TGFbeta-induced fibrogenesis of the pancreas

The biological cause of fibrosis is the accumulation of excessive amounts of extracellular matrix (ECM) which leads to tissue dysfunction and organ failure. A strong correlation can be found between pancreatic diseases and fibrotic processes, in particular chronic pancreatitis and pancreatic cancer. There is growing evidence that pancreatic fibrosis represents a dysregulation of the normal repair processes after injury. This concept is based on the findings that fibrosis and tissue repair involve similar biological reactions regulated by the same group of molecules. The best characterized example for these regulatory molecules are the members of the transforming growth factor beta family (TGFβ). TGFβ1 represents the prototype of this family of highly similar growth factors, with the unique ability to stimulate the expression and deposition of extracellular matrix and to inhibit its degradation. Growth factor-induced fibrotic events are targeted by a myofibroblast-like cell called pancreatic stellate cell (PSC). These cells show enhanced expression of all-important ECM proteins after TGFβ stimulation including collagen, fibronectin and proteoglycans. At the same time TGFβ inhibits the degradation of ECM by blocking the secretion of proteases and stimulating the production of naturally occurring protease inhibitors.     

TGF-beta signaling in vascular fibrosis

Transforming growth factor-beta (TGF-beta) participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy and heart failure. TGF-beta exerts pleiotropic effects on cardiovascular cells, regulating cell growth, fibrosis and inflammation. TGF-beta has long been believed to be the most important extracellular matrix regulator. We review the complex mechanisms involved in TGF-beta-mediated vascular fibrosis that includes the Smad signaling pathway, activation of protein kinases and crosstalk between these pathways. TGF-beta blockade diminishes fibrosis in experimental models, however better antifibrotic targets are needed for an effective therapy in human fibrotic diseases. A good candidate is connective tissue growth factor (CTGF), a downstream mediator of TGF-beta-induced fibrosis. Among the different factors involved in vascular fibrosis, Angiotensin II (AngII) has special interest. AngII can activate the Smad pathway independent of TGF-beta and shares with TGF-beta many intracellular signals implicated in fibrosis. Blockers of AngII have demonstrated beneficial effects on many cardiovascular diseases and are now one of the best options to block TGF-beta fibrotic responses. A better knowledge of the intracellular signals of TGF-beta can provide novel therapeutic approaches for fibrotic diseases.     

Glucocorticoids activate TGF-β induced PAI-1 and CTGF expression in rat hepatocytes

Background  

In addition to the activation of hepatic stellate cells TGF-β govern apoptosis and growth control of hepatocytes in liver injury. In non-parenchymal cells, TGF-β induces plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF) expression, which are involved in extra cellular matrix formation. Both genes were also regulated by glucocorticoids, which in certain cases showed antagonistic effects to the TGF-β-Smad 3 pathway. The purpose of our work was to investigate the influence of TGF-β and dexamethasone on PAI-1 and CTGF expression and secretion in primary hepatocytes.     

实验性肝纤维化大鼠肝组织CTGF基因DNA甲基化的变化

目的：研究实验性肝纤维化大鼠肝组织结缔组织生长因子（CTGF）启动子DNA甲基化变化及其对CTGF基因表达的影响。方法建立大鼠四氯化碳肝纤维化模型,以HE染色和Sirius red染色评估大鼠肝组织学变化;采用RT-PCR和Western blot法分别检测肝组织CTGF mRNA和蛋白质表达;采用亚硫酸氢盐修饰后测序评估肝组织CTGF基因DNA甲基化水平变化。结果在皮下注射四氯化碳6周时模型组大鼠肝组织呈III级纤维化改变;模型组大鼠肝组织CTGF mRNA及蛋白表达分别为（1.581±0.276）和（0.875±0.102）,均显著高于正常对照组分别为（0.415±0.041）和（0.115±0.083）,（P值均＆lt;0.01）;模型组CTGF基因启动子DNA甲基化率为（1.8±1.8）%,显著低于对照组（11.6±3.8）%,（P＜0.01）。结论肝纤维化大鼠肝组织CTGF基因表达上调,其机制可能与CTGF基因DNA低甲基化改变相关。     

TGF β1 and PDGF AA override Collagen type I inhibition of proliferation in human liver connective tissue cells

Background  

A marked expansion of the connective tissue population and an abnormal deposition of extracellular matrix proteins are hallmarks of chronic and acute injuries to liver tissue. Liver connective tissue cells, also called stellate cells, derived from fibrotic liver have been thoroughly characterized and correspond phenotypically to myofibroblasts. They are thought to derive from fat-storing Ito cells in the perisinusoidal space and acquire a contractile phenotype when activated by tissue injury. In the last few years it has become evident that several peptide growth factors such as PDGF AA and TGF-β are involved in the development of fibrosis by modulating myofibroblast proliferation and collagen secretion. The fact that during the development of chronic fibrosis there is concomitant deposition of collagen, a known inhibitory factor, and sustained cell proliferation, raises the possibility that stellate cells from chronic liver fibrosis patients fail to respond to normal physiologic controls.     

Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosis

Summary.  Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-β) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-β bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients ( n  =   138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients ( n  =   129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.     

Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options

Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. The extension of pathogenetic concepts of fibrosis will provide new therapeutic possibilities of interference with the fibrogenic mechanism in liver and other organs.     

Transforming growth factor-beta and connective tissue growth factor: key cytokines in scleroderma pathogenesis.

Evidence for a role for members of the transforming growth factor beta (TGF-beta) family of cytokines in the pathogensis of systemic sclerosis and other fibrotic conditions is provided from studies of TGF-beta protein and gene expression in lesional biopsy specimens, from altered responses of explanted fibroblasts to TGF-beta stimulation which are associated with increased receptor expression on these cells and from genetic data linking TGF-beta gene loci to the disease. Of the many effects of TGF-beta on fibroblast properties induction of the connective tissue growth factor/Cyr61/NOV (CCN) family members, connective tissue growth factor (CTGF) may be particularly relevant to fibrosis. Moreover, systemic sclerosis (SSc) fibroblasts demonstrate constitutive over expression of CTGF that promotes migration, proliferation and matrix production. Studies of mechanisms regulating constitutive expression of CTGF by SSc fibroblasts are currently being undertaken and indicate that a TGF-beta responsive element in the CTGF promoter is involved, although this appears to function independent of the Smad proteins, suggesting that other TGF-beta-regulated pathways may be involved. TGF-neutralizing strategies have now been shown to abrogate many animal models of fibrosis, and will soon reach the clinical arena for SSc. These agents will further clarify the role of this ligand in initiating or sustaining fibrosis and offer the exciting possibility of targeted therapy for this disease.     

The role of circulating fibrocytes in fibrosis

Fibrocytes are cells that circulate in the peripheral blood and produce connective tissue proteins such as vimentin and collagens I and III. Fibrocytes are associated with skin lesions, pulmonary fibrosis, and tumors and they contribute to the remodeling response by secreting matrix metalloproteinases. Fibrocytes can further differentiate, and they are a likely source of the contractile myofibroblast that appears in many fibrotic lesions. There is evidence in the skin for a prominent role for fibrocytes in the development of hypertrophic scars and keloids. In asthma or in experimental models of pulmonary fibrosis, fibrocytes have been shown to infiltrate areas of inflammation and tissue damage. Fibrocytes constitute part of the stromal response to tumor invasion, and there is evidence that these cells may be a prognosticator of malignant potential. IL-1, TGF-β, chemokines, and serum amyloid P modulate the appearance and function of fibrocytes. Fibrocytes themselves produce inflammatory cytokines, growth factors, and chemokines. The intercellular signals that modulate fibrocyte trafficking, proliferation, and differentiation are only partially defined, but a better understanding of these signals enable new therapies to prevent pathologic fibrosis or to improve the tissue repair response.     

结缔组织生长因子在慢性乙型肝炎和肝硬化中的表达研究

探讨结缔组织生长因子(CTGF)的肝内表达与肝炎肝硬化的关系。62例慢性乙肝或肝炎肝硬化的肝组织活检标本,采用免疫组化进行CTGF肝内表达研究。肝炎肝硬化患者CTGF的肝内表达明显高于慢性乙肝患者(P<0．01),慢性乙肝炎性活动期患者CTGF的表达高于非活动期患者(P<0．05)。CTGF的表达主要集中于汇管区和纤维化区,肝星状细胞、成纤维细胞和窦状隙内皮细胞染色常呈阳性。CTGF的表达与肝硬化密切相关,肝细胞的炎性活动可加强CTGF的表达。肝星状细胞、成纤维细胞和窦状隙内皮细胞是肝内CTGF的重要来源。     

Pediatric lung disease: from proteinases to pulmonary fibrosis

One distinctive outcome of interstitial lung diseases in childhood is the abnormal accumulation of pulmonary extracellular matrix. The clinical consequence of such excessive connective tissue accumulation is known as pulmonary fibrosis. While numerous aspects of its pathogenesis have become familiar, many key events involved in its inception and progression still remain unclear. There is now compelling evidence that lung damage due to uncontrolled proteolysis may help drive critical processes that regulate fibrotic matrix remodeling. In this regard, a number of proteinases have been implicated in promoting both the initial lung injury and the fibroproliferative repair that follows. This review summarizes the knowledge of how different matrix-targeting enzymes may act to influence the development of pediatric pulmonary fibrosis. Understanding the scientific basis of this complex process may highlight opportunities to limit unwanted proteolysis and the intensity of its fibrotic sequelae.     

Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damage

OBJECTIVE: Characteristics of osteoarthritis (OA) include cartilage damage, fibrosis, and osteophyte formation. Connective tissue growth factor (CTGF; also known as CCN2), is found in high levels in OA chondrocytes and is frequently involved in fibrosis, bone formation, and cartilage repair. The present study was therefore undertaken to investigate the potential role of CTGF in OA pathophysiology. METHODS: We transfected the synovial lining of mouse knee joints with a recombinant adenovirus expressing human CTGF and measured synovial fibrosis and proteoglycan content in cartilage on days 1, 3, 7, 14, and 28. Messenger RNA (mRNA) expression in synovium and cartilage was measured on days 3, 7, and 21. RESULTS: CTGF induced synovial fibrosis, as indicated by accumulation of extracellular matrix and an increase in procollagen type I-positive cells. The fibrosis reached a maximum on day 7 and had reversed by day 28. Levels of mRNA for matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), and transforming growth factor beta were elevated in the fibrotic tissue. TIMP-1 expression was elevated on day 3, while expression of other genes did not increase until day 7 or later. CTGF induced proteoglycan depletion in cartilage as early as day 1. Maximal depletion was observed on days 3-7. Cartilage damage was reduced by day 28. A high level of MMP-3 mRNA expression was found in cartilage. CTGF overexpression did not induce osteophyte formation. CONCLUSION: CTGF induces transient fibrosis that is reversible within 28 days. Overexpression of CTGF in knee joints results in reversible cartilage damage, induced either by the high CTGF levels or via factors produced by the CTGF-induced fibrotic tissue.     

Hypothesis: pathogenesis of systemic sclerosis

A hypothesis for the pathogenesis of systemic sclerosis (SSc) is proposed. Transforming growth factor-beta (TGF-beta) has received attention as an essential factor in the pathogenesis of various fibrotic disorders, including SSc, although some unknown additional factor has been sought as the second mediator of fibrotic disorders. Connective tissue growth factor (CTGF) has been shown to be closely related to the pathogenesis of SSc as follows: (1) CTGF mRNA expression was observed in the fibrotic lesions but not in the early nonfibrotic lesions or atrophic lesions. (2) Serum CTGF protein concentrations were significantly elevated, and correlated with skin sclerosis and lung fibrosis. (3) In our animal model, TGF-beta-induced subcutaneous fibrosis and subsequent CTGF application caused persistent fibrosis. Based on these data, we hypothesize that a 2-step process of fibrosis occurs in SSc: that is, TGF-beta induces fibrosis in the early stage and afterwards CTGF acts to maintain tissue fibrosis.     

Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine‐induced hepatic fibrosis in mice

Background: Hepatic fibrosis is characterized by a progressive accumulation of fibrillar extracellular matrix (ECM) proteins including collagen, which occurs in most types of chronic liver diseases. Transforming growth factor‐β (TGF‐β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of ECM accumulation. Aim: To evaluate the potential protective role of Smad3 deficiency in the pathogenesis of liver fibrosis induced by dimethylnitrosamine (DMN) in Smad3 null mice. Methods: Chronic hepatitis‐associated fibrosis was induced in 13 Smad3 null and 13 wild‐type (WT) mice by intraperitoneal DMN administration (10 μg/g body weight/day) for three consecutive days per week for 6 weeks. The liver was excised for macroscopic examination and histological, morphometric and immunohistochemical (IHC) analyses. For IHC, α‐smooth muscle actin (α‐SMA), collagen types I–III, TGF‐β1, connective tissue growth factor (CTGF), Smad3, Smad7 and CD3 antibodies were used. Results: At macroscopic examination, the liver of DMN‐treated Smad3 WT appeared harder with a dark brown colouring and necrotic areas compared with that from null mice. Histological and morphometric evaluation revealed a significantly higher degree of hepatic fibrosis and accumulation of connective tissue in the Smad3 WT compared with null mice. IHC evaluation showed a marked increase in α‐SMA, CTGF, collagen I‐III, TGF‐β and Smad3 staining in the liver of Smad3 WT compared with that in null mice, whereas Smad7 was increased only in null mice. Conclusions: The results indicate that Smad3 loss confers resistance to the development of DMN‐induced hepatic fibrosis. The reduced fibrotic response appears to be due to a reduction of fibrogenic myofibroblast activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of fibrosis complicating chronic hepatitis.     

Diffuse expression of heparan sulfate proteoglycan and connective tissue growth factor in fibrous septa with many mast cells relate to unresolving hepatic fibrosis of congenital hepatic fibrosis.

BACKGROUND AND AIMS: Congenital hepatic fibrosis (CHF) is characterized by dense portal/septal fibrosis and bile duct proliferation and tortuosity. In this study, the roles and significance of fibrosis-related cells and molecules in the process of progressive and unresolving fibrosis of CHF were examined in comparison with other fibrotic liver diseases. METHODS: Seven CHF livers were examined, and a total of 74 control livers (chronic viral hepatitis (CVH), alcoholic fibrosis/cirrhosis (F/C), extrahepatic biliary obstruction and livers showing non-specific reactive changes) were used as controls. All of these livers were wedge biopsied or surgically resected ones, and were formalin fixed and paraffin embedded. In addition to histologic observations, expression of heparan sulfate proteoglycan (HSPG), connective tissue growth factor (CTGF), mast cell-specific tryptase, alpha-smooth muscle actin for activated hepatic stellate cells (HSC) or myofibroblasts (MF) were immunohistochemically surveyed. HSPG and CTGF at mRNA were also examined by in situ hybridization. RESULTS: Portal/septal fibrosis of CHF were mature collagenous and elastic fiber poor, when compared with controls. HSPG and CTGF were diffusely abundant in fibrous portal tracts/septa in CHF, while they were more or less accentuated at periportal areas in alcoholic F/C and CVH. In CHF, the number of interface and portal/septal MF was increased from mild-to-moderate degree, while their increase was moderate to marked in alcoholic F/C and CVH, particularly F3/F4. While activated HSC were frequent in alcoholic F/C and CVH and they were continuous with interface MF, activated HSC in CHF were scanty. Instead, mast cells were increased in portal/septal fibrosis of CHF. Portal mononuclear cells and endothelial cells were positive for HSPG mRNA, and mononuclear cells for CTGF mRNA, and such cells were accentuated around proliferated bile ducts and ductules in CHF. CONCLUSIONS: Abundant CTGF retained diffusely in HSPG in the fibrous portal tracts/septa may be responsible for non-resolving hepatic fibrosis in CHF, and many mast cells and portal MF not related to HSC may causally relate to such characteristic finding in CHF.