Chronic liver injury, TGF-beta, and cancer.

Cells termed myofibroblasts are prominent in the injury response of all epithelial tissues. They exhibit proliferation, migration, production of collagen and other extracellular matrix (ECM) molecules, and contraction, all for containing the injury and closing the wound. When the injury is limited in time, the final stage of the repair involves a dismantling of the cellular apparatus and restoration of normal tissue structure. With multiple cycles of repair, however, there is net accumulation of ECM, to the detriment of tissue structure and function. Repair-related ECM coalesces into fibrous bundles and, over time, undergoes changes that render it resistant to degradation. The result is a scar. In the skin, a scar may have cosmetic importance only. In the liver, however, extensive scarring is the setting for unregulated growth and neoplasia; also, fibrous bands disrupt normal blood flow, leading to portal hypertension and its complications. With regard to therapy for fibrosis, the first consideration is elimination of the injury factor. However, given that many liver diseases do not have effective therapies at present, strategies targeting fibrogenesis per se are under development. The main source of myofibroblast-like cells and ECM production in the liver is the perisinusoidal stellate cell, which responds to injury with a pleiotypic change termed activation. Activation is orchestrated by cytokines and the ECM itself. Among the cytokines involved in this process, transforming growth factor-beta (TGF-beta) is particularly prominent. The early changes in ECM include de novo production of a specific "fetal" isoform of fibronectin, which arises from sinusoidal endothelial cells. It is stimulated by TGF-beta and acts directly on stellate cells to promote their activation. Based on these and other advances in understanding the fundamentals of the injury response, several strategies now exist for altering fibrogenesis, ranging from agents that block TGF-beta to traditional Chinese herbal extracts. Arrest of fibrogenesis, even with underlying cirrhosis, is likely to extend life or prolong the time to transplant. Whether it reduces the risk of hepatocellular carcinoma remains to be proven. Although TGF-beta antagonists are effective anti-fibrogenic agents, they will require detailed safety testing because of the finding that several forms of epithelial neoplasia are associated with altered regulation of TGF-beta.

Transforming growth factor-beta initiates wound repair in rat liver through induction of the EIIIA-fibronectin splice isoform.

A prominent feature of the hepatic response to injury is production of a fetal isoform of fibronectin, a splice variant containing the EIIIA region, which appears very early after injury and derives from sinusoidal endothelial cells. Previous studies have shown that it is instrumental in initiating the cellular response to injury, specifically the conversion of resting stellate cells to myofibroblast-like cells. The present work describes the regulation of this change in fibronectin expression. Using sinusoidal endothelial cells from normal or injured liver in primary culture, we show that exogenous transforming growth factor beta (TGFbeta) stimulates [EIIIA]Fn expression. To assess the role of TGFbeta in vivo, we used a chimeric IgG containing the extracellular portion of the TGFbeta type II receptor as a competitive inhibitor of the cytokine. Administered to animals at the time of injury, the inhibitor reduced expression of [EIIIA]Fn mRNA by 50% as compared to controls (P < 0.01). There was a corresponding decrease in [EIIIA]Fn protein production as judged by immunohistochemistry. Cell fractionation experiments indicated that the changes observed in whole-liver extracts were localized to sinusoidal endothelial cells. We conclude that TGFbeta initiates wound repair in part by stimulating endothelial expression of [EIIIA]Fn. Results with the soluble inhibitor of the TGFbeta type II receptor suggest a novel strategy for modulating wound repair in vivo.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis.

Transforming growth factor beta (TGF-beta) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-beta receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-beta type II receptor. This "soluble receptor" was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-beta receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Hepatic fibrosis as wound repair: a progress report.

The injury response of the liver fits within the general paradigm of wound repair. The overall repair response requires close coordination of several cell types and synthetic processes and is orchestrated by an interacting group of cytokines, extracellular matrix (ECM) proteins, and products of metabolism such as oxygen radicals. Many cytokines act over short distances, engaging specific receptors on their parent cell (autocrine) or on immediately adjacent cells (paracrine). The most prominent of these is transforming growth factor-beta (TGFbeta). The ECM also mediates cellular crosstalk and does so in two ways. Firstly, as a binder of cytokines, it is capable of concentrating, presenting, or sequestering these factors at specific locations. Secondly, like cytokines, individual ECM proteins interact with cells via specific receptors, many of which belong to the integrin family. Engagement of a receptor leads to its activation, followed by intracellular signaling events and modification of cell behavior. Recent work has identified a specific integrin on stellate cells (alpha1beta1) as critical to the contractility of these cells. Finally, contrary to the view once held of ECM as relatively inert "ground substance", this extracellular complex is highly dynamic, its cytokine- and cell-binding activities being subject to rapid change. Therefore, matrix proteinases also are important, both during the initiation of fibrosis in association with stellate cell activation, and during the resolution of the injury. As current research reveals the key elements of this regulatory network, new therapeutic modalities are emerging. The goal of therapy is to modify the extent of the repair response without deleting it. Strategies directed at several levels of regulation are under consideration, including agents that block cytokine effects (e.g., TGFbeta receptor antagonists) and direct inhibitors of stellate cell activation or contraction.

Porphyria and porphyrinology--the past fifteen years.

The porphyrias are diseases caused by defective biosynthesis of heme. Leavened by digressions on porphyria trivia, this article presents selected highlights from the last 15 years of research on the chemistry, diagnosis, and treatment of the porphyrias. Thanks largely to genetic analysis and new light shed on the magical chemistry of heme biosynthesis, this period has seen great advances in the understanding of porphyria. Sequence analyses of the genes for all of the enzymes required for heme biosynthesis have revealed the porphyrias as highly heterogeneous, with multiple mutations underlying each type. As a result of technical advances, clinical porphyrin analyses are easier and more detailed, but their misapplication to "multiple chemical sensitivity syndrome" or "intoxication porphyria" is unfortunate. The prospect of gene therapy shines ever brighter but is neither safe nor effective enough to be considered for porphyria. As practical spin-offs, porphyrins are in use increasingly for diagnosis and treatment of cancer and as herbicides and pesticides. Accounts of alleged porphyria in "Prominent People" in the popular press continue to appear, generating fanciful misconceptions, often at the expense of patients with these fascinating diseases.

Two novel antifibrotics, HOE 077 and Safironil, modulate stellate cell activation in rat liver injury: differential effects in males and females.

The perisinusoidal stellate cells of the liver in an injury milieu undergo activation, acquiring a myofibroblast-like phenotype. In this state, they are the principal source of collagen and related proteins in fibrosis. The present studies evaluate the mechanism of action of two novel antifibrotic compounds, HOE 077 and Safironil, which were designed as competitive inhibitors of collagen protein synthesis. Fibrosis was induced in rats by administration of carbon tetrachloride, and activation was monitored as the level of collagen I mRNA or smooth muscle alpha-actin. Both male and female rats were studied. Stellate cell activation, rather than collagen synthesis, proved to be the target of both HOE 077 and Safironil in the intact liver. In culture, the drugs not only prevented the activation of stellate cells but also accelerated their deactivation. They were no more effective in co-cultures containing hepatocytes than in pure stellate cell cultures, indicating that metabolic conversion of HOE 077 was not required. Interestingly, the response of cells from females was greater than that of male cells, leading to the conclusion that stellate activation is sexually dimorphic. This finding may be relevant to the observation that fibrosis in chronic viral hepatitis progresses less rapidly and that hepatocellular carcinoma is less frequent in females than in males.

Environmental chemical exposures and disturbances of heme synthesis.

Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria.

The role of alpha1beta1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture.

An unresolved question in wound contraction concerns the identity of integrins mediating the attachment of tissue myofibroblasts to matrix in the injury site. Previous studies with cell lines have focussed on alpha1beta1 and alpha2beta1, the principal collagen-binding integrins, but have yielded conflicting data. We have examined this issue in wound healing in the liver, isolating the myofibroblast population (activated stellate cells) and quantitating expression of the alpha1 and alpha2 integrin subunits during the in vivo injury. Normal stellate cells displayed alpha1 but no detectable alpha2. During injury, alpha1 expression was maintained; alpha2 became detectable at the mRNA level but at all times was <8% of alpha1 mRNA. Contraction of collagen lattices, studied with 24-h cultured cells and initiated by endothelin 1, was blocked 70% by anti-alpha1 and 30% by anti-alpha2 (both significant, p < 0.05). The inhibition by anti-alpha2, which was unexpected, was attributable to culture-induced change in integrin expression; both the mRNA and protein for alpha2 increased strikingly within 24 h of plating stellate cells on a collagen gel. We conclude that alpha1beta1 is the sole integrin utilized by contracting myofibroblasts in vivo. Although alpha2beta1 is capable of mediating contraction, its expression by myofibroblasts occurs largely, if not exclusively, in response to culture.

Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation.

Expression of the group of cytokines known as transforming growth factor-beta (TGF-beta 1, -beta 2 and -beta 3) is increased during liver regeneration induced by a 70% partial hepatectomy. The origin of these changes was examined in purified isolates of hepatocytes, sinusoidal endothelial cells, Kupffer cells (liver macrophages), and lipocytes (Ito or stellate cells) from normal and regenerating liver. In normal liver, TGF-beta 1 and -beta 2 levels were relatively high in sinusoidal endothelial cells and Kupffer cells. After partial hepatectomy, an early peak of TGF-beta 2 and -beta 3 was present in all four cell types, followed by a sustained increase in mRNA for TGF-beta 1, -beta 2, and -beta 3 primarily in the hepatocyte population. The specificity of these changes was established by examining a mechanistically different injury model, fibrosis induced by ligation of the biliary duct. In this model, TGF beta mRNA was increased only in lipocytes and the increase was progressive over a 7-d period of observation. Secretion of TGF beta protein was examined in cell isolates placed in short-term primary culture and generally reflected the corresponding mRNA level. The TGF beta released by hepatocytes was entirely in the latent form, whereas the individual nonparenchymal cell isolates released 50-90% active TGF beta. Hepatocyte-conditioned culture medium, after treatment to activate latent TGF beta, inhibited hepatocellular DNA synthesis as did the authentic factor. The data indicate that after injury TGF beta increases selectively in the cells that are the target of the factor, i.e., in hepatocytes after partial hepatectomy and in lipocytes in inflammation and fibrosis. We conclude that the effects of TGF beta in liver regeneration and fibrogenesis are predominantly, if not exclusively, autocrine.

Hepatic lipocytes: a major target for endothelin-1.

Lipocytes are the predominant target for ET-1 within the normal and fibrotic liver. ET-1-induced contraction of lipocytes becomes prominent in liver injury. In this setting, ET-1 may have an autocrine role in regulating sinusoidal flow, in association with eicosanoids and other endothelium derived factors such as nitric oxide (NO). In more advanced fibrosis, ET-1 may participate in contraction of collagen bands causing distortion of the lobular structure. A direct role of ET-1 in the activation of lipocytes is still hypothetical but could represent additional effects of ET-1 in various stages of liver fibrogenesis via paracrine and/or autocrine routes.

Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis.

We have examined the cell-specific expression of two fibronectin isoforms, EIIIA and EIIIB, during experimental hepatic fibrosis induced by ligation of the biliary duct. AT the mRNA level, EIIIA and EIIIB were undetectable in normal liver but expressed early injury, preceding fibrosis. The cellular sources of these changes were determined by fractionating the liver at various time points after bile duct ligation into its constituent cell populations and extracting RNA from the fresh isolates. EIIIA-containing fibronectin mRNA was undetectable in normal sinusoidal endothelial cells but increased rapidly within 12 h of injury. By contrast, the EIIIB form was restricted to hepatic lipocytes (Ito or fat-storing cells) and appeared only after a lag of 12-24 h: it was minimal in sinusoidal endothelial cells. Both forms were minimal in hepatocytes. At the protein level, EIIIA-containing fibronectin was markedly increased within two days of injury and exhibited a sinusoidal distribution. Secretion of this form by endothelial cells was confirmed in primary culture. Matrices deposited in situ by endothelial cells from injured liver accelerated the conversion ("activation") of normal lipocytes to myofibroblast-like cells, and pretreatment of matrices with monoclonal antibody to the EIIIA segment blocked this response. Finally, recombinant fibronectin peptide containing the EIIIA segment was stimulatory to lipocytes in culture. We conclude that expression of EIIIA fibronectin by sinusoidal endothelial cells is a critical early event in the liver's response to injury and that the EIIIA segment is biologically active, mediating the conversion of lipocytes to myofibroblasts.

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1.

The endothelins (ETs) form a group of three vasoactive peptides (ET-1, ET-2, and ET-3) for which two types of cellular receptors have been identified, types A and B ET receptors (ETA and ETB receptors, respectively). To address possible targets for ETs within the liver, we isolated the four principal liver cell populations and placed them in short-term primary culture. By ligand-binding assay and mRNA levels, expression of ET receptors was greatest on hepatic lipocytes (Ito cells or fat-storing cells), which are perisinusoidal cells exhibiting features of smooth muscle cells. Moreover, lipocytes expressed both ETA and ETB receptors. The mRNA for ETB receptor, but not for ETA receptor, was detectable in sinusoidal endothelial cells and Kupffer cells; neither mRNA was detectable in hepatocytes. Both ET-1 and ET-3 elicited contraction of activated lipocytes cultured on collagen lattices; the EC50 value for ET-1 was 3 +/- 1 nM and for ET-3 was 17 +/- 12 nM. In cell isolates from injured liver (after administration of carbon tetrachloride), expression of ET receptors was unchanged. However, mRNA for ET-1 was significantly increased in activated lipocytes, suggesting an autocrine loop for the initiation of lipocyte contraction. The findings imply that ET-1 may play a role in regulating sinusoidal perfusion through its effect on lipocytes, particularly in injury states.

Cell-matrix interactions in liver.

Studies of cell-matrix interaction in liver have demonstrated the biological impact of extracellular matrix on the structure and function of liver cells, both parenchymal and mesenchymal. Much of the work involves cell culture models, in which either hepatocytes or non-parenchymal liver cells are plated on various extracellular matrix proteins and the expression of tissue-specific function is assessed. The data suggest that a basement membrane-like matrix exists within the perisinusoidal space and is critical to the maintenance of normal liver function. There are reservations concerning the accuracy of cell-culture models with respect to the intact liver, in that the precise composition and structure of this matrix still is uncertain. Nonetheless, work to date has added a new dimension to the role of the extracellular matrix of the normal liver and a new appreciation of the potential impact of pathologically altered matrix ('fibrosis') in liver disease.