cells (Figure 3).

The transitional phenotype of PCI also has its own morphological characteristics. The cell acquires an elongated shape, the number of lipid inclusions decreases, and the number of invaginations of the nucleolemma decreases. The volume of the cytoplasm increases, containing numerous cisterns of the GES with bound ribosomes and free ribosomes, Mx. Hyperplasia of the components of the lamellar Golgi complex, represented by several stacks of 3-8 flattened cisterns, is observed; the number of lysosomes involved in the degradation increases.

Figure 3. - ZKIs in a transition state

a - ZKI (white arrows). Semi-thin slice. The color of azure II is basic magenta. Microphotography. Increased 1000; b - ZCI of an elongated shape and with a small number of lipid droplets; uv. 8,000; c - ZCI in contact with Kupffer cells (KC) and lymphocyte (Lc), uv. 6,000. (Hz - hepatocyte, l - lipid drops, E - erythrocyte); d - mitochondria (orange arrows), GRES (green arrows), Golgi cell (red arrow), lysosomes (blue arrows), level 20,000; b, c, d - electron diffraction patterns

tion of lipid droplets (Figure 3d). Hyperplasia of the components of the GRES and the Golgi complex is associated with the ability of fibroblasts to synthesize collagen molecules, as well as to model them through post-translational hydroxylation and glycosylation in the endoplasmic reticulum and elements of the Golgi complex.

In an undamaged liver, the PCI, being in a calm state, covers the sinusoidal capillary with its processes. The processes of the PCI are divided into 2 types: perisinusoidal (subendothelial) and interhepatocellular (Figure 4).

The first leave the cell body and extend along the surface of the sinusoidal capillary, covering it with thin finger-like branches. They are covered with short villi and have characteristic long micro-ejections that extend even further along the surface of the endothelial tube of the capillary. Interhepatocellular projections, having overcome the plate of hepatocytes and reaching the adjacent sinusoid, are divided into several perisinusoidal projections. Thus, the ZKI on average covers more than two adjacent sinusoids.

With liver damage, activation of the PCI and the process of fibrogenesis occurs, in which 3 phases are distinguished. They are designated initiation, prolongation and resolution (resolution of fibrous tissue). This process of transformation of “resting” HSCs into fibrosing myofibroblasts is initiated by cytokines (^-1,^-6,

Figure 4. - Perisinusoidal (subendothelial) and interhepatocellular processes (outgrowths) of the PCI

a - process of the PCI (yellow arrows) emerging from the cell body, uv. 30,000; b - extension of the ZCI, located along the surface of the sinusoidal capillary, containing a lipid droplet, uv. 30,000; c - subendothelially located processes of the PCI. Endothelial cell processes (pink arrows); d - interhepatocellular process of the PCI; area of destruction of the membranes of the HCI and hepatocyte (black arrows), uv. 10 000. Electron diffraction patterns

TOT-a), underoxidized metabolic products, reactive oxygen species, nitric oxide, endothelin, platelet-activating factor (PDGF), plasminogen activator, transforming growth factor (TGF-1), acetaldehyde and many others. Direct activators are hepatocytes in a state of oxidative stress, Kupffer cells, endotheliocytes, leukocytes, platelets producing cytokines (paracrine signals) and PCIs themselves (autocrine stimulation). Activation is accompanied by the expression (inclusion in work) of new genes, the synthesis of cytokines and proteins of the extracellular matrix (types I, III, U collagens).

At this stage, the process of activation of the PCI can be completed by stimulating the formation of anti-inflammatory cytokines in the PCI, inhibiting the production of TOT-a by macrophages in the damage area. As a result, the number of HCIs is sharply reduced, they undergo apoptosis and fibrosis processes in the liver do not develop.

In the second phase (prolonged), with prolonged constant paracrine and autocrine exposure to activating stimuli, the activated phenotype is “maintained” in the PCI, characterized by the transformation of the PCI into contractile myofibroblast-like cells that carry out the synthesis of extracellular fibrillar collagen.

The activated phenotype is characterized by proliferation, chemotaxis, contractility, loss of retinoid stores, and the formation of myofibroblast-like cells. Activated HSCs also show increased abundance of novel genes such as a-SMA, ICAM-1, chemokines, and cytokines. Cell activation indicates the onset of the early stages of fibrogenesis and precedes increased production of ECM proteins. Formed fibrous tissue undergo remodeling due to matrix breakdown with the help of matrix metalloproteinases (MMPs). In turn, matrix breakdown is regulated by tissue inhibitors of matrixmetaloproteinases (TIMPs). MMPs and TIMPs are members of the zinc-dependent enzyme family. MMPs are synthesized in the HCI in the form of inactive proenzymes, which are activated upon cleavage of the propeptide, but are inhibited upon interaction with endogenous TIMPs - TIMPs-1 and TIMPs-2. HCIs produce 4 types of membrane-type MMPs, which are activated by IL-1β. Among MMPs, particular importance is attached to MMPs-9, a neutral matrix metalloproteinase that has activity against collagen type 4, which is part of the basement membrane, as well as against partially denatured collagen types 1 and 5.

The increase in the PCI population in various types of liver damage is judged by the activity of a significant number of mitogenic factors, related tyrosine kinase receptors and other identified mitogens that cause the most pronounced proliferation of PCI: endothelin-1, thrombin, FGF - fibroblast growth factor, PDGF - endothelial growth factor blood vessels, IGF - insulin-like growth factor. The accumulation of HCI in areas of liver damage occurs not only due to the proliferation of these cells, but also due to their directed migration into these areas through chemotaxis, with the participation of chemoattractants such as PDGF and leukocyte chemoattractant-MCP (monocyte chemotactic protein-1).

In activated HSCs, the number of lipid droplets is reduced to 1-3 with their location at opposite poles of the cell (Figure 5).

Activated HSCs acquire an elongated shape, significant areas of the cytoplasm are occupied by the Golgi complex, and quite numerous GRES cisterns (an indicator of protein synthesis for export) are revealed. The number of other organelles is reduced: few free ribosomes and polysomes, single mitochondria, and irregularly lysosomes are found (Figure 6).

In 2007, HSCs were first called liver stem cells, since they express one of the markers of hematopoietic mesenchymal stem cells - CD133.

Figure 5. - ZKI in the activated state

a, b - HCI (blue arrows) with single lipid inclusions localized at opposite poles of the nucleus. The perisinusoidal connective tissue (in Fig. 6a) and the intercellular matrix layer around the hepatocyte (in Fig. 6b) are colored red. Cytotoxic lymphocytes (purple arrows). Endothelial cell (white arrow). Close contact between a plasma cell (red arrow) and a hepatocyte. Semi-thin sections. The color of azure II is basic magenta. Microphotographs. Increased 1000 ; c, d - ultrastructural components of the HCI: mitochondria (orange arrows), Golgi complex (red arrow), cisternae of its more osmiophilic cis-side facing the expanded elements of the granular endoplasmic reticulum (green arrows), lysosome (blue arrow) (magnitude 10,000 and 20,000, respectively); c, d - electron diffraction patterns

Myofibroblasts, which are absent in the normal liver, have three potential sources: first, during intrauterine development of the liver, in the portal tracts, myofibroblasts surround the vessels and bile ducts during their maturation, and after full development of the liver, they disappear and are replaced in the portal tracts by portal fibroblasts; second, when the liver is damaged, they are formed due to portal mesenchymal cells and resting HCI, less often due to transitional epithelial-mesenchymal cells. They are characterized by the presence of CD45-, CD34-, Desmin+, glial fibrillary-associated protein (GFAP)+ and Thy-1+.

Recent studies have shown that hepatocytes, cholangiocytes and endothelial cells can become myofibroblasts through epithelial or endothelial to mesenchymal transition (EMT). These cells include markers such as CD45-, albumin+ (ie hepatocytes), CD45-, CK19+ (ie cholangiocytes) or Tie-2+ (endothelial cells).

Figure 6. - High fibrotic activity of HCI

a, b - myofibroblast (MFB), the cell contains a large nucleus, elements of the GRES (red arrows), numerous free ribosomes, polymorphic vesicles and granules, single mitochondria and a bright visualization sign - a bundle of actin filaments in the cytoplasm (yellow arrows); took away 12,000 and 40,000; c, d, e, f - high fibrotic activity of HCI while retinoid-containing lipid droplets are preserved in the cytoplasm. Numerous bundles of collagen fibrils (white arrows), retaining (a) and losing (d, e, f) specific transverse striations; took away 25 000, 15 000, 8 000, 15 000. Electron diffraction patterns

In addition, bone marrow cells, consisting of fibrocytes and circulating mesenchymal cells, can transform into myofibroblasts. These are CD45+ cells (fibrocytes), CD45+/- (circulating mesenchymal cells), collagen type 1+, CD11d+ and MHC class 11+ (Figure 7).

Literary data confirm not only the close connection between the proliferation of oval cells and the proliferation of sinusoidal cells, but also data on the possible differentiation of HCI into the hepatic epithelium, which was called mesenchymal-epithelial transformation of perisinusoidal cells.

In a state of fibrogenic activation, myofibroblast-like PCIs, along with a decrease in the number and subsequent disappearance of lipid droplets, are characterized by focal proliferation (Figure 8), immunohistochemical expression of fibroblast-like markers, including smooth muscle α-actin, and the formation of pericellular collagen fibrils in the spaces of Disse.

During the developmental phase of fibrosis, increasing hypoxia of the liver tissue becomes a factor for additional overexpression of pro-inflammatory adhesion molecules in stem cells - 1CAM-1, 1CAM-2, VEGF, proinflammatory

Interaction of hepatic ductal progenitor cells with liver myofibroblasts

Myofibroblast-like HSCs in a state of fibrogenic activation.

Figure 7. - Participants in myofibroblastic activation of PCI

lytic chemoattractants - M-CSF, MCP-1 (monocyte chemotactic protein-1) and SGS (cytokine-mediated neutrophil chemoattractant) and others that stimulate the formation of pro-inflammatory cytokines (TGF-b, PDGF, FGF, PAF, SCF, ET-1 ) and enhance the processes of fibrogenesis in the liver, creating conditions for the self-sustaining induction of continuous activation of the PCI and fibrogenesis processes.

On microscopic preparations, pericapillary fibrosis manifests itself in the form of intense red coloring of the perisinusoidal connective tissue and the intercellular matrix layer around hepatocytes (often dying). On electron microscopic preparations, fibrotic changes are visualized either in the form of formed large bundles of collagen fiber fibrils that have retained transverse striations, or in the form of massive

deposits in the Disse space of fibrous mass, which are swollen collagen fibers that have lost their periodic striations (Figure 9).

According to modern concepts, fibrosis is a dynamic process that can progress and regress (Figure 10).

Recently, several specific markers of PCI have been proposed: vitamin A (VA) bloom into lipid droplets, GFAP, p75 NGF receptor, and synaptophysin. Research is being conducted on the participation of liver HCI in the proliferation and differentiation of liver stem cells.

We studied the content of retinol-binding protein (RSB-4), which forms a complex with VA, the concentration of which in the blood plasma normally correlates with the body's supply of VA, 80% of which is found in the PCI.

A relationship has been established between the contents

Figure 8. - Focal proliferation of PCI in a state of fibrogenic activation

a - hyperplasia of the PCI (white arrows) in the lumen of the dilated sinusoids; b - proliferation of transdifferentiated HSC (white arrows), endothelial cell (pink arrow). Semi-thin sections. The color of azure II is basic magenta. Microphotographs. Increased 1000

Figure 9. - Final stage of myofibroblastic activation of the PCI

a, b - perisinusoidal fibrosis (white arrows). Peri-sinusoidal connective tissue and the intercellular matrix layer around hepatocytes (b) are stained with basic fuchsin red. HCIs activated and transformed into fibroblasts (blue arrows). Hz in Fig. a - hepatocyte with devastated cytoplasm. Semi-thin sections. The color of azure II is basic magenta. Microphotographs. Increased 1000; c, d - perisinusoidal and perihepatocellular fibrosis in the liver lobule, increased electron density of collagen fiber fibrils; condensation of the mitochondrial matrix in the hepatocyte (orange arrow). UV.8,000 and 15,000, respectively. Electron diffraction patterns

Table 1. - Indicators of RSB-4 content in patients with liver cirrhosis (LC) and chronic hepatitis (CH) of various etiologies, ng/ml (M±t)

Group n M±m р

Liver cirrhosis 17 23.6±2.29<0,05

CG, AST normal 16 36.9±2.05* >0.05

CG, AST >2 norms 13 33.0±3.04* >0.05

CG, ALT normal 13 37.5±3.02* >0.05

CG, ALT >2 norms 21 35.9±2.25* >0.05

Control 15 31.2±2.82

Note: p - significant differences with control (p<0,05); * - достоверные различия между ЦП и ХГ (р<0,05)

A false lobule surrounded by a fibrous septum. Masseau staining - circle of false lobule. Painting according to Nu.Uv.x50 Masson. UV.x200

Figure 10. - Dynamics of events in the false lobule of a patient with viral cirrhosis 6 months after transplantation of autologous mesenchymal stem cells into the liver

We eat RSB-4 and the 4th stage of fibrosis (cirrhosis), in contrast to chronic hepatitis, in which such a dependence was not observed, regardless of biochemical markers of inflammatory activity in the liver.

This fact must be taken into account when justifying replacement therapy to eliminate VA deficiency in the body, which may be due to the depletion of the potential of PCI caused by the progression of fibrosis in the liver.

1. The maximum effectiveness of assessing the structural and functional state of the PCI is ensured by a morphological study of an intravital biopsy with the simultaneous use of a set of cellular visualization techniques (light, electron microscopy of ultrathin sections and original methods of fixation and staining).

2. The results of a morphological study of PCI make it possible to improve the quality of intravital diagnosis of fibrosis, monitor it and predict the outcomes of chronic diffuse liver lesions at a higher modern level.

3. The results of morphological conclusions will allow the clinician to additionally include in the formulation of the final diagnosis updated data on the stage of chronicity (stabilization, progression or resolution of fibrosis) during therapy.

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CLINICAL CYTOLOGY OF THE LIVER: ITO STELLATE CELLS (HEPATIC STELLATE CELLS)

Tsyrkunov V. M., Andreev V. P., Kravchuk R. I., Kandratovich I. A. Educational Establishment "Grodno State Medical University", Grodno, Belarus

The aim of the work is to present the structural and functional characteristics of HSC based on the findings of cytological identification of intravital liver biopsy samples.

Materials and methods. Classical methods of light and electron microscopy of biopsy samples within the original technique of using ultrathin sections, fixation and staining were applied.

Results. The structural characteristics of the HSC of the liver biopsy samples from patients with chronic hepatitis C are presented on photo illustrations of light and electron microscopy. HSC are depicted at different stages (rest, activation) and during the process of transformation into myofibroblasts.

Conclusions. The use of original methods of clinical and morphological identification and evaluation of the functional status of HSC allows to improve the quality of diagnosis and prognosis of liver fibrosis.

Could perisinusoidal cells be regional stem cells of the liver? Study of the influence of liver cells on stem cells Stellate cells

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

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annotation scientific article on fundamental medicine, author of the scientific work - Tsyrkunov V.M., Andreev V.P., Kravchuk R.I., Kondratovich I.A.

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