Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats

Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone-an inhibitor of collagen synthesis-on thioacetamide (TAA)-induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin-positive cells expressing high levels of the collagen alpha1(I) gene and containing high levels of TIMP-2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen alpha1(I) gene, resulting in low levels of collagen. The level of TIMP-2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen alpha1(I) gene expression, TIMP-2 content, and smooth muscle actin-positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen alpha1(I) gene expression in the SV40-immortalized rat HSC-T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.     

How can transforming growth factor beta be targeted usefully to combat liver fibrosis?

Transforming growth factor beta-1 (TGF-beta 1) plays a pivotal role in tissue fibrogenesis. Understanding the factors that control resolution of fibrosis is critical to devising means to combat clinical fibrosis. Future challenges would include designing ways to block the fibrosis-specific actions of TGF-beta.Blockade of transforming growth factor beta (TGF-beta) activity in vivo in animal models has proven to be an effective means of inhibiting the fibrotic response to injury in various organs. Similarly, transgenic animals in which TGF-beta 1 expression is artificially enhanced show marked spontaneous fibrosis or increased fibrotic response to injury. TGF-beta is known to effect fibroplasias, not only by its well known action of increasing extracellular matrix synthesis but also by coordinately regulating key proteins which mediate connective tissue homeostasis. This includes down-regulation of interstitial collagenase and other matrix metalloproteinases and up-regulation of antiproteases such as tissue inhibitor of metalloproteinase I and plasminogen activator inhibitor. Whilst inhibition of TGF-beta activity appears to be well tolerated in rodents over several weeks, the ultimately lethal phenotype of TGF-beta 1 knockout mice warns us that this pluripotent cytokine is essential for normal health. Therefore, downstream pathways activated by TGF-beta, which might be specific for its fibrotic effects, might be more useful targets for human fibrotic disease therapy. For example, the TGF-beta response protein connective tissue growth factor may be a good target for antifibrotics but definitive evidence awaits development of suitable genetically modified animal models and specific inhibitors.     

Halofuginone, an inhibitor of collagen synthesis by rat stellate cells, stimulates insulin-like growth factor binding protein-1 synthesis by hepatocytes

BACKGROUND/AIMS: Halofuginone, an inhibitor of collagen synthesis, prevented and caused resolution of established hepatic fibrosis. A genomic approach in vivo was used to search for additional genes responsible for halofuginone mode of action. METHODS: Fibrosis was induced in rats by thioacetamide (TAA) and evaluated by collagen type I gene expression and the levels of collagen, tissue inhibitors of metalloproteinases-2 and smooth-muscle actin. Halofuginone was given in the diet. cDNA from liver biopsies was hybridized on Atlas arrays comprising of 588 genes. The results were confirmed by Northern blots and in situ hybridization. RESULTS: Insulin-like growth factor binding protein-1 (IGFBP-1) was one of the 13 genes differentially expressed in the fibrotic liver after halofuginone treatment. After 2 and 4 weeks, halofuginone prevented the TAA-induced down-regulation of IGFBP-1 gene expression. Halofuginone also prevented the TAA-dependent changes in IGFBP-3 gene expression. Halofuginone affected IGFBP-1 synthesis in rat hepatocytes and cells of hepatocyte origin and caused time- and dose-dependent increases in the IGFBP-1 gene expression and synthesis by HepG2 cells. The IGFBP-1 secreted by HepG2-inhibited stellate cell motility. CONCLUSIONS: Halofuginone is an anti-fibrotic drug that inhibits collagen synthesis by stellate cells and preventing alteration in the synthesis of IGFBPs by hepatic cells.     

Animal models of systemic sclerosis: insights into systemic sclerosis pathogenesis and potential therapeutic approaches

PURPOSE OF REVIEW: Animal models have been extremely valuable in contributing to a better understanding of the pathogenesis of systemic sclerosis. Discussed here are recent studies that have examined the molecular pathways and potential therapeutic approaches for systemic sclerosis using animal models. RECENT FINDINGS: Reported evidence further indicates that the immune system plays a role in modulating the fibrosis observed in the tight skin-1/+ mouse model for systemic sclerosis. CD19, interleukin-6, and interleukin-4 are involved. The injection of spleen cells into immune-compromised mice resulted in fibrotic, vascular, and immunologic alterations quite similar to those of systemic sclerosis. Transforming growth factor-beta and its signaling pathway (JAK kinase and STAT-6, Smad2/3, and Smad7) appear to play a central role in the development of fibrosis as well as monocyte chemoattractant protein-1, CCR-2, platelet-derived growth factor C, and excessive apoptosis. Viruses were shown to be possible cofactors. The therapeutic agents hepatocyte growth factor and halofuginone were shown to prevent fibrosis in animal models of systemic sclerosis. SUMMARY: The transforming growth factor-beta signaling pathway is a common mechanism of tissue fibrosis in animal models of systemic sclerosis, although numerous additional molecules modulate this pathway or have a direct effect on fibrosis.     

碱性成纤维细胞生长因子与肺纤维化

碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)是一种重要的多肽生长因子,也是成纤维细胞因子(fibroblast growth factor,FGF)家族原型成员之一.随着对细胞因子在特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)发病中作用认识的逐渐深入,近年研究发现bFGF在纤维化的肺组织、血清、肺泡灌洗液中有高表达并与肺纤维化程度呈正相关,这提示bFGF表达上调与肺纤维化关系密切,抑制促纤维化的细胞因子为IPF的治疗提供了一条新的有效途径[1].     

Bronchiolitis obliterans

Bronchiolitis obliterans in the adult patient is a relatively uncommon and vexing clinical entity. This confusion results because this pathologic finding occurs in a variety of diverse clinical settings. Bronchiolitis obliterans is a fibrotic process that primarily affects the small conducting airways. The lesion results from damage to the bronchiolar epithelium and the repair process leads to excessive proliferation of granulation tissue. The alveoli adjacent to the small airway are almost always involved; however, a considerable portion of the interstitium is usually spared. The findings in these patients may physiologically and radiographically mimic chronic obstructive pulmonary disease (COPD). On the other hand, some of the processes associated with bronchiolitis obliterans result in restrictive or mixed restrictive and obstructive ventilatory defects; consequently, they may be confused with other diffuse infiltrative lung disorders. This review will focus principally on bronchiolitis obliterans in adults, which, until recently, was considered rare. There has been heightened interest in this process in adults because of its association with the connective tissue diseases, its development following toxic fume exposure, its occurrence as a result of chronic graft versus host reactions, and the increasing recognition of patients with idiopathic forms of the disease that have an insidious onset often confused with more common problems such as COPD or idiopathic pulmonary fibrosis.     

Cytokine directed therapy in scleroderma: rationale,current status,and the future

The hallmark of scleroderma is cutaneous and visceral fibrosis characterized and by increased biosynthesis of multiple matrix proteins by interstitial fibroblasts. Studies over recent years have delineated pathways involved in promoting matrix synthesis and elucidated the molecular pathways of regulation. Central to the regulation of fibrosis are extracellular mediators, called cytokines, which are elaborated by a variety of cells, including those in the immune system, vascular cells, and fibroblasts themselves. The concept that inhibiting or promoting the action of these naturally occurring profibrotic or antifibrotic molecules, respectively, is a rational therapeutic approach to treating scleroderma and other fibrotic diseases finds support in animal studies and anticytokine therapy conducted in relation to rheumatoid arthritis and other disorders. This review looks at cytokines known or thought to play a role in scleroderma and/or other fibrotic states and at potential therapy directed at these mediators. Potential targets for therapy include transforming growth factor beta (TGF-beta), connective tissue growth factor (CTGF), IL-4, IL-13, MCP-1, and endothelin, among others.     

Blood platelets and inflammation: their relationship with liver and digestive diseases

An expansion of knowledge from basic and clinical research has highlighted the critical role of platelets in inflammation and tissue repair in addition to their established contribution to hemostasis. Activated platelets are a rich source of mediators participating to inflammation and tissue regeneration. Platelet-derived microparticles recapitulate essential platelet functions and their contribution to the pathogenesis of inflammatory diseases has been emphasized. Recent findings suggest that platelets are both friends and foes for the liver. Platelets are essential to liver regeneration, platelet-derived serotonin being critical. However platelets can also exacerbate liver damage, as in immune-mediated injury. The dual role of platelets has recently been exemplified in animal models of liver fibrosis. Platelets release profibrogenic mediators, such as CXC Chemokine Ligand 4, that is instrumental in the progression of liver fibrosis. On the other hand, thrombocytopenia aggravates liver fibrosis, an outcome linked to the downregulation of hepatic stellate cell collagen production by platelet derived hepatocyte growth factor. CD154, a key molecule in inflammation, is expressed by platelets and is a pathogenic mediator in inflammatory bowel disease. Here, we summarize some of the mechanisms linking platelets with inflammation and comment few recent articles indicating why platelets may prove to be important pathogenic mediators in liver and gastrointestinal diseases.     

Interplay between inflammation, immune system and neuronal pathways: effect on gastrointestinal motility

Sepsis is a systemic inflammatory response representing the leading cause of death in critically ill patients,mostly due to multiple organ failure.The gastrointestinal tract plays a pivotal role in the pathogenesis of sepsisinduced multiple organ failure through intestinal barrier dysfunction,bacterial translocation and ileus.In this review we address the role of the gastrointestinal tract,the mediators,cell types and transduction pathways involved,based on experimental data obtained from models of inflamma...     

Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of pro‐angiogenic and prometastatic factors in murine liver fibrosis

Background: Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy. We questioned whether a pre‐existing fibrosis affects tumour development of implanted syngenic hepatoma cells. To further investigate a selected panel of factors involved in tumour growth, tumour organ samples were characterized for gene expression of vascular endothelial growth factor (VEGF)‐A/‐C, VEGF receptors Flt1, Flk‐1, Flt‐4 and for VEGF‐A protein levels. Results: The presented data show that tumour sizes were 3.7‐fold increased and fibrotic livers had numerous satellites. Increased tumour sizes were associated with elevated intratumoral VEGF‐A protein amounts and intratumoral increased VEGF receptor gene expression levels in tumour tissue from fibrotic livers as compared with non‐fibrotic livers. Additionally, intratumoral gene expression levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 were elevated in fibrotic mice. Conclusion: Our results indicate that liver fibrosis stimulates tumour development of implanted syngenic hepatoma cells. Accelerated tumour growth was going along with elevated intratumoral VEGF‐A and VEGF‐A receptor status, which most probably mediated pro‐angiogenic and prometastatic effects in this model. Furthermore, advanced tumour spread was associated with increased MMP‐2/‐9 expression. These data suggest that the intratumoral VEGF‐A proteins levels and VEGF receptor status contribute to accelerated hepatocellular carcinoma development in fibrotic mice and that elevated MMP‐2, MMP‐9 and VEGF‐C levels could promote tumour metastasis in this model.     

What we should know about portal vein thrombosis in cirrhotic patients: A changing perspective

Portal vein thrombosis (PVT) is one of the most common complications occurring during the natural course of liver cirrhosis. Even though PVT is often asymptomatic, the worsening of liver function, an unexpected episode of gastrointestinal bleeding or ascitic decompensation may be landmarks of PVT development. Beyond these clinical manifestations, it is debated whether PVT really has an impact on liver cirrhosis natural history or rather represents only one of its consequences. Probably PVT development should not only be considered as a matter of impaired blood flow or pro-coagulation tendency. On one hand, PVT seems a consequence of the worsening in portal vein outflow due to the increased hepatic resistance in cirrhotic livers. On the other hand, vascular microthrombosis secondary to necroinflammation may cause liver ischemia and infarction, with loss of hepatic tissue (parenchymal extinction) which is replaced by fibrotic tissue. Therefore, PVT might also be considered as the overt manifestation of the liver fibrosing process evolution and anticoagulant therapy may thus have microscopic indirect effects also on the progression of liver disease. At present, a connection between PVT development and the progression of liver fibrosis/cirrhosis has not yet been demonstrated. Nevertheless, it is not clear if PVT development may worsen cirrhotic patients’ outcome by itself. Some authors tried to assess liver transplant benefit in PVT cirrhotic patients but data are contrasting. In this review, we will try to answer these questions, providing a critical analysis of data reported in literature.     

Severe fibrosis and increased expression of fibrogenic cytokines in the gastric wall of systemic sclerosis patients

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and internal organs. Although the esophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the fibrotic process and the expression of fibrogenic cytokines in the gastric wall of SSc patients with gastroesophageal involvement. METHODS: Full-thickness surgical and endoscopic gastric biopsy samples were obtained from 14 SSc patients and 10 controls. Tissue sections were either stained with Masson's trichrome or by immunohistochemistry and analyzed for the expression of types I, III, and IV collagen, alpha-smooth muscle actin (alpha-SMA), transforming growth factor beta (TGFbeta), connective tissue growth factor (CTGF), and endothelin 1 (ET-1). RESULTS: In the gastric wall of SSc patients, Masson's trichrome staining and immunohistochemistry for types I and III collagen revealed a high amount of collagen in the lamina propria that increased toward the muscularis mucosae. In addition, muscle layers showed features of atrophy, with wide areas of focal fibrosis surrounding smooth muscle cells. Type IV collagen was present around glands and small vessels, suggesting a thickening of the basal lamina. The expression of the fibrogenic cytokines TGFbeta and CTGF, ET-1, and the myofibroblast marker alpha-SMA was stronger in SSc patients than in controls. CONCLUSION: A pronounced deposition of collagen, the presence of myofibroblasts, and increased expression of several profibrotic factors are important hallmarks in the stomach of patients with SSc. The fibrotic involvement of the gastric wall may account for muscle atrophy leading to stomach hypomotility in SSc.     

Molecular and clinical basis for the regeneration of human gastrointestinal epithelia

In the gastrointestinal tract, rapid renewal of the epithelium continues throughout life. Therefore, it is believed that the gastrointestinal epithelium has a prominent capacity for regeneration when tissue damage occurs. However, we face some clinical conditions in which regeneration of the gastrointestinal epithelia is severely disturbed. One example is the refractory ulcers seen in the intestine of inflammatory bowel disease patients, and a novel therapy to regenerate damaged intestinal epithelia is earnestly desired in those conditions. Little is known about the maintenance and regeneration of the intestinal epithelia, and a molecular or clinical basis for regenerative medicine is totally lacking at the moment. In this review, we discuss recent findings of the molecules regulating the proliferation and differentiation of epithelial cells. Further study of these molecules may lead to the identification and purification of intestinal stem cells that may be used as a source for transplantation in diseased patients. Endogenous stem cells also could be manipulated to correct dysregulated or prolonged regeneration in diseased patients. Alternatively, we will raise bone marrow cells as another novel source for regenerating the intestinal epithelia. Bone marrow-derived cells are the only cells of extragastrointestinal origin that are shown to contribute to the regeneration of the gastrointestinal epithelia. In bone marrow transplant recipients, donor-derived epithelial cells substantially repopulated the gastrointestinal tract during epithelial regeneration after graft-versus-host disease or ulcer formation. Utilization of these cells may also lead to a novel therapy to regenerate the damaged gastrointestinal epithelia, whether by bone marrow transplantation or by the administration of humoral factors.     

Novel functions of platelets in the liver

Platelets contain not only proteins needed for hemostasis but also many growth factors that are required for organ development, tissue regeneration, and repair. Thrombocytopenia, which is frequently observed in patients with chronic liver disease (CLD) and cirrhosis, is due to various causes, such as decreased thrombopoietin production and accelerated platelet destruction caused by hypersplenism; however, the relationship between thrombocytopenia and hepatic pathogenesis and the role of platelets in CLD are poorly understood. Thus, in this paper, the experimental evidence for platelets improving liver fibrosis and accelerating liver regeneration is summarized and addressed based on studies conducted in our laboratory and current progress reports from other investigators. Platelets improve liver fibrosis by inactivating hepatic stellate cells to decrease collagen production. The level of intracellular cAMP is increased by adenosine through its receptors on hepatic stellate cells, thereby resulting in inactivation of these cells. Adenosine is produced by degradation of adenine nucleotides, which are stored in abundance within the dense granules of platelets. The regenerative effect of platelets in the liver consists of three mechanisms: a direct effect on hepatocytes, a cooperative effect with liver sinusoidal endothelial cells, and a collaborative effect with Kupffer cells. Based on these experiments, a clinical trial suggested that the increase in platelets induced by platelet transfusion improved liver function in patients with CLD in a clinical setting.We highlight the current knowledge concerning the role of platelets in CLD and expect to open a novel avenue for application of these clinical therapies to treat liver disease.     

Epidemiology and Risk Factors for Pulmonary Hypertension in Systemic Sclerosis

The morphogen pathways Wnt, hedgehog, and Notch are key regulators of organ development and tissue homeostasis. In adults, the tightly regulated activity of morphogen pathways is essential for cell renewal and tissue regeneration. Loss of control and persistent activation of morphogen pathways, however, can lead to a variety of diseases, including malignancy and fibrotic disorders. In recent years, pathological activation of Wnt, hedgehog, and Notch pathways have been described in systemic sclerosis (SSc) and other fibrotic diseases. Experimental models reveal that morphogen pathways drive fibroblast activation and collagen release. In these model systems, genetic or pharmacological blockade of morphogen pathways inhibits collagen release and reduces experimental fibrosis. Importantly, inhibitors for Wnt, hedgehog, and Notch are already in clinical evaluation, thereby emphasizing the translational implications of these findings. Further experimental studies, however, should deepen our knowledge before initiating clinical trials with inhibitors of morphogen pathways.     

Interplay between bone marrow and liver in the pathogenesis of hepatic fibrosis

Recent advances in the technologies of both molecular biology and regenerative medicine have made it possible to identify bone marrow (BM)-derived cells migrating into various fibrotic organs including the liver. A number of studies have reported that BM-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. On the other hand, it has also been shown that BM-derived cells express matrix metalloproteinases and contribute to the regression of experimental liver fibrosis. These contradictory results may arise, at least in part, from the uncertainty of various different methods that have been used in those studies. In this review article, we describe the interplay between BM and liver in the progression and regression of liver fibrosis, with an emphasis on the necessity of qualified methods with high specificity and sensitivity to evaluate the role of BM-derived cells in collagen production.     

Effect of gamma-interferon on collagen synthesis by normal and fibrotic human lung fibroblasts.

Increased lung collagen and increased collagen synthesis by lung fibroblasts is well recognized in pulmonary fibrosis. gamma-Interferon has been shown to inhibit collagen synthesis by fibroblasts. To understand its effect on lung fibroblasts we compared how this lymphokine affects the growth and collagen synthesis of normal and fibrotic human lung fibroblasts. The results showed that gamma-IFN inhibited DNA synthesis in all fibroblast strains examined. Both collagen production and type 1 mRNA levels were reduced in three normal and two fibrotic cell strains exposed to gamma-IFN, while they were not affected in one strain from fibrotic lung. Even though an occasional cell was unaffected by the gamma-IFN, collagen mRNA level was reduced in most cells and it remained reduced for 48 h after removing the gamma-IFN. These results show that gamma-IFN inhibits the growth of fibroblast cultures derived from normal and fibrotic human lungs and suppresses collagen synthesis in most of these cells.