Inflammation of bronchial smooth muscle in allergic asthma.

BACKGROUND: Recent observations in asthma suggest that bronchial smooth muscle is infiltrated by inflammatory cells including mast cells. Such an infiltration may contribute to airway remodelling that is partly due to an increase in smooth muscle mass. Whether muscle increase is the result of smooth muscle cell hypertrophy remains controversial and has not been studied by ultrastructural analysis. A morphometric analysis of airway smooth muscle (ASM) was undertaken in asthmatic patients using electron microscopy to examine the interactions between ASM cells and inflammatory cells. METHODS: ASM specimens were obtained from 14 asthmatic subjects and nine non-asthmatic controls undergoing fibreoptic endoscopy. Inflammatory cell counts were assessed by immunohistochemistry, and ultrastructural parameters were measured using electron microscopy in a blinded fashion on smooth muscle cells and inflammatory cells. RESULTS: ASM from asthmatic patients was infiltrated by an increased number of mast cells and lymphocytes. Smooth muscle cells and their basal lamina were thicker in asthmatic patients (9.5 (0.8) and 1.4 (0.2) microm) than in controls (6.7 (0.4) and 0.7 (0.1) microm). In asthmatics the extracellular matrix was frequently organised in large amounts between ASM cells. Myofibroblasts within smooth muscle bundles were only observed in asthmatics, some of them displaying a close contact with ASM cells. CONCLUSION: In asthma, airway myositis is characterised by a direct interaction between ASM cells and mast cells and lymphocytes. Smooth muscle remodelling was present, including cell hypertrophy and abnormal extracellular matrix deposition moulding ASM cells.

A role for thrombin in liver fibrosis.

BACKGROUND/AIM: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. METHODS: Fibrosis was induced in rats by administration of CCl4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin (ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. RESULTS: After three weeks of CCl4, treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p = 0.03) and the expression of TIMP-1 mRNA by 52% (p = 0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl4, treatment with SSR182289 resulted in a significant decrease in fibrosis (-30%, p = 0.04) and ASMA positive areas (-35%, p = 0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl4 as shown by measuring levels of serum aminotransferases and the area of necrosis. CONCLUSIONS: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.

Expression and cellular localization of fibrillin-1 in normal and pathological human liver.

BACKGROUND: The expression and the distribution of fibrillin-1 and elastin were studied in normal and pathological human liver samples. METHODS: As controls, histologically normal/subnormal liver samples (n = 24) were used. Pathological samples corresponded to seven cirrhosis and eight hepatocellular carcinomas (HCC) developed on cirrhotic (four) or noncirrhotic (four) liver. RESULTS: In normal liver, fibrillin-1 and elastin co-localized in vessel walls and portal tract connective tissue. Fibrillin-1 alone was detected along sinusoids and in portal spaces at the interface with the limiting hepatocytic plates and close to the basement membrane of bile ducts. By transmission electron microscopy, typical bundles of microfibrils were detected both in Disse space and in portal zones. Cirrhotic nodules were usually rich in fibrillin-1 along sinusoids; fibrillin-1 and elastin were co-localized in fibrotic septa surrounding nodules. In HCC, fibrillin-1 was present between tumoral hepatocytes; stromal reaction around the tumors contained both fibrillin-1 and elastin. CONCLUSIONS: Fibrillin-1 was associated with elastin in portal mesenchyme and vessel walls of normal liver, in fibrotic septa around cirrhotic nodules and stromal reaction around HCC, but was expressed alone in the perisinusoidal space. The functional roles for fibrillin-1 in non-elastic tissues, such as the liver, remain to be elucidated.

Deactivation of cultured human liver myofibroblasts by trans-resveratrol, a grapevine-derived polyphenol.

Liver myofibroblasts are major actors in the development of liver fibrosis and cancer progression. There is a large interest in drugs that might deactivate these cells. Many studies have shown that the grapevine-derived polyphenol, trans-resveratrol, and other stilbenes have therapeutic potential in some diseases. In this work, we have studied the effect of grapevine polyphenols on cultured human liver myofibroblasts. We have shown that trans-resveratrol profoundly affects myofibroblast phenotype. Trans-resveratrol induced morphological modifications. It markedly reduced proliferation of myofibroblasts in a dose-dependent manner. Trans-resveratrol also decreased the expression of alpha smooth muscle actin (alpha-SMA) without affecting vimentin or beta-cytoplasmic actin expression. It decreased myofibroblast migration in a monolayer wounding assay. We also showed that trans-resveratrol inhibited the messenger RNA (mRNA) expression of type I collagen. Finally, it decreased the secretion of matrix metalloproteinase 2 (MMP-2). We conclude that trans-resveratrol can deactivate human liver myofibroblasts. In the second part of this study, we have shown that neither trans-piceid (a glycosylated analog) nor trans-piceatannol (a hydroxylated analog) reproduces trans-resveratrol effects on liver myofibroblasts. We finally show that, although trans-resveratrol decreases the proliferation of skin fibroblast and vascular smooth muscle cells, it does not affect their expression of alpha-SMA, which indicates some cell specificity.

Expression and cellular localization of the urokinase-type plasminogen activator and its receptor in human hepatocellular carcinoma.

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in tumour invasion. Previous studies have shown by RT-PCR that uPA and uPAR mRNAs are expressed in human hepatocellular carcinoma (HCC). Here, in situ hybridization, immunohistochemistry, and double immunofluorescence were used to identify the cells expressing uPA and uPAR in 26 HCCs. The results indicate that uPA and uPAR were expressed in every case, almost exclusively in stromal cells, mostly myofibroblasts and macrophages, except for rare tumoural hepatocytes expressing cytokeratin 7. These results show the important role of stromal cells of HCC in the pericellular proteolysis which facilitates cancer cell invasion.

Activation of cultured rat hepatic stellate cells by tumoral hepatocytes.

Hepatocellular carcinoma (HCC) is the main type of primary liver cancer, and it develops from hepatocytes. The stroma of HCC is infiltrated by myofibroblasts. In other settings, such as liver fibrosis, myofibroblasts are derived mainly from the activation of hepatic stellate cells (HSC). In this study, we investigated whether tumoral hepatocytes were able to activate HSC. HSC were isolated from normal rats and were plated in dishes coated with Matrigel, to prevent their spontaneous activation. HSC were exposed to conditioned medium (CM) from the rat HCC lines Fao and H5. Tumor cell CM elicited major morphologic changes, such as spreading and generation of cytoplasmic processes. Fao and H5 CM increased HSC proliferation to 1.60 and 1.76 times control values, respectively. The expression of alpha-smooth muscle actin was low or undetectable in control cells and was markedly increased by both tumor cell CM but not by normal rat hepatocyte CM. Desmin expression was also enhanced. Gelatinase A secretion was significantly increased 1.20-fold by Fao CM and 1.55-fold by H5 CM. Expression of beta-type platelet-derived growth factor receptor mRNA was increased 5.8-fold by H5 CM but was decreased to 13% of control levels by Fao CM. HSC activation by tumor cell CM was not prevented by urokinase or matrix metalloproteinase inhibitors, suggesting that Matrigel degradation was not central to the activation process. Finally, a blocking antibody to transforming growth factor-beta1 did not impede Fao CM-induced activation but significantly blocked the increase in matrix metalloproteinase-2 expression induced by H5 CM. Our results show that tumoral rat hepatocyte CM is able to induce the activation of rat HSC in culture. The lack of induction of beta-type platelet-derived growth factor receptor mRNA by Fao CM indicates that, in some cases, tumor-induced activation differs from classic fibrosis-type activation. Our data thus suggest that HSC recruitment and activation in HCC could be under the control of tumor cells.

Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase.

BACKGROUND/AIMS: We have shown that hepatocyte growth factor secreted by human hepatic myofibroblasts increased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. METHODS: Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay. Cell invasion was assayed on Matrigel-coated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-beta chain-specific antibody. RESULTS: HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. CONCLUSION: Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.

Aspirin and heparin prevent hepatic blood stasis and thrombosis induced by the toxic glycoprotein Bolesatine in mice.

Bolesatine is a toxic glycoprotein isolated from Boletus satanas Lenz, which inhibits protein synthesis in vivo and in vitro. The LD50 (24 h) is 1 mg /kg bw (i.p.), in mice and rats. When given i.p. to mice (0.1 - 1.0 mg/kg bw) bolesatine induced thrombi and blood stasis in the liver, 5 - 21 h after injection, and modifications of the number of blood corpuscles in peripheral blood. These effects were efficiently reversed by aspirin, ticlopidin and heparin (as attested by histology and electron microscopy) which however failed to prevent death in animals given lethal doses. Together, these results showed that the death of bolesatine poisoned animals given high doses, was rather due to a combination of thrombosis and other toxic effects. In addition, they suggest that these antithrombotic drugs may overcome cases of human poisoning, with low exposures of this boletus, showing a hypertension probably due to mechanical obstruction which resists normal therapy.

Characterization of a new human liver myofibroblast cell line: transcriptional regulation of plasminogen activator inhibitor type I by transforming growth factor beta 1.

Myofibroblasts (MF) are a major effector cell type in liver fibrogenesis, where they are thought to derive from the activation of hepatic stellate cells. Cultured human MF, grown from liver explants, retain most of the in vivo characteristics of liver MF but are in limited supply. A continuous MF cell line would therefore be valuable in studying human liver fibrogenesis. For this purpose, we sought to immortalize human liver MF with polyoma virus large T antigen. MF were obtained from explants of human liver and transfected with a plasmid containing the coding sequence of polyoma virus large T antigen. This procedure yielded an activity growing cell line, designated GREF-X, which did not express large T antigen. Nevertheless, this cell line has been passaged repeatedly for almost 1 year and is thus likely immortalized. The morphology of GREF-X resembles that of primary liver MF. These cells have a doubling time of approximately 72 hours and are density-inhibited, and their growth is serum-dependent. Moreover, GREF-X cells do not grow in soft agar or induce tumors in nude mice, suggesting that they are not transformed. They stain positively for MF markers, such as smooth muscle alpha-actin and vimentin; express collagens type I, IV, V, and VI, fibronectin, and laminin: and secrete matrix-metalloproteinase-2. In addition, GREF-X cells are able to take up and esterify [3H]retinol, suggesting that they actually derive from hepatic stellate cells. Finally, these cells respond to transforming growth factor-beta 1, a major mediator of liver fibrogenesis, by increasing secretion of fibronectin and plasminogen activator-inhibitor type 1. Transient transfection experiments showed that plasminogen activator-inhibitor type 1 regulation, by transforming growth factor-beta 1, was transcriptional. We believe, therefore, that GREF-X would be a useful tool for studying the pathophysiology and pharmacology of liver fibrogenesis.

Ultrastructure of human Kupffer cells maintained in culture.

Sinusoidal cells were isolated by collagenase perfusion and metrizamide gradient centrifugation, from liver resected for partial hepatectomy performed under warm ischemic conditions. Kupffer cells were then separated from this population by centrifugal elutriation. Isolated Kupffer cells showed good viability, with the typical features of Kupffer cells and were engaged in the endocytosis of foreign particles. They showed numerous morphological criteria of activation. However, cultured Kupffer cells were no longer in an activated state. Kupffer cells were preserved in maintenance cultures for 2 weeks. Purity of these cultures was to 93-97%. During culture, Kupffer cells retained their ultrastructural characteristics and were active in the endocytosis of latex beads and opsonized zymosan particles. It is thus possible that partial hepatectomy performed under warm ischemia could provide valuable material for the study of Kupffer cells in vitro.

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.

Severe portal hypertensive gastropathy and antral vascular ectasia are distinct entities in patients with cirrhosis.

BACKGROUND/AIMS: Whereas severe portal hypertensive gastropathy and gastric antral vascular ectasia (GAVE) have been separately defined in patients with cirrhosis, there is much confusion in the literature because they are both characterized by red spots at endoscopy. This prospective study compared clinical, biochemical, and pathological features of these syndromes. METHODS: Three groups of patients with cirrhosis and either GAVE (n = 14), severe portal hypertensive gastropathy (n = 14), or no gastric features at endoscopy (controls; n = 10) were included. RESULTS: No difference was found between patients with gastropathy and controls. Patients with GAVE presented with the following significant differences compared with other patients: a higher Child-Pugh score, a lower blood level of hemoglobin and gastrin, and a higher intestinal blood loss. At pathological examination, these patients more frequently had vascular ectasia (P = 0.04), spindle cell proliferation (P < 0.01), fibrohyalinosis (P = 0.004), and Gilliam's score of > or = 2 (P < 0.05); thrombi were encountered only in patients with GAVE (P = 0.006). Using discriminant analysis, spindle cell proliferation and fibrohyalinosis were the only significant variables yielding a diagnostic accuracy of 85% for GAVE and gastropathy. CONCLUSIONS: GAVE and severe portal hypertensive gastropathy are two distinct entities.

Lynxacarus radovskyi infestation in a cat.

An adult domestic short-hair cat from south Texas was examined because of excessive dandruff on the back, neck, thorax, and hind limbs. Removal of a few hairs for microscopic evaluation revealed Lynxacarus radovskyi, the cat fur mite. The small (< 0.5 mm) mite could be readily identified by its laterally compressed body and its characteristic grasping of the hair shaft between the gnathosoma and palpi. Thus far, this mite has been identified as a parasite of cats in warm, humid environments. The number of parasites and apparent discomfort in cats varies considerably, from massive infestation with little discomfort to few mites and marked pruritus. Acaricides that are effective against other ectoparasites of cats apparently are effective in controlling L. radovskyi.

Nodular regenerative hyperplasia in the rat induced by a selenium-enriched diet: study of a model.

Weaned male rats were fed a 4 ppm selenium diet. Compared after 2 mo with a control group fed a 0.4 ppm diet, the rats' body weights had not significantly decreased and liver function was normal, but portal pressure was 1.8 times higher (p less than 0.05). Liver weight was slightly increased (10.3%; p less than 0.05). All livers had an abnormal appearance. In the less severe cases the surface was only slightly irregular, but in the more severe cases, atrophic micronodular lobes and hypertrophic lobes, with mildly irregular surfaces, were present. On light microscopy, atrophic lobes displayed a peripheral nodular zone with micronodules separated by rows of atrophic hepatocytes without fibrosis, characteristic of nodular regenerative hyperplasia, and a central atrophic zone that was sometimes peliotic. Hypertrophic lobes and livers in the less severe cases had only minor and relatively localized evidence of nodular regenerative hyperplasia; occasional peliosis was seen. In all cases portal veins, hepatic veins and hepatic arteries were normal. By electron microscopy, in nonnodular zones with no obvious evidence of parenchymal atrophy, the endothelial wall showed signs of complete or incomplete capillarization with frequent enlargement of the Disse space. The selenium-enriched diet is a reproducible model of liver nodular regenerative hyperplasia. In this model, damage to the sinusoidal wall could represent the primum movens of microcirculatory disturbances.

Characterization of liver-associated natural killer cells in patients with liver tumors.

The existence of a marginal lymphocyte population in rat liver sinusoids has already been demonstrated using the sinusoidal lavage method. We used the same technique to study the lymphocyte population in human liver obtained ex vivo after partial hepatectomy for benign or malignant tumors and compared it with peripheral and portal blood lymphocyte populations. Percentages of lymphocyte surface phenotypes were evaluated by flow cytometry. The lymphocyte population obtained from human liver is mainly made up of CD56+ (35%) cells. This percentage is three times greater than that found in peripheral and portal blood. Two-color flow cytometry analysis showed that within the CD56+ liver cell population, at least three distinct subsets could be found: (a) CD3+/CD56+/CD16-; (b) CD3-/CD56+/CD16-; and (c) CD3-/CD56+/CD16+. Although these subsets were also present in peripheral and portal blood, the percentage distribution was completely different because most CD56+ cells in peripheral and portal blood belonged to the CD3-/CD56+/CD16+ subset. These results show the existence of a heterogeneous natural killer cell population in human livers with tumors. The functional significance of this heterogeneity still needs to be explained.

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.

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.

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.

Hyperplastic foci in the atrophic liver of rats after portacaval anastomosis.

Hyperplastic focal areas were investigated in livers of male rats 1 and 9 months after portacaval anastomosis (PCA) by light and electron microscopy. These alterations, predominantly found in periportal areas, were characterized by light microscopy as clusters of enlarged hepatocytes along narrowed sinusoids, contrasting in the remaining liver acinus with smaller hepatocytes along widened sinusoids. No differences were observed between 1 and 9 months PCA except for glycogen content, which was homogeneously distributed in the liver at 1 month but completely lacking in foci at 9 months. The most striking ultrastructural alterations were the sinusoids delimited in these hyperplastic areas by a thickened barrier consisting of thick endothelial cells encircled by numerous subendothelial processes of the perisinusoidal fat-storing cells. Deep and widened recesses of the sinusoidal lumen separated the two-cell-thick plates of the hyperplastic cells. Hepatocytes in foci, thought to represent regenerative areas, tend to increase their exchange surface. Their progressive loss in glycogen and their two-cell-thick plates architecture should be in favour of a potential malignancy. However, the spontaneous evolution of these foci which do not necessarily give rise to nodules, as well as the lack of other features of transformation, do not support this possibility.