Cholangiocyte endothelin 1 and transforming growth factor beta1 production in rat experimental hepatopulmonary syndrome

BACKGROUND & AIMS: Hepatic production and release of endothelin 1 plays a central role in experimental hepatopulmonary syndrome after common bile duct ligation by stimulating pulmonary endothelial nitric oxide production. In thioacetamide-induced nonbiliary cirrhosis, hepatic endothelin 1 production and release do not occur, and hepatopulmonary syndrome does not develop. However, the source and regulation of hepatic endothelin 1 after common bile duct ligation are not fully characterized. We evaluated the sources of hepatic endothelin 1 production after common bile duct ligation in relation to thioacetamide cirrhosis and assessed whether transforming growth factor beta1 regulates endothelin 1 production. METHODS: Hepatopulmonary syndrome and hepatic and plasma endothelin 1 levels were evaluated after common bile duct ligation or thioacetamide administration. Cellular sources of endothelin 1 were assessed by immunohistochemistry and laser capture microdissection of cholangiocytes. Transforming growth factor beta1 expression and signaling were assessed by using immunohistochemistry and Western blotting and by evaluating normal rat cholangiocytes. RESULTS: Hepatic and plasma endothelin 1 levels increased and hepatopulmonary syndrome developed only after common bile duct ligation. Hepatic endothelin 1 and transforming growth factor beta1 levels increased over a similar time frame, and cholangiocytes were a major source of each peptide. Transforming growth factor beta1 signaling in cholangiocytes in vivo was evident by increased phosphorylation and nuclear localization of Smad2, and hepatic endothelin 1 levels correlated directly with liver transforming growth factor beta1 and phosphorylated Smad2 levels. Transforming growth factor beta1 also stimulated endothelin 1 promoter activity, expression, and production in normal rat cholangiocytes. CONCLUSIONS: Cholangiocytes are a major source of hepatic endothelin 1 production during the development of hepatopulmonary syndrome after common bile duct ligation, but not in thioacetamide-induced cirrhosis. Transforming growth factor beta1 stimulates cholangiocyte endothelin 1 expression and production. Cholangiocyte-derived endothelin 1 may be an important endocrine mediator of experimental hepatopulmonary syndrome.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFβ3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells

Chondrogenic promotion by rhGDF5 with or without rhTGFβ3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFβ3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFβ1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFβtype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFβRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFβ1 or rhTGFβ3 treatment. ALK6 protein expression also showed increase by rhTGFβ3. rhTGFβ1/rhTGFβ3 induced ALK6 up-regulation was inhibited by SD-208, a TGFβ type I receptor inhibitor. Chondrogenesis by rhTGFβ1/rhTGFβ3 or the combination between rhTGFβ1/rhTGFβ3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFβ1/rhTGFβ3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFβ3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFβs in the synergy between rhTGFβs and rhBMPs.     

TGF-β1／Smad信号转导通路在子痫前期患者胎盘组织中的表达及其临床意义

目的检测子痫前期患者胎盘组织中TGF—β1／Smad的表达,探求TGF—β1／Smad信号转导通路在子痫前期发病中的作用。方法采用免疫组织化学法检测40例子痫前期（轻、重度）和正常孕妇15例正常妊娠妇女胎盘中TGF-β1以及Smad3、Smad4、Smad7的分布和表达,采用TUNEL法检测三组胎盘滋养细胞凋亡的情况。结果TGF-β以及Smad2、Smad3、Smad4、Smad7在正常妊娠和子痫前期患者胎盘的滋养细胞、内皮细胞、基质细胞中均有表达,以滋养细胞表达为主。TGF-β1以及Smad3、Smad4在正常妊娠组、子痫前期轻度组和重度组的表达强度及范围均呈逐渐升高趋势,Smad7在正常妊娠组、子痫前期轻度和重度组表达强度及范围依次呈逐渐下降趋势。正常妊娠和子痫前期患者胎盘的滋养细胞、内皮细胞、基质细胞均存在凋亡,以滋养细胞凋亡为主。重度子痫前期组胎盘滋养细胞凋亡（1．02）％显著高于子痫前期轻度组（0．73）％,子痫前期轻度组显著高于正常妊娠组（0．38）％,P〈0．05。滋养细胞TGF—β1的表达与滋养细胞凋亡具有相关性（r=0．96,P〈0．05）。结论子痫前期患者胎盘组织中的TGF—β1可能是通过Smad信号转导蛋白抑制滋养细胞浸润,诱导细胞凋亡。     

Collagen gel contraction as a measure of fibroblast function in an animal model of subsynovial connective tissue fibrosis

Carpal tunnel syndrome (CTS) is a peripheral neuropathy characterized by non‐inflammatory fibrosis of the subsynovial connective tissues (SSCT). A rabbit model of CTS was developed to test the hypothesis that SSCT fibrosis causes the neuropathy. We used a cell‐seeded collagen‐gel contraction model to characterize the fibrosis in this model in terms of cellular mechanics, specifically to compare the ability of SSCT cells from the rabbit model and normal rabbits to contract the gel, and to assess the effect of transforming growth factor‐β1,which is upregulated in CTS, on these cells. SSCT fibrosis was induced in six retired breeder female rabbits which were sacrificed at 6 weeks (N = 3) and 12 weeks (n = 3). An additional two rabbits served as controls. SSCT was harvested according to a standard protocol. Gels seeded with SSCT cells from rabbits sacrificed at 6 weeks had significantly higher tensile strength (p < 0.001) and Young's modulus (p < 0.001) than gels seeded with cells from rabbits sacrificed at 12 weeks or control animals. TGF‐β1 significantly increased the decay time constant (p < 0.001), tensile strength (p < 0.001), and Young's modulus (p < 0.001) regardless of the cell source. This model may be useful in screening therapeutic agents that may block SSCT fibrosis, identifying possible candidates for CTS treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:668–674, 2015.     

Dysregulated TGF‐β signaling alters bone microstructure in a mouse model of Loeys‐Dietz syndrome

Loeys‐Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor‐β (TGF‐β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF‐β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein‐labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three‐point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF‐β1–induced Smad2 and Erk1/2 phosphorylation and upregulated TGF‐β1 ligand mRNA expression, compatible with G357W as a loss‐of‐function mutation in the TGF‐β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross‐activation of TGF‐β–related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF‐β receptor signaling in bone. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1447–1454, 2015.     

Biomarkers for the Prediction and Diagnosis of Fibrostenosing Crohn’s Disease: A Systematic Review

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NO-releasing xanthine KMUP-1 bonded by simvastatin attenuates bleomycin-induced lung inflammation and delayed fibrosis

Background and purposePulmonary fibrosis (PF) is a progressing lung injury initiated by pulmonary inflammation (PI). Bleomycin (BLM) is the most common pathogenesis of PF through early PI and extensive extracellular matrix deposition. This study is aimed to determine whether NO-releasing KMUP-1 inhibits PI and PF, and if so, the benefits of KMUP-1S resulted from simvastatin (SIM)-bonding to KMUP-1.Experiment approachC57BL/6 male mice were intra-tracheally administered BLM (4 U/kg) at day 0. KMUP-1 (1–5 mg/kg), KMUP-1S (2.5 mg/kg), SIM (5 mg/kg), Plus (KMUP-1 2.5 mg/kg + SIM 2.5 mg/kg), and clarithromycin (CAM, 10 mg/kg) were orally and daily administered for 7 and 28 days, respectively, to mice, sacrificed at day-7 and day-28 to isolate the lung tissues, for examining the inflammatory and fibrotic signaling and measuring the cell population and MMP-2/MMP-9 activity in broncholaveolar lavage fluid (BAL).Key resultsKMUP-1 and KUP-1S significantly decreased neutrophil counts in BAL fluid. Fibroblastic foci were histologically assessed by H&E and Masson's trichrome stain and treated with KMUP-1 and references. Lung tissues were determined the contents of collagen and the expressions of TGF-β, α-SMA, HMGB1, CTGF, eNOS, p-eNOS, RhoA, Smad3, p-Smad3, MMP-2 and MMP-9 by Western blotting analyses, respectively. These changes areregulated by NO/cGMP and inhibited by various treatments. KMUP-1 and KMUP-1S predominantly prevented HMGB1/MMP-2 expression at day-7 and reduced TGF-β/phosphorylated Smad3 and CTGF at day-28.Conclusions and implicationsKMUP-1 and KMUP-S restore eNOS, inhibit iNOS/ROCKII/MMP-2/MMP-9, attenuate histologic collagen disposition and reduce BALF inflammatory cells, potentially useful for the treatment of BLM-lung PF.