Ito-cell gene expression and collagen regulation.

Ito cells are perisinusoidal cells thought to be a major source of collagen in normal and fibrotic livers. These cells appear to have features similar to several cell types but when cultured assume a fibroblast-like morphology. In this study we evaluated the phenotype of both freshly isolated and cultured Ito cells by examining their gene expression. To better define the modulators of Ito-cell collagen synthesis, we also examined the effect of transforming growth factor-beta 1, tumor necrosis factor-alpha and dexamethasone on collagen synthesis by these cells. Northern hybridization analysis revealed that cultured Ito cells expressed different types of procollagen mRNAs than did freshly isolated cells. Cultured cells contained large amounts of type I procollagen mRNA and lesser amounts of types III and IV, whereas freshly isolated cells contained more type IV procollagen mRNA than types I and III. Treatment of cultured cells with either transforming growth factor-beta 1 or tumor necrosis factor-alpha resulted in a greater than three-fold increase in total collagen content, and the effects of these cytokines on Ito-cell collagen synthesis involved different levels of gene regulation. Transforming growth factor-beta 1-treated cells had an approximately threefold increase in their type I procollagen mRNA levels, whereas no increase in this mRNA level was found in tumor necrosis factor-alpha-treated cells. Transforming growth factor-beta 1 treatment induced a twofold increase in transforming growth factor-beta 1 mRNA content in cultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma-interferon treatment inhibits collagen deposition in murine schistosomiasis.

Since interferons have been shown to affect the synthesis of matrix proteins such as collagen in several in vitro systems, the potential role of gamma-interferon in inhibiting hepatic fibrosis was investigated. Hepatic cells, consisting primarily of hepatocytes, were treated with recombinant gamma-interferon for 24 hr. Northern blot hybridization showed that gamma-interferon treatment caused a profound decrease in pro-alpha 2(I)collagen mRNA levels but an increase in beta-actin mRNA content. The effects of gamma-interferon were then studied in an in vivo model of hepatic fibrogenesis, murine schistosomiasis. Schistosoma-infected mice were treated with daily i.m. injections of gamma-interferon for a 4-week period starting 4 weeks after the initial infection. gamma-Interferon treatment decreased collagen deposition as determined by histologic evaluation and measurement of total liver collagen content. Northern blots showed Types I and III procollagen mRNA levels for treated, infected animals to be only 32 and 29% that of infected controls, but beta-actin mRNA levels were significantly elevated. These results indicate a potential role for gamma-interferon as an antifibrogenic agent in vivo.

In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis.

Despite extensive efforts, little progress has been made in identifying the factors that induce hepatic fibrosis. Transforming growth factor-beta (TGF-beta) has been shown to enhance collagen production, therefore its role in hepatic fibrosis was investigated. Treatment of cultured hepatic cells with TGF-beta 1 increased type I procollagen mRNA levels 13-fold due to post-transcriptional gene regulation. When two animal models of hepatic fibrosis, murine schistosomiasis and CCl4-treated rats, were examined, they both exhibited increased levels of TGF-beta 1 gene expression at times that somewhat preceded the increase in collagen synthesis. In contrast, in murine schistosomiasis, mRNA levels of tumor necrosis factor and interleukin-1 peaked early in the fibrogenic process. Immunohistochemical analysis showed TGF-beta 1 to be present in normal mouse liver and to be markedly increased in mice infected with schistosomiasis. TGF-beta 1 appeared in the hepatic parenchyma, primarily in hepatocytes. These findings strongly suggest a role for TGF-beta 1 in a pathophysiological state.

Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization.

Previous studies investigating the cellular origins of several collagens in young adult rat hearts (Eghbali et al., 1988) demonstrated that the mRNAs for types I and III collagen occurred in non-myocyte cells, mostly fibroblasts, whereas the mRNA for type IV collagen was observed in both myocytes and non-myocyte cells. In the present study, cellular localization of collagen mRNAs has been achieved by in situ hybridization in rat heart tissue and in isolated heart cells. Frozen tissue sections, isolated cardiomyocytes, cultured neonatal cardiomyocytes and fibroblasts were hybridized with DNA probes for type-specific collagens, actin, and myosin heavy chain. Silver grains were visualized by dark field imaging. In heart sections, types I and III mRNAs were observed predominantly adjacent to myocytes and in the interstitium, where fibroblasts are known to be present. In contrast, type IV collagen mRNA was identified both within the myocytes and the interstitium. In freshly isolated adult cardiomyocytes and in cultured neonatal cardiomyocytes, collagen type IV mRNA was observed but type I collagen mRNA was not. In cultured neonatal fibroblasts, both types IV and I collagen mRNAs were abundant.

Molecular studies of ceruloplasmin deficiency in Wilson's disease.

Deficiency of serum ceruloplasmin is a characteristic biochemical abnormality of Wilson's disease, although the mechanism of this finding is unknown. Ceruloplasmin messenger RNA (mRNA) levels were therefore examined in five patients with Wilson's disease and five controls with other types of hepatic disease. Northern and dot blot hybridizations showed that detectable ceruloplasmin mRNA was present in all of the patients with Wilson's disease, including one patient with no detectable serum ceruloplasmin. However, the ceruloplasmin mRNA levels in the Wilson's disease patients were only 33% that of controls (P less than 0.001). In contrast, albumin mRNA levels in the Wilson's disease patients averaged 161% that of controls. In an attempt to better delineate the level of gene expression responsible for this decrease in ceruloplasmin mRNA, the nuclear run-on assay was used to analyze transcriptional rates. The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that the diminished serum ceruloplasmin levels in patients with Wilson's disease are due at least in part to a decrease in ceruloplasmin gene transcription.

Differential effects of gamma-interferon on collagen and fibronectin gene expression.

The interferons are a group of endogenous proteins that exhibit a variety of biological functions in addition to their ability to induce resistance to viruses. In order to evaluate the anti-fibrogenic actions of interferon, we have delineated the level of regulation responsible for gamma-interferon-induced changes in collagen and fibronectin gene expression in cultured fibroblasts. Confluent human skin fibroblasts were exposed to 500 anti-viral units/ml of gamma-interferon. RNA was then extracted from the cells, and steady-state mRNA levels were determined by Northern and dot blot hybridization studies. Cells exposed to interferon had type I procollagen mRNA levels that were 23% of control and type III procollagen mRNA levels only 7% of control. The interferon-treated cells also had beta-actin mRNA levels that were decreased to 51% that of untreated cells but had fibronectin steady-state mRNA levels that were 560% of control levels. Nuclear run-on assays revealed that interferon did not affect the transcriptional rates of types I and III collagen or beta-actin, but it did increase the transcriptional rate of fibronectin to 670% of control levels. These findings demonstrate that gamma-interferon causes a marked decrease in types I and III procollagen mRNA levels in vitro by a post-transcriptional mechanism while inducing fibronectin expression at a transcriptional level.

The effects of dexamethasone on in vitro collagen gene expression.

Glucocorticoids have been shown to be useful in the treatment of certain types of chronic liver disease both by inhibiting fibrosis and by improving liver function. We have previously demonstrated in an in vivo model of hepatic fibrogenesis that dexamethasone inhibits the synthesis of types I and IV collagen. In the present study we have evaluated the level of regulation responsible for the dexamethasone-induced changes in collagen gene expression in a defined in vitro system. Primary cultures of adult rat hepatocytes treated with and without dexamethasone under classical cell culture conditions or using defined media were evaluated for synthesis and abundance of procollagen and beta-actin mRNAs. Cells treated with dexamethasone had decreased types I and IV procollagen mRNA steady state levels due in part to diminished transcription rates of the genes. On the other hand, beta-actin mRNA levels were unaffected by dexamethasone. Transient expression experiments were performed to more precisely define the mechanism whereby dexamethasone affects type I procollagen gene transcription. The recombinant plasmid, pAZ1009, containing the mouse alpha 2(I) procollagen gene promoter linked to the chloramphenicol acetyltransferase gene, was transfected into mouse fibroblast cell lines. Cells transfected with the pAZ1009 plasmid in the presence of dexamethasone had a significant decrease in chloramphenicol acetyltransferase activity when compared to cells not exposed to dexamethasone. These data suggest that dexamethasone inhibits collagen synthesis through a direct effect on the collagen gene promoter and appears also to have a post-transcriptional effect on procollagen mRNA content.

Transcriptional and posttranscriptional effects of dexamethasone on albumin and procollagen messenger RNAs in murine schistosomiasis.

We have previously shown that dexamethasone increases albumin mRNA and decreases procollagen steady-state mRNA levels in rat hepatocyte cultures. These studies were extended by evaluating an in vivo model of fibrogenesis (murine schistosomiasis) and by determining a more precise level of gene expression responsible for these changes. Control mice and litter mates infected with Schistosomiasis mansoni were evaluated at 8 weeks postinfection when the livers of the infected mice had become fibrotic and their serum albumin levels significantly decreased. The addition of 4 micrograms/mL dexamethasone to the drinking water of half of the infected mice led to a 75% decrease in the liver collagen content as determined by high-performance liquid chromatography. RNA was extracted from the livers of mice under three conditions: control and infected +/- dexamethasone. This RNA was then hybridized with cDNA probes to determine steady-state levels of specific mRNAs. In the infected mice, albumin mRNA levels were decreased compared to control; however, infected mice treated with dexamethasone increased their albumin mRNA content by 3-fold at 8 weeks. Types I and IV procollagen steady-state mRNA levels in infected mice were increased compared to control while dexamethasone suppressed the mRNA level of collagen in infected mice by 50%. The level of gene expression responsible for these steady-state changes was evaluated by nuclear run-on analysis. While the effect of schistosomiasis on these genes was primarily at a transcriptional level, dexamethasone exerted its effect on different genes in the injured liver by diverse mechanisms, i.e., decreasing collagen synthesis at a transcriptional level and increasing albumin by posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of molecular hybridization technology to evaluate albumin and procollagen mRNA content in baboons and man.

We have developed the methodology for evaluating the effects of pathophysiological conditions on the molecular mechanisms of hepatic protein synthesis and fibrogenesis in baboons and man. Total RNA was extracted from percutaneous liver biopsies of five baboons who were chronically fed an ethanol-rich liquid diet, their pair-fed controls and from humans with a variety of liver abnormalities. Chronic alcohol administration in baboons with liver fibrosis and normal serum albumin levels increased in vitro protein synthesis as measured by [35S]methionine incorporation, albumin mRNA content and Type I procollagen mRNA content. There was no difference in the beta-actin (a constitutive protein) mRNA content. In humans, serum albumin levels correlated with albumin mRNA content as indicated by the intensity of dot blot hybridization and Type I procollagen mRNA levels correlated with the activity of liver fibrosis. The use of RNA-DNA hybridization to investigate procollagen mRNA from human biopsies appears to be a valuable tool for evaluating the potential for collagen synthesis and the future course of liver disease. Besides the use of RNA-DNA hybridization, we describe other methodologies which are useful in delineating the levels of gene expression responsible for hepatic mRNA regulation in normal liver and disease states in man. The use of molecular techniques to evaluate human liver disease provides an opportunity to develop clinically relevant information while at the same time offering the additional advantage of providing fundamental knowledge about fibrogenesis.

Increased type I procollagen mRNA levels and in vitro protein synthesis in the baboon model of chronic alcoholic liver disease.

We have investigated the molecular mechanisms responsible for the histologic changes induced by ethanol in the baboon model of alcoholic liver disease. Eleven ethanol-fed baboons and their pair-fed controls had histology evaluated and RNA extracted from percutaneous liver biopsy specimens. In 6 of the ethanol-fed animals, fatty liver developed, but no significant differences were found when the RNA from the control and ethanol-fed livers was translated in the reticulocyte lysate system or analyzed with specific cDNA probes. Five of the baboons given ethanol, however, developed significant fibrosis. Molecular evaluation revealed that the RNA from these livers was more active in in vitro protein synthesis, and the type I procollagen mRNA content was significantly higher per microgram of liver RNA as determined by hybridization analysis (183% +/- 23% SEM of control, p less than 0.02). In addition, there were higher levels of albumin mRNA content in the livers of ethanol-fed baboons that developed fibrosis (180% +/- 21% SEM of control, p less than 0.05). There was no change, however, in the levels of beta-actin mRNA, a representative constitutive protein. These findings in the baboon model of alcoholic fibrosis show that ethanol consumption increases type I procollagen mRNA, which may foster fibrogenesis; increases albumin mRNA content without causing an increase in serum albumin; and induces no change in levels of beta-actin mRNA. These studies also show that percutaneous needle biopsy can supply sufficient tissue to evaluate molecular changes in human liver disease.

Ascorbate-generated endogenous extracellular matrix affects cell protein synthesis in calf aortic smooth muscle cells.

Ascorbate supplementation of cultured fetal calf aortic smooth muscle cells leads to increased deposition of extracellular matrix proteins and stimulation of cellular protein synthesis (E. Schwartz et al., J cell biol 92 (1983) 462) [7]. In the present study, we have investigated this phenomenon at the level of gene expression. Cells were grown for three weeks on tissue culture plastic with or without ascorbate (50 micrograms/ml). When compared to controls, cells grown in presence of ascorbate had twice as much poly(A+) RNA per microgram of total RNA, and ascorbate led to a 50% increase in [35S]methionine incorporation when the total RNA was translated in the reticulocyte lysate system. SDS-PAGE revealed no change in the protein pattern under the two conditions. "Northern" hybridization revealed a two- to fivefold increase in the sequence content of beta-actin, alpha-tubulin and type I pro alpha 1-collagen in total RNA of ascorbate-supplemented cells, but no difference was observed in the mRNA sequence content for the three specific proteins when equal amounts of poly(A+) RNA from ascorbate and control cells were hybridized with the three cloned cDNAs. To evaluate the effect of an exogenous matrix, cells were also plated on collagen gels. RNA isolated from cells grown on collagen without added ascorbate exhibited translational activity and mRNA sequence content similar to cells grown with ascorbate on tissue culture plastic. In contrast, no differences from controls were found in cells grown for one week in the presence of ascorbate, at which time no significant deposition of collagen occurs in the extracellular matrix. These results suggest that the stimulation in protein synthesis in fetal calf smooth muscle cells supplemented with ascorbate is associated with an increase in the proportion of poly(A+) RNA in the total RNA pool, and that the production of an endogenous collagen-rich matrix in the presence of ascorbate may be the basis for these pretranslational changes.

Effects of dexamethasone on albumin and collagen gene expression in primary cultures of adult rat hepatocytes.

To further our studies on collagen gene expression, we have evaluated the molecular basis for the finding that steroids decrease collagen synthesis in cultured hepatocytes. We studied the effects of dexamethasone on primary cultures of adult rat hepatocytes grown on tissue culture plastic in either serum-supplemented medium or a serum-free hormonally defined medium. Cells were plated and allowed to attach for 24 hr in a mixture of serum-supplemented medium + hormonally defined medium. Cultures were then fed every 24 hr for 4 days under 1 of 4 conditions: serum-supplemented medium, serum-supplemented medium + dexamethasone, hormonally defined medium or hormonally defined medium + dexamethasone. On the fifth day, RNA was extracted. Dexamethasone did not affect the amount of RNA isolated; nor did it influence the quantitative translation of the mRNA in the rabbit reticulocyte lysate mRNA-dependent system. Employing hybridization analysis, dexamethasone resulted in increased albumin mRNA content in hepatocytes grown in serum-supplemented medium but had no affect on hormonally defined medium, and decreased type I in collagen mRNA content in cells grown in either serum-supplemented medium or hormonally defined medium. In cells cultured in hormonally defined medium, the beta-actin and procollagen mRNA levels were lower than those in serum-supplemented medium, but albumin mRNA levels were higher, and in fact equivalent to those in vivo. beta-Actin mRNA levels were not affected by dexamethasone in either serum-supplemented medium or hormonally defined medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of the collagen in hepatic fibrosis in advanced murine schistosomiasis.

Livers from CF 1 mice infected with Schistosoma mansoni, 50 cercariae each, were examined 8 weeks postinfection. The collagen content of the livers was increased about 18-fold over that for normal control animals. Quantitation of the collagen types in pepsin-solubilized collagen showed a change in the ratio of Type I to Type III from 2:1 in normal mice to 1:1 in the fibrotic animals. The change in ratio resulted from a very large accumulation of Type III collagen and, accordingly, may provide an important model for study of the possible reversibility of fibrotic lesion.

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.

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.