Smad Signaling Research Articles

Dachengqi decoction ameliorates sepsis-induced liver injury by inhibiting the TGF-β1/Smad3 pathways

BackgroundSepsis-induced acute liver injury (ALI) is a major contributor to mortality in septic patients. Exploring the pathogenesis and developing effective treatment strategies for sepsis-induced ALI is critical for improving patient outcomes. Dachengqi decoction (DCQD), which is a classic Chinese herbal medicine, has been shown to possess potent anti-inflammatory properties. However, the protective effects and underlying mechanisms of DCQD against sepsis-induced ALI remain unclear. This study aimed to investigate the protective effect of DCQD on sepsis-induced ALI and elucidate the involvement of the TGF-1β/Smad3 pathways. MethodsA septic mouse model was established using caecal ligation and puncture (CLP) to evaluate the protective effect of DCQD on sepsis-induced ALI in vivo. An in vitro cellular inflammation model was established using LPS-stimulated LO2 cells to further investigate the underlying mechanism. ResultsDCQD (2.5, 5.0, and 10.0 g/kg body weight) was administered twice daily for 2 days and exerted a dose-dependent protective effect against sepsis-induced ALI. DCQD treatment significantly inhibited inappropriate inflammatory responses and oxidative stress in liver tissue. Moreover, DCQD maintained liver homeostasis by inhibiting hepatocyte apoptosis and improving sepsis-induced liver damage. In vivo and in vitro studies indicated that the TGF-β1/Smad3 signalling pathway played an important role in sepsis-induced ALI, and DCQD treatment significantly inhibited the activation of this pathway. ConclusionsDCQD can effectively suppress excessive inflammatory responses and oxidative stress, leading to a substantial reduction in hepatocyte apoptosis in sepsis-induced ALI.

Longitudinal assessment of bleomycin-induced pulmonary fibrosis by evaluating TGF-β1/Smad2, Nrf2 signaling and metabolomic analysis in mice

IntroductionPulmonary fibrosis (PF) is characterized by an increase in collagen synthesis and deposition of extracellular matrix. Several factors, including transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog family proteins (Smad), and alpha-smooth muscle actin (α-SMA) trigger extracellular matrix (ECM) accumulation, fibroblast to myofibroblasts conversion, and epithelial-to-mesenchymal-transition (EMT) leading to PF. However, the role of cellular defense mechanisms such as the role of nuclear factor erythroid 2–related factor 2 (Nrf2) signaling during the onset and progression of PF is not understood completely. AimThe present study aims to analyze the involvement of TGF-β1/Smad signaling, and Nrf2 in the EMT and metabolic alterations that promote fibrosis in a time-dependent manner using bleomycin (BLM)-induced PF model in C57BL/6 mice. Key findingsHistopathological studies revealed loss of lung architecture and increased collagen deposition in BLM-exposed mice. BLM upregulated TGF-β1/Smad signaling and α-SMA at all time-points. The gradual increase in the accumulation of α-SMA and collagen implied the progression of PF. BLM exposure raises Nrf2 throughout each specified time-point, which suggests that Nrf2 activation might be responsible for TGF-β1-induced EMT and the development of PF. Further, metabolomic studies linked the development of PF to alterations in metabolic pathways. The pentose phosphate pathway (PPP) was consistently enriched across all the time-points. Additionally, alterations in 22 commonly enriched pathways, associated with fatty acid (FA) and amino acid metabolism were observed in 30- and 60-days. SignificanceThis study elucidates the association of TGF-β1/Smad and Nrf2 signaling in the EMT and metabolic alterations associated with the etiology and progression of PF.

FOXM1 Promotes Mesothelioma Cell Migration and Invasion via Activation of SMAD Signaling.

Forkhead box M1 (FOXM1) is a transcription factor closely associated with various human malignancies and is considered an attractive target for cancer therapy. Mesothelioma is a malignancy primarily due to asbestos exposure and certain genetic factors, requiring a better understanding of tumorigenesis for improved treatment. Asbestos-exposed human mesothelial cells have been reported to up-regulate FOXM1 expression in a dose-dependent manner. FOXM1 expression was evaluated in mesothelioma tissues and cell lines. FOXM1 small interfering RNA was transfected into mesothelioma cell lines to analyze its biological functions and regulatory mechanisms. FOXM1 was over-expressed in mesothelioma tissues and cell lines. Knock-down of FOXM1 in mesothelioma cell lines inhibited cell proliferation, migration, and invasion. These results suggest that up-regulation of FOXM1 expression promotes mesothelioma tumorigenesis and progression. We previously reported that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) promotes the proliferation, migration, and invasion of mesothelioma cell lines. In this study, IGF2BP3 knock-down suppressed FOXM1 expression in mesothelioma cell lines. Our results suggest that IGF2BP3, an upstream regulator, contributes to increased FOXM1 expression. Furthermore, IGF2BP3 and FOXM1 knock-down suppressed SMAD signaling by inhibiting SMAD2/3 phosphorylation in mesothelioma cell lines. IGF2BP3/FOXM1 promotes mesothelioma cell migration and invasion via SMAD signaling, highlighting IGF2BP3/FOXM1 as a potential target for mesothelioma treatment.

A cysteine proteinase inhibitor ALLN alleviates bleomycin-induced skin and lung fibrosis

BackgroundSystemic sclerosis (SSc) is a connective tissue disease that is characterized by fibrosis in the skin and internal organs, such as the lungs. Activated differentiation of progenitor cells, which are mainly resident fibroblasts, into myofibroblasts is considered a key mechanism underlying the overproduction of extracellular matrix and the resultant tissue fibrosis in SSc. Calpains are members of the Ca2+-dependent cysteine protease family, whose enzymatic activities participate in signal transduction and tissue remodeling, potentially contributing to fibrosis in various organs. However, the roles of calpain in the pathogenesis of SSc remain unknown. This study aimed to examine the anti-fibrotic properties of N-acetyl-Leu-Leu-norleucinal (ALLN), one of the cysteine proteinase inhibitors that primarily inhibit calpain, in vitro and in vivo, to optimally translate into the therapeutic utility in human SSc.MethodsNormal human dermal and lung fibroblasts pretreated with ALLN were stimulated with recombinant transforming growth factor beta 1 (TGF-β1), followed by assessment of TGF-β1/Smad signaling and fibrogenic molecules.ResultsALLN treatment significantly inhibited TGF-β1-induced phosphorylation and nuclear transport of Smad2/3 in skin and lung fibroblasts. TGF-β1-dependent increases in α-smooth muscle actin (αSMA), collagen type I, fibronectin 1, and some mesenchymal transcription markers were attenuated by ALLN. Moreover, our findings suggest that ALLN inhibits TGF-β1-induced mesenchymal transition in human lung epithelial cells. Consistent with these in vitro findings, administering ALLN (3 mg/kg/day) three times a week intraperitoneally remarkably suppressed the development of skin and lung fibrosis in a SSc mouse model induced by daily subcutaneous bleomycin injection. The number of skin- and lung-infiltrating CD3+ T cells decreased in ALLN-treated mice compared with that in control-treated mice. Phosphorylation of Smad3 and/or an increase in αSMA-positive myofibroblasts was significantly inhibited by ALLN treatment on the skin and lungs. However, no adverse effects were observed.ConclusionsOur results prove that calpains can be a novel therapeutic target for skin and lung fibrosis in SSc, considering its inhibitor ALLN.

Fusobacterium nucleatum promotes colorectal cancer metastasis by excretion of miR-122-5p from cells via exosomes

Fusobacterium nucleatum (Fn) infection and microRNAs (miRNAs) are closely associated with colorectal cancer (CRC) development, but the mechanism by which Fn regulates tumor-suppressive miRNAs via exosomes and facilitates CRC metastasis remains unclear. Here, we identified that Fn infection significantly increased exosomal miR-122-5p levels in the serum of CRC patients and CRC cell culture supernatants through two miRNA panels of high-throughput sequencing and RT-qPCR analysis. In Fn-infected patients, the serum exosomal levels of miR-122-5p were negatively associated with their expression levels of tissues. Downregulated miR-122-5p was demonstrated to enhance the migration, invasion, and metastasis abilities of CRC cells invivo and invitro. Secretion of miR-122-5p into exosomes is mediated by hnRNPA2B1. Mechanistically, Fn activated the TGF-β1/Smads signaling pathway to promote EMT by regulation of the miR-122-5p/FUT8 axis. In conclusion, Fn infection may stimulate CRC cells to excrete exosome-wrapped miR-122-5p, and activate the FUT8/TGF-β1/Smads axis to promote metastasis.

Liraglutide Suppresses Myocardial Fibrosis Progression by Inhibiting the Smad Signaling Pathway.

Liraglutide is a commonly used hypoglycemic agent in clinical practice, and has been demonstrated to have protective effects against the development of cardiovascular disease. However, its potential role in myocardial fibrosis remains unexplored. The present study aims to assess the impact of liraglutide on the activation of cardiac fibroblasts. Primary rat adult fibroblasts were isolated, cultured, and randomly allocated into 4 groups: control group, transforming growth factor beta1 (TGFβ1) stimulation group, liraglutide group, and TGFβ1+liraglutide group. Fibroblast activation was induced by TGFβ1. Cell proliferation activity was assessed using the CKK-8 kit, and cellular activity was determined using the MTT kit. Reverse transcrition-quantitative polymerase chain reaction (RT-qPCR) was utilized to quantify the level of collagen transcription, immunofluorescence staining was performed to detect the expression level of type III collagen and α-smooth muscle protein (α-SMA), and immunoblotting was conducted to monitor alterations in signal pathways. The addition of 10, 25, 50 and 100 nmol/L of liraglutide did not induce any significant impact on the viability of fibroblasts (P>0.05). The rate of cellular proliferation was significantly higher in the TGFβl stimulation group than in the control group. However, the treatment with 50 and 100 nmol/L of liraglutide resulted in the reduction of TGFβl-induced cell proliferation (P<0.05). The RT-qPCR results revealed that the transcription levels of type I collagen, type III collagen, and α-SMA were significantly upregulated in the TGFβl stimulation group, when compared to the control group (P<0.05). However, the expression levels of these aforementioned factors significantly decreased in the TGFβl+liraglutide group (P<0.05). The immunofluorescence staining results revealed a significant increase in the expression levels of type III collagen and α-SMA in the TGFβl stimulation group, when compared to the control group (P<0.05). However, these expression levels significantly decreased in the TGFβl+liraglutide group, when compared to the TGFβl stimulation group (P<0.05). The Western blotting results revealed that the expression levels of phosphorylated smad2 and smad3 significantly increased in the TGFβl stimulation group, when compared to the control group (P<0.05), while these decreased in the TGFβl+liraglutide group (P<0.05). Liraglutide inhibits myocardial fibrosis development by suppressing the smad signaling pathway, reducing the activation and secretion of cardiac fibroblasts.

Retracted: Exosomes Derived from SW480-Resistant Colon Cancer Cells are Promote Angiogenesis via BMP-2/Smad5 Signaling Pathway.

[This retracts the article DOI: 10.1155/2022/6124374.].

Adipose-derived mesenchymal stem cell therapy for reverse bleomycin-induced experimental pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive respiratory disease. Arguably, the complex interplay between immune cell subsets, coupled with an incomplete understanding of disease pathophysiology, has hindered the development of successful therapies. Despite efforts to understand its pathophysiology and develop effective treatments, IPF remains a fatal disease, necessitating the exploration of new treatment options. Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of IPF, but further investigation is needed to understand its therapeutic effect. This study aimed to assess the therapeutic effect of adipose-derived mesenchymal stem cells in a bleomycin-induced pulmonary fibrosis model. First, MSC cells were obtained from mice and characterized using flow cytometry and cell differentiation culture methods. Then adult C57BL/6 mice were exposed to endotracheal instillation of bleomycin and concurrently treated with MSCs for reversal models on day 14. Experimental groups were evaluated on days 14, 21, or 28. Additionally, lung fibroblasts challenged with TGF-β1 were treated with MSCs supernatant or MSCs to explore the mechanisms underlying of pulmonary fibrosis reversal. Mesenchymal stem cells were successfully isolated from mouse adipose tissue and characterized based on their differentiation ability and cell phenotype. The presence of MSCs or their supernatant stimulated the proliferation and migration of lung fibrotic cells. MSCs supernatant reduced lung collagen deposition, improved the Ashcroft score and reduced the gene and protein expression of lung fibrosis-related substances. Bleomycin-challenged mice exhibited severe septal thickening and prominent fibrosis, which was effectively reversed by MSCs treatment. MSC supernatant could suppress the TGF-β1/Smad signaling pathway and supernatant promotes fibroblast autophagy. In summary, this study demonstrates that MSCs supernatant treatment is as effective as MSCs in revert the core features of bleomycin-induced pulmonary fibrosis. The current study has demonstrated that MSCs supernatant alleviates the BLM-induced pulmonary fibrosis in vivo. In vitro experiments further reveal that MSC supernatant could suppress the TGF-β1/Smad signaling pathway to inhibit the TGF-β1-induced fibroblast activation, and promotes fibroblast autophagy by Regulating p62 expression. These findings contribute to the growing body of evidence supporting the therapeutic application of MSCs in cell therapy medicine for IPF.

Flavokawain A ameliorates pulmonary fibrosis by inhibiting the TGF-β signaling pathway and CXCL12/CXCR4 axis

Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease characterized by myofibroblast proliferation and extracellular matrix deposition that has a high mortality rate and limited therapeutic options. Flavokawain A(FKA) is the major component of chalcone in kava extract. FKA has been reported to inhibit TGF-β1-induced cardiomyocyte fibrosis by suppressing ROS production in A7r5 cells, but the role and mechanism of FKA in pulmonary fibrosis are unknown. In this study, we evaluated the effect of FKA on pulmonary fibrosis using an animal model of bleomycin-induced pulmonary fibrosis and showed that FKA alleviated the development of pulmonary fibrosis in a dose-dependent manner and improved lung function as well as collagen deposition and extracellular matrix accumulation in mice. In vitro studies showed that FKA inhibited myofibroblast activation and lung fibrosis progression by inhibiting TGF-β1/Smad signaling in a dose-dependent manner. In addition, we identified CXCL12 as a potential target of FKA through target prediction. Molecular docking, CETSA(cellular thermal displacement assay) and silver staining assays further demonstrated that FKA could interact with CXCL12 and that FKA could inhibit CXCL12 dimerization in vitro. Further analysis revealed that FKA could inhibit fibroblast activation and reduce extracellular matrix (ECM) production and collagen deposition by blocking CXCL12/CXCR4 signaling, and knocking down CXCR4 expression could weaken the inhibitory effect of FKA on CXCL12/CXCR4 signal transduction. In conclusion, our study showed that FKA inhibited CXCL12/CXCR4 signaling by inhibiting CXCL12 dimerization, blocked the CXCL12/CXCR4 signaling pathway and inhibited the TGF-β1-mediated signaling pathway to ameliorate pulmonary fibrosis, and FKA is a promising therapeutic agent for pulmonary fibrosis.

Kinetin inhibits hepatic stellate cell activation and induces apoptosis via interactions with the TGF-β1/Smad signaling pathway

Hepatic fibrosis is the pathological repair response of the liver to chronic injury; hepatic stellate cell (HSC) activation is the central link in the pathogenesis of hepatic fibrosis. Previously, we showed that kinetin, a plant cytokinin hormone, has a protective effect on CCl4-induced liver injury in mice. However, the role of kinetin in liver fibrosis remains unclear. We aimed to study these protective effects and to determine the mechanisms by which kinetin mediates HSC activation and apoptosis. For this purpose, the human HSC line LX-2 was treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 24 h to stimulate activation. We found that treatment with kinetin at the sub-cytotoxic dose of 40 μg/ml for 48 h reduced the expression of the HSC activation marker α-SMA and inhibited the secretion of extracellular matrix proteins. In addition, kinetin was found to inhibit the proliferation and migration of LX-2 cells. We found that kinetin induced apoptosis in LX-2 cells by increasing the level of cleaved-caspase 3 and the Bax-to-Bcl-2 ratio. Interestingly, these effect were not observed in quiescent HSCs, suggesting that they are activation-dependent. Further study showed that kinetin attenuates activation and promotes apoptosis of LX-2 cells in vitro in part by suppressing the TGF-β1/Smad signaling pathway.

Pacritinib is a potent ACVR1 inhibitor with significant anemia benefit in patients with myelofibrosis

AbstractIn patients with cytopenic myelofibrosis, treatment with the JAK2/IRAK1 inhibitor pacritinib was associated with anemia benefit in the phase 3 PERSIST-2 study. The impact of pacritinib on transfusion independence (TI) has not been previously described, nor has the mechanism by which pacritinib improves anemia been elucidated. Because it has been previously postulated that inhibition of activin A receptor, type 1 (ACVR1)/activin receptor-like kinase-2 improves anemia in patients with myelofibrosis via suppression of hepcidin production, we assessed the relative inhibitory potency of pacritinib compared with other JAK2 inhibitors against ACVR1. Pacritinib inhibited ACVR1 with greater potency (half-maximal inhibitory concentration [IC50] = 16.7 nM; Cmax:IC50 = 12.7) than momelotinib (IC50 = 52.5 nM; Cmax:IC50 = 3.2), fedratinib (IC50 = 273 nM; Cmax:IC50 = 1.0), or ruxolitinib (IC50 > 1000; Cmax:IC50 < 0.01). Pacritinib’s inhibitory activity against ACVR1 was corroborated via inhibition of downstream SMAD signaling in conjunction with marked suppression of hepcidin production. Among patients on PERSIST-2 who were not transfusion independent at baseline based on Gale criteria, a significantly greater proportion achieved TI on pacritinib compared with those treated on best available therapy (37% vs 7%, P = .001), and significantly more had a ≥50% reduction in transfusion burden (49% vs 9%, P < .0001). These data indicate that the anemia benefit of the JAK2/IRAK1 inhibitor pacritinib may be a function of potent ACVR1 inhibition.

KLF10 Inhibits TGF-β-Mediated Activation of Hepatic Stellate Cells via Suppression of ATF3 Expression.

Liver fibrosis is a progressive and debilitating condition characterized by the excessive deposition of extracellular matrix proteins. Stellate cell activation, a major contributor to fibrogenesis, is influenced by Transforming growth factor (TGF-β)/SMAD signaling. Although Krüppel-like-factor (KLF) 10 is an early TGF-β-inducible gene, its specific role in hepatic stellate cell activation remains unclear. Our previous study demonstrated that KLF10 knockout mice develop severe liver fibrosis when fed a high-sucrose diet. Based on these findings, we aimed to identify potential target molecules involved in liver fibrosis and investigate the mechanisms underlying the KLF10 modulation of hepatic stellate cell activation. By RNA sequencing analysis of liver tissues from KLF10 knockout mice with severe liver fibrosis induced by a high-sucrose diet, we identified ATF3 as a potential target gene regulated by KLF10. In LX-2 cells, an immortalized human hepatic stellate cell line, KLF10 expression was induced early after TGF-β treatment, whereas ATF3 expression showed delayed induction. KLF10 knockdown in LX-2 cells enhanced TGF-β-mediated activation, as evidenced by elevated fibrogenic protein levels. Further mechanistic studies revealed that KLF10 knockdown promoted TGF-β signaling and upregulated ATF3 expression. Conversely, KLF10 overexpression suppressed TGF-β-mediated activation and downregulated ATF3 expression. Furthermore, treatment with the chemical chaperone 4-PBA attenuated siKLF10-mediated upregulation of ATF3 and fibrogenic responses in TGF-β-treated LX-2 cells. Collectively, our findings suggest that KLF10 acts as a negative regulator of the TGF-β signaling pathway, exerting suppressive effects on hepatic stellate cell activation and fibrogenesis through modulation of ATF3 expression. These results highlight the potential therapeutic implications of targeting the KLF10-ATF3 axis in liver fibrosis treatment.

Retracted] Inhibition airway remodeling and transforming growth factor‑β1/Smad signaling pathway by astragalus extract in asthmatic mice.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the P‑smad2 western blotting data shown in Fig.7 were strikingly similar to data appearing in different form (namely, the bands appeared in the reverse orientation) in Fig.4A in another article [LvZ‑D, NaD, LiuF‑N, Du Z‑M, Sun Z, Li Z, MaX‑Y, WangZ‑N and XuH‑M: Induction of gastric cancer cell adhesion through transforming growth factor‑beta1‑mediated peritoneal fibrosis. J Exp Clin Cancer Res29: 139, 2010], which was written by mostly different authors at different research institutes (the author Zheng‑Hai Qu did appear as an author on both papers). Owing to the fact that the contentious data in the above article had already been published prior to its submission to International Journal of Molecular Medicine, and due to a lack of overall confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 29: 564‑568, 2012; DOI: 10.3892/ijmm.2011.868].

CL4-modified exosomes deliver lncRNA DARS-AS1 siRNA to suppress triple-negative breast cancer progression and attenuate doxorubicin resistance by inhibiting autophagy

Triple-negative breast cancer (TNBC) is a fatal disease. Drug resistance and the lack of effective drugs are the leading causes of death in patients with TNBC. Recently, long non-coding RNAs have been proven to be effective drug design targets owing to their high tissue specificity; however, an effective drug delivery system is necessary for their clinical application. In this study, we constructed a novel nanodrug delivery system based on the epidermal growth factor receptor (EGFR)-targeted aptamer CL4-modified exosomes (EXOs-CL4) for the targeted delivery of aspartyl-tRNA synthetase-antisense RNA 1 (DARS-AS1) small interfering RNA (siRNA) and doxorubicin (DOX) to TNBC cells in vitro and in vivo. This delivery system exerted potent anti-proliferation, anti-migration, and pro-apoptotic effects on TNBC cells. Silencing DARS-AS1 increased the sensitivity of TNBC cells to DOX by suppressing the transforming growth factor-β (TGF-β)/Smad3 signaling pathway-induced autophagy, thereby enhancing the synergetic antitumor effects. Collectively, our findings revealed that EXOs-CL4-mediated delivery of DARS-AS1 siRNA can be used as a new treatment strategy for DOX-resistant TNBC. Moreover, EXOs-CL4 can be used as effective drug delivery systems for targeted TNBC therapy.

Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation

Objectives: Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts. Methods and Results: To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2 CreER driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation. Conclusions: Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.

Abstract P2011: Reduced Activity Of Activin Like Kinase 1 (ALK1) Increases Mortality And Myocardial Rupture After Acute Myocardial Infarction

Introduction: Acute myocardial infarction (AMI) is a primary cause of heart failure (HF). Transforming growth factor beta (TGFβ) plays a central role in cardiac remodeling after AMI. Reduced activity of the TGFβ Type I receptor, activin like kinase (ALK)-1 promotes cardiac fibrosis in murine models of heart failure. A functional role for ALK1 in post-infarct cardiac remodeling remains poorly understood. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after AMI. Methods: AMI was induced by left coronary ligation in wild type (WT), and ALK+/- mice. After 14 days, LV function was assessed using conductance catheters and myocardial ALK1 signaling was quantified. Using siRNAs targeting ALK1 in in human cardiac fibroblasts (H-CFs), we further tested the role of ALK1 in cardiac fibrosis. Results: Reduced ALK1 activity significantly reduced survival compared to WT (p= 0.008 respectively) after AMI. ALK1 mice demonstrated a higher rate of cardiac rupture compared to WT after AMI (0% vs 85.7%). Both WT ad ALK1+/- mice demonstrated reduced LV ejection fraction and stroke work compared to sham controls (p=0.001 and p=0.0001 respectively). Profibrotic SMAD3 signaling was increased and antifibrotic SMAD1 signaling decreased in ALK1-/+ (pSMAD3, p=0.03, pSMAD1, p=0.003) mice 14 days after AMI. Reduced SMAD1 signaling was directly associated with increased myocardial Type 1 collagen protein levels in ALK1-/+ (R2=0.42, p=0.01) mice compared to WT. Myocardial levels of the TGFb1 co-receptor, endoglin, were increased significantly in ALK+/- as compared to WT (p=0.001) after AMI. Activity of MMP9 was increased in ALK1+/- mice, but protein levels were not affected 2 weeks after MI. Conclusion: We identify for the first time that loss of ALK1 activity increases cardiac rupture and mortality after AMI. These findings identify a novel functional role for ALK1 as a determinant of post-infarct survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after AMI.

Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer’s disease risk gene SORL1

SORL1 is implicated in the pathogenesis of Alzheimer's disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain.Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor β (TGF-β)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

Bromodomain protein 4 mediates the roles of TGFβ1-induced Stat3 signaling in mouse liver fibrogenesis

Epigenetic reader Bromodomain protein 4 (BrD4) functions as a global genomic regulator to direct hepatic stellate cell (HSC) activation (a key step in liver fibrogenesis) and liver fibrosis. The pivotal pro-fibrotic cytokine transforming growth factor-β1 (TGFβ1) signals through both Smad and Stat3 to elicit a wide array of biological effects. Stat3 is widely acknowledged as a regulator of gene transcription and is involved in fibrosis of multiple tissues. The present study focused on BrD4 function implication in the roles of TGFβ1-induced Stat3 signaling in HSC activation and liver fibrosis by using heterozygous TGFβ1 knockout mice and HSC culture. Results showed that Stat3 was required for TGFβ1-induced BrD4 expression in HSCs. BrD4 expression paralleled Stat3 activation in activated HSCs in human cirrhotic livers. BrD4 was involved in the roles of TGFβ1-induced Stat3 in HSC activation and liver fibrogenesis. Smad3 bound to phosphorylated-Stat3 and contributed to TGFβ1-induced Stat3 signaling. BrD4 expression induced by Stat3 signaling required the early-immediate gene Egr1. Egr1 had a positive feedback on Stat3 activation. In conclusion, a network consisting of Stat3 signaling, Smad3 signaling, Egr1, and BrD4 was involved in the effects of TGFβ1 on liver fibrosis, providing new toxicological mechanisms for TGFβ1 in liver fibrogenesis.

Integrating network pharmacology and experimental verification to explore the mechanisms of salidroside against myocardial fibrosis

Myocardial fibrosis (MF) is the manifestation of a variety of cardiovascular diseases. Salidroside (SAL) has been proved to have a certain effect on anti-fibrosis in various organs. However, the mechanism of SAL in the treatment of MF remains unclear. Network pharmacology showed that there were 1228 SAL-related target genes and 2793 MF-related target genes. The intersection of these genes resulted in 271 drug-disease interactions, and 15 core active targets were filtered from protein-protein interaction mapping. The top 20 Gene ontology biological processes analysis showed that the involved processes were close to the pathogenesis of MF. Among the top 20 enriched KEGG pathways, Wnt/β-catenin and TGF-β1/Smad3 signaling pathways were identified. In vivo, MI rats exhibited thinning of the myocardial region and the formation of fibrous scars, the expression of smad3 and β-catenin were increased. After SAL treatment, there was a significant reduction in collagen area and a decrease in the ratio of collagen type I to type III. The expression of smad3 and β-catenin was suppressed and positively correlated with the dosage of SAL. SAL may contribute to the progression of MF through the TGF-β1/Smad3 and Wnt/β-catenin signaling pathways.

Genetic and pharmacological inhibition of METTL3 alleviates renal fibrosis by reducing EVL m6A modification through an IGF2BP2-dependent mechanism.

N6 -methyladenosine (m6A) is of great importance in renal physiology and disease progression, but its function and mechanism in renal fibrosis remain to be comprehensively and extensively explored. Hence, this study will explore the function and potential mechanism of critical regulator-mediated m6A modification during renal fibrosis and thereby explore promising anti-renal fibrosis agents. Renal tissues from humans and mice as well as HK-2 cells were used as research subjects. The profiles of m6A modification and regulators in renal fibrosis were analysed at the protein and RNA levels using Western blotting, quantitative real-time polymerase chain reaction and other methods. Methylation RNA immunoprecipitation sequencing and RNA sequencing coupled with methyltransferase-like 3 (METTL3) conditional knockout were used to explore the function of METTL3 and potential targets. Gene silencing and overexpression combined with RNA immunoprecipitation were performed to investigate the underlying mechanism by which METTL3 regulates the Ena/VASP-like (EVL) m6A modification that promotes renal fibrosis. Molecular docking and virtual screening with in vitro and in vivo experiments were applied to screen promising traditional Chinese medicine (TCM) monomers and explore their mechanism of regulating the METTL3/EVL m6A axis and anti-renal fibrosis. METTL3 and m6A modifications were hyperactivated in both the tubular region of fibrotic kidneys and HK-2 cells. Upregulated METTL3 enhanced the m6A modification of EVL mRNA to improve its stability and expression in an insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2)-dependent manner. Highly expressed EVL binding to Smad7 abrogated the Smad7-induced suppression of transforming growth factor-β (TGF-β1)/Smad3 signal transduction, which conversely facilitated renal fibrosis progression. Molecular docking and virtual screening based on the structure of METTL3 identified a TCM monomer named isoforsythiaside, which inhibited METTL3 activity together with the METTL3/EVL m6A axis to exert anti-renal fibrosis effects. Collectively, the overactivated METTL3/EVL m6A axis is a potential target for renal fibrosis therapy, and the pharmacological inhibition of METTL3 activity by isoforsythiaside suggests that it is a promising anti-renal fibrosis agent.