The collagenase of Entamoeba histolytica.

The present work was designed to investigate the capacity of trophozoites of Entamoeba histolytica to adhere to and digest human collagen types I and III in vitro. The time-course of binding of ameba to both human collagen types I and III was similar. However, the kinetics of detachment were different for each collagen type. Trophozoites of E. histolytica cultured on heat-reconstituted type I collagen gels produced a well-defined area of lysis. Quantitative studies using 14C-labeled collagen revealed that after 24 h of incubation, Entamoeba digested three and a half times more type I than type III collagen, thus suggesting the presence of a collagenase with higher specificity for type I collagen. This activity was optimum with trophozoites harvested after 42 h in culture (1.5 X 10(5) trophozoites/ml). The digestion of type I collagen was a function of the number of trophozoites, and was inhibited by EDTA, L-cysteine, and serum, but not by soybean trypsin inhibitor, phenylmethanesulfonyl fluoride, or N-ethylmaleimide (NEM). Electrophoretic analysis of the type I collagen fragments revealed three main classes of polypeptides of 75,000, 50,000, and 25,000 daltons. Subsequent proteolysis of these collagen fragments was probably carried out by other proteases derived from trophozoites. This activity was inhibited with 10 mM NEM. Collagenase activity appeared to be located at the plasma membrane and direct contact of the ameba with the substrate is required for collagen digestion. The results suggest that collagenase activity of E. histolytica may play an important role in tissue invasion.

Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity.

Injury to cardiac myocytes often leads to the production of anti-myosin antibodies. While these antibodies are a marker of myocardial injury, their contribution to pathogenesis in diseases such as autoimmune myocarditis or rheumatic fever is much less clear. We demonstrate in this report that monoclonal anti-myosin antibodies can mediate myocarditis in a susceptible mouse strain. Additionally, we show disease susceptibility depends on the presence of myosin or a myosin-like molecule in cardiac extracellular matrix. This study demonstrates that susceptibility to autoimmune heart disease depends not only on the activation of self-reactive lymphocytes but also on genetically determined target organ sensitivity to autoantibodies.

Connective tissue biomatrix: its isolation and utilization for long-term cultures of normal rat hepatocytes.

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance). Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans). Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, long-term survival (months) and retention of some hepatocyte-specific functions.

Inhibition of liver fibrosis by 1-azetidine-2-carboxylic acid in rats treated with carbon tetrachloride.

L-Azetidine-2-carboxylic acid (AZC), an analogue of proline, has been shown to partially ameliorate hepatic cirrhosis induced in rats by CCl(4). AZC caused a diminution in formation of collagen in the liver accompanied by a relative decrease in the pool of free proline. The synthesis of noncollagenous proteins in the livers of treated rats did not appear to be affected.

The relationship between the free pool of proline and collagen content in human liver cirrhosis.

The free proline, free glutamic acid, and total collagen contents of the livers of cirrhotic and noncirrhotic patients were determined. The amounts of free proline in the sera of the patients were also determined. The results indicated that certain metabolic changes occurred in cirrhotic livers of humans that were similar to the metabolic changes observed previously in CCl(4)-induced cirrhosis in the rat. The amount of free proline was coordinate with the increase in total collagen, and both were inversely related to the amount of free glutamic acid. The average proline concentration in sera of cirrhotic patients was not higher than that of non cirrhotic patients, suggesting that the metabolic alteration noted above is a local event in the liver related to fibrogenesis. These and other results suggest that the pool size of free proline may play a prime role in regulation of collagen biosynthesis in liver cirrhosis.

Liver collagen synthesis in murine schistosomiasis.

Collagen synthesis was measured in liver slices obtained from mice with hepatosplenic schistosomiasis. Enlarged fibrotic livers from these mice contained 20 times more collagen than normal. This model of hepatic fibrosis results from an inflammatory granulomatous host response to Schistosoma mansoni ova in portal tracts, rather than from direct lover cell injury as with carbon tetrachloride-induced liver fibrosis. Collagen synthesis, as measured by the formation of labeled protein-bound hydroxyproline, occurred in granulomas isolated from fibrotic livers. Labeled collagen that cochromatographed with type I collagen was extracted with neutral salt solution from liver slices incubated with labeled proline. The free proline pool of the liver was doubled in infected mice; coordinately, liver slices from these animals showed maximal collagen production when the concentration of free proline in the medium was raised to 0.4 mM, the same level measured in the fibrotic livers. Under such conditions, collagen synthesis was at a rate equivalent to the formation of 5.4 nmol of protein-bound hydroxyproline per g liver in 6 h. In comparative incubations in medium containing 0.2 mM proline, fibrotic liver slices produced 16-fold more collagen than normal slices. The proline analogue, L-azetidine 2-carboxylic acid, effectively inhibited synthesis of labeled collagen by fibrotic liver slices. These studies show the synthesis of collagen in a reproducible animal model of the most prevalent form of human liver fibrosis. Difinitition of the controlling factors in this system is of interest for the general problem of fibrosis produced by immunological responses.

Propranolol ameliorates the development of portal-systemic shunting in a chronic murine schistosomiasis model of portal hypertension.

We investigated the role of early portal hypotensive pharmacotherapy in preventing the development of portal-systemic shunting in a portal hypertensive model of chronic murine schistosomiasis induced by infecting C3H mice with 60 cercariae of Schistosoma mansoni. Propranolol was administered in drinking water to 20 animals for a period of 6 wk at a dose of 10 mg.kg-1d-1, starting at 5 wk of schistosomal infection. 32 age-matched mice with chronic schistosomal infection served as controls. All animals were studied 11 wk after the infection. Compared with controls the portal pressure (10.8 +/- 0.40 mmHg) was significantly lower (P less than 0.001) in the propranolol-treated animals (7.9 +/- 0.80 mmHg). Portal-systemic shunting was decreased by 79%, from 12.2 +/- 3.34% in controls to 2.5 +/- 0.99% in the propranolol group (P less than 0.05). Portal venous inflow was reduced by 38% in the propranolol treated animals (2.50 +/- 0.73 ml/min; n = 6) compared with controls (4.00 +/- 0.34 ml/min; n = 8; P less than 0.05). The worm burden, the granulomatous reaction, the collagen content of the liver, and the serum bile acid levels were not significantly different between the two groups of animals. These results demonstrate that in chronic liver disease induced by schistosomiasis, the development of portal-systemic shunting can be decreased or prevented by the reduction of flow and pressure in the portal system.

Accelerated development of liver fibrosis in CCl4-treated rats by the weekly induction of acute phase response episodes: upregulation of alpha1(I) procollagen and tissue inhibitor of metalloproteinase-1 mRNAs.

Patients with alcoholic hepatitis have several manifestations of the acute phase response (APR) and have elevated blood levels of interleukin-1, interleukin-6 and tumor necrosis factor-alpha. We have previously shown that liver stellate cells express interleukin-6 mRNA and protein and respond to this cytokine with increased expression of alpha1(I) procollagen mRNA. We further showed that the production of an APR episode stimulates a transient expression of alpha1(I) procollagen mRNA in the liver. In this communication we demonstrate that the concomitant induction of a weekly APR episode in rats with a schedule of CCl4 to produce cirrhosis, accelerates the development of liver fibrosis. We show that the enhancement of liver fibrosis is due, in part, to further upregulation in the expression of alpha1(I) procollagen and tissue inhibitor of metalloproteinases-1 mRNAs above values observed in control rats receiving only CCl4. The effect of the APR appears to have specificity since not all the mRNAs measured were equally affected. Altogether, these results suggest that increased blood or liver levels of APR cytokines, whether induced by APR episodes, endotoxin or other unrelated causes, may contribute to the development of liver fibrosis by enhancing the expression of type I collagen and of tissue inhibitor of metalloproteinases-1 mRNAs.

Effect of colchicine on collagen, albumin and transferrin synthesis by cirrhotic rat liver slices.

Collagen synthesis was found to be increased in liver slices of rats made cirrhotic by chronic administration of CCl4. The liver function was impaired, as determined by an increased retention of conjugated bilirubin and low serum albumin values. However, when animals received colchicine simultaneously with CCl4, collagen synthesis and deposition were inhibited, and the liver function appeared normal. When a group of rats was made cirrhotic by chronic administration of CCl4, and then kept for 30 days without further treatment, fibrosis persisted and collagen synthesis was very low. However, the liver function was severely impaired. When similar rats received L-azetidine-2-carboxylic acid during the 30-days period following CCl4 administration, there was a slight but not significant improvement in liver function. The collagen synthesis and the extent of fibrosis were similar to the controls. However, if similar rats received colchicine during the 30 days period, collagen synthesis was almost negligible, there was a slight decrease in fibrosis and there was a great improvement in liver function. In all the cirrhotic animals studied, transferrin biosynthesis remained constant.

Ethanol induces the expression of alpha 1(I) procollagen mRNA in a co-culture system containing a liver stellate cell-line and freshly isolated hepatocytes.

To study the fibrogenic action of ethanol in vitro we used a co-culture system of freshly isolated hepatocytes and a liver stellate cell line (CFSC-2G) developed in our laboratory. Our results show that in this co-culture system ethanol induces the expression of alpha 1(I) procollagen mRNA in a dose- and time-dependent manner. This effect of ethanol was due to its metabolism by alcohol dehydrogenase since 4-methylpyrazole prevented the ethanol-mediated increase in alpha 1(I) procollagen mRNA. Ethanol was more efficient than acetaldehyde in inducing alpha 1(I) procollagen mRNA expression and its effect was protein synthesis-independent. Transfection of either hepatocytes or liver stellate cells with a reporter gene, chloramphenicol acetyl transferase (CAT), driven by 3700 bp of the mouse alpha 1(I) procollagen promoter demonstrated that only LSC expressed significant CAT activity and that this activity was enhanced by ethanol. Overall, our results suggest that this co-culture system is a useful model to study alcohol-induced fibrogenesis in vitro and that mechanisms other than acetaldehyde formation may also play an important role in alcohol-induced fibrogenesis.

Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy.

To evaluate scar-type and matrix connective tissue and to assess their role in the diastolic dysfunction of hypertrophic cardiomyopathy, surgically resected subaortic myectomy specimens and several autopsy hearts from patients with hypertrophic cardiomyopathy were studied. Eighteen specimens were differentially stained by a newly developed method that precisely determines relative collagen content; these tissues were compared with postmortem hypertrophied and normal control subaortic specimens. Quantitation revealed a 72% higher level (36.5 vs. 22.1 micrograms collagen/mg protein) of stainable collagen in the hearts with hypertrophic cardiomyopathy than in hypertrophied control hearts. The endocardial plaque was quantitated morphometrically, and it constituted only 4.6 +/- 1.7% of the total increased collagen content in the cardiomyopathy specimens. For the matrix studies, the cardiomyopathy specimens were stained by a silver impregnation technique that identifies connective tissue elements not normally visible with routine histologic methods. There was a marked increase in content of all matrix components, both in areas of pathologic scarring and in "normal" zones. Whorls of matrix connective tissue were noted in regions of myocyte whorls, as well as independent of them. Thus, these studies revealed a striking increase of both scar-type and matrix connective tissue in hypertrophic cardiomyopathy. The extensive scarring and the pronounced interstitial and intercellular matrix connective tissue may contribute to the increased ventricular chamber stiffness and impaired relaxation in this disease.

Fibrogenesis in cirrhosis. Potential for therapeutic intervention.

Liver cirrhosis is an end stage of several diseases that affect the liver chronically. It is characterized, among other things, by excess collagen deposition, distortion of liver architecture, tissue malfunction and hemodynamic alterations. Many of the complications of cirrhosis may result from excess matrix-deposition. Therefore, prevention of collagen accumulation or removal of collagen deposits could ameliorate the disease. In this article we discuss the pathophysiology of liver fibrosis and we describe various compounds with antiinflammatory and antifibrogenic activity. We discuss their possible mechanism of action and we describe animal and clinical studies in which these compounds have been utilized.

p38 MAPK mediates the regulation of alpha1(I) procollagen mRNA levels by TNF-alpha and TGF-beta in a cell line of rat hepatic stellate cells(1).

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections.

A simple, sensitive, and quantitative procedure is described for the measurement of collagen and protein content in tissue sections prepared from formalin-fixed paraffin-embedded samples. The method can detect as little as 5.7 micrograms of collagen per mg of protein. It is based on the selective binding of Sirius red F3BA and Fast green FCF to collagen and noncollagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid. Both dyes are eluted readily and simultaneously with NaOH-methanol and the absorbances obtained at 540 and 605 nm can be used to determine the amount of collagen and protein. The color equivalence of each dye was determined after destaining the sections and measuring the collagen content by hydroxyproline analysis and the amount of protein by the micro-Kjeldahl procedure. When several sections prepared from five rat tissues were analyzed first by the dye binding method and then by the chemical procedure, comparable results were obtained. This method could be of use in measuring collagen in tissue specimens and could be helpful in assessing the degree of fibrosis in tissue samples and in evaluating the effects of antifibrogenic drugs currently in use.

Lipid quantitation in formalin-fixed liver sections.

We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.

Increased stimulation of alkaline phosphatase by small doses of colchicine entrapped in liposomes. A biochemical test to detect effective liposome-hepatocyte interaction.

The subcutaneous administration of colchicine encapsulated in small unilamellar vesicles reduced the initial toxicity peak and maintained for several days an adequate level of the drug in the liver. Colchicine is an excellent marker for effective liposome-hepatocyte interaction since it fulfills the following criteria: (a) When taken up by the hepatocytes within liposomes, it is active and induces the synthesis of alkaline-phosphatase two to three times over control values. The injection of at least ten times more free colchicine is necessary to attain a similar induction. (b) If released from extracellular liposomes, colchicine is cleared rapidly from the circulation. The results show that liposomes, in spite of their reduced aqueous compartment (approximately 1.0 microliter/mumole of lipid), can achieve clinical utility when administered subcutaneously because of their efficient interaction with parenchymal cells and their continual arrival from the injection site.

Liver fibroblast proliferation in murine schistosomiasis.

Liver fibrosis in schistosomiasis is associated with prominent accumulations of fibroblasts. Primary cell cultures were prepared from the fibrotic livers of Schistosoma mansoni-infected mice, and cells with the appearance of fibroblasts by light microscopy were isolated from these cultures. Proliferation of these cells was examined in coculture experiments with syngeneic inflammatory cells. T cell-enriched mononuclear cells from spleens of S. mansoni-infected or normal mice, and Kupffer cell/macrophages from fibrotic liver all stimulated the proliferation of liver fibroblasts, as measured by 3H-thymidine uptake. Primary cultures of mouse skin fibroblasts showed similar responses to coculture, but an established fibroblast line, 3T3, was unresponsive. Cell-free supernatant medium from coculture experiments did not affect fibroblast proliferation, perhaps because of the requirement for serum in the culture medium. Liver fibroblasts derived from this disease model may be especially suitable for study of the interaction between tissue inflammation and fibrosis.

Resistance to reinfection in experimental murine schistosomiasis: role of porto-hepatic hemodynamics.

Experiments were conducted to evaluate critically, and independently of the immune system, the possible role of hemodynamic mechanisms in resistance to schistosomal reinfection. The effects of a challenge schistosomal infection were compared in groups of mice which were either previously infected with schistosomiasis, vaccinated with irradiated cercariae, or underwent partial portal vein ligation for the induction of portal hypertension and porto-systemic shunting. Following infection with 60 cercariae, the appearance of portal hypertension preceded by approximately 2 weeks the development of porto-systemic shunting, which reached maximal values 11 weeks postinfection. Such a primary infection conferred on C3H mice an estimated 90% protection to a 2nd infection, measured by the reduction of worm burden. Worm burdens were also reduced in vaccinated and ligated animals as compared to normal controls. The protection amounted to 30% and 56%, respectively, in the C3H strain and 63% and 75-85%, respectively, in the C57Bl/6 strain. Reduction in worm burden in the ligated animals is believed to be due to the extrahepatic porto-systemic vascular shunts. Hemodynamic as well as immunological factors may account for the resistance to reinfection observed in chronic murine schistosomiasis.

Liver collagen-type characterization in human schistosomiasis. A histological, ultrastructural, and immunocytochemical correlation.

Liver biopsies of four patients with hepatosplenic schistosomiasis, two patients with schistosomiasis and chronic active hepatitis, two patients with chronic active hepatitis and four control patients with no clinical evidence of either disease, were examined by standard light microscopic techniques, electron microscopy and immunocytochemical staining for collagen type I, III and B. Pure schistosomiasis showed the classical "clay-pipe stem fibrosis" and granulomata composed of eosinophils, macrophages and lymphocytes. In that group, the hepatocellular damage was less conspicuous than in the groups with chronic hepatitis and was usually confined to the granulomatous or fibrotic areas. Destruction of the normal architecture and infiltration by macrophages and lymphocytes with severe damage of hepatocytes was found only in the cases of chronic active hepatitis, with or without associated schistosomiasis. Increased collagen deposits were demonstrated in all three groups. Types I, III and B were found in the enlarged portal triads and fibrotic septa. The intranodular or intralobular collagen stained negatively for type I and strongly positive for types III and B.

Colchicine improves the alterations in the liver adenylate cyclase system of cirrhotic rats.

The adenylate cyclase system and the number and affinities of receptors for insulin and glucagon were studied in rats treated with CCl4 and in rats that received colchicine in addition to CCl4. Liver glycogen, cAMP and total collagen content were also measured in those animals. Rats received only mineral oil or only colchicine were used as controls. In this latter group, all parameters measured were normal. Differences in liver collagen content between groups treated with CCl4 and CCl4 + colchicine were not statistically significant. Basal adenylate cyclase activity was 2-fold increased in the CCl4 group. In the animals receiving CCl4 and colchicine, adenylate cyclase activity was normal. Adenylate cyclase activity stimulated by fluoride or by glucagon was also increased in the CCl4 group. However, this increase was due to to the enhanced basal activity. The number of receptors and the affinities of the receptors of glucagon and insulin were normal in all groups. cAMP levels were found increased in the CCl4 treated animals and this was accompanied by a 90% reduction in liver glycogen. In the group treated with CCl4 + colchicine, cAMP was normal and liver glycogen was only reduced 25%. These results suggest that part of the clinical and biochemical improvements observed in the colchicine treated animals were due in part to a reversal of the alterations of the adenylate cyclase system induced by CCl4.