Endoglin in liver fibrogenesis: Bridging basic science and clinical practice

Endoglin, also known as cluster of differentiation CD105, was originally identified 25 years ago as a novel marker of endothelial cells. Later it was shown that endoglin is also expressed in pro-fibrogenic cells including mesangial cells, cardiac and scleroderma fibroblasts, and hepatic stellate cells. It is an integral membranebound disulfide-linked 180 kDa homodimeric receptor that acts as a transforming growth factor-β(TGF-β) auxiliary co-receptor. In humans, several hundreds of mutations of the endoglin gene are known that give rise to an autosomal dominant bleeding disorder that is characterized by localized angiodysplasia and arteriovenous malformation. This disease is termed hereditary hemorrhagic telangiectasia type Ⅰ and induces various vascular lesions, mainly on the face, lips, hands and gastrointestinal mucosa. Two variants of endoglin(i.e., S- and L-endoglin) are formed by alternative splicing that distinguishes from each other in the length of their cytoplasmic tails. Moreover, a soluble form of endoglin, i.e.,sol-Eng, is shedded by the matrix metalloprotease-14 that cleaves within the extracellular juxtamembrane region. Endoglin interacts with the TGF-β signaling receptors and influences Smad-dependent and-independent effects. Recent work has demonstrated that endoglin is a crucial mediator during liver fibrogenesis that critically controls the activity of the different Smad branches. In the present review, we summarize the present knowledge of endoglin expression and function, its involvement in fibrogenic Smad signaling, current models to investigate endoglin function, and the diagnostic value of endoglin in liver disease.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence,apoptosis and attenuated TGF-β signaling

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Distribution of endoglin in early human development reveals high levels on endocardial cushion tissue mesenchyme during valve formation

Endoglin is a component of the receptor complex for transforming growth factor (TGF)-β1 and TGF-β3. We analysed its expression by immunohistochemistry in human embryos at 4–8 weeks of gestation and in hearts ranging from 4–13 weeks old. We compared endoglin distribution with that of TGF-β receptors type I (TβR-I), type II (TβR-II) and betaglycan. Endoglin was found on endothelial cells in all tissues examined, consistent with its expression in adult blood vessels. TβR-I, TβR-II and betaglycan were observed on most cell types and had an overall similar pattern of distribution. Endoglin was detected on the endocardium as early as 4 weeks, but was absent from myocardium. It was present at high levels on the endocardial cushion tissue mesenchyme from 5–8 weeks’ gestation, during heart septation and valve formation, and subsequently decreased as the valves matured. Endoglin expression in heart extracts was confirmed by Western blot analysis. TβR-I, TβR-II and betaglycan were mostly found on cardiac myocytes, but were detectable at low levels on endocardium. They were expressed transiently on cushion mesenchyme, albeit at much lower levels than endoglin. All four components of the TGF-β receptor complex were detected by RT-PCR in embryonic heart. Thus transient up-regulation of the components of the TGF-β receptor complex, and particulartly of endoglin, is associated with heart septation and valve formation during early human development.     

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

Endoglin is an endothelial-specific transforming growth factor beta (TGF-β) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-β signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified β-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and β-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-β-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/β-arrestin2 interaction is disrupted. Given that TGF-β-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.     

Tenascin-C promotes migration of hepatic stellate cells and production of type I collagen

Tenascin-C (TN-C) is an extracellular matrix glycoprotein markedly upregulated during liver fibrosis. The study is performed to explore the role of TN-C during the growth and activation of hepatic stellate cells (HSCs). We found that TN-C was accumulated accompanying with the HSC activation. Our data on cell migration assay revealed that the rTN-C treatment enhanced HSC migration in a dose- and time-dependent manner, but did not influence their proliferation. HSCs transfected with pTARGET-TN-C overexpression vector displayed increased the type I collagen (Col I) production. TN-C overexpression enhanced the process of HSC activation through TGF-β1 signaling. Moreover, the anti-α9β1 integrin antibody treatment blocked the TN-C-driven Col I increase in rat HSCs. Collectively, TN-C had a positive role in activation of HSCs mediated by TGF-β1 and α9β1 integrin, manifesting elevation of Col I production and promotion of cell migration. Our results provide a potential insight for the therapy of hepatic fibrosis.     

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Peroxynitrite-mediated matrix metalloproteinase-2 activation in human hepatic stellate cells

To investigate whether hepatic stellate cells (HSCs) alter their expression of MMPs after exposure to nitrogen oxide intermediate (NOI), a human hepatic stellate cell line, LI90 cells, was stimulated with an NO donor, SNAP, or a peroxynitrite donor, SIN-1, and the culture supernatants were analyzed by gelatin zymography or anti-MMPs immunoblot. Although SIN-1 did not enhance the secretions of MMP-1 and 13, SIN-1 induced the NF-kappaB activation, MT1-MMP expression and the secretion of activated MMP-2 in LI90 cells. These results suggest that peroxynitrite may contribute to the remodeling of the extracellular matrix in liver by activating pro-MMP-2.     

WDNM1 is Associated with Differentiation and Apoptosis of Mammary Epithelial Cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFα treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFα induced WDNM1 expression showed the NFκB-dependent mechanism since it's expression was abrogated in IκBαM (super-repressor of NFκB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFα-NFκB signal pathway, is associated with HC11 cell apoptosis.     

Involvement of KLF14 and egr-1 in the TGF-beta1 action on Leydig cell proliferation

Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-β1 exerts stimulatory and inhibitory effect depending on the type I receptor involved in the signaling pathway. The aim of the present work was to study the signaling mechanisms and the intermediates involved in the action of TGF-β1 on TM3 Leydig cell proliferation in the presence or absence of progesterone.The MTT assay showed that the presence of progesterone in the culture media lead to a proliferative effect that was blocked by Ru 486, an inhibitor of progesterone receptor; and ALK-5 did not participate in this effect.TGF-β1 (1 ng/ml) increased the expression of p15 (an inhibitor of cell cycle) in TM3 Leydig cells, and this effect was blocked by progesterone (1 μM). The expression of PCNA presented a higher increase in the cell cultured with TGF-β1 plus progesterone than in cells cultured only with TGF-β1.Progesterone induced the gene expression of endoglin, a cofactor of TGF-β1 receptor that leads to a stimulatory signaling pathway, despite of the absence of progesterone response element in endoglin gene. In addition, the presence of progesterone induced the gene expression of egr-1 and also KLF14, indicating that this steroid channels the signaling pathway into a non-canonical mechanism. In conclusion, these findings suggest that the proliferative action of TGF-β1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone.     

PPARγ/NF-κB and TGF-β1/Smad pathway are involved in the anti-fibrotic effects of levo-tetrahydropalmatine on liver fibrosis

Liver fibrosis is a necessary stage in the development of chronic liver diseases to liver cirrhosis. This study aims to investigate the anti-fibrotic effects of levo-tetrahydropalmatine (L-THP) on hepatic fibrosis in mice and cell models and its underlying mechanisms. Two mouse hepatic fibrosis models were generated in male C57 mice by intraperitoneal injection of carbon tetrachloride (CCl4) for 2 months and bile duct ligation (BDL) for 14 days. Levo-tetrahydropalmatine was administered orally at doses of 20 and 40 mg/kg. An activated LX2 cell model induced by TGF-β1 was also generated. The results showed that levo-tetrahydropalmatine alleviated liver fibrosis by inhibiting the formation of extracellular matrix (ECM) and regulating the balance between TIMP1 and MMP2 in the two mice liver fibrosis models and cell model. Levo-tetrahydropalmatine inhibited activation and autophagy of hepatic stellate cells (HSCs) by modulating PPARγ/NF-κB and TGF-β1/Smad pathway in vivo and in vitro. In conclusion, levo-tetrahydropalmatine attenuated liver fibrosis by inhibiting ECM deposition and HSCs autophagy via modulation of PPARγ/NF-κB and TGF-β1/Smad pathway.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Characterization of chicken endoglin, a member of the zona pellucida family of proteins, and its tissue expression

Endoglin is a TGF-β co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates.     

Physalin D attenuates hepatic stellate cell activation and liver fibrosis by blocking TGF-β/Smad and YAP signaling

BackgroundHepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-β1 (TGF-β1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-β/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy.PurposeThe present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo.Study design/methodsWe conducted a series of experiments using carbon tetrachloride (CCl₄)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD.ResultPD decreased TGF-β1-induced COL1A1 promoter activity. PD inhibited TGF-β1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl₄- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells.ConclusionThese results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-β/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.     

H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-β signaling in both hepatic stellate cells and hepatocytes

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial-mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl₄-induced mice, a toxicant-induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor-β (TGF-β) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR-148a and subsequently sustained the level of ubiquitin-specific protease 4 (USP4), which was an identified target of miR-148a and was able to stabilize TGF-β receptor I. In conclusion, our findings revealed a novel H19/miR-148a/USP4 axis which promoted liver fibrosis via TGF-β pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.     

Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia

Endoglin is a type I membrane protein expressed as a disulphide-linked homodimer on human vascular endothelial cells whose haploinsufficiency is responsible for the dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia (HHT). Structurally, endoglin belongs to the zona pellucida (ZP) family of proteins that share a ZP domain of ∼ 260 amino acid residues at their extracellular region. Endoglin is a component of the TGF-β receptor complex, interacts with the TGF-β signalling receptors types I and II, and modulates cellular responses to TGF-β. Here, we have determined for the first time the three-dimensional structure of the ∼ 140 kDa extracellular domain of endoglin at 25 Å resolution, using single-particle electron microscopy (EM). This reconstruction provides the general architecture of endoglin, which arranges as a dome made of antiparallel oriented monomers enclosing a cavity at one end. A high-resolution structure of endoglin has also been modelled de novo and found to be consistent with the experimental reconstruction. Each subunit comprises three well-defined domains, two of them corresponding to ZP regions, organised into an open U-shaped monomer. This domain arrangement was found to closely resemble the overall structure derived experimentally and the three modelled de novo domains were tentatively assigned to the domains observed in the EM reconstruction. This molecular model was further tested by tagging endoglin's C terminus with an IgG Fc fragment visible after 3D reconstruction of the labelled protein. Combined, these data provide the structural framework to interpret endoglin's functional domains and mutations found in HHT patients.     

Mechanism of thymosin β4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway

Liver fibrosis is a grievous global challenge, where hepatic stellate cells (HSCs) activation is a paramount step. This study analyzed the mechanism of Tβ4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway. The liver fibrosis mouse models were established via bile duct ligation (BDL) and verified by HE and Masson staining. TGF-β1-induced activated LX-2 cells were employed in vitro experiments. Tβ4 expression was determined using RT-qPCR, HSC activation markers were examined using Western blot analysis, and ROS levels were tested via DCFH-DA kits. Cell proliferation, cycle, and migration were examined by CCK-8, flow cytometry, and Transwell assays, respectively. Effects of Tβ4 on liver fibrosis, HSC activation, ROS production, and HSC growth were analyzed after transfection of constructed Tβ4-overexpressing lentiviral vectors. MAPK/NF-κB-related protein levels were tested using Western blotting and p65 expression in the nucleus was detected through immunofluorescence. Regulation of MAPK/NF-κB pathway in TGF-β1-induced LX-2 cells was explored by adding MAPK activator U-46619 or inhibitor SB203580. Furthermore, its regulating in liver fibrosis was verified by treating BDL mice overexpressing Tβ4 with MAPK inhibitor or activator. Tβ4 was downregulated in BDL mice. Tβ4 overexpression inhibited liver fibrosis. In TGF-β1-induced fibrotic LX-2 cells, Tβ4 was reduced and cell migration and proliferation were enhanced with elevated ROS levels, while Tβ4 overexpression suppressed cell migration and proliferation. Tβ4 overexpression blocked the MAPK/NF-κB pathway activation by reducing ROS production, thus inhibiting liver fibrosis in TGF-β1 induced LX-2 cells and BDL mice. Tβ4 ameliorates liver fibrosis by impeding the MAPK/NF-κB pathway activation.