Ultrastructure of Sinusoidal Cells in a Benign Liver Cell Adenoma

A typical benign liver cell adenoma was removed from a 25-year-old female taking an oral contraceptive. The biopsy was perfusion-fixed, allowing good visualization of sinusoids and sinusoidal cells. Kupffer cells were seldom seen. Endothelial cells were of irregular thickness; most of them had few fenestrae and were attached to each other by well visible junctions. Normal perisinusoidal cells (PSC) were not seen but were replaced by myofibroblast-like cells with thick subendothelial processes. A basement membrane often underlaid endothelial cells and the processes of PSC. The enlarged Disse space with a flattened sinusoidal hepatocyte membrane contained occasional red blood cells and an abundant extracellular matrix but few collagen fibers. These sinusoidal abnormalities leading to the capillarization of sinusoids, either the cause or the consequence of the disease, indicated a loss of normal hepatocyte homeostasis.     

人肝贮脂细胞表达结蛋白的免疫细胞化学观察

用免疫细胞化学法观察了正常人肝贮脂细胞表达结蛋白中间丝。发现结蛋白阳性细胞位于血窦旁旁狄氏间隙内,呈星形或纺锤形,于肝小叶内大致均匀分布,有些细胞内含有空泡,每高倍(400X)视野细胞数为15～25个。汇管区结缔组织内及肝静脉周围散在少数与小叶内形态相似的结蛋白阳性细胞。结果表明正常人肝贮脂细胞表达结蛋白,可作为研究该细胞的良好标志,并支持了贮脂细胞的肌源性学说。[关键词]贮脂细胞,结     

Benign Recurrent Cholestasis: A Light and Electron Microscopic Study with Emphasis on Sinusoidal Cells

A liver biopsy was performed on a patient with benign recurrent cholestasis. Cholestasis was mainly centrolobular with infiltration by sinusoidal macrophages. There was no necrosis. All the classic and specific ultrastructural criteria of cholestasis were observed in hepatocytes under electron microscopy. Perfusion-fixation of the biopsy allowed in addition a good visualization of sinusoids and sinusoidal cells. Numerous macrophages (Kupffer cells) with intense phagocytic activity were present in the lumen; some formed the sinusoidal barrier or were infiltrated in the Disse space. Endothelial cells contained numerous dense bodies and had few fenestrae. Cellular debris of hepatocytic origin which were not phagocytized in the Disse space were extruded in the lumen either through enlarged endothelial pores or by progressive invagination in the endothelial wall followed by outpouching in the sinusoid. In an enlarged Disse space containing amorphous material and collagen fibrils some perisinusoidal cells were transitional cells. These results indicate that pure cholestasis leads not only to hepatocyte injury with intense phagocytic activity but also to some degree of sinusoidal cell damage and extracellular matrix changes.     

Desmin-containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury

Using immunohistochemical methods, we have confirmed that the perisinusoidal cells in rat liver express the intermediate filament protein, desmin, and we have used this marker for identification of the cells on light microscopy. The study has been extended to quantify the response of perisinusoidal cells to acute liver injury induced by carbon tetrachloride. A significant increase in desmin-positive cells was observed in areas of necrosis as early as 48 h following the administration of a single bolus of carbon tetrachloride. This reached a peak at 72 h, with a five-fold increase in desmin-positive cells in areas of necrosis. These observations are consistent with the hypothesis that perisinusoidal cells are involved in the response to acute liver injury. Anti-desmin antibodies are of potential value in further characterizing the functional role of perisinusoidal cells in normal and diseased liver.     

Pathology of the liver sinusoids

The hepatic sinusoids comprise a complex of vascular conduits to transport blood from the porta hepatis to the inferior vena cava through the liver. Under normal conditions, portal venous and hepatic artery pressures are equalized within the sinusoids, oxygen and nutrients from the systemic circulation are delivered to the parenchymal cells and differentially distributed throughout the liver acini, and proteins of liver derivation are carried into the cardiac/systemic circulation. Liver sinusoid structures are lined by endothelial cells unique to their location, and Kupffer cells. Multifunctional hepatic stellate cells and various immune active cells are localized within the space of Disse between the sinusoid and the adjacent hepatocytes. Flow within the sinusoids can be compromised by physical or pressure blockage in their lumina as well as obstructive processes within the space of Disse. The intimate relationship of the liver sinusoids to neighbouring hepatocytes is a significant factor affecting the health of hepatocytes, or transmission of the effects of injury within the sinusoidal space. Pathologists should recognize several patterns of injury involving the sinusoids and surrounding hepatocytes. In this review, injury, alterations and accumulations within the liver sinusoids are illustrated and discussed.     

The sinusoidal barrier in rats with portacaval anastomosis: A morphometric study

Summary Shunting of portal blood in the rat leads to liver atrophy and to an increase in arterial blood flow with microcirculatory disturbances. The aim of this study was to investigate the effects of these disturbances on the liver sinusoidal barrier (endothelial and perisinusoidal cells) using morphometric techniques. Rats with portacaval anastomosis (PCA) and sham operated pair-fed controls were studied 3 months after the shunt. Sinusoidal volume density in PCA increased but not significantly and the volume density (V_v) of total endothelial (EC) and perisinusoidal cells (PSC) increased by 104.54% compared to sham operated pair-fed rats. The increase of EC V_v was not associated with an increase in surface density (S_v) suggesting a fall in the number of small fenestrations and an increase in cell thickness. This interpretation supports the morphological observations. The increase of PSC V_v was mainly related to the increase in their subendothelial processes V_v and not to that of the cell body V_v. Lipids V_v and RER S_v expressed per sinusoidal cells remained unchanged suggesting that the balance between the 2 hypothetical functions of the PSC, namely fibrogenesis and storage of vitamin A, was maintained.In conclusion, changes of EC and PSC after PCA result mainly in thickening of the sinusoidal barrier. This increase may impair exchanges between the sinusoidal lumen and Disse space and contribute to functional abnormalities.This work was supported by a grant from INSERM CRL n^o 807003     

Liver Fibrosis in Elderly Cadavers: Localization of Collagen Types I,III, and IV, α‐Smooth Muscle Actin,and Elastic Fibers

We have shown a high prevalence of liver fibrosis in elderly cadavers with diverse causes of death by Sirius red stain; however, the various collagen types in these samples have yet to be evaluated. To further characterize the histopathology of the fibrotic lesions in the livers of these elderly cadavers, this study used immunohistochemistry and histochemistry to identify the principal collagens produced in liver fibrosis, fibrogenic cells and elastic fibers. Collagen I and III immunoreactions were found to colocalize in collagen fibers of fibrotic central veins, perisinusoidal fibrotic foci, portal tract stroma, and fibrous septa. α‐Smooth muscle actin‐expressing perisinusoidal hepatic stellate cells (HSCs), as well as perivenular, portal, and septal myofibroblasts, were closely associated with collagen fibers, reflecting their fibrogenic functions. HSCs and myofibroblasts were also noted to express collagen IV, which may contribute to production of basal lamina‐like structures. In fibrotic livers, the sinusoidal lining showed variable immunostaining for collagen IV. Collagen IV immunostaining revealed vascular proliferation and atypical ductular reaction at the portal–septal parenchymal borders, as well as capillary‐like vessels in the lobular parenchyma. While elastic fibers were absent in the space of Disse, they were found to codistribute with collagens in portal tracts, fibrous septa and central veins. Our combined assessment of collagen types, HSCs, myofibroblasts, and elastic fibers is significant in understanding the histopathology of fibrosis in the aging liver. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Synaptophysin: A novel marker for human and rat hepatic stellate cells

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

Basement membrane proteins in the space of Disse: a reappraisal.

The distribution of two major basement membrane components, type IV collagen and laminin, was studied within the perisinusoidal space of Disse in normal human liver using (i) an immunoperoxidase method for light microscopy and (ii) immunogold labelling for ultrastructural localisation. Although immunoreactivity depended on the mode of tissue fixation, both proteins could be identified at this site using a panel of affinity purified antibodies. These findings indicate that these proteins are normal constituents of the perisinusoidal extracellular matrix, and refute the hypothesis that capillarization of the sinusoids in chronic liver disease results from neo-expression of laminin in the space of Disse.     

“Sternzellen” in the liver: Perisinusoidal cells with special reference to storage of vitamin A

The “Sternzellen” (von Kupffer, 1876) in the liver of normal animals and of those injected with excess vitamin A were examined with the light and electron microscopes. These cells were stellate perisinusoidal cells located in the space of Disse and were separated from the sinusoidal lumen by the endothelium. Their cytoplasm contained a number of lipid droplets which reacted intensely with gold chloride. These lipid droplets imparted intense vitamin A fluorescence under the fluorescence microscope. Following the administration of excess vitamin A, these lipid droplets increased remarkably. The “Sternzellen” were identical with cells described as “interstitial” or “fat-storing” cells by others, and were quite different from the so-called Kupffer cells of the liver reticulo-endothelial system.     

Modifications of the sinusoidal barrier in a model of postsinusoidal hypertension in the rat. An ultrastructural study of endothelial cells

Sinusoids and sinusoidal cells were examined by light and electron microscopy, using a rat model of postsinusoidal hypertension. One month after partial ligation of the vena cava (PLVC) above the hepatic veins, subcapsular hemorrhagic areas were visible with proliferation of hepatic veins; in non hemorrhagic areas, sinusoidal congestion was found. Postsinusoidal hypertension led to a significant increase in sinusoidal volume and to major abnormalities of the endothelium such as endothelial processes and pouches with numerous diaphragmed fenestrae; some red blood cells could be seen in these pouches. Endothelial cells sent out processes in between hepatocytes. Complete and incomplete pseudo-neolumens were found near sinusoids. Numerous Kupffer cells were located either in the sinusoidal barrier or infiltrating the Disse space close to extravasated red blood cells and perisinusoidal cell processes. 18 months after PLVC, lesions were much the same except for the presence of red blood cells in the Disse space.     

Uptake Behavior of Embryonic Chick Liver Cells

The capacity of selective uptake by liver cells, focusing particularly on the parenchymal and perisinusoidal stellate cells during chick liver development (8–18 days of incubation), was ultrastructurally examined after injection of 240‐nm‐diameter lecithin (phosphatidylcholine) ‐coated or noncoated beads into the extraembryonic circulation. Cytoplasmic projections of both cells as well as extrasinusoidal macrophages reached into the sinusoid‐like vascular spaces. The primitive perisinusoidal stellate cells were identified by immunocytochemistry as being rich in desmin‐positive cytoplasmic intermediate filaments. The cells demonstrated selective uptake of noncoated beads by means of their cytoplasmic projections. These findings were significant in the early period of incubation, indicating that the phagocytic activity is a characteristic and transient phenomenon of developmental differentiation. Large numbers of coated and a few noncoated beads penetrated into the perivascular spaces. The parenchymal cells incorporated only the coated beads that passed through the endothelial lining, suggesting that these cells express selective but limited phagocytic capacity against large “foreign” substances even long before their maturation. The cell projections were not engaged in uptake function. Extrasinusoidal macrophages, Kupffer cells, and intraluminal primitive macrophages all took up both beads; however, lecithin coating of the beads clearly suppressed their uptake function. These data suggest that the uptake function of large “foreign” substances appears to be intrinsic to liver cells and lecithin coating would be useful for delivering large substances to parenchymal cells. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Early activation of hepatic stellate cells and perisinusoidal extracellular matrix changes during ex vivo pig liver perfusion

In previous works, we observed during liver transplantation procedure, the early activation of hepatic stellate cells (HSC) which acquire alpha-smooth muscle (SM) actin expression. In this study, we evaluated changes in HSC and in perisinusoidal extracellular matrix during ex vivo pig liver perfusion. Under general anesthesia, pig livers were flushed and removed, and then perfused ex vivo for 6 h with homologous blood. Liver biopsies were taken before and after washout, at 5 min perfusion, and then hourly. Tissues were processed for immunohistochemistry, immunofluorescence, confocal microscopy, in situ hybridization and electron microscopy. Before and after liver washout, alpha-SM actin was present in vessel walls but in very few lobular HSC. After 1 h perfusion, a strong reactivity for alpha-SM actin was present in HSC, particularly along dilated sinusoids. At the ultrastructural level, numerous microfilament bundles appeared in HSC cytoplasmic processes. During perfusion, type I and type IV collagens, type III procollagen, and fibronectin acquired a looser organisation in relation with the enlargement of perisinusoidal spaces; laminin appeared in perisinusoidal spaces around portal areas and fibrillin deposits increased. In situ hybridization studies showed an increase of the type I procollagen mRNA expression mainly in portal tracts and septa. Ex vivo liver perfusion induces: 1) an early activation of HSC which acquire the expression of alpha-SM actin, and 2) significant changes in the perisinusoidal extracellular matrix. These results are compatible with the view that HSC function as liver specific pericytes participating in the regulation of sinusoidal blood pressure.     

Removal of cellular debris formed in the Disse space in patients with cholestasis

Summary Using electron microscopy, we investigated how cellular debris, formed in the Disse space during cholestasis, was cleared. Ten patients with cholestasis of varied origin and severity were studied and compared with 10 controls without liver disease. In cholestatic patients, sinusoidal cells contained variable amounts of amylase PAS-positive material. In clean perfusion-fixed sinusoids the endothelial cells often appeared swollen and active, with few fenestrations. Hepatocyte blebs and cellular debris were sometimes seen in the Disse space. Two mechanisms were apparently involved in the clearing process: phagocytosis by macrophages either infiltrated into the Disse space, or forming the barrier; and the passage of debris from the Disse space into the sinusoidal lumen through the endothelial wall. Debris was either forced through enlarged pores or through the wall, with a progressive invagination followed by an outpouching in the lumen. The force, possibly provided by endothelial massage, may not be sufficient to push out cellular debris from the Disse space; morphological data seemed to indicate that endothelial damage may be a necessary factor. Debris present in the lumen was phagocytized by numerous active macrophages. Cellular debris was not observed in the Disse space of control patients.This work was supported by a grant from the CNRS (RCP 724)     

Perisinusoidal fibrosis of the liver in patients with thrombocytopenic purpura

Summary 10 patients with thrombocytopenic purpura (TP) underwent splenectomy. Eight of these patients had idiopathic TP (certain or probable). All had normal liver function tests. Liver histology of the surgical biopsy was normal with the exception of a non specific mild portal infiltration in 6 cases. On Sirius red staining the perisinusoidal network was normal in 3 cases, mildly or moderately increased in 5 cases and often associated with perivenular fibrosis. Collagen types I, III, IV, laminin and fibronectin were increased in the 8 biopsies tested. On semi-thin sections, numerous Kupffer cells were observed. Under the electron microscope, sinusoidal abnormalities were very similar in all 7 patients studied: numerous Kupffer cells containing abundant lysosomes, numerous collagen bundles in the Disse space, active endothelial cells, transformation of some perisinusoidal cells into cells with some of the characteristics of fibroblasts (increased RER) and myofibroblasts (peripheral condensations of the filamentous network), increased fragments of basement membrane-like material. In two cases there was an increase in the number of perisinusoidal cells loaded with lipids. The similarity of the lesions and the absence of other fibrogenic causes (except in 2 cases) suggest that TP may represent another group of diseases with perisinusoidal fibrosis. The aetiology of fibrosis remains unknown but platelet derived growth factor and activated macrophages may play a major role.     

Morphologic features in the regenerating liver—a comparative intravital,lightmicroscopical and ultrastructural analysis with focus on hepatic stellate cells

Different cell types play a role in the liver regeneration. The present study reveals morphological key steps of liver regeneration by correlating intravital, light, and electron microscopic with immunohistochemistry results focusing on hepatic stellate cells (HSCs). In Lewis rats, liver regeneration was induced by a 2/3-hepatectomy. Animals (n = 7 each) were killed after 0, 1, 2, 3, 4, 7, and 14 days. Morphological features were investigated by light microscopy, immunohistochemistry [α-smooth muscle actin (α-SMA), Desmin, vascular endothelial growth factor (VEGF)/VEGF receptor, Ki-67, ssDNA], intravital microscopy (sinusoid density, number of hepatocytes, and HSC), and electron microscopy focussed on cell-to-cell interactions. During liver regeneration, HSC were activated at day 3 showing a loss of autofluorescence and simultaneously an increased α-SMA expression and direct cell contact to hepatocytes. HSC activation was followed by increasing VEGF expression and sinusoid density. After 14 days, liver architecture and ultrastructure was restored and HSCs were deactivated showing decreased α-SMA expression as well as increased apoptosis and no more direct cell contact to hepatocytes. HSCs play a central role in the regenerating liver by governing angiogenesis and extracellular matrix remodeling. A direct cell contact to hepatocytes seems to be essential for HSC activation, whereas deactivation is accompanied by loosening of hepatocyte contact and increased apoptosis.     

Transforming growth factor-β induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway

Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis.

Transforming growth factor (TGF)-β, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75^NTR expression was weak in prPSC. In contrast to ihPSC TGF-β activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1.

We conclude that under conditions of upregulated TGF-β, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.     

Spongiosis hepatis: chemical induction, pathogenesis, and possible neoplastic fate in a teleost fish model.

Spongiosis hepatis (SH), first reported as a distinct lesion associated with certain forms of hepatic neoplasia in rats, has also been induced with chemicals, in a predictable fashion, in small teleost fishes being studied as carcinogenesis research models. The sheepshead minnow (Cyprinodon variegatus), exposed to N-nitrosodiethylamine (DENA) in sea water, provided the model for this study. The fish developed SH and presented a spectrum of developmental or progressive stages of the lesion over a 140 week holding period following a 6 week exposure to / 57 mg/L DENA. The origin of SH in the fish model is homologous to that in the rat model, both species having the perisinusoidal cell (stellate cells of Ito) in the space of Disse as the cell of origin. Light (LM) and electron microscopy (EM) studies characterized the different pathogenetic stages of SH in liver of the sheepshead minnow and revealed a possible late transition of SH to putative polymorphic cell neoplasms. The possible preneoplastic or neoplastic nature of SH from its time of origin in chemically exposed fish to time of appearance of associated presumptive neoplasms is discussed. SH may be a bioindicator of exposure to certain chemicals in some vertebrate species, from fishes to mammals.     

An undifferentiated embryonal sarcoma of the liver containing adipophilin-positive vesicles in an adult with massive sinusoidal invasion

Undifferentiated embryonal sarcoma of the liver (UESL) is a malignant mesenchymal tumor that occurs typically in children and rarely in adults. Here we describe a case of UESL in a 51-year-old woman who presented with a cystic lesion in the liver. Because it grew slowly, the anterior segment of the liver was resected to check the lesion. Histologically, the lesion looked like a telangiectatic hepatic adenoma. Two years after resection, the tumor recurred, and she died 3 years later due to liver failure. The autopsy revealed that these lesions were UESL with massive sinusoidal invasion, and a review of the case indicated the primary lesion was also UESL. We also confirmed these tumor cells by staining with CD56, alpha-smooth muscle actin (SMA), and adipophilin, suggesting that they have a character similar to that of stellate cells in the space of Disse. The histological result of our patient revealed atypical UESL. Therefore, UESL should be considered when a hepatic lesion with degeneration is seen, even in an adult. In addition, the immunohistochemical appearance of this case implies that UESL is perhaps derived from stellate cells or stellate cells with myofibroblast differentiation in the space of Disse.     

Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.

It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs.