Codistribution of Collagen Type IV and Laminin in Liver Fibrosis of Elderly Cadavers: Immunohistochemical Marker of Perisinusoidal Basement Membrane Formation

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane. Formation of perisinusoidal basement membranes beneath the endothelium is an integral feature of capillarization of sinusoids that is a significant pathology found in advanced fibrosis. Liver fibrosis is prevalent in elderly cadavers; however, basement membrane formation in these liver samples has yet to be studied. Collagen type IV and laminin are major basement membrane proteins and their codistribution around sinusoids provides an immunohistochemical marker of basement membrane formation. Here, we examined the intralobular sites of perisinusoidal basement membrane formation in elderly cadaveric livers having various stages of fibrosis. Collagen IV and laminin codistributed in basement membranes of portal and septal ductular and vascular structures, providing a positive control. In the parenchyma, collagen IV immunostaining of sinusoids was panlobular in all stages of fibrosis, and the stain was continuous along the sinusoids. In contrast, laminin was not detected in livers, showing minimal fibrotic change. It was rarely seen in perisinusoidal/pericellular fibrosis, but frequently in septa formation, bridging fibrosis, and cirrhosis. The laminin stain was patchy, occurring principally in sinusoids of periportal and periseptal areas, less commonly in mid‐lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin codistributed with collagen IV in these sinusoidal locations, thus marking the sites of perisinusoidal basement membrane formation in aged fibrotic livers. This development is presumably related to aging of the liver and exacerbated by liver injury caused by advanced liver fibrosis, possibly resulting in sinusoidal capillarization. Anat Rec, 296:953–964, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Liver perisinusoidal fibrosis in BB rats with or without overt diabetes.

Perisinusoidal fibrosis is a vascular lesion observed in the liver of type I diabetic patients. To investigate whether this liver lesion is secondary to hyperglycemia or whether it represents a separate collagen vascular disorder, the authors studied the structure of liver sinusoids in genetically susceptible BB rats in which a spontaneous diabetes develops similar to human type I diabetes. Seven diabetic insulin-treated BB rats, 7 nondiabetic BB rats, and 6 control non-BB rats were studied. Histologic abnormalities of the collagen network were detected on trichrome-stained sections. Perisinusoidal collagen fibers were quantified ultrastructurally by the point-counting method. All control non-BB rats had normal livers; 86% of the diabetic as well as 71% of the nondiabetic BB rats displayed localized sinusoidal thickening corresponding ultrastructurally to perisinusoidal fibrosis; in these abnormal rats the percentage of collagen fibers per sinusoid unit was significantly higher than that in controls. Fibrous septa (2 diabetic and 5 nondiabetic BB rats) and liver nodulation (3 diabetic and 1 nondiabetic BB rats) were also observed. Perisinusoidal fibrosis is a frequent liver vascular abnormality in a strain of rats genetically predisposed to the development of type I diabetes. The lesion is independent of the presence of diabetes. These observations suggest that liver perisinusoidal fibrosis in patients with type I diabetes might be linked to a genetic abnormality rather than to hyperglycemia per se.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, III AND IV (BASEMENT MEMBRANE) COLLAGENS IN THE LIVER

The tissue distribution of type I, III and IV (basement membrane) collagens in normal human and bovine livers was examined by indirect immunofluorescence microscopy, by using type-specific rabbit antibodies to individual types of bovine collagen. Type I and III collagens were found to distribute in the interstitium of portal tracts as thick fiber bundles and in perisinusoidal spaces as thin fibers like reticulin fibers, both in human and bovine livers. No significant distribution differences of type I and III collagens in the livers was observed under the experimental conditions employed, indicating that both collagens are Involved in in vivo collagen fibrillogenesis in the tissue, regardless of the sizes of collagen fibers, as in the skin (Conn. Tiss. Res. 7: 157–163, 1980). Type IV collagen, when examined with bovine liver, was located in hepatic arteries, portal veins and bile ducts of portal tracts, and was also distributed in the perisinusoidal spaces In a linear fasion.     

Portal and parenchymal alterations of the liver in idiopathic portal hypertension: a histological and immunochemical study

Idiopathic portal hypertension (IPH) is characterized by presinusoidal portal hypertension owing to the intrahepatic, presinusoidal portal venous block, whereas the primary cause and initial vascular lesions(s) remain only speculative. In this study, a total of 97 IPH livers were histopathologically and immunohistochemically examined, placing emphasis on hepatic parenchymal fibrosis and atrophy as well as on portal tract fibrosis. Alcoholic cirrhosis and normal livers were used as controls. When compared with normal livers, the expression of connective tissue growth factor (CTGF) in periductal mononuclear cells was significant. Matrix metalloproteinase (MMP)9-positive mononuclear cells were fewer in number in the portal tract of IPH liver, when compared with alcoholic cirrhosis. These findings suggest a possible pathogenesis of collagen and elastin deposition because of increased CTGF expression and decreased MMP-9 expression in portal tracts of IPH. Sinusoidal dilatation associated with hepatocellular atrophy and apoptosis occurred frequently, but focally in 20% of the IPH cases. These changes were most often found in hyperplastic hepatocellular areas and in the perivenular areas of hepatic lobules. In these areas, pericellular fibrosis and thin fibrous septa were also frequently seen. In these fibrotic areas, there were deposited not only collagen fibers, but also elastic fibers, in which alpha-smooth muscle actin-positive sinusoidal cells, reflecting activated hepatic stellate cells, were frequently detected. It is possible that in IPH cases, continuing portal venous blood insufficiency may be responsible for hepatic parenchymal damage, which may be followed by hepatocellular apoptotic dropout and then by hepatic parenchymal atrophy and fibrosis.     

DISTRIBUTION OF COLLAGEN TYPES I, III, AND V IN FIBROTIC AND NEOPLASTIC HUMAN LIVER

The distribution of collagen types I, III, and V in normal and fibrotic human livers and hepatocellular carcinoma was studied by indirect immunofluorescence procedure using type specific antibodies. Type I collagen as well as type III collagen was present in normal liver within the portal tracts and along the perisinusoidal spaces. Basement membrane collagen, type V collagen, was demonstrated only around the bile ducts and vessels of the portal tracts and central veins. In fibrotic liver, both type I and III collagens were found in increased amounts in fibrotic areas. In fibrous septa of active cirrhosis, however, type I collagen as well as type III collagen was abundant, whereas in inactive cirrhosis type I fibers were predominant. Type V collagen was observed in the walls of proliferative bile ductules and vessels in the fibrotic liver, and also along the sinusoids in the periportal areas. In hepatocellular carcinoma, each type of collagen was distributed regularly along the sinusoid-like vascular channels within the tumor.     

Nonparenchymal liver cells and granulomas during lamprey biliary atresia

Transmission (thin sections and freeze-fracture replicas) and scanning electron microscopy were used to describe the nonparenchymal liver cells during the seven (1–7) stages of metamorphosis in the sea lamprey, Petromyzon marinus L., when bile ducts and canaliculi degenerate. The biliary atresia is accompanied by an increased diameter of fenestrae in the endothelium, an active phagocytosis by Kupffer cells in the sinusoids, and large lipid inclusions in perisinusoidal lipocytes (fat-storing or Ito cells). Plasma-like cells and foci of nonparenchymal cells (granulomas) are present in the liver interstitium during at least four stages of metamorphosis. The fenestrae in the sinusoidal wall are wider (up to 2.8-μm diameter) than normally reported for vertebrate livers but are likely a reflection of the morphogenetic and physiological events and consequences of the biliary atresia. Kupffer cells are involved in an extensive erythrophagocytosis, the storage of iron, and perhaps the incorporation of cellular components from hepatocytes. Lipocytes are the vitamin A-storing cells of the transforming liver and may be responsible for some perisinusoidal fibrosis. Granulomas are present during stages 3–6 and are focal areas where mononuclear leukocytes (lymphocytes and plasmalike cells), macrophages, and neutrophils have infiltrated the hepatic parenchyma. The function of the granulomas is not known; but their presence may be related to the porous nature of the sinusoidal wall, the tissue degeneration, and/or the physiological change (e.g, bile stasis) during biliary atresia.     

Increased Serum Type IV Collagen Peptide in Carbon Tetrachloride-treated Rats

We developed a competitive enzyme immunoassay for serum type IV collagen peptide as a marker of fibrogenesis, and examined the relationship between serum type IV collagen peptide and hepatic disorder in CCI, treated rats. The rats were treated for 8 weeks and signs of liver damage began to appear from about week 2. With the progression of these signs to liver fibrosis, type IV collagen increased in the fibrous septa and especially in the perisinusoidal walls, where the increase was manifested as development of a real basement membrane beneath the sinusoidal endothelial cells. In CCI₄-treated rats, serum type IV collagen peptide significantly increased with the progression of liver fibrosis. When CCI₄ administration was stopped, the collagen peptide rapidly decreased without any rebound rise. An intimate relationship was found between the production of serum type IV collagen peptide and liver prolyl hydroxylase activity and the amount of collagen deposited in the liver. These results suggest that serum type IV collagen peptide will be a uset ul biochemical marker for the early detection of fibrogenesis in the liver.     

An immunohistochemical study of tumour vessels in metastatic liver cancers and the surrounding liver tissue

To elucidate the phenotype of the blood vessels and the expression of the growth factors involved in angiogenesis in metastatic liver cancers, we carried out an immunohistochemical study of 57 surgically resected livers with metastatic cancer. Blood vessels in the metastatic liver cancers frequently expressed von Willebrand factor (vWF), Ulex europaeus agglutinin I (UEA I)-binding sites, α-smooth muscle actin (α-SMA), type IV collagen and laminin. Sinusoidal endothelial cells around the metastatic liver cancers were positive for vWF in 33.3% of the specimens examined and for UEA I in 28.1%. α-SMA-positive perisinusoidal cells accumulated in the vicinity of the metastatic liver cancers in 68.4% of the specimens. Type IV collagen was detected in the perisinusoidal space close to the metastatic cancers as well as distant from them (91.2%). Laminin was detected in the perisinusoidal space in only one specimen (1.8%). Tumour cells of the metastatic liver cancers were positive for vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and acidic fibroblast growth factor (aFGF) in 78.9%, 38.4% and 7.0% of the specimens, respectively. Hepatocytes close to the metastatic liver cancers expressed bFGF more strongly than those distant from the metastatic liver cancers, and their expression of bFGF was more intense than that in the tumour cells. These results suggest that: (1) tumour vessels in metastatic liver cancers consist of endothelium, basement membrane and pericytes, (2) the sinusoids adjacent to tumours undergo capillarization, and (3) vascular endothelial growth factor may contribute to angiogenesis in metastatic liver cancer. Basic fibroblast growth factor may be responsible for the sinusoidal capillarization and the peritumoral fibrosis.     

Ultrastructure of the hepatic perisinusoidal cells in man and other mammalian species.

Perisinusoidal (P.S.) cells occurring in the spaces of Disse in the livers of normal cats, dogs, miniature pigs, albino rats, human adults and children were examined by electron microscopy. The ultrastructural details of the P.S. cells and their topographic relationships with hepatocytes, sinusoidal lining cells and reticulum fibers are described. Species differences between P.S. cells were primarily a dissimilarity in lipid content: the main ultrastructural features were the same in all species studied. The P.S. cells of the rat liver displayed only low endocytotic activity, and no phagosome formation following intravenous administration of horseradish peroxidase. The close topographic relationship of the P.S. cells with the intralobular reticulum fibers was reminiscent of the intimate connection between fibroblasts and collagen fibers, or, in cat liver, of the reticulum cell--reticulum fiber association seen in lymphoid organs. Fibroblasts were not found inside the hepatic lobules. These findings support the conclusion that the reticulum fibers of hepatic lobules are produced by perisinusoidal cells which, however, display also other functions.     

Increased serum type IV collagen peptide in carbon tetrachloride-treated rats

We developed a competitive enzyme-immunoassay for serum type IV collagen peptide as a marker of fibrogenesis, and examined the relationship between serum type IV collagen peptide and hepatic disorder in CCl4-treated rats. The rats were treated for 8 weeks and signs of liver damage began to appear from about week 2. With the progression of these signs to liver fibrosis, type IV collagen increased in the fibrous septa and especially in the perisinusoidal walls, where the increase was manifested as development of a real basement membrane beneath the sinusoidal endothelial cells. In CCl4-treated rats, serum type IV collagen peptide significantly increased with the progression of liver fibrosis. When CCl4 administration was stopped, the collagen peptide rapidly decreased without any rebound rise. An intimate relationship was found between the production of serum type IV collagen peptide and liver prolyl hydroxylase activity and the amount of collagen deposited in the liver. These results suggest that serum type IV collagen peptide will be a useful biochemical marker for the early detection of fibrogenesis in the liver.     

Ultrastructure of the hepatic perisinusoidal cells in man and other mammalian species

Perisinusoidal (P.S.) cells occurring in the spaces of Disse in the livers of normal cats, dogs, miniature pigs, albino rats, human adults and children were examined by electron microscopy. The ultrastructural details of the P.S. cells and their topographic relationships with hepatocytes, sinusoidal lining cells and reticulum fibers are described. Species differences between P.S. cells were primarily a dissimilarity in lipid content: the main ultrastructural features were the same in all species studied. The P.S. cells of the rat liver displayed only low endocytotic activity, and no phagosome formation following intravenous administration of horseradish peroxidase. The close topographic relationship of the P.S. cells with the intralobular reticulum fibers was reminiscent of the intimate connection between fibroblasts and collagen fibers, or, in cat liver, of the reticulum cell - reticulum fiber association seen in lymphoid organs. Fibroblasts were not found inside the hepatic lobules. These findings support the conclusion that, the reticulum fibers of hepatic lobules are produced by perisinusoidal cells which, however, display also other functions.     

Peliosis-like ultrastructural changes of the hepatic sinusoids in human chronic hypervitaminosis A: Report of three cases

In addition to hypertrophy of Ito cells and perisinusoidal fibrosis, previously unrecognized ultrastructural abnormalities of the hepatic sinusoids were observed in three patients with chronic hypervitaminosis A: 1) large areas of communication between the sinusoidal lumina and the perisinusoidal spaces, allowing extravasation of blood cells; 2) marked dilation of the perisinusoidal spaces; and 3) swelling and clarification of endothelial cells. Most of these changes, along with some other sinusoidal barrier alterations previously reported in chronic hypervitaminosis A (i.e., bleb formation on the sinusoidal membrane of the hepatocytes and the presence of multiple cellular layers lining the sinusoids), are strikingly similar to those observed in peliosis hepatis. The present findings suggest that sinusoidal barrier abnormalities might constitute a major event in the pathophysiology of vitamin A-induced liver injury as well as of peliosis hepatis.     

Immunocytochemical localization of type B collagen: a component of basement membrane in human liver

An indirect immunofluorescent method for localizing specific types of collagen was applied to unfixed cryostat sections of autopsy and biopsy specimens of normal and fibrotic human livers. Monospecific antibodies to Types I, III, and B collagens were raised in goats by injecting collagens extracted from normal and fibrotic human livers. The antibody against B collagen stained the delicate sinusoidal meshwork within the lobule that closely paralleled in distribution the staining of reticulin fibers as seen in classical silver preparations. Antibody to Type III collagen stained at the sinusoidal aspect of the hepatocytes in a pattern similar to anti-B antibody. All three antibodies tested strongly stained the portal tracts and the fibrotic bands in specimens with cirrhosis. These observations indicate that reticulin fibers within the liver lobule contain both Type III and Type B collagen.     

Ultrastructure of initial stages of perivenular fibrosis in alcohol-fed baboons.

Perivenular fibrosis was studied in baboons pair-fed with alcohol containing or isocaloric control diets for up to 8.5 years, and biopsies were performed on the animals one to two times a year. The number and types of mesenchymal cells surrounding the terminal hepatic venules were examined at various stages of thickening of the rim of the terminal hepatic venules by light and electron microscopy. The number of mesenchymal cells increased with progression of fibrosis and showed good correlation with the thickness of the rim (r = 0.7435, P less than 0.001, n = 56). myofibroblasts were the most common mesenchymal cells. They were present around the terminal hepatic venules in the control animals and proliferated after alcohol. This was associated with increased deposition of collagen fibers around the terminal hepatic venules. This fibrotic process extended into the perisinusoidal space of the centrolobular areas, sometimes connecting with pericellular fibrosis and/or fat granuloma, which developed in the lobule. Thus, in baboons fed alcohol, perivenular fibrosis is associated with myofibroblast proliferation followed by collagen deposition. Myofibroblast proliferation appears to represent the earliest detectable precursor lesion leading to hepatic fibrosis in alcoholic liver injury.     

Expression of alpha-smooth muscle actin in liver diseases.

To evaluate the distribution of alpha-smooth muscle actin (alpha-SMA) positive cells in various liver diseases, we undertook an immunohistochemical study of liver diseases including chronic persistent hepatitis, chronic active hepatitis, liver cirrhosis, intrahepatic cholelithiasis and hepatocellular carcinoma. As a control, fetal livers (gestational age: 22-26 weeks) showed alpha-SMA positive cells along the blood vessels of the portal area, terminal hepatic venules and at perisinusoidal spaces. Perisinusoidal alpha-SMA positive cells were bipolar shaped and had round nuclei. In chronic persistent hepatitis, a few alpha-SMA positive cells were admixed with the inflammatory infiltrates mostly along the intact limiting plate. They were also detected multifocally in a linear pattern along the dilated sinusoid. In chronic active hepatitis, very strong alpha-SMA staining was detected at the site of piecemeal necrosis and adjacent lobules. A-SMA expression was decreased in some cases after interferon treatment. In cases of transplanted liver biopsies, expression of intralobular alpha-SMA was diffusely increased but showed no correlation with degree of acute rejection. Cirrhotic livers revealed strong alpha-SMA positivity in fibrous septae as well as in the perisinusoidal space of intact hepatocytes at the leading edge of fibrosis. Interlobular bile ducts were concentrically circumscribed by alpha-SMA positive cells in cases of intrahepatic cholelithiasis. In trabecular type hepatocellular carcinomas, most sinusoidal lining cells were positive for alpha-SMA. Most intralobular alpha-SMA positive cells represent, if not all, perisinusoidal cells (PSCs) which are involved in intralobular fibrogenesis in various liver diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural Changes in Livers of Two Patients with Hypervitaminosis A

The principal distinctive ultrastructural changes observed in the livers of 2 patients with chronic hypervitaminosis A were perisinusoidal fibrosis and massive accumulation of lipid-storing cells (Ito cells). The fibrosis consisted of a network of basement membranes with numerous bundles of collagen and reticulum fibrils. This network contained numerous Ito cells, and moderate numbers of lymphocytes, macrophoges and other mesenchymal cells. Impairment of blood flow by perisinusoidal fibrosis probably resulted in the secondary alterations in hepatocytes which included cellular atrophy and formation of cytoplasmic bullae.     

Morphogenesis of intralobular liver fibrosis in obstructive jaundice patients

The results of ultrastructural study of the intralobular liver fibrosis in 38 patients with obstructive jaundice of various duration are presented. It is established that the appearance of fibroblast-like sinusoid cells plays a leading role in the development of intralobular liver fibrosis. The morphologic characteristics of poorly differentiated and differentiated cells of this type and their involvement in collagen fiber synthesis within liver lobules are given. It is shown that in obstructive jaundice a significant disturbance of the sinusoidal wall takes place due to the formation of collagen fibers, basal membrane that results in the worsening of the exchange between the blood and hepatocytes.     

Abnormal elastic system fibers in fibrotic human liver

The network of elastic system fibers in human fibrotic liver was investigated by histological methods, immunohistochemical staining, and electron microscopy. Type III collagen was seen not only in regions of portal fibrosis but also in the sinusoidal wall. However, elastic system fibers were not found in the Disse space of the sinusoidal wall. Elastic system fibers including oxytalan, elaunin, and elastic fibers were found successively in the course of elastogenesis. A few normal oxytalan fibers and abnormal oxytalan fibers were observed in the periportal tracts. Few normal elaunin and abnormal elaunin fibers were observed in regions of portal fibrosis but not in the surrounding margin. Elastic fibers, only in scarce amounts, were observed around the portal veins in the case of chronic active hepatitis but not in acute hepatitis. Abnormal oxytalan fibers were seen as a bundle of wavelike microfibrils and had an irregular arrangement. Abnormal elaunin fibers were not associated with bundles of microfibrils. Abnormal elaunin fibers in large amounts were found interspersed with spiraled collagen, which most likely indicates that the oxytalan fibers degenerated in the course of elastogenesis. Thus, in a fibrotic liver it is possible that synthesis of normal elaunin and elastic fibers does not occur or that the quantity of such fibers synthesized may be small because of the effect of the degenerated oxytalan fibers. As a characteristic of liver fibrosis, the composition of abnormal elastic system fibers and spiraled collagen differs from that in other fibrotic organs.     

Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver

To identify Ito cells in normal and pathological adult human livers, immunohistochemical studies were performed by the avidin-biotin-peroxidase complex method using monoclonal antibodies for -smooth muscle actin (ASMA), desmin, and vimentin. Fifty one needle biopsies, 7 surgically resected specimens, and 5 autopsy specimens were studied. In the normal adult liver vascular smooth muscle cells and pericytes, together with perisinusoidal cells with thin cytoplasmic processes were positive for ASMA. These latter cells formed a loose and discontinuous layer along the sinusoidal walls. Immunoelectron microscopy showed that the ASMA-positive perisinusoidal cells were Ito cells containing fat droplets. The other sinusoidal lining cells were negative for ASMA. In chronic liver disease, ASMA-positive Ito cells showed an increase in number, size, and the intensity of immunostaining in areas of piecemeal necrosis), and formed a continuous cellular network. These cells were dendritic in shape with irregularly elongated cytoplasmic processes and contained an increased amount of microfilaments, in association with loss of the characteristic fat droplets. Thus, their ultrastructural features corresponded to those of myofibroblastic cells. Ito cells showed no staining for desmin in both normal and pathological livers. These results indicate that immunohistochemistry using an anti-ASMA antibody is a sensitive and reliable method for the identification of both normal and transformed Ito cells in adult human livers.