Perisinusoidal fibrosis and basement membrane-like material in the livers of diabetic patients.

A direct correlation exists between collagenization of Disse's space and the presence of diabetic microangiopathy in type I diabetes. To confirm and extend this finding, we studied four liver biopsy samples from two patients with type I diabetes (one with retinopathy) and two patients with type II diabetes (no retinopathy). All had normal or subnormal results on liver function tests and normal liver architecture. Levels of collagen types I, III, and IV, laminin, and fibronectin, as determined by immunocytochemical techniques, appeared increased in all patients. Liver biopsy samples were perfusion fixed for electron microscopy of sinusoids and sinusoidal cells. Numerous and thick collagen bundles could be seen in Disse's space, as could the increase of basement membrane-like material underlying the endothelial cells, perisinusoidal cells, and sinusoidal membrane of hepatocytes. Perisinusoidal cells were active and had abundant rough endoplasmic reticula and thick processes. This preliminary study indicates that collagenization of Disse's space is not specific to a certain type of diabetes. The increase of basement membrane-like material raises the question of whether liver sinusoids are truly different from other capillaries as far as diabetic microangiopathy is concerned.

Hepatocellular carcinoma developed on noncirrhotic livers. Sinusoids in hepatocellular carcinoma.

In hepatocellular carcinoma, there is modification of cell-to-cell and cell-to-extracellular matrix interactions. Two cases of well-differentiated hepatocellular carcinoma that developed in noncirrhotic livers were explored by light and electron microscopy on perfusion-fixed liver biopsy specimens. In addition, immunocytolocalization of collagen types I, III, and IV, laminin, and fibronectin was assessed. The main features included the following: absence of collagen staining inside the tumor with Sirius red; decrease of collagen types I and III and the increase of collagen type IV, laminin, and fibronectin; widening of Disse's spaces containing numerous, discontinuous, and thick fragments of basement membrane-like material piled up beneath endothelial cells and around perisinusoidal cells; transformation of perisinusoidal cells into cells with the characteristics of fibroblasts and myofibroblasts; decreased numbers of fenestrae for endothelial cells with processes often overlapping and attached with tight junctions; and rarefaction of Kupffer's cells. Expression of cellular and extracellular material abnormalities are related to the tumor differentiation. The study of these abnormalities may have implications in prognosis.

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.

Congenital portacaval shunt in rats: liver adaptation to lack of portal vein--a light and electron microscopic study.

In five rats with congenital portacaval shunt, liver atrophy, hyperplastic foci in the periportal zone, atrophic hepatocytes in the centrolobular zone, well-preserved hepatocyte ultrastructure with abundant rough endoplasmic reticulum, packed mitochondria and numerous peroxisomes were observed as in surgical portacaval shunt. However, portal triads were abnormal in contrast to surgical shunt. In large portal triads, hepatic arteries were prominent, bile ducts numerous and portal veins were lacking. Instead, small or large capillaries were seen in the portal tracts usually at the periphery. These capillaries appeared to be in continuity with nearby sinusoids presenting the ultrastructural characteristics of capillaries. These observations suggest that absence of the portal vein is compensated by formation of neocapillaries. It is assumed that these capillaries originate from periportal sinusoids and are necessary to distribute blood to all sinusoids and form a reservoir to lower arterial pressure.

Hypertrophy of biliary epithelium in rats with portacaval shunt.

Bile ducts of rats with 3-month-old portacaval shunt were shown by light microscopy to present hypertrophy of the biliary epithelium. This hypertrophy could be linked to the increased hepatic arterial flow following portacaval shunt.

Extracellular matrix composition and integrin expression in early hepatocarcinogenesis in human cirrhotic liver.

Extracellular matrix (ECM) plays a major role in cell differentiation, proliferation, and gene expression, both in physiological and in pathological conditions. Immunohistochemistry has been used to investigate modifications of ECM and related receptors, the integrins, in 26 small nodular lesions developed in human cirrhotic livers, on the basis that these lesions could represent sequential steps of hepatocarcinogenesis: the lesions were 16 macroregenerative nodules (MRNs), either of ordinary (n = 5) or atypical (n = 11) type, and ten small (< 15 mm) hepatocellular carcinomas (HCCs). Data were compared with those obtained in the surrounding cirrhotic tissue, in large HCCs, and in normal liver. The results indicate similarities between ordinary MRNs and cirrhosis, on the one hand, and between atypical MRNs and small HCCs, on the other. Strong and homogeneous deposition of collagen type IV and laminin in sinusoids and overexpression of alpha 6 integrin by sinusoidal cells and hepatocytes were especially noticeable in dysplastic areas characteristic of atypical MRNs, as in small HCCs. In addition, the staining of alpha 2 and alpha 6 integrins in MRNs revealed the presence of widespread atypical ductular proliferation expanding from periportal and perinodular areas, containing epithelial cells with transitional (hepato-biliary) phenotype. These findings suggest a transition from atypical MRNs to small HCCs and a possible role for liver epithelial precursor cells ('stem cells') in the development and evolution of MRNs.

Transformation of sinusoids into capillaries in a rat model of selenium-induced nodular regenerative hyperplasia: an immunolight and immunoelectron microscopic study.

The oral administration of selenium (Se) to young rats induces, over a 2-month period, the formation of nodular regenerative hyperplasia with sinusoidal damage around nodules. Perinodular areas located in zone 1 comprise atrophic hepatocytes and capillarized sinusoids without fibrosis. We used this unique model of capillarization without fibrosis to investigate the temporal relationship between the process of capillarization and changes occurring in the deposition of components of the extracellular matrix. After 2 weeks of intoxication, type III collagen and fibronectin were stable, but laminin and type IV collagen had increased in zone 1, resulting in the formation of septae between portal tracts. Even at 8 weeks, these two components still formed the principal deposits in perinodular zones. Electron microscopy showed already at 1 week in zone 1 that part of the endothelial wall had detached from hepatocytes. Sinusoidal endothelial cells progressively acquired certain of the characteristics of a vascular endothelium, some proliferated, and perisinusoidal cells transformed into myofibroblasts, surrounded by deposits of laminin and type IV collagen. These results indicate that both laminin and type IV collagen are involved in capillarization without fibrosis and in angiogenesis; fibronectin would not seem to play a role.

Expression of hepatocyte growth factor in human hepatocellular carcinoma.

BACKGROUND/AIMS: We have shown that hepatocyte growth factor, secreted by human liver myofibroblasts, promoted in vitro invasion of human hepatocellular carcinoma cell lines. The aim of this work was to measure hepatocyte growth factor expression in 29 human hepatocellular carcinomas and the corresponding peri-tumoral livers. METHODS: We used reverse transcription-polymerase chain reaction, in situ hybridization, ELISA and Western blot. RESULTS: Sixty-two of tested hepatocellular carcinomas were positive by reverse transcription-polymerase chain reaction. With in situ hybridization, a signal was found in every sample. In many cases, the signal was localized in cells labeled with an anti-smooth muscle alpka-actin antibody, while hepatocytes were mostly non-labeled. ELISA, performed in 15 pairs of hepatocellular carcinomas and surrounding livers, detected hepatocyte growth factor in every sample with wide variations. Hepatocellular carcinomas that had developed in non-cirrhotic livers contained essentially the same amount of hepatocyte growth factor as the matching non-tumoral liver. In cirrhotic livers, the hepatocyte growth factor content of the tumors was significantly lower than that of the surrounding cirrhotic livers. CONCLUSIONS: These data indicate that hepatocyte growth factor is expressed at significant levels in every hepatocellular carcinoma tested and that its expression takes place in the stromal myofibroblasts.

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

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.

Perisinusoidal fibrosis of the liver in patients with thrombocytopenic purpura.

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.

Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study.

BACKGROUND/AIMS: Marked changes in extracellular matrix occur in the stroma of hepatocellular carcinoma, as compared to normal or cirrhotic liver. The cell types responsible for extracellular matrix synthesis within hepatocellular carcinoma have not been clearly identified. METHODS: In vivo collagen synthesis was studied by in situ hybridization and immunohistochemistry for types I, IV, V and VI collagen, together with immunolabeling of alpha-smooth muscle actin, a myofibroblast marker, and CD34, an endothelial cell marker. In vitro, extracellular matrix deposition by cultured myofibroblasts was studied by reticulin staining, immunocytochemistry and RNase protection. RESULTS: All collagens studied were expressed in the stroma of the tumor, with a higher level of type VI and IV collagens than of type I and V. The majority of the cells expressing collagen transcripts in human hepatocellular carcinoma stroma were alpha-actin positive and CD 34 negative. In vitro experiments demonstrated that the hepatocellular carcinoma cell lines HepG2, HuH7 and Hep3B markedly increased extracellular matrix deposition by human liver myofibroblasts. This increase was mediated by a soluble mediator present in tumor cell conditioned medium. It was not explained by an increase in mRNA levels of extracellular matrix components, nor by a decrease in the secretion of matrix-degrading proteinases by myofibroblasts. CONCLUSIONS: Myofibroblasts are the main source of collagens in the stroma of hepatocellular carcinoma. Our data also indicate that tumoral hepatocytes increase extracellular matrix deposition by cultured myofibroblasts, probably by post-transcriptional mechanisms. The generation of hepatocellular carcinoma stroma by myofibroblasts could thus be under control of tumoral cells.

Osteonectin (SPARC) expression in human liver and in cultured human liver myofibroblasts.

Osteonectin/SPARC is a glycoprotein involved in the regulation of cell shape, adhesion, migration, and proliferation. It also has complex effects on extracellular matrix synthesis and turnover. We found that osteonectin mRNA was very abundant in a human liver myofibroblast library. Using Northern and Western blot, immunoprecipitation, and radioimmunoassay, we found that cultured liver myofibroblasts actively secreted osteonectin. Myofibroblasts are very rare in normal liver but proliferate during liver fibrosis where they synthesize extracellular matrix components. Thus, we studied the distribution of osteonectin in normal and fibrotic human liver using in situ hybridization. Osteonectin mRNA expression was weak in normal liver but very high in fibrotic liver within fibrous septae and scattered sinusoidal cells. Serial sectioning and double staining experiments with an antibody to smooth muscle alpha-actin showed that osteonectin transcripts were mostly co-localized with myofibroblasts. In conclusion, osteonectin is highly expressed in human liver myofibroblasts in culture as well as in human liver fibrosis in vivo. The many biological properties of osteonectin make it a candidate effector of human liver fibrogenesis.

Osteonectin/SPARC is overexpressed in human hepatocellular carcinoma.

Osteonectin (ON)/SPARC is a glycoprotein involved in extracellular matrix remodelling. ON expression by myofibroblasts has been reported in fibrotic human liver. As ON also plays a role in cell adhesion, differentiation, and proliferation, this study was designed to document its expression in human hepatocellular carcinoma (HCC). Tissues from 26 HCCs of various histological grades and architecture and from surrounding non-tumour liver (23 cirrhotic or fibrotic, three non-fibrotic) were tested by in situ hybridization and immunohistochemistry. Immunohistochemical detection of alpha-smooth muscle actin (alpha-SMA) was performed on serial sections or in combination with hybridization. Large amounts of ON mRNA and protein were detected in the tumour capsule, in the fibrous bands, and along capillaries within HCCs. The signal was located in cells suggestive of myofibroblasts, as confirmed by positive staining for alpha-SMA. In HCC, ON protein was always detectable, with strong staining in high-grade tumours, whereas it was mostly undetectable in non-tumour tissues. A clear difference was also shown for ON transcripts, except in a few cases with chronic active hepatitis, where ON transcripts were also expressed at a high level. Overexpression of ON transcripts in HCC vs. non-tumour liver was confirmed by RNA blot in 20/22 patients tested. In conclusion, ON is strongly expressed by the stromal myofibroblasts of human HCC, especially of high grade. This expression could play a role in tumour progression.

Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFbeta1) in biliary atresia and paucity of intrahepatic bile ducts during infancy.

BACKGROUND/AIMS: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts. METHODS: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization. RESULTS: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. CONCLUSIONS: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.