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1
Takeuchi, H., S. Kubota, E. Murakashi, et al. "Nicotine-induced CCN2: from Smoking to Periodontal Fibrosis." Journal of Dental Research 89, no. 1 (2009): 34–39. http://dx.doi.org/10.1177/0022034509353403.
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Since fibrosis is observed in smokers’ gingiva, it was hypothesized that fibrosis was caused by nicotine in the periodontium. Therefore, in this study, we investigated the effects of nicotine on the induction of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF), in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells. With 1 μg/mL nicotine, vacuolization and attenuated proliferation were observed. Interestingly, 1 μg/mL nicotine increased the production of CCN2/CTGF protein in both cells without increasing mRNA expression. Furthermore, type I collagen mRNA and protein were also increased and were significantly blocked by a CCN2/CTGF neutralizing antibody. This is the first report to describe a relationship between nicotine and CCN2/CTGF in periodontal tissue cells. Analysis of our data also indicated that nicotine was cytotoxic, while it increased CCN2/CTGF and, eventually, type I collagen production. These findings suggest that periodontal fibrosis can be promoted by nicotine from smoking via effects on CCN2/CTGF.
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Romero, Jose R., Dennis A. Ricupero, Alicia Rivera, Ronald H. Goldstein, and Paul R. Conlin. "Activation of Na + /Ca 2+ Exchanger in Kinin B 1 Receptor-Stimulated Human Fibroblast Is Associated with Collagen Production." Hypertension 36, suppl_1 (2000): 710–20. http://dx.doi.org/10.1161/hyp.36.suppl_1.710.
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P148 The arterial wall in hypertension is characterized by thickening of the media, in part due to increased deposition of connective tissue. Autocrine and paracrine factors may participate in this process; including products of the kallikrein-kinin system. We evaluated early signal transduction events and effects on collagen formation in B 1 -stimulated human myofibroblast cells (IMR-90). We measured cytosolic calcium (Ca cyt ) levels in cells loaded with FURA-2AM. Gene expression of connective tissue growth factor (CTGF) and α1(I) collagen was determined by estimating mRNA levels using Northern analysis of B 1 stimulated cells. Activation of the B 1 receptor with des-arg 10 -kallidin stimulated a three-fold increase in CTGF mRNA by increasing its stability. Furthermore, B 1 receptor activation caused an increase in α1(I) collagen mRNA and a four-fold increase in type I collagen synthesis in these cells; events not observed in B 2 receptor-stimulated cells. Activation of the B 1 receptor stimulated a dose dependent rise in Ca cyt (EC 50 =1.9nM) which was completely inhibited by des-arg 10 -[leu 9 ]-kallidin (100nM), a B 1 receptor antagonist. Isosmotic replacement of extracellular Na + with N -methyl,D-glucamine blocked > 90% of the B 1 stimulated rise in Ca cyt . A similar effect was observed when Ca 2+ was removed from the extracellular media, suggesting a role for the plasma membrane Na + /Ca 2+ exchanger (NCX). To further define a role for the NCX on CTGF formation we used dichlorobenzamil (DCB) and KB-R7943, two specific NCX inhibitors. DCB completely blocked the activation of B 1 receptor induced increase in CTGF mRNA stability while not affecting basal CTGF mRNA levels. In contrast, preincubation with EIPA, an amiloride analog, did not affect basal or stimulated CTGF mRNA levels. Furthermore, 60μM KB-R7943 blocked the B 1 stimulated rise in Ca cyt . NCX isoform 1 was identified in these cells using RT-PCR and immuno-detection. Thus, B 1 receptor stimulation increases fibrogenesis through a mechanism that involves modulation of cation metabolism via reverse-mode activation of NCX.
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Chen, Chung-Ming, Hsiu-Chu Chou, Leng-Fang Wang, and Yaw-Dong Lang. "Experimental Oligohydramnios Decreases Collagen in Hypoplastic Fetal Rat Lungs." Experimental Biology and Medicine 233, no. 11 (2008): 1334–40. http://dx.doi.org/10.3181/0803-rm-99.
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Neonates with premature rupture of the membrane and oligohydramnios have an increased risk of acute respiratory morbidity. The aims of this study are to investigate the effects of experimental oligohydramnios on transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) expressions and collagen level in fetal rat lungs. On day 16 of gestation, we anesthetized timed pregnant Sprague-Dawley dams, punctured the uterine wall and fetal membranes of each amniotic sac which resulted in oligohydramnios. Fetuses in the opposite uterine horn served as controls. On days 19 and 21 of gestation, fetuses were delivered by cesarean section. Rats exposed to oligohydramnios exhibited significantly lower lung weight/body weight ratios on days 19 and 21 of gestation than did the control rats. Lung type I collagen and TGF-β1 mRNA expressions and lung collagen levels were significantly decreased in rats exposed to oligohydramnios on days 19 and 21 of gestation. Type I collagen and inhibitors of metalloproteinase-1 (TIMP-1) proteins were decreased and matrix metalloproteinase-1 (MMP-1) was increased in oligohydramnios-exposed rats on days 19 and 21 of gestation. CTGF mRNA expressions were comparable between control and oligohydramnios-exposed rats on days 19 and 21 of gestation. These data suggest that downregulation of collagen might be involved in the pathogenesis of oligohydramnios-induced respiratory morbidity.
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Zhou, Hong, Caixia Fang, Lihui Zhang, Yonggui Deng, Mian Wang, and Fengling Meng. "Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, ameliorates hepatic fibrosis in rats with type 2 diabetes." Chinese Medical Journal 127, no. 2 (2014): 225–31. http://dx.doi.org/10.3760/cma.j.issn.0366-6999.20131917.
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Background Hyperglycemia may accelerate liver fibrosis. Currently, there is no effective treatment for liver fibrosis induced by type 2 diabetes. The study aim was to investigate whether RhoA/Rho kinase (ROCK) pathway is involved in liver fibrosis in the rats with type 2 diabetes and define the protective effects of fasudil on livers. Methods A rat model of type 2 diabetes was established by high fat diet combined with streptozotocin (30 mg/kg, intraperitoneal injection). Animals were randomly assigned to 3 groups: control rats, untreated diabetic rats that received vehicle and fasudil-treated diabetic rats that received ROCK inhibitor fasudil hydrochloride hydrate (10 mg/kg per day, intraperitoneal injection, for 14 weeks). The morphological features of liver were observed by HE staining. Accumulation of collagen in livers was determined by Masson staining and the measurement of hydroxyproline. The mRNA expression of transforming growth factor-β1 (TGFβ1), connective tissue growth factor (CTGF), type-I, and type-III procollagen was assessed with real-time polymerase chain reaction. The phosphorylation of myosin phosphatase target subunit-1 (MYPT1) and the protein levels of TGFβ1 and α-smooth muscle actin (α-SMA) were evaluated by Western blotting. Results Compared with control rats, untreated diabetic rats showed higher values of collagen and hydroxyproline in livers (P <0.01), the phosphorylation of MYPT1 and the protein levels of TGFβ1 and α-SMA were increased (P <0.01), and the mRNA expression of TGFβ1, CTGF, type-I, and type-III procollagen was upregulated (P <0.01); compared with untreated diabetic rats, treatment with fasudil signifcantly reduced values of collagen and hydroxyproline (P <0.01), and decreased the phosphorylation of MYPT1 and the levels of TGFβ1 and α-SMA (P <0.01), concomitant with the downregulation of TGFβ1/CTGF, type-I, and type-III procollagen mRNA expression (P <0.01). Conclusions Fasudil ameliorates liver fibrosis in rats with type 2 diabetes at least partly by inhibiting TGFβ1/CTGF pathway and α-SMA expression. Inhibition of RhoA/ROCK may be a novel therapeutic target for liver fibrosis in diabetic non-alcoholic steatohepatitis.
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Wang, Shinong, та Raimund Hirschberg. "BMP7 antagonizes TGF-β-dependent fibrogenesis in mesangial cells". American Journal of Physiology-Renal Physiology 284, № 5 (2003): F1006—F1013. http://dx.doi.org/10.1152/ajprenal.00382.2002.
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Exogenous administration of recombinant human bone morphogenetic protein (BMP)-7 was recently shown to ameliorate renal glomerular and interstitial fibrosis in rodents with experimental renal diseases. We tested the hypothesis that BMP7 functions by antagonizing profibrogenic events that are induced by transforming growth factor (TGF)-β in cultured mesangial cells. Incubation of murine mesangial cells with TGF-β (50–200 pM) increased cell-associated collagen type IV and fibronectin, soluble collagen type IV, thrombospondin, and connective tissue growth factor (CTGF). Coincubation with recombinant human BMP7 (200 pM) reduced the increase of these ECM proteins and CTGF. The changes in collagen type IV and fibronectin proteins occurred without concomitant changes in collagen type α1IV and fibronectin mRNA levels, suggesting that TGF-β and BMP7 act primarily by affecting ECM protein degradation. Indeed, TGF-β decreases the levels and activity of matrix metalloprotease (MMP)-2, the major metalloprotease that is secreted by mesangial cells. Moreover, BMP7 inhibits TGF-β-induced activation of MMP2. Because TGF-β reduces the activity of MMPs through increasing plasminogen activator inhibitor (PAI)-1, we tested whether BMP7 interferes with this TGF-β effect. BMP7 reduces, by about two-thirds, the activation of a PAI-1 promoter/luciferase reporter in cells stably transfected with this construct. The findings from these studies indicate that BMP7 reduces TGF-β-induced ECM protein accumulation in cultured mesangial cells primarily by maintaining levels and activity of MMP2 partially through prevention of TGF-β-dependent upregulation of PAI-1.
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Chaqour, Brahim, Catherine Whitbeck, Ji-Soo Han, et al. "Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction." American Journal of Physiology-Endocrinology and Metabolism 283, no. 4 (2002): E765—E774. http://dx.doi.org/10.1152/ajpendo.00131.2002.
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Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.
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Zhang, Lin, Jin Tan, Yi-Ping Liu, Xun Liu та Mang Luo. "Curcumin relieves the arecoline-induced fibrosis of oral mucosal fibroblasts via inhibiting HIF-1α/TGF-β/CTGF signaling pathway: an in vitro study". Toxicology Research 10, № 3 (2021): 631–38. http://dx.doi.org/10.1093/toxres/tfab046.
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Abstract Oral submacosal fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis. The current treatments for OSF have failed to achieve the desired curative effect. Here, we propose that curcumin has excellent therapeutic effect on OSF and explore its specific mechanism. Transwell assay was performed to detected cell migration. Flow cytometry was used to measured apoptosis. And MTT assay was performed to test cell viability. Gene and protein levels were detected by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Our results displayed that curcumin treatment reduced fibrosis-related molecules (collagen type I alpha 1, collagen type III alpha 1, tissue inhibitor of metalloprotease 2) in arecoline-treated oral mucosal fibroblasts and elevated matrix metalloproteinase 2 expression. Additionally, curcumin could suppress cell proliferation and migration, and enhance the apoptosis of arecoline-treated normal oral mucosal fibroblasts. Most importantly, the hypoxia-inducible factor-1α (HIF-1α), transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) expressions in arecoline-treated normal oral mucosal fibroblasts were reduced after exposure to curcumin, whereas the activation of HIF-1α/TGF-β/CTGF axis reversed curcumin’s effect on improving fibrosis of arecoline-treated normal oral mucosal fibroblasts. Therefore, curcumin alleviated oral submucosal fibrosis via inhibiting HIF-1α/TGF-β/CTGF axis. In summary, curcumin effectively inhibited the migration and proliferation and promoted apoptosis in arecoline-induced normal oral mucosal fibroblasts by inactivating HIF-1α/TGF-β/CTGF pathway. And curcumin might be a potential therapeutic drug for OSF treatment.
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Hillege, Michèle, Ricardo Galli Caro, Carla Offringa, Gerard de Wit, Richard Jaspers та Willem Hoogaars. "TGF-β Regulates Collagen Type I Expression in Myoblasts and Myotubes via Transient Ctgf and Fgf-2 Expression". Cells 9, № 2 (2020): 375. http://dx.doi.org/10.3390/cells9020375.
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Transforming Growth Factor β (TGF-β) is involved in fibrosis as well as the regulation of muscle mass, and contributes to the progressive pathology of muscle wasting disorders. However, little is known regarding the time-dependent signalling of TGF-β in myoblasts and myotubes, as well as how TGF-β affects collagen type I expression and the phenotypes of these cells. Here, we assessed effects of TGF-β on gene expression in C2C12 myoblasts and myotubes after 1, 3, 9, 24 and 48 h treatment. In myoblasts, various myogenic genes were repressed after 9, 24 and 48 h, while in myotubes only a reduction in Myh3 expression was observed. In both myoblasts and myotubes, TGF-β acutely induced the expression of a subset of genes involved in fibrosis, such as Ctgf and Fgf-2, which was subsequently followed by increased expression of Col1a1. Knockdown of Ctgf and Fgf-2 resulted in a lower Col1a1 expression level. Furthermore, the effects of TGF-β on myogenic and fibrotic gene expression were more pronounced than those of myostatin, and knockdown of TGF-β type I receptor Tgfbr1, but not receptor Acvr1b, resulted in a reduction in Ctgf and Col1a1 expression. These results indicate that, during muscle regeneration, TGF-β induces fibrosis via Tgfbr1 by stimulating the autocrine signalling of Ctgf and Fgf-2.
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McDonald, Emily A., Ling Cheng, Blanca Jarilla, et al. "Maternal Infection with Schistosoma japonicum Induces a Profibrotic Response in Neonates." Infection and Immunity 82, no. 1 (2013): 350–55. http://dx.doi.org/10.1128/iai.01060-13.
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ABSTRACTThe global burden of schistosomiasis is significant, with fibrosis a major associated morbidity and the primary cause of mortality. We have previously shown that schistosomiasis during pregnancy upregulates proinflammatory cytokines in the cord blood. In this study, we extend these findings to include a large panel of fibrosis-associated markers. We developed a multiplex bead-based assay to measure the levels of 35 proteins associated with fibrosis. Cord blood from 109 neonates born to mothers residing in an area ofSchistosoma japonicumendemicity was assessed for these molecules. Ten mediators were elevated in the cord blood from schistosome-infected pregnancies, including insulin-like growth factor 1 (IGF-1), tumor growth factor β1 (TGF-β1), connective tissue growth factor (CTGF), procollagen I carboxy-terminal propeptide (PICP), amino-telopeptide of type 1 collagen (ICTP), collagen VI, desmosine, matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP-1), and TIMP-4. Many of these were also positively correlated with preterm birth (PICP, ICTP, MMP-2, TGF-β1, desmosine, CTGF, TIMP-1). In addition, birth weight was 168 g lower for infants with detectable levels of CTGF than for those with CTGF levels below the level of detection. Maternal schistosomiasis results in upregulation of fibrosis-associated proteins in the cord blood of the neonate, a subset of which are also associated with adverse birth outcomes. As the first report of fibrosis-associated molecules altered in the newborn of infected mothers, this study has broad implications for the health of the fetus, stretching from gestation to adulthood.
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Roestenberg, Peggy, Frans A. van Nieuwenhoven, Jaap A. Joles, et al. "Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice." American Journal of Physiology-Renal Physiology 290, no. 6 (2006): F1344—F1354. http://dx.doi.org/10.1152/ajprenal.00174.2005.
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Connective tissue growth factor (CTGF) is overexpressed in diabetic nephropathy (DN) and has therefore been implicated in its pathogenesis. The objective of the present study was to determine the tissue distribution of increased CTGF expression and the relationship of plasma, urinary, and renal CTGF levels to the development and severity of DN. We studied the relationship between CTGF and renal pathology in streptozotocin (STZ)-induced diabetes in C57BL/6J mice. Diabetic and age-matched control mice were killed after 1, 2, 4, and 9 wk of diabetes. In addition, key parameters of diabetes and DN were analyzed in 10-mo-old diabetic ob/ob mice and their ob/+ littermates. STZ-induced diabetic mice showed a significantly increased urinary albumin excretion after 1 wk and increased mesangial matrix score after 2 wk. Increased renal fibronectin, fibronectin ED-A, and collagen IVα1 expression, as well as elevated plasma creatinine levels, were observed after 9 wk. After 2 wk, CTGF mRNA was upregulated threefold in the renal cortex. By 9 wk, CTGF mRNA was also increased in the heart and liver. In contrast, transforming growth factor-β1 mRNA content was significantly increased only in the kidney by 9 wk. Renal CTGF expression was mainly localized in podocytes and parietal glomerular epithelial cells, and less prominent in mesangial cells. In addition, plasma CTGF levels and urinary CTGF excretion were increased in diabetic mice. Moreover, albuminuria strongly correlated with urinary CTGF excretion ( R = 0.83, P < 0.0001). Increased CTGF expression was also demonstrated in type 2 diabetic ob/ob mice, which points to a causal relationship between diabetes and CTGF and thus argues against a role of STZ in this process. The observed relationship of podocyte and urinary CTGF to markers of DN suggests a pathogenic role of CTGF in the development of DN.
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Ploeg, Meike C., Chantal Munts, Tayeba Seddiqi та ін. "Culturing of Cardiac Fibroblasts in Engineered Heart Matrix Reduces Myofibroblast Differentiation but Maintains Their Response to Cyclic Stretch and Transforming Growth Factor β1". Bioengineering 9, № 10 (2022): 551. http://dx.doi.org/10.3390/bioengineering9100551.
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Isolation and culturing of cardiac fibroblasts (CF) induces rapid differentiation toward a myofibroblast phenotype, which is partly mediated by the high substrate stiffness of the culture plates. In the present study, a 3D model of Engineered Heart Matrix (EHM) of physiological stiffness (Youngs modulus ~15 kPa) was developed using primary adult rat CF and a natural hydrogel collagen type 1 matrix. CF were equally distributed, viable and quiescent for at least 13 days in EHM and the baseline gene expression of myofibroblast-markers alfa-smooth muscle actin (Acta2), and connective tissue growth factor (Ctgf) was significantly lower, compared to CF cultured in 2D monolayers. CF baseline gene expression of transforming growth factor-beta1 (Tgfβ1) and brain natriuretic peptide (Nppb) was higher in EHM-fibers compared to the monolayers. EHM stimulation by 10% cyclic stretch (1 Hz) increased the gene expression of Nppb (3.0-fold), Ctgf (2.1-fold) and Tgfβ1 (2.3-fold) after 24 h. Stimulation of EHM with TGFβ1 (1 ng/mL, 24 h) induced Tgfβ1 (1.6-fold) and Ctgf (1.6-fold). In conclusion, culturing CF in EHM of physiological stiffness reduced myofibroblast marker gene expression, while the CF response to stretch or TGFβ1 was maintained, indicating that our novel EHM structure provides a good physiological model to study CF function and myofibroblast differentiation.
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Wu, Shi-Bei, Tzu-Yu Hou, Hui-Chuan Kau та Chieh-Chih Tsai. "Effect of Pirfenidone on TGF-β1-Induced Myofibroblast Differentiation and Extracellular Matrix Homeostasis of Human Orbital Fibroblasts in Graves’ Ophthalmopathy". Biomolecules 11, № 10 (2021): 1424. http://dx.doi.org/10.3390/biom11101424.
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Pirfenidone is a pyridinone derivative that has been shown to inhibit fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Its effect on orbital fibroblasts remains poorly understood. We investigated the in vitro effect of pirfenidone in transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation and extracellular matrix (ECM) homeostasis in primary cultured orbital fibroblasts from patients with Graves’ ophthalmopathy (GO). The expression of fibrotic proteins, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin, and collagen type I, was determined by Western blots. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for the ECM homeostasis were examined. After pretreating the GO orbital fibroblasts with pirfenidone (250, 500, and 750 μg/mL, respectively) for one hour followed by TGF-β1 for another 24 h, the expression of α-SMA, CTGF, fibronectin, and collagen type I decreased in a dose-dependent manner. Pretreating the GO orbital fibroblasts with pirfenidone not only abolished TGF-β1-induced TIMP-1 expression but recovered the MMP-2/-9 activities. Notably, pirfenidone inhibited TGF-β1-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), the critical mediators in the TGF-β1 pathways. These findings suggest that pirfenidone modulates TGF-β1-mediated myofibroblast differentiation and ECM homeostasis by attenuating downstream signaling of TGF-β1.
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Liu, Mengxuan, Megumi Honjo, Reiko Yamagishi, and Makoto Aihara. "Effects of Brimonidine, Latanoprost, and Omidenepag on Tunicamycin-Induced Endoplasmic Reticulum Stress and Fibrosis in Human Trabecular Meshwork Cells." Biomolecules 15, no. 3 (2025): 389. https://doi.org/10.3390/biom15030389.
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This study evaluated the effects of α2-adrenergic agonist, prostaglandin F2α analog, and EP2 receptor agonist on tunicamycin-induced endoplasmic reticulum (ER) stress and fibrosis in human trabecular meshwork (TM) cells. Human TM cells were treated with tunicamycin for 24 h, followed by cotreatment with brimonidine (BRI), latanoprost (LAT), or omidenepag (OMD). Immunocytochemistry was used to assess expressions of collagen type I alpha 1 chain (COL1A1), fibronectin, F-actin, and alpha-smooth muscle actin (α-SMA). Western blotting was performed to evaluate levels of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Real-time qPCR was used to examine the mRNA expressions of COL1A1, connective tissue growth factor (CTGF), fibronectin, α-SMA, CHOP, GRP78, and sXBP-1. Expressions of COL1A1, CTGF, F-actin, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1 significantly increased after tunicamycin treatment. BRI cotreatment significantly downregulated the mRNA and protein expressions of GRP78, and LAT or OMD cotreatment significantly reduced the CHOP and sXBP-1 expressions compared to the tunicamycin-treated group. BRI, LAT, or OMD cotreatment significantly attenuated cellular cytoskeletal changes and the increase of fibrosis markers such as COL1A1, CTGF, fibronectin, and α-SMA. In addition, COL1A1 mRNA expression was significantly lowered with LAT or OMD cotreatment compared to the BRI-cotreated group. Cotreatment with α2-adrenergic agonist, prostaglandin F2α analog, or EP2 receptor agonist alleviates tunicamycin-induced ER stress in human TM cells.
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Yang, Guimin, Yupeng Deng, Guangming Cao, and Chongdong Liu. "Galectin-3 promotes fibrosis in ovarian endometriosis." PeerJ 12 (February 14, 2024): e16922. http://dx.doi.org/10.7717/peerj.16922.
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Objective This study aimed to investigate the potential role of galectin-3 (Gal-3) in the pathogenesis of fibrotic alterations in ovarian endometriosis (OVE). Methods In this study, we collected the ectopic endometrial tissues and eutopic endometrial tissues from 31 OVE patients treated by laparoscopy, and the eutopic endometrial tissues from 23 non-OVE patients with leiomyoma or other benign diseases were used as control. Hematoxylin and eosin (H&E) and Masson’s trichrome staining were utilized for histopathological assessment. The primary normal endometrial stromal cells (NESC), ectopic endometrial stromal cells (ECSC), and eutopic endometrial stromal cells (EUSC) were isolated. Gal-3 overexpression plasmids (Gal-OE) and short hairpin RNA targeting Gal-3 (Gal-3-shRNA) were transfected into the immortalized human endometriotic cell line 12Z, respectively. RT-qPCR, Western blot analysis, and immunohistochemistry were used to detect the mRNA and protein expression levels of Gal-3, type I collagen (COL-1), connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA), respectively. Results H&E and Masson staining showed that ovarian ectopic endometrium exhibited glandular hyperplasia, high columnar glandular epithelium, apical plasma secretion, more subnuclear vacuoles, and obvious fibrosis, compared with normal endometrium. The mRNA and protein levels of Gal-3 , CTGF, α-SMA, and COL-1 were all upregulated in the ectopic endometrial tissues of OVE patients compared to the eutopic endometrial tissues from OVE patients and non-OVE patients. Moreover, ECSC expressed higher levels of Gal-3, CTGF, α-SMA, and COL-1 than EUSC and NESC. Follow-up investigations demonstrated that the Gal-3 overexpression substantially increased fibrosis-related markers including CTGF, α-SMA, and COL-1 within the 12Z cell line. Conversely, Gal-3 knockdown showed the opposite effects. Conclusion Gal-3 promotes fibrosis in OVE, positioning it as a prospective therapeutic target for mitigating fibrosis in endometriosis.
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García-Bañuelos, Jesús, Edén Oceguera-Contreras, Ana Sandoval-Rodríguez, et al. "AdhMMP8 Vector Administration in Muscle: An Alternate Strategy to Regress Hepatic Fibrosis." Cells 12, no. 17 (2023): 2127. http://dx.doi.org/10.3390/cells12172127.
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The development of several vaccines against the SARS-CoV2 virus and their application in millions of people have shown efficacy and safety in the transfer of genes to muscle turning this tissue into a protein-producing factory. Established advanced liver fibrosis, is characterized by replacement of hepatic parenchyma by tissue scar, mostly collagen type I, with increased profibrogenic and proinflammatory molecules gene expression. Matrix metalloproteinase 8 (MMP-8) is an interstitial collagen-degrading proenzyme acting preferentially on collagen type I when activated. This study was carried out to elucidate the effect of an intramuscularly delivered adenoviral vector containing proMMP-8 gene cDNA (AdhMMP8) in male Wistar rats with experimental advanced liver fibrosis induced by thioacetamide. Therapeutic effects were monitored after 1, 2, or 3 weeks of a single dose (3 × 1011 vp/kg) of AdhMMP8. Circulating and liver concentration of MMP-8 protein remained constant; hepatic fibrosis decreased up to 48%; proinflammatory and profibrogenic genes expression diminished: TNF-α 2.28-fold, IL-1 1.95-fold, Col 1A1 4-fold, TGF-β1 3-fold and CTGF 2-fold; and antifibrogenic genes expression raised, MMP-9 2.8-fold and MMP-1 10-fold. Our data proposes that the administration of AdhMMP8 in muscle is safe and effective in achieving liver fibrosis regression at a comparable extent as when the adenoviral vector is delivered systemically to reach the liver, using a minimally invasive procedure.
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DU, Li-qun, Hong-ling YANG, Xin-yi WU, Shen-guo WANG, and Yun LI. "Effect of poly(DL-lactide-co-glycolide) on scar formation after glaucoma filtration surgery." Chinese Medical Journal 126, no. 23 (2013): 4528–35. http://dx.doi.org/10.3760/cma.j.issn.0366-6999.20131766.
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Background Glaucoma filtering surgery (GFS) is the most common procedure performed in the treatment of glaucoma. Although antiscarring agents help prevent postsurgical scarring and improve glaucoma surgical outcomes, they may be associated with an increased incidence of severe and potentially blinding complications. Poly(DL-lactide-co-glycolide) (PDLLA/GA) is a bioresorbable polymer, which can be prepared with a large range of physical, mechanical, and biological properties and has been widely used in medicine, including as an absorbable suture and a drug carrier and especially as a scaffold in tissue engineering. This study aimed to evaluate the effect of PDLLA/GA on scar formation after glaucoma filtration surgery (GFS). Methods Forty-eight New Zealand white rabbits were divided into two groups randomly and GFS was performed on the right eye of each. PDLLA/GA membranes were put under the sclera flap for evaluation. GFS with no membrane inserted served as control. Clinical evaluations of intraocular pressure (IOP) and the presence of a filtration bleb were performed at intervals (3 days, 1, 2, 4, 8, 12, 20, and 24 weeks) postoperatively. At each time point, three eyes per group were excised to observe histological changes such as inflammation and scar formation and the expression of collagen type IV, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). The expression of connective tissue growth factor (CTGF) mRNA was determined by reverse transcription-polymerase chain reaction. Results The lower IOP level and an effective bleb were maintained for a long time after GFS in the PDLLA/GA group. The histological analysis showed less inflammation and scar formation, weaker expression of collagen type IV and PCNA, more intense MMP-9 and TIMP-1, slightly elevated ratio of MMP-9 and TIMP-1, and a smaller increase in CTGF mRNA postoperatively in the PDLLA/GA group but less than the control group (P <0.05). Conclusion PDLLA/GA membranes may be promising for preventing fibrosis after GFS.
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Correll, Kelly A., Karen E. Edeen, Rachel L. Zemans, et al. "Transitional human alveolar type II epithelial cells suppress extracellular matrix and growth factor gene expression in lung fibroblasts." American Journal of Physiology-Lung Cellular and Molecular Physiology 317, no. 2 (2019): L283—L294. http://dx.doi.org/10.1152/ajplung.00337.2018.
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Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-β (TGF-β)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-β may provide another approach to limiting the development of fibrosis after alveolar injury.
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Sheykhhasan, Mohsen, Hoda Fazaeli, Azar Sheikholeslami, Seyed Abbas Seyedebrahimi, Seyed Jalal Eshagh Hoseini, and Naser Kalhor. "Identification of Diagnostic Biomarkers by Bioinformatics Analysis in the Inflamed and Non-inflamed Intestinal Mucosa of Patients With Crohn’s Disease." Research in Molecular Medicine 9, no. 3 (2021): 209–20. http://dx.doi.org/10.32598/rmm.9.3.1069.4.
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Background: Crohn’s Disease (CD) is a type of inflammatory bowel disease that, despite its unknown etiology, is generally associated with genetics, immune system, and environmental factors. In this study, we uncover transcriptional signatures in patients with CD and subsequently explain the putative molecular pathways in the inflamed and non-inflamed intestinal mucosa. Materials and Methods: We obtain GSE83448 gene expression profiles from the Omnibus gene expression database. Also, for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of Differentially Expressed Gene (DEG) pathways, we used DAVID software. DEGs were detected in the inflamed and non-inflamed intestinal mucosa of CD patients compared to the control group using the GEO2R instrument. Significant modules and hub genes were identified after producing protein-protein interaction (PPIs) networks of DEGs using Cytoscape software. Results: The 10 specific hub genes of CD, including Matrix Metallopeptidase 2 (MMP2), Cadherin 1 (CDH1), Periostin (POSTN), Collagen type I alpha 2 chain (COL1A2), C-X-C motif chemokine ligand 8 (CXCL8), Collagen type III alpha 1 chain (COL3A1), JUN, Serine Protease Inhibitor clade E member 1 (SERPINE1), Integrin alpha M (ITGAM), and Connective Tissue Growth Factor (CTGF), were used as biomarkers to discriminate between inflamed and non-inflamed intestinal mucosa groups in patients. Conclusion: These findings could lead to new molecular targets and diagnostic biomarkers for both inflamed and non-inflamed intestinal mucosa in CD patients.
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Trundle, Jessica, Viktorija Cernisova, Alexis Boulinguiez, Ngoc Lu-Nguyen, Alberto Malerba, and Linda Popplewell. "Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy." Biomedicines 12, no. 1 (2024): 216. http://dx.doi.org/10.3390/biomedicines12010216.
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Duchenne muscular dystrophy (DMD) is characterised by fibrotic tissue deposition in skeletal muscle. We assessed the role of periostin in fibrosis using mdx mice, an established DMD murine model, for which we conducted a thorough examination of periostin expression over a year. RNA and protein levels in diaphragm (DIA) muscles were assessed and complemented by a detailed histological analysis at 5 months of age. In dystrophic DIAs, periostin (Postn) mRNA expression significantly exceeded that seen in wildtype controls at all timepoints analysed, with the highest expression at 5 months of age (p < 0.05). We found Postn to be more consistently highly expressed at the earlier timepoints compared to established markers of fibrosis like transforming growth factor-beta 1 (Tgf-β1) and connective tissue growth factor (Ctgf). Immunohistochemistry confirmed a significantly higher periostin protein expression in 5-month-old mdx mice compared to age-matched healthy controls (p < 0.01), coinciding with a significant fibrotic area percentage (p < 0.0001). RT-qPCR also indicated an elevated expression of Tgf-β1, Col1α1 (collagen type 1 alpha 1) and Ctgf in mdx DIAs compared to wild type controls (p < 0.05) at 8- and 12-month timepoints. Accordingly, immunoblot quantification demonstrated elevated periostin (3, 5 and 8 months, p < 0.01) and Tgf-β1 (8 and 12 months, p < 0.001) proteins in the mdx muscle. These findings collectively suggest that periostin expression is a valuable marker of fibrosis in this relevant model of DMD. They also suggest periostin as a potential contributor to fibrosis development, with an early onset of expression, thereby offering the potential for timely therapeutic intervention and its use as a biomarker in muscular dystrophies.
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Xu, Xuefeng, Sa Luo, Biyun Li, Huaping Dai, and Jinglan Zhang. "IL-25 contributes to lung fibrosis by directly acting on alveolar epithelial cells and fibroblasts." Experimental Biology and Medicine 244, no. 9 (2019): 770–80. http://dx.doi.org/10.1177/1535370219843827.
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Interleukin (IL)-25 is shown to potentiate type-2 immunity and contribute to chronic airway inflammation and remodeling in allergic airway diseases. However, the role of IL-25 in idiopathic pulmonary fibrosis (IPF), dominated by nonatopic type-2 immunity, still remains largely unclear. Herein, we detected the expression levels of IL-25 and IL-17BR (IL-25’s receptor) by using lung tissue samples gained from IPF patients and normal subjects. Also, by directly intranasal (IN) instillation of IL-25 to mice, we examined the potential roles and mechanisms of IL-25 in the development of lung fibrosis. Furthermore, we tested whether IL-25 can directly activate human lung fibroblast by in vitro cell culture. Immunohistochemical, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the mRNA and protein levels of IL-25 and IL-17BR are significantly higher in IPF patients when compared with normal controls. Intranasal instillation of IL-25 to mice markedly induces the expressions of alveolar IL-5 and IL-13. Furthermore, immunohistochemical analysis showed that the main components of the extracellular matrix including collagen I, collagen III and fibronectin are notably induced by IL-25 instillation in lung parenchyma (especially in alveolar epithelial cells [AECs]). Also, IL-25 potentiates the expression of connective tissue growth factor (CTGF) in AECs and the recruitment of lung fibroblast. By using Cell Counting Kit-8 and EDU incorporation assay, we found that IL-25 markedly enhances the proliferation of lung fibroblast. Finally, IL-25 potentiates fibroblast to produce several fibrogenic genes including collagen I/III, fibronectin, CTGF, α smooth muscle (α-SMA) and tissue inhibitor of metalloproteinase (TIMP)-1 as determined by RT-PCR assay. Collectively, we concluded that IL-25 is increased in IPF lungs and contributes to lung fibrosis by directly mediating AECs/fibroblast activation. Impact statement Our work focused on alveolar epithelial cells (AECs)-derived type-2 cytokine (interleukin [IL]-25) in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We showed that IL-25 and IL-17BR (IL-25’s receptor) is upregulated in lung tissues (especially in AECs and lung fibroblasts) of IPF patients and contributes to lung fibrosis by directly activating lung fibroblasts and modulating epithelial–mesenchymal transition (EMT) of AECs. We suggest that IL-25 may be one of the master switches hidden in the milieu of abnormal epithelial–mesenchymal crosstalk. Treatment targeting IL-25 may be the potential and novel method for IPF patients.
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McCurdy, Sarah M., Qiuxia Dai, Jianhua Zhang, et al. "SPARC mediates early extracellular matrix remodeling following myocardial infarction." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 2 (2011): H497—H505. http://dx.doi.org/10.1152/ajpheart.01070.2010.
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Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT ( n=28) and null ( n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null ( P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 μl ( P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 μl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to 19 ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). Fibroblasts isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in fibroblast gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.
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Wu, Qiuqian, and Jason H. Huang. "Ectopic expression of Smurf2 and acceleration of age-related intervertebral disc degeneration in a mouse model." Journal of Neurosurgery: Spine 27, no. 1 (2017): 116–26. http://dx.doi.org/10.3171/2016.11.spine16901.
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OBJECTIVELumbar intervertebral disc degeneration, an age-related process, is a major cause of low-back pain. Although low-back pain is a very common clinical problem in the aging population, no effective treatment is available, largely owing to lack of understanding of the molecular mechanisms underlying disc degeneration. The goal of this study was to characterize how ectopic expression of Smurf2 driven by the collagen Type II alpha 1 (Col2a1) promoter alters disc cell phenotype and associated cellular events, matrix synthesis, and gene expression during disc degeneration in mice.METHODSTo characterize how ectopic expression of Smurf2 in Col2a1-promoter working cells affects the disc degeneration process, the authors performed histological and immunohistochemical analysis of lumbar spine specimens harvested from wild-type (WT) and Col2a1-Smurf2 transgenic mice at various ages (n ≥ 6 in each age group). To elucidate the molecular mechanism underlying Smurf2-mediated disc degeneration, the authors isolated cells from WT and Col2a1-Smurf2 transgenic lumbar intervertebral discs and performed Western blot and real-time RT-PCR (reverse transcription polymerase chain reaction) to examine the protein and mRNA levels of interesting targets.RESULTSThe authors demonstrated that approximately 30% of WT mice at 10–12 months of age had started to show disc degeneration and that the disc degeneration process was accelerated by 3–6 months in Col2a1-Smurf2 transgenic mice. Chondrocyte-like cell proliferation, maturation, and fibrotic tissue formation in the inner annulus were often accompanied by fibroblast-to-chondrocyte differentiation in the outer annulus in transgenic discs. The chondrocyte-like cells in transgenic discs expressed higher levels of connective tissue growth factor (CTGF) than were expressed in WT counterparts.CONCLUSIONSThe findings that ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated disc degeneration in Col2a1-Smurf2 transgenic mice, and that higher levels of CTGF protein and mRNA were present in Col2a1-Smurf2 transgenic discs, indicate that Smurf2 accelerates disc degeneration via upregulation of CTGF.
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Morinaga, Jun, Yutaka Kakizoe, Taku Miyoshi, et al. "The antifibrotic effect of a serine protease inhibitor in the kidney." American Journal of Physiology-Renal Physiology 305, no. 2 (2013): F173—F181. http://dx.doi.org/10.1152/ajprenal.00586.2012.
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Interstitial fibrosis is a final common pathway for the progression of chronic kidney diseases. Activated fibroblasts have an extremely important role in the progression of renal fibrosis, and transforming growth factor (TGF)-β1 is a major activator of fibroblasts. Since previous reports have indicated that serine protease inhibitors have a potential to inhibit TGF-β1 signaling in vitro, we hypothesized that a synthetic serine protease inhibitor, camostat mesilate (CM), could slow the progression of renal fibrosis. TGF-β1 markedly increased the phosphorylation of TGF-β type I receptor, ERK 1/2, and Smad2/3 and the levels of profibrotic markers, such as α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1, in renal fibroblasts (NRK-49F cells), and they were all significantly reduced by CM. In protocol 1, 8-wk-old male Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO) and were concurrently treated with a slow-release pellet of CM or vehicle for 14 days. Protocol 2 was similar to protocol 1 except that CM was administered 7 days after UUO. CM substantially improved renal fibrosis as determined by sirius red staining, collagen expression, and hydroxyproline levels. The phosphorylation of ERK1/2 and Smad2/3 and the levels of α-SMA, CTGF, promatrix metalloproteinase-2, and matrix metalloproteinase-2 were substantially increased by UUO, and they were all significantly attenuated by CM. These antifibrotic effects of CM were also observed in protocol 2. Our present results suggest the possibility that CM might represent a new class of therapeutic drugs for the treatment of renal fibrosis through the suppression of TGF-β1 signaling.
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Yang, Ru, Jawaria Amir, Haibo Liu, and Brahim Chaqour. "Mechanical strain activates a program of genes functionally involved in paracrine signaling of angiogenesis." Physiological Genomics 36, no. 1 (2008): 1–14. http://dx.doi.org/10.1152/physiolgenomics.90291.2008.
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Studies were performed to examine the extent to which mechanical stimuli mediate control of angiogenesis in bladder cells both in vitro and in vivo. Differential gene expression between control nonstretched and cyclically stretched bladder smooth muscle cells was assessed using oligonucleotide microarrays and pathway analysis by the web tool Fast Assignment and Transference of Information (FatiGO). Data showed that a substantial proportion (33 of 86) of mechanically responsive genes were angiogenesis-related and include cytokines, growth-related factors, adhesion proteins, and matricellular, signal transduction, extracellular matrix (ECM), and inflammatory molecules. Integrative knowledge of protein-protein interactions revealed that 12 mechano-sensitive gene-encoded proteins have interacting partner(s) in the vascular system confirming their potential role in paracrine regulation of angiogenesis. Angiogenic genes include matricellular proteins such as Cyr61/CCN1, CTGF/CCN2 and tenascin C, components of the VEGF and IGF systems, ECM proteins such as type I collagen and proteoglycans, and matrix metalloproteinases. In an in vivo model of bladder overdistension, 5 of 11 mechano-responsive angiogenic genes, independently tested by real-time PCR, were upregulated as a result of pressure overload including Cyr61/CCN1, CTGF/CCN2, MCP-1, VEGF-A, MMP-1, and midkine. Meanwhile, the molecular anatomy of angiogenic gene promoters reveals the presence of GA box-binding for the myc-associated zinc finger protein, MAZ, often found adjacent to binding sites for mechano-responsive transcription factors (e.g., NF-κB), suggesting that the coordinated activity of these factors may induce selective angiogenic gene transcription. These data suggest that mechanical control of angiogenic genes is an integral part of the adaptive and plasticity responses to mechanical overload.
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Zhou, Xuan, Jun Xiong, Shi Lu, et al. "Inhibitory Effect of Corilagin on miR-21-Regulated Hepatic Fibrosis Signaling Pathway." American Journal of Chinese Medicine 47, no. 07 (2019): 1541–69. http://dx.doi.org/10.1142/s0192415x19500794.
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Corilagin is a polyphenol that can be extracted from many medicinal plants and shows multiple pharmacological effects. We aimed to investigate the role of corilagin on miR-21-regulated hepatic fibrosis, especially miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway, in hepatic stellate LX2 cell line and Sprague–Dawley rats. The mRNA or protein levels of miR-21, Smad7, connective tissue growth factor (CTGF), [Formula: see text]-smooth muscle actin ([Formula: see text]-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), collagen type I alpha 1 (COL1A1), Smad2, Smad3, Smad2/3, p-Smad2, p-Smad3, p-Smad2/3, and transforming growth factor-[Formula: see text]1 (TGF-[Formula: see text]1) in LX2 cells and liver tissues were determined. Furthermore, gain-of and loss-of function of miR-21 in miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway were analyzed in LX2 cells. Liver tissues and serum were collected for pathological analysis, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Corilagin treatment reduced mRNA or protein levels of miR-21, CTGF, [Formula: see text]-SMA, TIMP-1, TGF-[Formula: see text]1, COL1A1, p-Smad2, p-Smad3, and p-Smad2/3 both in vitro and in vivo. While corilagin increased mRNA and protein levels of Smad7 and MMP-9. After gain-of and loss-of function of miR-21, the downstream effectors of miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway in LX2 cells changed accordingly, and the changes were inhibited by corilagin. Simultaneously, administration of corilagin not only ameliorated pathological manifestation of liver fibrosis but also reduced levels of [Formula: see text]-SMA and COL1A1 in liver tissues and TGF-[Formula: see text]1, ALT levels in serum. Corilagin is able to potentially prevent liver fibrosis by blocking the miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway in LX2 cells and CCl4-induced liver fibrosis rats, which may provide a novel therapeutic strategy for liver fibrosis.
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Huang, Xiao R., Arthur C. K. Chung, Xiao J. Wang, Kar Neng Lai та Hui Y. Lan. "Mice overexpressing latent TGF-β1 are protected against renal fibrosis in obstructive kidney disease". American Journal of Physiology-Renal Physiology 295, № 1 (2008): F118—F127. http://dx.doi.org/10.1152/ajprenal.00021.2008.
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Transforming growth factor (TGF)-β1, once activated, binds to its receptors and mediates renal fibrosis via the downstream Smad signaling pathway. We reported here that mice overexpressing latent TGF-β1 in keratinocytes were protected against renal fibrosis in a model of obstructive kidney disease. In normal mice, both transgenic (Tg) and wild-type (WT) mice had normal renal histology and function, despite a 10-fold increase in plasma latent TGF-β1 in Tg mice. A severe renal fibrosis was developed in WT mice at 7 days after urinary obstruction. Unexpectedly, renal fibrosis was prevented in Tg mice, although levels of latent TGF-β1 in both circulation and renal tissues remained high. Compared with the WT mice, quantitative real-time PCR showed that upregulation of renal α-smooth muscle actin (SMA), collagen I, and collagen III mRNA was inhibited in Tg mice (60–70% reduced, all P < 0.01). These were further confirmed by immunohistochemistry with a marked inhibition of tubulointerstitial accumulation of α-SMA+ fibroblasts, collagen I, and collagen III matrix in Tg mice (all P < 0.001). Further studies showed that inhibition of renal fibrosis in Tg mice was associated with a significant reduction in renal TGF-β1 and CTGF (60% reduced, P < 0.05), an increase in renal Smad7, a suppression of TSP-1 (a critical factor for TGF-β1 activation), and an inhibition of Smad2/3 activation (all P < 0.001). In conclusion, latent TGF-β may play a protective role in renal fibrosis. Inhibition of renal TGF-β1 expression and activation, thereby blocking the downstream TGF-β signaling pathway, may be a critical mechanism by which latent TGF-β1 protects against renal fibrosis.
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Lee, Da-Young, Sun-Mi Yun, Moon-Young Song, Sang-Deok Ji, Jong-Gon Son та Eun-Hee Kim. "Administration of Steamed and Freeze-Dried Mature Silkworm Larval Powder Prevents Hepatic Fibrosis and Hepatocellular Carcinogenesis by Blocking TGF-β/STAT3 Signaling Cascades in Rats". Cells 9, № 3 (2020): 568. http://dx.doi.org/10.3390/cells9030568.
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Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide and the majority of HCC patients occur with a background of hepatic fibrosis and cirrhosis. We have previously reported the hepatoprotective effects of steamed and freeze-dried mature silkworm larval powder (SMSP) in a chronic ethanol-treated rat model. Here, we assessed the anti-fibrotic and anti-carcinogenic effects of SMSP on diethylnitrosamine (DEN)-treated rats. Wistar rats were intraperitoneally injected with DEN once a week for 12 or 16 weeks with or without SMSP administration (0.1 and 1 g/kg). SMSP administration significantly attenuated tumor foci formation and proliferation in the livers of the rats treated with DEN for 16 weeks. SMSP administration also inhibited hepatic fibrosis by decreasing the levels of collagen fiber and the expression of pro-collagen I and alpha-smooth muscle actin (α-SMA). Moreover, SMSP supplementation improved the major parameters of fibrosis such as transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and collagen type I (Col1A1) in the livers from the rats treated with DEN for 16 weeks. As s possible mechanisms, we investigated the effects of SMSP on the TGF-β and signal transducer and activator of transcription 3 (STAT3)-mediated signaling cascades, which are known to promote hepatic fibrosis. We found that SMSP treatment inhibited the activation of TGF-β and the phosphorylation of STAT3 pathway in DEN-treated rats. Moreover, SMSP administration suppressed the expressions of the target genes of TGF-β and STAT3 induced by DEN treatment. Our findings provide experimental evidences that SMSP administration has inhibitory effects of hepatic fibrosis and HCC induced by DEN In Vivo and could be a promising strategy for the prevention or treatment of hepatic fibrosis and hepatocellular carcinogenesis.
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van Beusekom, Cyrina D., and Tanja M. Zimmering. "Profibrotic effects of angiotensin II and transforming growth factor beta on feline kidney epithelial cells." Journal of Feline Medicine and Surgery 21, no. 8 (2018): 780–87. http://dx.doi.org/10.1177/1098612x18805862.
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Objectives The aim of this study was to evaluate the role of angiotensin II (AT-II) and its main mediator, transforming growth factor beta 1 (TGF-β1), in the development of feline renal fibrosis. Methods Expression of marker genes indicating epithelial-to-mesenchymal transition (EMT), profibrotic mediators and matricellular proteins was measured in feline kidney epithelial cells (Crandell Rees feline kidney [CRFK] cells) after incubation with AT-II and/or TGF-β1. Results Cells incubated with TGF-β1 or the combination of TGF-β1 with AT-II showed clear EMT with more stretched fibroblastic cells, whereas the cells incubated without TGF-β1 and AT-II (control) showed more epithelial cells. Gene expression of collagen type I ( COL1), tenascin-C ( TNC), trombospondin-1 ( TSP-1), connective tissue growth factor ( CTGF) and alpha-smooth muscle actin ( α-SMA) increased significantly after incubation of the CRFK cells with TGF-β1 or TGF-β1 in combination with AT-II for 12 h. As incubation of the CRFK cells with only AT-II did not show any significant rise in gene expression of the above-mentioned genes, this was further investigated. In contrast to healthy feline kidney tissue, CRFK cells showed almost no expression of the AT-II type 1 (AT1) receptor. Conclusions and relevance TGF-β1 significantly induced expression of the EMT marker gene α-SMA, profibrotic mediator CTGF, and fibrogenic proteins COL1, TNC and TSP-1 in CRFK cells. The effect of TGF-β1 on myofibroblast formation was also observed by the stretched appearance of the CRFK cells. As CRFK cells expressed almost no AT1 receptors, this cell line proved not suitable for testing the efficacy of drugs that interact with the AT1 receptor. As AT-II stimulates the effects of TGF-β1 in mammals, the results of this study suggest an indirect profibrotic effect of AT-II besides the demonstrated profibrotic effect of TGF-β1 and thus the development of feline renal fibrosis. Modulation of EMT or proliferation of myofibroblasts could serve as a diagnostic tool and a novel therapeutic target to inhibit renal fibrogenesis, and could possibly serve in the therapy of feline renal fibrosis.
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Lubel, John S., Chandana B. Herath, Jorge Tchongue, et al. "Angiotensin-(1–7), an alternative metabolite of the renin–angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat." Clinical Science 117, no. 11 (2009): 375–86. http://dx.doi.org/10.1042/cs20080647.
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Ang-(1–7) (angiotensin-1–7), a peptide product of the recently described ACE (angiotensin-converting enzyme) homologue ACE2, opposes the harmful actions of AngII (angiotensin II) in cardiovascular tissues, but its role in liver disease is unknown. The aim of the present study was to assess plasma levels of Ang-(1–7) in human liver disease and determine its effects in experimental liver fibrosis. Angiotensin peptide levels were measured in cirrhotic and non-cirrhotic patients with hepatitis C. The effects of Ang-(1–7) on experimental fibrosis were determined using the rat BDL (bile-duct ligation) model. Liver histology, hydroxyproline quantification and expression of fibrosis-related genes were assessed. Expression of RAS (renin–angiotensin system) components and the effects of Ang-(1–7) were examined in rat HSCs (hepatic stellate cells). In human patients with cirrhosis, both plasma Ang-(1–7) and AngII concentrations were markedly elevated (P<0.001). Non-cirrhotic patients with hepatitis C had elevated Ang-(1–7) levels compared with controls (P<0.05), but AngII concentrations were not increased. In BDL rats, Ang-(1–7) improved fibrosis stage and collagen Picrosirius Red staining, and reduced hydroxyproline content, together with decreased gene expression of collagen 1A1, α-SMA (smooth muscle actin), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), ACE and mas [the Ang-(1–7) receptor]. Cultured HSCs expressed AT1Rs (AngII type 1 receptors) and mas receptors and, when treated with Ang-(1–7) or the mas receptor agonist AVE 0991, produced less α-SMA and hydroxyproline, an effect reversed by the mas receptor antagonist A779. In conclusion, Ang-(1–7) is up-regulated in human liver disease and has antifibrotic actions in a rat model of cirrhosis. The ACE2/Ang-(1–7)/mas receptor axis represents a potential target for antifibrotic therapy in humans.
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Schrör and Hohlfeld. "Mechanisms of anti-ischemic action of prostaglandin E1 in peripheral arterial occlusive disease." Vasa 33, no. 3 (2004): 119–24. http://dx.doi.org/10.1024/0301-1526.33.3.119.
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The mechanisms of anti-ischemic effects of PGE1 in patients with peripheral arterial occlusive disease (PAD) are probably complex and clearly not limited to a direct vasodilator action. In addition to the known effects on blood flow, viscosity, fibrinolysis and platelet aggregation, the compound also inhibits monocyte and neutrophil function, suggesting that PGE1 will also have anti-inflammatory effects. Recent research has detected additional actions of PGE1 and prostacyclin analogs which might be relevant to its clinical efficacy. This includes inhibition of expression of adhesion molecules (E-selectin, ICAM-1, and VCAM-1), release of inflammatory cytokines (TNFalpha, MCP-1), matrix components and generation and release of growth factors (CYR61, CTGF). These actions may also contribute to the long-term effects of PGE1, particularly in more advanced stages of PAD. Gene-expression experiments with chemically stable prostacyclins and PGE1 suggest that several genes in vascular smooth muscle cells and fibroblasts are modified by prostaglandins at the transcriptional level. This includes TNFalpha-induced VCAM expression in vascular smooth muscle cells which appears to be inhibited via the prostaglandin EP2 receptor as well as IL-1-induced expression of the type-I collagen gene in fibroblasts. Thus, regulation of gene transcription by PGE1 may contribute to tissue protection in critical ischemia of the lower limbs.
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Qiu, Ming, Huanyu Shu, Lu Li та ін. "Interleukin 10 Attenuates Angiotensin II-Induced Aortic Remodelling by Inhibiting Oxidative Stress-Induced Activation of the Vascular p38 and NF-κB Pathways". Oxidative Medicine and Cellular Longevity 2022 (26 квітня 2022): 1–15. http://dx.doi.org/10.1155/2022/8244497.
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Interleukin 10 (IL-10) is a probable anti-inflammatory factor that can attenuate hypertrophic remodelling caused by overloaded pressure and improve cardiac function. In this study, IL-10 was decreased in both the plasma of hypertensive patients and the aortic vessels of angiotensin II (Ang II)-induced hypertensive mice. IL-10 was unable to alter blood pressure in the case of Ang II-induced hypertension. The aortic thickness, collagen deposition, and the levels of fibrosis-associated markers, including collagen type I α 1 (Col1α1), connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β), and matrix metalloproteinase 2 (MMP2), were significantly reduced in the IL-10 treatment group compared with the vehicle group after Ang II treatment. Moreover, IL-10 treatment significantly inhibited the number of CD45+ positive cells and the mRNA expression levels of proinflammatory cytokines in the vascular tissue of Ang II-infused mice. Furthermore, dihydroethidium (DHE) and 4hydroxynonenal (4-HNE) staining showed that IL-10 decreased Ang II-induced vascular oxidative stress and lipid peroxidation. Furthermore, IL-10 suppressed Ang II-induced proliferation, fibrosis, and inflammation of mouse vascular adventitial fibroblasts (mVAFs). Mechanistically, IL-10 suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase and nuclear factor-κB (NF-κB) in Ang II-induced vascular fibrosis. In summary, our data indicated that IL-10, as a potential therapeutic target treatment, could limit the progression of Ang II-induced aortic remodelling.
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Qiu, Ming, Huanyu Shu, Lu Li та ін. "Interleukin 10 Attenuates Angiotensin II-Induced Aortic Remodelling by Inhibiting Oxidative Stress-Induced Activation of the Vascular p38 and NF-κB Pathways". Oxidative Medicine and Cellular Longevity 2022 (26 квітня 2022): 1–15. http://dx.doi.org/10.1155/2022/8244497.
Full textAbstract:
Interleukin 10 (IL-10) is a probable anti-inflammatory factor that can attenuate hypertrophic remodelling caused by overloaded pressure and improve cardiac function. In this study, IL-10 was decreased in both the plasma of hypertensive patients and the aortic vessels of angiotensin II (Ang II)-induced hypertensive mice. IL-10 was unable to alter blood pressure in the case of Ang II-induced hypertension. The aortic thickness, collagen deposition, and the levels of fibrosis-associated markers, including collagen type I α 1 (Col1α1), connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β), and matrix metalloproteinase 2 (MMP2), were significantly reduced in the IL-10 treatment group compared with the vehicle group after Ang II treatment. Moreover, IL-10 treatment significantly inhibited the number of CD45+ positive cells and the mRNA expression levels of proinflammatory cytokines in the vascular tissue of Ang II-infused mice. Furthermore, dihydroethidium (DHE) and 4hydroxynonenal (4-HNE) staining showed that IL-10 decreased Ang II-induced vascular oxidative stress and lipid peroxidation. Furthermore, IL-10 suppressed Ang II-induced proliferation, fibrosis, and inflammation of mouse vascular adventitial fibroblasts (mVAFs). Mechanistically, IL-10 suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase and nuclear factor-κB (NF-κB) in Ang II-induced vascular fibrosis. In summary, our data indicated that IL-10, as a potential therapeutic target treatment, could limit the progression of Ang II-induced aortic remodelling.
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Peterson, Joshua M., Anesh Prasai, Jayson W. Jay, Steven E. Wolf, and Amina El Ayadi. "143 Galunisertib Exerts Targeted Anti-Fibrotic Effects in In Vitro Models of Burn Wound Healing." Journal of Burn Care & Research 42, Supplement_1 (2021): S95. http://dx.doi.org/10.1093/jbcr/irab032.147.
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Abstract Introduction Inhibition of TGF-β has shown promising in vitro and in vivo results for reduction of hypertrophic scarring after burn injury. However, TGF-β regulates diverse cellular pathways apart from fibrosis, including physiologic wound healing, cell cycle control, and homeostasis. Galunisertib, a novel small molecular tyrosine kinase inhibitor of TGF-beta receptor type 1, specifically targets downstream pro-fibrotic pathways of TGF-β signaling, has an excellent adverse effect profile, and minimal off-target effects. We hypothesized that galunisertib diminishes fibrotic phenotypes in a targeted fashion, making it a promising candidate drug for prevention of hypertrophic burn scar and contracture. Methods Commercially available fibroblasts were treated with TGF-β at concentrations typical of burn wounds to induce fibrotic phenotype fibroblasts (FPF). FPF cells were treated with galunisertib for 0, 1, 2, 3, and 7 days at concentrations ranging from 0.01 μM to 100 μM. FPF viability and proliferation were assessed with MTT assay. Modulation of FPF cell fibrotic gene and was analyzed using quantitative real-time PCR (qRT-PCR) of COL1A1, COL3A1, FN1, αSMA, CTGF, DCN, MMP1, and MMP13. Analysis of phenotype modulation was performed by western blotting of P-Smad2/3, collagen-1, fibronectin, and αSMA. Statistical analysis performed with students t-test and ANOVA. Results TGF-β treated FPF cells had significantly increased proliferation relative to commercially available fibroblasts, which was attenuated by treatment with 2.5–10 μM galunisertib at 2 or 3 days after treatment in a concentration dependent manner (p &lt; 0.05) while also not causing a relative decrease in proliferation. Expression of FPF downstream pro-fibrotic genes underwent significant fold changes (FN1, αSMA, CTGF: p &lt; 0.05). Protein expression showed decrease in both SMAD2/3 phosphorylation and fibrotic protein expression. Scratch assay analysis is ongoing. Conclusions Galunisertib exerts targeted dose-dependent inhibition of fibrotic gene expression and phenotypes in vitro without hindering cellular proliferation.
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Lu, H. K., H. P. Chou, C. L. Li, M. Y. Wang та L. F. Wang. "Stimulation of Cells Derived from Nifedipine-induced Gingival Overgrowth with Porphyromonas gingivalis, Lipopolysaccharide, and Interleukin-1β". Journal of Dental Research 86, № 11 (2007): 1100–1104. http://dx.doi.org/10.1177/154405910708601115.
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The purpose of this study was to clarify the main contributory factor of nifedipine-induced gingival overgrowth either by Porphyromonas gingivalis lipopolysaccharide ( Pg-LPS) or interleukin-1beta (IL-1β). Human gingival fibroblasts from healthy tissues and nifedipine-induced gingival overgrowth tissues were stimulated with nifedipine, IL-1β, Escherichia coli lipopolysaccharide ( Ec-LPS), and Pg-LPS, and the gene expressions were analyzed by RT-PCR. Analysis of the data showed no strong evidence of a synergistic effect of nifedipine and Pg-LPS on IL-6, connective tissue growth factor (CTGF), and type 1 collagen gene expression of either healthy cells or nifedipine-induced gingival overgrowth cells. Among the three stimulants—IL-1β, Pg-LPS, and Ec-LPS—androgen receptor and IL-6 gene expressions in both the healthy and nifedipine-induced gingival overgrowth groups were strongly up-regulated by the presence of IL-1β only. Furthermore, the responses to IL-1β in the nifedipine-induced gingival overgrowth group were stronger than those of the healthy group. It can be concluded that IL-1β is an important mediator responsible for the higher IL-6 and androgen receptor expression of nifedipine-induced gingival overgrowth cells.
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Trink, Jackie, Renzhong Li, Yaseelan Palarasah, et al. "Activated Alpha 2-Macroglobulin Is a Novel Mediator of Mesangial Cell Profibrotic Signaling in Diabetic Kidney Disease." Biomedicines 9, no. 9 (2021): 1112. http://dx.doi.org/10.3390/biomedicines9091112.
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Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients’ serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFβ1). In patients with established DKD, urinary α2M* and TGFβ1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.
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Castillero, Estibaliz, Hirokazu Akashi, Marc Najjar, et al. "Activin type II receptor ligand signaling inhibition after experimental ischemic heart failure attenuates cardiac remodeling and prevents fibrosis." American Journal of Physiology-Heart and Circulatory Physiology 318, no. 2 (2020): H378—H390. http://dx.doi.org/10.1152/ajpheart.00302.2019.
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Myostatin (MSTN) is a transforming growth factor (TGF)-β superfamily member that acts as a negative regulator of muscle growth and may play a role in cardiac remodeling. We hypothesized that inhibition of activin type II receptors (ACTRII) to reduce MSTN signaling would reduce pathological cardiac remodeling in experimental heart failure (HF). C57BL/6J mice underwent left anterior descending coronary artery ligation under anesthesia to induce myocardial infarction (MI) or no ligation (sham). MI and sham animals were each randomly divided into groups ( n ≥ 10 mice/group) receiving an ACTRII or ACTRII/TGFβ receptor-signaling inhibiting strategy: 1) myo-Fc group (weekly 10 mg/kg Myo-Fc) or 2) Fol + TGFi group (daily 12 µg/kg follistatin plus 2 mg/kg TGFβ receptor inhibitor), versus controls. ACTRII/TGFBR signaling inhibition preserved cardiac function by echocardiography and prevented an increase in brain natriuretic peptide (BNP). ACTRII/TGFBR inhibition resulted in increased phosphorylation (P) of Akt and decreased P-p38 mitogen-activated protein kinase (MAPK) in MI mice. In vitro, Akt contributed to P-SMAD2,3, P-p38, and BNP regulation in cardiomyocytes. ACTRII/TGFBR inhibition increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) levels and decreased unfolded protein response (UPR) markers in MI mice. ACTRII/TGFBR inhibition was associated with a decrease in cardiac fibrosis and fibrosis markers, connective tissue growth factor (CTGF), type I collagen, fibronectin, α-smooth muscle actin, and matrix metalloproteinase (MMP)-12 in MI mice. MSTN exerted a direct regulation on the UPR marker eukaryotic translation initiation factor-2α (eIf2α) in cardiomyocytes. Our study suggests that ACTRII ligand inhibition has beneficial effects on cardiac signaling and fibrosis after ischemic HF. NEW & NOTEWORTHY Activin type II receptor ligand inhibition resulted in preserved cardiac function, a decrease in cardiac fibrosis, improved SERCA2a levels, and a prevention of the unfolded protein response in mice with myocardial infarction.
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Yoon, Jung Joo, Yun Jung Lee, Dae Gill Kang, and Ho Sub Lee. "Protective Role of Oryeongsan Against Renal Inflammation and Glomerulosclerosis in db/db Mice." American Journal of Chinese Medicine 42, no. 06 (2014): 1431–52. http://dx.doi.org/10.1142/s0192415x14500906.
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Diabetic nephropathy is characterized by renal hardening and interstitial fibrosis caused by extracellular matrix (ECM) accumulation. The most distinctive diabetic lesion in the glomeruli is mesangial expansion and hyperplasia, which ultimately leads to diabetic nephrosclerosis. Oryeongsan (ORS), a traditional Chinese herbal medication, is widely used to treat nephrosis, dropsy, and uremia. In this study, type 2 diabetic animals (db/db mice) were administered ORS (100 mg/kg/day) for 8 weeks to examine the potential beneficial effects on metabolic abnormalities and diabetic nephropathy progression, including renal fibrosis. The body weight, total-cholesterol, triglyceride, and LDL-c levels were significantly decreased in ORS-treated db/db mice compared to untreated db/db mice. In addition, the blood glucose, insulin, glucose tolerance, and the homeostasis model assessment of insulin resistance index (HOMA-IR) were significantly improved in ORS-treated db/db mice compared to untreated db/db mice. Creatinine clearance (Ccr), urine albumin, and BUN levels were also improved by ORS treatment. The ratio of mesangial matrix/glomerular area was markedly higher in db/db mice than in db/m mice, but ORS significantly reduced this expansion. TGF-β1, Smad-2/-4, Collagen IV, CTGF, and TIMP decreased with ORS treatment, as were Smad-7 and MT1-MMP in ORS-treated db/db mice. Furthermore, ICAM-1 and MCP-1 expression were suppressed in ORS-treated db/db mice. Therefore, these findings suggest that ORS ameliorated insulin resistance and diabetes-associated glomerulosclerosis in db/db mice, possibly by disturbing the TGF-β1/Smads pathway. ORS may be a new therapeutic option for treating diabetic nephropathy.
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Subramanian, Umadevi, Chandramohan Ramasamy, Samivel Ramachandran, Joshua M. Oakes, Jason D. Gardner та Kailash N. Pandey. "Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-β1/SMAD Signaling Pathway". International Journal of Molecular Sciences 23, № 19 (2022): 11487. http://dx.doi.org/10.3390/ijms231911487.
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The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1−/−, 0-copy), heterozygous (Npr1+/−, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-β1 receptor (TGF-β1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1−/− (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-βRI (4.3-fold), TGF-βRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-β1-mediated SMAD-dependent pathway.
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Scavo, Maria Principia, Giuseppe Lisco, Nicoletta Depalo, et al. "Semaglutide Modulates Extracellular Matrix Production of LX-2 Cells via Exosomes and Improves Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)." International Journal of Molecular Sciences 25, no. 3 (2024): 1493. http://dx.doi.org/10.3390/ijms25031493.
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Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely related to some metabolic disorders, such as central obesity and type 2 diabetes (T2D). Glucagon-like peptide 1 receptor agonists (GLP-1RAs), such as semaglutide, may have therapeutic roles in MASLD associated with T2D. This study aims to investigate the molecular mechanisms underlying the effectiveness of semaglutide on MASLD in terms of progression from liver steatosis to fibrosis. We characterized exosomes from ten patients with type 2 diabetes (T2D) before (T0) and after 12 months (T12) of treatment with once-weekly subcutaneous semaglutide. Six of ten patients were considered responders to therapy (R) based on MASLD severity downgrading by at least one class according to a validated ultrasonographic (US) score. Normal hepatocytes (HEPA-RG) and stellate (LX-2) cells were challenged with exosomes from R and NR patients, isolated before and after 12 months of therapy. Exosomes from both R and NR patients isolated at T0 significantly affected LX-2 viability. After 12 months of treatment, only those isolated from R patients restored cell viability, whereas those from NR patients did not. No effects were observed on HEPA-RG cells. Exosomes at T12 from R but not from NR patients significantly decreased the production of α-SMA, a marker of LX-2 activation, a liver stellate cell model, and ph-SMAD2 and CTGF, involved in fibrosis processes. TGF-β1 was not modulated by the exosomes of R and NR patients. As a downstream effect, Vimentin, Collagen 1A1, and Fibronectin extracellular matrix components were also downregulated, as measured by droplets digital PCR. In conclusion, these results shed light on the potential effectiveness of semaglutide in preventing liver fibrosis in MASLD.
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Sánchez-López, Elsa, Juan Rodriguez-Vita, Cecile Cartier та ін. "Inhibitory effect of interleukin-1β on angiotensin II-induced connective tissue growth factor and type IV collagen production in cultured mesangial cells". American Journal of Physiology-Renal Physiology 294, № 1 (2008): F149—F160. http://dx.doi.org/10.1152/ajprenal.00129.2007.
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Connective tissue growth factor (CTGF) is overexpressed in kidney diseases associated with extracellular matrix accumulation. Angiotensin II (ANG II) participates in renal fibrosis by the upregulation of growth factors, including CTGF, and extracellular matrix proteins, such as type IV collagen. During renal injury, ANG II and the macrophage-produced cytokine interleukin-1β (IL-1β) may be present simultaneously in the glomerular environment. However, there are no studies about the interaction between ANG II and IL-1β in renal fibrosis. For this reason, in cultured mesangial cells (MC), we investigated whether IL-1β could regulate ANG II-mediated collagen accumulation and the mechanisms underlying this process. In MC, CTGF is a downstream mediator of type IV collagen production induced by ANG II. IL-1β did not increase the production of CTGF and type IV collagen but significantly inhibited ANG II-induced CTGF and type IV collagen overexpression. Moreover, IL-1β also inhibited type IV collagen upregulation caused by exogenous recombinant CTGF. Matrix metalloproteinase-9 (MMP-9) is the main enzyme involved in type IV collagen degradation. In MC, coincubation of IL-1β and ANG II caused a synergistic increase in MMP-9 gene expression and activity, associated with type IV collagen inhibition. The described IL-1β effects were dependent on activation of ERK/MAPK but independent p38-MAPK, JNK, phosphatidylinositol 3-kinase/Akt, and Rho-associated kinase pathways. In summary, these data indicate that IL-1β inhibited ANG II-mediated type IV collagen production, via CTGF downregulation, and increased type IV collagen degradation, through MMP-9 upregulation. Our in vitro data show that the proinflammatory cytokine IL-1β abrogates ANG II-induced CTGF production, describing antagonistic activities of proinflammatory cytokines on ANG II actions.
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Kennedy, David, Fatimah Khalaf, Brendan Sheehy, et al. "Telocinobufagin, a Novel Cardiotonic Steroid, Promotes Renal Fibrosis via Na+/K+-ATPase Profibrotic Signaling Pathways." International Journal of Molecular Sciences 19, no. 9 (2018): 2566. http://dx.doi.org/10.3390/ijms19092566.
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Cardiotonic steroids (CTS) are Na+/K+-ATPase (NKA) ligands that are elevated in volume-expanded states and associated with cardiac and renal dysfunction in both clinical and experimental settings. We test the hypothesis that the CTS telocinobufagin (TCB) promotes renal dysfunction in a process involving signaling through the NKA α-1 in the following studies. First, we infuse TCB (4 weeks at 0.1 µg/g/day) or a vehicle into mice expressing wild-type (WT) NKA α-1, as well as mice with a genetic reduction (~40%) of NKA α-1 (NKA α-1+/−). Continuous TCB infusion results in increased proteinuria and cystatin C in WT mice which are significantly attenuated in NKA α-1+/− mice (all p < 0.05), despite similar increases in blood pressure. In a series of in vitro experiments, 24-h treatment of HK2 renal proximal tubular cells with TCB results in significant dose-dependent increases in both Collagens 1 and 3 mRNA (2-fold increases at 10 nM, 5-fold increases at 100 nM, p < 0.05). Similar effects are seen in primary human renal mesangial cells. TCB treatment (100 nM) of SYF fibroblasts reconstituted with cSrc results in a 1.5-fold increase in Collagens 1 and 3 mRNA (p < 0.05), as well as increases in both Transforming Growth factor beta (TGFb, 1.5 fold, p < 0.05) and Connective Tissue Growth Factor (CTGF, 2 fold, p < 0.05), while these effects are absent in SYF cells without Src kinase. In a patient study of subjects with chronic kidney disease, TCB is elevated compared to healthy volunteers. These studies suggest that the pro-fibrotic effects of TCB in the kidney are mediated though the NKA-Src kinase signaling pathway and may have relevance to volume-overloaded conditions, such as chronic kidney disease where TCB is elevated.
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Ding, Luo-Bin, Yao Li, Guang-Yuan Liu та ін. "Long non-coding RNA PVT1, a molecular sponge of miR-26b, is involved in the progression of hyperglycemia-induced collagen degradation in human chondrocytes by targeting CTGF/TGF-β signal ways". Innate Immunity 26, № 3 (2019): 204–14. http://dx.doi.org/10.1177/1753425919881778.
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The current study was conducted to investigate the role of long non-coding RNA PVT1 in hyperglycemia-triggered human osteoarthritis (OA) chondrocytes. Cartilage from knee OA patients with and without diabetes, as well as normal cartilage, was obtained. Isolated human chondrocytes were treated with 30 nM of Glc with or without pioglitazone. The expression levels of PVT1, miR-26b, and type II collagen were determined by RT-PCR. Type II collagen was detected by immunocytochemistry and chondrocytes were stained with Alcian blue. Moreover, the interaction among PVT1, miR-26b, and CTGF was examined using bioinformatics, FISH, RIP, RNA-pull down, and luciferase reporter assays. Over-expression of PVT1 and miR-26b were performed and expressions of CTGF, TGF-β1, SMAD3, MMP-13, and type II collagen proteins were examined. Significantly higher expression of PVT1 was observed in diabetic OA. High Glc induced the elevated expression of PVT1, CTGF, TGF-β1, IL-6, and MMP-13, as well as decreased expression of type II collagen and miR-26b. These alterations could be reversed by pioglitazone. PVT1 acted as a sponge for miR-26b to facilitate CTGF expression. Over-expression of PVT1 increased the expressions of CTGF, TGF-β1, SMAD3, and MMP-13 and decreased expression of type II collagen. Our findings confirmed that PVT1 is involved in the hyperglycemia-induced collagen degradation, via the miR-26b-CTGF-TGF-β1-axis.
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Chiu, Y. H., J. Spierings, J. M. Van Laar, J. De Vries-Bouwstra, M. Van Dijk, and R. Goldschmeding. "POS0469 ENDOTHELIAL TO MESENCHYMAL TRANSITION AND SENESCENCE ARE PART OF THE FIBROTIC PATHOGENESIS IN SYSTEMIC SCLEROSIS." Annals of the Rheumatic Diseases 81, Suppl 1 (2022): 488.3–489. http://dx.doi.org/10.1136/annrheumdis-2022-eular.76.
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BackgroundSystemic sclerosis (SSc) is a systemic autoimmune disease characterised by inflammation, vasculopathy and fibrosis. Several mechanisms, including endothelial to mesenchymal transition (EndMT) and cellular senescence, may be included in the fibrosis process, especially in response to inflammation and vasculopathy.ObjectivesOur study aimed to identify how EndMT and cellular senescence manifest in SSc skin fibrosis.MethodsWe performed a cross-sectional biobank study on formalin-fixed, paraffin-embedded skin biopsies from patients meeting the ACR/EULAR 2013 classification criteria for SSc. The extent of dermal fibrosis and inflammatory cell infiltration was scored on haematoxylin-eosin stained samples. Fibrosis severity was semi-quantified by the compactness of collagen bundles and scored from one to three. The presence of senescence was defined by P21 or P16 positivity without co-localised Ki-67. The expression of P16, P21, and CTGF on fibroblasts and endothelia was scored from zero to two. The semi-quantitative score was evaluated independently in the superficial and deep dermis by agreement of two researchers (MRD and YHC). EndMT was assessed by co-localisation of CD31 and α-SMA double staining by immunofluorescence, and enclosure of ERG positive endothelial cell nuclei by α-SMA stained cytoplasm under bright field. Lymph vessels were identified by D2-40 (podoplanin) staining, and density was assessed.ResultsIn total, skin biopsies from 18 SSc patients and four age-matched healthy controls were investigated. The characteristics of the patients are listed in Table 1. The dermal fibrosis score revealed a correlation with modified Rodnan skin score (Figure 1A).Table 1.Clinical features of 18 patients with systemic sclerosisAge, median (IQR)47 (21)Female, n (%)11 (61)Biopsy site, n (%) Upper limb3 (17) Trunk6 (33) Lower limb8 (44)Type of SSc, n (%) Limited cutaneous SSc13 (72) Diffuse cutaneous SSc5 (28)mRSS, median (IQR)4 (9)Anti-topoisomerase I antibody positive, n (%)3 (17)Anti-RNA polymerase 3 positive, n (%)2 (11)Anti-centromere positive, n (%)3 (17)SSc, systemic sclerosis; IQR, interquartile range; mRSS, modified Rodnan skin score.Figure 1.(A) The modified Rodnan skin score (mRSS) was higher in skin biopsies with severe fibrosis. (B) While merging superficial and deep dermis semi-quantitative scores, increased senescence marker was associated with increased CTGF expression on fibroblasts. (C) Endothelial to mesenchymal transition (EndMT) was more in systemic sclerosis skin without difference between groups with various fibrosis severity. (D)EndMT was associated with increased senescence markers on fibroblasts.Senescence markers showed higher expression on fibroblasts from highly fibrotic dermis than healthy control. The median (IQR) percentage of P16 positive fibroblasts in dermis was 0.7 (0.5) in healthy controls, 0.9 (0.7) in grade one fibrosis, 0.5 (0.5) in grade two fibrosis, and 6.0 (4.6) in grade three fibrosis (p = 0.144). The median (IQR) percentage of P21 positive fibroblasts in dermis was 3.3 (7.0) in healthy controls, 10.3 (11.8) in grade one fibrosis, 33.2 (19.2) in grade two fibrosis, and 32.9 (40.7) in grade three fibrosis (p = 0.042). Moreover, increase of senescence markers on fibroblasts was associated with increased CTGF expression (Figure 1B).The density of blood and lymphatic vessels did not correlate with fibrosis severity. Still, the frequency of EndMT was significantly higher in patients with SSc, although it did not differ between groups with various fibrosis severity (Figure 1C). EndMT was not observed in lymphatic vessels (i.e. there was no co-localisation of D2-40 and α-SMA). Finally, the frequency of EndMT was increased with stronger fibroblast senescence (Figure 1D).ConclusionEndMT and fibroblast senescence were more prominent in SSc skin compared to healthy control skin. Strong fibroblast senescence was associated with EndMT. Fibroblast senescence might facilitate EndMT and be involved in SSc fibrosis.Disclosure of InterestsYu-Hsiang Chiu: None declared, Julia Spierings Grant/research support from: J. Spierings has received a grant from Boehringer., Jacob M. van Laar Consultant of: J. M. van Laar has received honoraria from Abbvie, Arxx Tx, Galapagos, Gesyntha, Leadiant, and Roche., Grant/research support from: J.M. van Laar has received grants from Boehringer, Astra Zeneca, MSD, and Roche., Jeska de Vries-Bouwstra: None declared, Marijke van Dijk: None declared, Roel Goldschmeding: None declared.
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Wasson, C., E. Clavane, R. Ross та ін. "OP0113 THE BETA SECRETASE BACE1 DRIVES SYSTEMIC SCLEROSIS FIBROBLASTS ACTIVATION THROUGH Β-CATENIN AND NOTCH SIGNALLING". Annals of the Rheumatic Diseases 82, Suppl 1 (2023): 75.1–75. http://dx.doi.org/10.1136/annrheumdis-2023-eular.2236.
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BackgroundThe beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is well known for its role in the development of Alzheimer’s disease via the generation of β-amyloid. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic inflammatory diseases, including type 2 diabetes and cardiovascular disease. However, to date there has been no studies looking into the role of BACE1 in the autoimmune condition Systemic Sclerosis (SSc).ObjectivesThe aim of this study was to assess the expression profile of BACE1 in SSc patient samples and investigate the effects of BACE1 inhibitors and siRNA on SSc fibroblast activation.MethodsPatient fibroblasts were obtained from full thickness forearm skin biopsies from healthy and early diffuse SSc patients. BACE1 was inhibited with 2 specific small molecule inhibitors and siRNA specific to BACE1. Morphogen signalling was activated with recombinant TGF-β, Wnt-3a or the smoothened agonist SAG. A xenotransplant bleomycin mouse model using patient pDC was used to interrogatein vivoexpression of BACE1 in fibrosis.ResultsHere we show that BACE1 protein levels are elevated in SSc patient skin biopsies. In particular BACE1 was increased in the fibroblasts and endothelial cells of the SSc skin. BACE1 was elevated in isolated dermal fibroblasts grown in culture (2.3 fold increase, N=4). BACE1 protein levels were elevated in the bleomycin skin fibrosis model. Interestingly BACE1 mRNA levels were unaffected in cultured SSc fibroblasts, suggesting a post-translational modification led to the elevated protein levels.Inhibition of BACE1 with small molecule inhibitors (that have been proven safe in phase 1 clinical trials for Alzheimer’s) or siRNA blocked pro-fibrotic gene (alpha SMA, Collagen Type 1 and CTGF) expression in SSc fibroblasts. In addition overexpression of BACE1 in healthy fibroblasts resulted in myofibroblast activation (2-fold increase in alpha SMA protein expression). Interestingly overexpression of a BACE1 mutant construct which disrupts the secretase activity of the protein, was unable to induce fibroblast activation.Disruption of BACE1 (with both the inhibitors and siRNA) blocked morphogen mediated fibroblasts activation. The BACE1 inhibitors and siRNA blocked TGF-β, Wnt-3a and Hedgehog mediated alpha-SMA expression in healthy fibroblasts. Furthermore, we show that BACE1 regulation of dermal fibroblast activation was dependent on the β-catenin and Notch signalling pathways. BACE1 ability to regulate non-canonical Wnt receptors led to elevated β-catenin expression which in turn activated the Notch signalling pathway.ConclusionThis is the first evidence that BACE1 and in particular its secretase activity, plays a role in SSc and fibrosis in general. The ability of BACE1 to regulate SSc fibroblast activation reveals an exciting new therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in phase 1 clinical trials for Alzheimer’s disease. Future work includes investigating the role of BACE1 in vascular/endothelial cell dysfunction in SSc.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
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Ouyang, Xiaosen, Thu H. Le, Carlos Roncal, et al. "Th1 inflammatory response with altered expression of profibrotic and vasoactive mediators in AT1A and AT1B double-knockout mice." American Journal of Physiology-Renal Physiology 289, no. 4 (2005): F902—F910. http://dx.doi.org/10.1152/ajprenal.00141.2005.
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AT1 double receptor (AT1A and AT1B) knockout mice have lower blood pressure, impaired growth, and develop early renal microvascular disease and tubulointerstitial injury. We hypothesized that there would be an increased expression of vasoactive, profibrotic, and inflammatory mediators expressed in the kidneys of AT1 double-knockout mice. We examined the renal expression of various mediator systems in control ( n = 6) vs. double-knockout mice ( n = 6) at 3–5 mo of age by real-time PCR, immunohistochemistry, and Western blot analysis. AT1 double-knockout mice show activation of Th1-dependent pathways (with increased expression of IFN-α, IL-2 mRNA) with increased expression of both monocyte (MCP-1 mRNA) and T cell (RANTES mRNA) chemokines, infiltration of CD4+ and CD11b+ cells, increased fibrosis-associated mediators (CTGF, TGF-β and TNF-α mRNA) and extracellular matrix (collagens I and III mRNA and protein) deposition compared with controls ( P < 0.05 for all markers). These changes were associated with increased mRNA expression of endothelin (ET)-1 and ET-A receptor ( P < 0.05), cyclooxygenase (COX)-2/TXA2 synthase ( P < 0.05), NADPH oxidase (p40-phox, p67-phox, P < 0.05) and iNOS and nNOS ( P < 0.05). COX-2 and nNOS protein were also increased in the kidneys of AT1 double-knockout mice by Western blot analysis ( P < 0.05). Although renin and angiotensinogen mRNA expression were increased in the knockout mice, AT2 receptor mRNA expression was not significantly different from wild-type mice. In conclusion, the absence of the AT1 receptor is associated with marked renal alterations in vasoactive, profibrotic, and immune mediators with an inflammatory pattern favoring a Th1 phenotype.
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Meng, YH, C. Tian, L. Liu, L. Wang, and Q. Chang. "Elevated expression of connective tissue growth factor, osteopontin and increased collagen content in human ascending thoracic aortic aneurysms." Vascular 22, no. 1 (2013): 20–27. http://dx.doi.org/10.1177/1708538112472282.
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Little is known about the molecular mechanisms of ascending thoracic aortic aneurysms (ATAAs). Abnormal extracellular matrix changes and variations of vascular smooth muscle cells (VSMCs) have been implicated in abdominal aortic aneurysm formation. Our objective was to investigate the alterations of collagen, stimulators of collagen synthesis and synthetic VSMCs in patients with ATAA. Surgical samples from ATAA were taken from 20 patients, and 18 control aortas were obtained during coronary artery bypass surgery. All aortic wall specimens were fixed for histology and immunohistochemistry for collagen, connective tissue growth factor (CTGF) and osteopontin. Realtime polymerase chain reaction was used to determine their mRNA expression. Histology and semi-quantitative analysis demonstrated that protein levels of collagen, CTGF and osteopontin significantly increased by 1.9-, 1.4- and 2.2-fold, respectively ( P < 0.01 for all) in the ATAA group than in the control group. Similar results were shown in mRNA levels of type Iα1and IIIα1 collagen, CTGF and osteopontin. The protein levels of CTGF and osteopontin were positively correlated with aortic diameter ( r = 0.67, r = 0.73; P < 0.01 for both). In conclusion, overexpression of aortic CTGF and synthetic VSMCs marker (osteopontin), which is likely to be responsible for elevated aortic collagen content, may provide a potential mechanism for aneurysmal enlargement.
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Del Papa, N., M. Lorini, V. Carbonelli, et al. "AB0153 ADIPOSE-DERIVED STROMAL/STEM CELLS FROM SYSTEMIC SCLEROSIS PATIENTS SUCCESSFULLY EXERT A PARACRINE ANTI-FIBROTIC ACTIVITY AND INDUCE A PRO-ANGIOGENIC PHENOTYPE OF SCLERODERMA FIBROBLASTS IN VITRO." Annals of the Rheumatic Diseases 79, Suppl 1 (2020): 1377.2–1377. http://dx.doi.org/10.1136/annrheumdis-2020-eular.3248.
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Background:Adipose-derived stromal/stem cells (ADSCs) are multipotential non-hematopoietic progenitor cells with anti-inflammatory, immunomodulatory and regenerative effects. They have the advantage of accessibility and potent pro-angiogenic effects when compared with other stem cells, such as bone-marrow derived stem cells. Recent studies have shown that autologous fat grafting may be effective in the treatment of fibrotic and vascular complications in systemic sclerosis (SSc), despite a pro-fibrotic signature.Objectives:Aim of the study was to better characterize the proliferative and secretive profile of ADSCs in normoxic and hypoxic conditions, and to evaluate the mechanisms by which ADSCs exert these observed clinical effects.Methods:Adipose tissue samples were obtained by liposuction from 12 SSc patients and 10 healthy donors (HD). ADSCs were purified according to their adherence to the plastic and characterized to express specific MSC surface antigens by flow cytometry analysis. Proliferation of ADSCs from SSc patients and normal controls was evaluated in normoxic and hypoxic conditions. Fibroblasts and ADSCs derived from SSc patients were co-cultured in direct and indirect culture systems and compared to HD. Fibroblasts proliferation, mRNA expression and protein secretion of VEGF and known fibrotic mediators including TGF β-1, TGFR, CTGF, Collagen type I (Col I) were analyzed in the same conditions.Results:Normoxic and hypoxic culture conditions did not modify the proliferation rate of both normal and SSc ADSCs. Hypoxia significantly increased mRNA expression of VEGF by HD and SSc ADSCs but had no effect on the mRNA expression of pro-fibrotic mediators, ie TGFβ and TGFR. Normal and SSc fibroblast proliferation was significantly reduced in both co-culture systems (p < 0.001) and by treatment with normoxic and hypoxic conditioned medium (CM) (p=0.001 and p=0.002). In the same conditions, mRNA expression and protein secretion of TGF-β1, CTGF and Col I were significantly reduced (p = 0.003, p =0.02, p=0.04). These results were confirmed when normal and SSc fibroblasts were cultured in the presence of ADSCs normoxic and hypoxic CM (p=0.02 and p=0.01). Furthermore, a significant increase in mRNA expression and production of VEGF was observed in SSc fibroblasts cultured in the presence of normoxic and hypoxic CM (p = 0.002 and p< 0.0001, respectively).Conclusion:We found that treatment with the medium from normoxic and hypoxic preconditioned ADSCs has an anti-fibrotic effect through both the inhibition of fibroblast proliferation and key mediators of fibrosis. The increased expression of VEGF by SSc fibroblasts in the presence of normoxic and, even more, hypoxic ADSCs CM, suggests that ADSCs can induce a paracrine pro-angiogenic phenotype, even in fibroblasts with a pro-fibrotic signature. Altogether these data show that ADSCs may exert their anti-fibrotic and pro-angiogenetic effects on SSc fibroblasts by the secretion of paracrine factors, partly explaining the mechanisms underlining beneficial clinical results of fat graft in SSc patients.Disclosure of Interests:Nicoletta Del Papa: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Antonina Minniti: None declared, Francesca Pignataro: None declared, Wanda Maglione: None declared, Gabriele Di Luca: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB
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Ong, V., X. Shiwen, F. Zhang, et al. "POS0145 CLINICAL AND PATHOGENIC SIGNIFICANCE OF S100A4 OVEREXPRESSION IN SYSTEMIC SCLEROSIS." Annals of the Rheumatic Diseases 82, Suppl 1 (2023): 293–94. http://dx.doi.org/10.1136/annrheumdis-2023-eular.581.
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BackgroundS100A4 belongs to a family of proteins originally defined by solubility of the prototypic members in 100% saturated ammonium sulphate. Preclinical studies suggest that extracellular S100A4 as a Damage Associated Molecular Pattern (DAMP) protein is upregulated upon stress or injury and may be a target for antifibrotic therapy.ObjectivesWe explored expression and function of S100A4 as a driver of fibroblast activation in systemic sclerosis (SSc).MethodsS100A4 protein concentration was measured by ELISA in serum of SSc (n=94) and healthy controls (n=15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n=6) and healthy controls (NF, n=6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate the profibrotic molecular, functional and genomic phenotype in SSc and control fibroblasts.ResultsMedian [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p=0.027). There was significant association with SSc-ILD (p=0.025, n=55), scleroderma renal crisis (p=0.026, n=4) and trend of increase in pulmonary hypertension (p=0.075, n=7) and a trend for S100A4 to fall in longer disease duration (r=-0.18, p=0.09) and a weak correlation (r=0.28, p=0.07) with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF (0.28 [0.02-3.29]; p<0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF 0.50 [0.91]; SScF 8.16 [2.49]; p<0.0001). S100A4 induced a fibrotic phenotype in NF with concentration-dependent increase in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p<0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR<0.001 and FC>1.5) induced in NF by S100A4, were also constitutively overexpressed, and downregulated by AX-202, in SScF (Figure 1A). Pathway mapping of these S100A4 dependent genes in SSc showed the most significant enriched Kegg pathways (FDR<0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold) (Figure 1B).ConclusionOur findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. This study supports examining the therapeutic potential of targeting S100A4 in SSc are warranted.Figure 1.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsVoon Ong Speakers bureau: Boehringer Ingelheim, Xu Shiwen: None declared, Fenge Zhang: None declared, Rory Maclean: None declared, Kristina Clark: None declared, Signe Borchert Employee of: Arxx Therapeutics, Rizwan I Hussain Employee of: Arxx Therapeutics, Jörg Klingelhöfer Employee of: Arxx Therapeutics, Jonas Hallén Employee of: Arxx Therapeutics, Christopher P Denton Speakers bureau: Janssen; Boehringer Ingelheim, Consultant of: GSK; CSL Behring; Boehringer Ingelheim; Merck; Roche; Sanofi, Grant/research support from: GSK; CSL Behring; Inventiva; Horizon; Arxx Therapeutics.
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Dean, Rachael G., Leanne C. Balding, Riccardo Candido, et al. "Connective Tissue Growth Factor and Cardiac Fibrosis after Myocardial Infarction." Journal of Histochemistry & Cytochemistry 53, no. 10 (2005): 1245–56. http://dx.doi.org/10.1369/jhc.4a6560.2005.
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The temporal and spatial expression of transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-β1, CTGF, and procollagen α1(I) mRNA were localized by in situ hybridization, and TGF-β1 and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-β1, CTGF, and collagen after MI. Procollagen α1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-β1 mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-β1 or CTGF. TGF-β1 is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.
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Qi, W., S. Twigg, X. Chen та ін. "Integrated actions of transforming growth factor-β1 and connective tissue growth factor in renal fibrosis". American Journal of Physiology-Renal Physiology 288, № 4 (2005): F800—F809. http://dx.doi.org/10.1152/ajprenal.00179.2004.
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Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-β1 (TGF-β1) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-β1 is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values ( P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-β or to the TGF-β type II receptor (TβRII). TGF-β1 induced a greater increase in fibronectin and collagen IV secretion in both PTC ( P < 0.01) and CF ( P < 0.01) compared with that observed with CTGF alone. The combination of TGF-β1 and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-β1 (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein ( P < 0.05), whereas CTGF had no effect on TGF-β1 mRNA or protein expression. TGF-β1 (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-β. It has further uniquely demonstrated that CTGF requires TGF-β, signaling through the TβRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.