CTGF Expression Research Articles

Effects of salvianolic acid A and salvianolic acid B in renal interstitial fibrosis via PDGF-C/PDGFR-α signaling pathway

BackgroundRenal interstitial fibrosis (RIF) is the main pathological feature of end-stage renal disease (ESRD) caused by various chronic kidney diseases (CKD), and is closely related to renal dysfunction and patient prognosis. Salvianolic acid A (Sal A) and salvianolic acid B (Sal B), isolated from traditional Chinese medicine Salviae miltiorrhizae, have been confirmed to have anti-fibrotic effects on liver, cardiac and kidney. However, the precise molecular mechanism underlying the nephroprotective effects of Sal A and Sal B, and whether there is a difference between the two in RIF are still unclear. PurposeThis study investigated the pharmacological effects of Sal A and Sal B in RIF and explore the underlying mechanisms by in vivo and in vitro experiments. MethodsThe nephroprotective effects of Sal A, Sal B and Sal A+B were evaluated by assessing the parameters related to kidney function such as renal histology, renal function, urinary protein NAG, urinary β2 microglobulin. In addition, RIF-related markers such as CTCF and Par3 were also detected. Thereafter, the related protein or gene levels of PDGF-C/PDGFR-α signaling pathways, apoptosis and endoplasmic reticulum stress (ERS) were determined by western blot, real-time PCR, flow cytometry or immunofluorescence staining. ResultsIn vivo, the results showed that Sal A, Sal B and Sal A+B partially improved kidney dysfunction, increased the expression of Par-3 and reduced the expression of CTGF, PDGF-C and PDGFR-α. In vitro, the results also showed that Sal A, Sal B and Sal A+B reversed apoptosis and ERS in HSA-induced HK-2 cells via regulating PDGF-C/PDGFR-α signaling pathway. ConclusionThis article revealed a novel mechanism linking PDGF-C/PDGFR-α signaling pathway to RIF and suggested that Sal A, Sal B and Sal A+B were considered as potential therapeutic agents for the amelioration of RIF.

HMOX1 silencing prevents doxorubicin-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis by downregulating CTGF.

Doxorubicin is a type of effective antitumor drug but can contribute to cardiomyocyte injuries. We aimed to dissect the mechanism of the HMOX1/CTGF axis in DOX-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis. Bioinformatics analysis was conducted to retrieve differentially expressed genes in a DOX-induced mouse model. Mouse cardiomyocytes, HL-1 cells, were induced with l µM DOX, after which gain- or loss-of-function assays were applied. CCK-8, fluorescent probe assay, flow cytometry, and corresponding kits were employed to detect cell viability, ROS levels, mitochondrial membrane potential and cell apoptosis, and GSH and Fe2+ contents, respectively. qRT-PCR or Western blot assay was adopted to test HMOX1, CTGF, BCL-2, Caspase3, Cleaved-Caspase3, and GPX4 expression. Bioinformatics analysis showed that HMOX1 and CTGF were highly expressed in DOX-induced mice and correlated with each other. Also, HMOX1 and CTGF expression was high in HL-1 cells after DOX treatment, along with an obvious decrease in cell viability and GSH and GPX4 expression, an increase in ROS levels, apoptosis, and Fe2+ contents, and mitochondrial membrane potential dysfunction or loss. HMOX1 or CTGF silencing diminished cell apoptosis, Cleaved-Caspase3 expression, Fe2+ contents, and ROS levels, enhanced cell viability and the expression of GSH, GPX4, and BCL-2, and recovered mitochondrial membrane potential in DOX-induced HL-1 cells. Nevertheless, the effects of HMOX1 silencing on the viability, apoptosis, ferroptosis, and mitochondrial dysfunction of DOX-induced HL-1 cells were counteracted by CTGF overexpression. In conclusion, HMOX1 silencing decreased CTGF expression to alleviate DOX-induced injury, mitochondrial dysfunction, and ferroptosis of mouse cardiomyocytes.

Association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment

In this study, we have investigated the association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment. Twenty-seven patients aged between 15 and 57 years underwent periapical surgery. The retrieved periapical tissue sample was used for mRNA expression analysis of COL1A1, VTN, ITGA5, IL-4, TNF, ANGPT, VEGFA, and CTGF. All patients were recalled after 6 and 12 months for periapical healing evaluation. Healing was then correlated with baseline gene expression. Healing was observed in 15 patients at the end of 6 months, which increased to 21 patients after 12 months. Six patients showed no healing even after 12 months. Analysis of baseline expression levels of the tested genes with healing status showed the mean relative expression of VTN, VEGFA, ANGPT, TNF, and CTGF to be significantly different (p < 0.05) between the healing group (6 and 12 months) (72.99%) and the non-healing (94.42%) group. Periapical Index scores 3–5 exhibited a positive correlation with ITGA-5 expression. Overexpression of ANGPT and a strong positive correlation between ITGA5 and PAI scores in the non-healing group of patients may suggest these genes to be a potential prognostic biomarker for periapical wound non-healing after surgical endodontic treatment.

Intrarenal Single-Cell Sequencing of Hepatitis B Virus Associated Membranous Nephropathy.

To date, the pathogenesis of hepatitis B virus (HBV)-associated membranous nephropathy (MN) remains elusive. This study aimed to decipher the etiopathogenesis of HBV-associated MN by performing single-cell RNA sequencing (scRNA-seq) of kidney biopsy specimens from a patient with HBV-associated MN and two healthy individuals. We generated 4,114 intrarenal single-cell transcriptomes from the HBV-associated MN patient by scRNA-seq. Compared to healthy individuals, podocytes in the HBV-associated MN patient showed an increased expression of extracellular matrix formation-related genes, including HSPA5, CTGF, and EDIL3. Kidney endothelial cells (ECs) in the HBV-associated MN were enriched in inflammatory pathways, including NF-kappa B signaling, IL-17 signaling, TNF signaling and NOD-like receptor signaling. Gene ontology (GO) functional enrichment analysis and Gene Set Variation Analysis (GSVA) further revealed that differentially expressed genes (DEGs) of ECs from the HBV-associated MN patients were enriched in apoptotic signaling pathway, response to cytokine and leukocyte cell-cell adhesion. The up-regulated DEGs in glomerular ECs of HBV-associated MN patients were involved in biological processes such as viral gene expression, and protein targeting to endoplasmic reticulum. We further verified that the overexpressed genes in ECs from HBV-associated MN were mainly enriched in regulation of protein targeting to endoplasmic reticulum, exocytosis, viral gene expression, IL-6 and IL-1 secretion when compared with anti-phospholipase A2 receptor (PLA2R)-positive idiopathic membranous nephropathy (IMN). The receptor-ligand crosstalk analysis revealed potential interactions between endothelial cells and other cells in HBV-associated-MN. These results offer new insight into the pathogenesis of HBV-associated MN and may identify new therapeutic targets for HBV-associated MN.

Forced Overexpression of Signal Transducer and Activator of Transcription 3 (STAT3) Activates Yes-Associated Protein (YAP) Expression and Increases the Invasion and Proliferation Abilities of Small Cell Lung Cancer (SCLC) Cells.

Background: We sought to investigate the interaction between signal transducer and activator of transcription 3 (STAT3) and the Yes-associated protein (YAP) signaling pathway in human small cell lung cancer (SCLC) cells. Methods: The STAT3-overexpressing SCLC cell lines H146 and H446 were established by plasmid DNA transfection for in vitro and in vivo experiments. Results: Overexpression of STAT3 increased YAP protein expression in H146 and H446 cells. STAT3 overexpression significantly increased YAP mRNA expression and the mRNA expression of the YAP signaling downstream genes CTGF and CYR61 in H146 and H446 cells (p < 0.05). We showed that STAT3 overexpression promoted EMT (epithelial–mesenchymal transition) with increased matrix metalloproteinase (MMP)-2 and MMP9 expression. Transwell assays showed that STAT3 overexpression increased the invasion ability of H146 and H446 cells. In addition, STAT3-overexpressing H146 cells grew significantly more rapidly than control H146 cells in the xenograft mouse model (p < 0.05). Immunohistochemistry (IHC) staining and Western blotting (WB) showed that STAT3-overexpressing H146 tumors had increased p-STAT3 and YAP staining and protein expression compared with control tumors. Increased EMT was also observed in STAT3-overexpressed xenograft tumors. Conclusions: The results of our study suggest that the overexpression of STAT3 promotes SCLC EMT, invasion, and proliferation through the activation of the YAP signaling pathway.

Identification of Potential miRNA-mRNA Regulatory Network in Denervated Muscular Atrophy by Bioinformatic Analysis.

Muscle atrophy caused by long-term denervation leads to the loss of skeletal muscle mass and strength, resulting in a poor recovery of functional muscles and decreasing quality of life. Increasing differentially expressed microRNAs (DEMs) have been reported to be involved in the pathogenesis of denervated muscle atrophy. However, there is still insufficient evidence to explain the role of miRNAs and their target genes in skeletal muscle atrophy. Therefore, an integrative exploration of the miRNA-mRNA regulatory network in denervated muscle atrophy is necessary. A total of 21 (16 upregulated and 5 downregulated) DEMs were screened out in the GSE81914 dataset. Med1, Myod1, Nfkb1, Rela, and Camta1 were predicted and verified to be significantly upregulated in denervated muscle atrophy, from which 6 key TF-miRNA relationship pairs, including Med1-mir-1949, Med1-mir-146b, Myod1-mir-29b, Nfkb1-mir-21, Rela-mir-21, and Camta1-mir-132, were obtained. 60 target genes were then predicted by submitting candidate DEMs to the miRNet database. GO and KEGG pathway enrichment analysis showed that target genes of DEMs were mainly enriched in the apoptotic process and PI3K/Akt signaling pathway. Through the PPI network construction, key modules and hub genes were obtained and potentially modulated by mir-29b, mir-132, and mir-133a. According to the qRT-PCR results, the expression of COL1A1 and Ctgf is opposite to their related miRNAs in denervated muscle atrophy. In the study, a potential miRNA-mRNA regulatory network was firstly constructed in denervated muscle atrophy, in which the mir-29b-COL1A1 and mir-133a-Ctgf pathways may provide new insights into the pathogenesis and treatment.

HLMSC Secretome Affects Macrophage Activity Differentially Depending on Lung-Mimetic Environments.

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs.

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway

To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action. SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells. Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus. Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

Downregulation of lncRNA Miat contributes to the protective effect of electroacupuncture against myocardial fibrosis

BackgroundMyocardial fibrosis changes the structure of myocardium, leads to cardiac dysfunction and induces arrhythmia and cardiac ischemia, threatening patients’ lives. Electroacupuncture at PC6 (Neiguan) was previously found to inhibit myocardial fibrosis. Long non-coding RNAs (lncRNAs) play a variety of regulatory functions in myocardial fibrosis, but whether electroacupuncture can inhibit myocardial fibrosis by regulating lncRNA has rarely been reported.MethodsIn this study, we constructed myocardial fibrosis rat models using isoproterenol (ISO) and treated rats with electroacupuncture at PC6 point and non-point as control. Hematoxylin–eosin, Masson and Sirius Red staining were performed to assess the pathological changes and collagen deposition. The expression of fibrosis-related markers in rat myocardial tissue were detected by RT-qPCR and Western blot. Miat, an important long non-coding RNA, was selected to study the regulation of myocardial fibrosis by electroacupuncture at the transcriptional and post-transcriptional levels. In post-transcriptional level, we explored the myocardial fibrosis regulation effect of Miat on the sponge effect of miR-133a-3p. At the transcriptional level, we studied the formation of heterodimer PPARG–RXRA complex and promotion of the TGF-β1 transcription.ResultsMiat was overexpressed by ISO injection in rats. We found that Miat can play a dual regulatory role in myocardial fibrosis. Miat can sponge miR-133a-3p in an Ago2-dependent manner, reduce the binding of miR-133a-3p target to the 3ʹUTR region of CTGF mRNA and improve the protein expression level of CTGF. In addition, it can also directly bind with PPARG protein, inhibit the formation of heterodimer PPARG–RXRA complex and then promote the transcription of TGF-β1. Electroacupuncture at PC6 point, but not at non-points, can reduce the expression of Miat, thus inhibiting the expression of CTGF and TGF-β1 and inhibiting myocardial fibrosis.ConclusionWe revealed that electroacupuncture at PC6 point can inhibit the process of myocardial fibrosis by reducing the expression of lncRNA Miat, which is a potential therapeutic method for myocardial fibrosis.

MO242: Intrarenal Single Cell Sequencing of Hepatitis B Virus Associated Membranous Nephropathy

Abstract BACKGROUND AND AIMS The pathogenesis underlying hepatitis B virus (HBV)-associated membranous nephropathy (MN) remains incompletely understood. METHOD To decipher the etiopathogenesis of HBV-associated MN, intrarenal single-cell RNA sequencing (scRNA-seq) was applied to the kidney biopsy from a HBV-associated MN patient and a health control to define the transcriptomic landscape at single-cell resolution. Differentially expressed genes (DEGs) and enrichment analysis were compared between HBV-associated MN and anti-PLA2R positive idiopathic membranous nephropathy (IMN) using intrarenal scRNAseq. RESULTS We generated 4114 intrarenal single-cell transcriptomes from a HBV-associated MN patient by single-cell RNA-sequencing. We found that kidney podocytes cells in the HBV-associated MN patient displayed increased expression of several genes, including EDIL3, GPC6, HSPA5 and CTGF, which were closely related to extracellular matrix formation. Kidney endothelial cells of HBV-associated MN were mainly enriched in inflammatory pathways including TNF signaling, IL-17 signaling, NOD-like receptor signaling, NF-kappa B signaling pathway and Toll-like receptor signaling pathway. TNF signaling pathway and IL-17 signaling pathway activation were confirmed in proximal tubule cells from the HBV-associated MN patient. Moreover, ligand-receptor analysis highlighted the extensive communication of mesangial cells, which infers great importance in HBV-associated MN. Furthermore, gene ontology (GO) function enrichment analysis revealed that DEGs of endothelial cells from the HBV-associated MN patient were enriched in interleukin-6 secretion and interleukin-1 secretion when compared with anti-PLA2R positive IMN. CONCLUSION These results offer new insight into the pathogenesis of HBV-associated MN.

Abstract 130: Thymidine Phosphorylase Enhances Abdominal Aortic Aneurysm Formation In Mice

Abdominal aortic aneurysm (AAA) is affected by multiple factors, including vessel wall cells, extracellular matrix, genetic disorders, etc.; however, the pathophysiology of AAA remains unclear. We recently found that thymidine phosphorylase (TYMP) is a signaling protein, and an increase of TYMP inhibits the proliferation of VSMCs, the major cell population that affects AAA progression. TYMP enhances platelet activity, thrombosis, and inflammation. These data suggest that TYMP may play a role in AAA formation. By using human AAA samples, we found that TYMP expression was significantly increased in AAA vessel walls than in healthy aortas. We thus further examined the role of TYMP in AAA formation using a mouse model. WT C57BL/6J and Tymp -/- mice were fed a western diet (TD.88137), started at 4 weeks (wks) old for 8 wks, and then received implantation of Ang II mini pump, which delivers Ang II to mice at a dose of 1 μg/kg/min for 4 wks. By echography, we found that Ang II perfusion dramatically increased inner aorta diameter at the diastolic phase in WT, but not Tymp -/- mice. Mice were sacrificed 4 wks after Ang II perfusion. AAA was confirmed in 3 of the 12 WT mice, but no AAA was found in all 11 Tymp -/- mice used. The AAA was mainly formed at the suprarenal artery level. Histological examination confirmed the presence of freshly formed or fibrotic hematoma. By using TYMP overexpressing rat VSMC cell line, C2, and its control cells, PC, we found that TYMP increased MMP2 production, secretion, and activation, which was diminished by tipiracil, a selective TYMP inhibitor. Ang II did not affect extracellular MMP2 activity in both PC and C2. TYMP facilitated TNF-α induced MMP2 activation. TYMP induced constitutive AKT phosphorylation, which was further enhanced by TNF-α. TYMP increased expression of active TGFβ1. Consequently, TYMP significantly enhanced thrombospondin-1 type 1 repeat domain (TSR) stimulated TGFβ1 signaling activation and expression of CTGF, which is correlated with severe fibrotic disorders and AAA. In summary, our study, for the first time, demonstrated that TYMP, a pyrimidine salvage pathway enzyme, plays an important role in regulating vascular biology, especially, VSMC function. TYMP-mediated microenvironment changes enhance the development of AAA.

SIRT6 Protects Against Liver Fibrosis by Negatively Regulating YAP/TAZ

Hepatic fibrosis often occurs after chronic liver injury, and it is largely attributable to the activation of hepatic stellate cells (HSCs). It is a determining step in the pathophysiology of non‐alcoholic steatohepatitis (NASH), an advanced form of fatty liver disease. The Hippo pathway and its effectors Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), have been implicated in hepatic fibrogenesis. However, how YAP and TAZ are regulated in HSCs remains incompletely understood. Here in this work, we have identified Sirtuin 6 (SIRT6), a NAD+ dependent deacetylase, as a regulator of YAP and TAZ in HSCs. Using primary HSCs deficient in SIRT6 from mice fed a moderately‐high‐fat‐cholesterol‐cholate diet (HFCC) for one week as well as in vitro models using transforming growth factor‐β (TGF‐β) treated LX‐2 cells, we have revealed that YAP and TAZ were acetylated during the HSC activation. Moreover, expression of YAP and TAZ target genes including connective tissue growth factor (Ctgf), cysteine‐rich angiogenic inducer 61 (Cyr61) and ankyrin repeat domain 1 (Ankrd1) was increased in those HSCs. Immunofluorescence microscopy analysis of liver sections obtained from Sirt6 HSC‐specific knockout mice fed HFCC diet for six weeks showed elevated YAP nuclear localization in the HSCs, suggesting an activation of YAP during fibrosis. Further in vitroanalysis of YAP/TAZ activity upon gain‐ and loss‐of‐function of SIRT6 in TGF‐β treated LX‐2 cells showed a decrease or increase of Ctgf, Cyr61 and Col3a1gene expression,respectively. To understand how SIRT6 regulates YAP and TAZ, we performed immunoprecipitation and lysine acetylation analysis, and our data showed that SIRT6 indeed interacted with YAP and TAZ and deacetylated them. Taken together, our study has identified a key regulatory axis in which SIRT6 interacts with YAP/TAZ and deacetylates them at several lysine residues, thereby suppressing their transcriptional co‐activator activity during fibrosis.

Oncogenic Role of Connective Tissue Growth Factor Is Associated with Canonical TGF-β Cascade in Colorectal Cancer.

TGF-β signaling pathways promote tumour development and control several downstream genes such as CTGF and MMPs. This study aimed to investigate the association between CTGF and MMP-1 mRNA expressions with clinicopathological status and survival rate in colorectal cancer patients. We investigated expression levels of CTGF and MMP-1 genes in paraffin-embedded tumours and adjacent normal tissue blocks (ADJ) by Real Time-PCR. Then, the expression of Smad2 and Smad4 proteins in the TGF-β canonical pathway was evaluated by immunohistochemistry. Finally, the correlation between CTGF, MMP-1, and the canonical TGF-β-signalling pathway with the clinicopathological features was investigated. Expression levels of MMP-1and CTGF were higher in tumours compared with adjacent normal tissues. Overexpression levels of MMP-1 and CTGF were associated with lymph node metastasis, distant metastasis, tumour histopathological grading, advanced stage, and poor survival (p < 0.05). Additionally, a significant association between the upregulation of MMP-1 and tumour location was noted. Upregulation of Smad2 and Smad4 proteins were also significantly correlated with lymph node metastasis, distant metastasis, advanced stage, and poor survival (p < 0.0001). This study showed that canonical TGF-β signalling regulates both CTGF and MMP-1 expression and CRC progression. Moreover, TGF-β signalling and its downstream genes could be used as novel biomarkers and novel approaches for targeted therapy in CRC.

Mechanical Cues, E-Cadherin Expression and Cell "Sociality" Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

Intervention Study of Dictyophora Polysaccharides on Arsenic-Induced Liver Fibrosis in SD Rats.

Long-term arsenic (As) exposure can cause liver injury, hepatic cirrhosis, and cancer. Meanwhile, Dictyophora polysaccharides (DIP) have excellent antioxidation, anti-inflammation, and immune protection effects. There are currently few reports on the protection effects of DIP on As-induced hepatotoxicity and its pharmacological value. Therefore, this study was aimed at elucidating the protection of DIP on As-induced hepatotoxicity and exploring its preventive role in antifibrosis. In our study, the SD rat As poisoning model was established by the feeding method to explore the influence of As exposure on liver fibrosis. Then, DIP treatment was applied to the rats with As-induced liver fibrosis, and the changes of serum biochemical indexes and liver tissue pathology were observed. And the expression of fibrosis-related proteins TGF-β1, CTGF, and α-SMA levels was then determined to explore the DIP intervention function. The results demonstrated that through reduced pathological changes of hepatic and increased serum AST, ALT, TP, ALB, and A/G levels, DIP ameliorated liver fibrosis induced by As as reflected. And the administration of DIP decreased the concentration of HA, LN, PCIII, CIV, TBIL, and DBIL. In addition, the synthesis of TGF-β1 inhibited by DIP might regulate the expression of CTGF and decrease the proliferation of fibrinogen and fibroblasts, which reduced the synthesis of fibroblasts to transform into myofibroblasts. And a decrease of myofibroblasts downregulated the expression of α-SMA, which affected the synthesis and precipitation of ECM and alleviated the liver fibrosis caused by exposure to As. In conclusion, based on the pathological changes of liver tissue, serum biochemical indexes, and related protein expression, DIP can improve the As-induced liver fibrosis in rats and has strong medicinal value.

Molecular markers of cardiac fibrosis after myocardial infarction

Aim. To perform a screening for molecular markers of cardiac fibrosis upon myocardial infarction.Materials and Methods. We carried out echocardiography-guided endomyocardial biopsy of affected and intact interventricular septum segments of 7 patients with anterior myocardial infarction. Fibrotic and adjacent intact cardiac tissue was dissected into 2 equal segments and: 1) homogenized with the further RNA extraction, reverse transcription, and quantitative polymerase chain reaction; 2) fixed in formalin and embedded into paraffin with the further van Gieson staining for the histological verification of cardiac fibrosis.Results. We found that the expression of ACTA2, VIM, CTGF, COL1A1, TGFB1, TGFBR1, AGTR1, CCL2 and TNF genes in fibrotic cardiac tissue was ≥ 3-fold higher as compared with the adjacent intact myocardium reflective of active extracellular matrix production by fibroblast-derived myofibroblasts.Conclusion. We have for the first time shown AGTR1, CCL2, and TNF genes as candidates for post-infarction cardiac fibrosis in addition to ACTA2, VIM, CTGF, COL1A1, TGFB1, and TGFBR1 genes.

Identification of Small-molecule YAP-TEAD inhibitors by High-throughput docking for the Treatment of colorectal cancer

The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth, proliferation, and apoptosis. Dysregulation of the Hippo pathway due to overexpression of YAP has been reported in various cancers. Inhibition of TEAD represses the expression of associated genes, proving the value of this transcription factor for the development of novel anti-cancer therapies. We retrieved a promising hit compound L06 which is a potent TEAD4 inhibitor through docking-based virtual screening. L06 inhibits TEAD autopalmitoylation, interrupts YAP-TEAD interaction, and reduces the YAP-TEAD transcriptional activity. Moreover, L06 reduces the expression of CTGF, inhibits HCT 116 colorectal cancer cell proliferation, migration and invasion. The YAP–TEAD complex is a viable drug target, and L06 is a lead compound for the development of more potent TEAD inhibitors to treat colorectal cancer and other hyperproliferative pathologies.

Disruption of LTBP4 Inhibition-Induced TGFβ1 Activation Promoted Cell Proliferation and Metastasis in Skin Melanoma by Inhibiting the Activation of the Hippo-YAP1 Signaling Pathway.

Melanoma is a malignant tumor derived from melanocytes, which is the most fatal skin cancer. The present study aimed to explore and elucidate the candidate genes in melanoma and its underlying molecular mechanism. A total of 1,156 differentially expressed genes were obtained from the GSE46517 dataset of Gene Expression Omnibus database using the package “limma” in R. Based on two algorithms (LASSO and SVM-RFE), we obtained three candidate DEGs (LTBP4, CDHR1, and MARCKSL1). Among them, LTBP4 was identified as a diagnostic marker of melanoma (AUC = 0.985). Down-regulation of LTBP4 expression was identified in melanoma tissues and cells, which predicted poor prognosis of patients with melanoma. Cox analysis results discovered that LTBP4 with low expression was an independent prognostic factor for overall survival in patients with melanoma. LTBP4 inhibition reduced cell apoptosis and promoted cell proliferation and metastasis. These changes were correlated with the expression levels of caspase-3, Ki67 and E-cadherin. Further, as indicated by tumor formation study of nude mice, LTBP4 silencing improved the tumorigenic ability of melanoma cells. Knockdown of LTBP4 increased the percentage of active TGFβ1 secreted by melanoma cells. CTGF, Gyr61, and Birc5 expression levels were reduced, YAP1 phosphorylation was inhibited, and YAP1 was translocated from the cytoplasm to the nucleus in melanoma cells treated with TGF-β1. These effects were reversed by LTBP4 overexpression. As evidenced by immunofluorescent staining, Western blotting and luciferase reporter assay, LTBP4 overexpression activated the Hippo signaling pathway, which was characterized by the increased nuclear-cytoplasmic translocation of YAP1 and the enhanced phosphorylation of YAP1, MST1, and MOB1. In addition, the effects of LTBP4 overexpression on inhibiting CTGF, Cyr61 and Birc5 expression, promoting the apoptosis, and inhibiting the metastasis and proliferation of melanoma cells were reversed by the overexpression of YAP1 or MST1. In conclusion, the LTBP4-TGFβ1-Hippo-YAP1 axis is a critical pathway for the progression of skin melanoma.

MicroRNA-122-5p Aggravates Angiotensin II-Mediated Myocardial Fibrosis and Dysfunction in Hypertensive Rats by Regulating the Elabela/Apelin-APJ and ACE2-GDF15-Porimin Signaling.

Hypertension is the leading risk factor for cardiovascular disorders. This study aimed to explore roles of microRNA (miR)-122-5p in hypertension. Angiotensin II (Ang II; 1.5 mg/kg/day) with an osmotic minipump was used to induce hypertensive rats pretreated by rAAV-miR-122-5p or rAAV-GFP, respectively. Notably, Ang II infusion caused marked increases in myocardial fibrosis, inflammation, oncosis, and oxidant injury in rats, which were aggravated by rAAV-miR-122-5p. RAAV-miR-122-5p exacerbated Ang II–mediated cardiac dysfunction and structural injury in hypertensive rats, with downregulated levels of apelin, elabela, ACE2, and GDF15, as well as upregulated expression of porimin and CTGF. In cultured rat cardiac fibroblasts, Ang II contributed to augmentation of cellular oncosis, migration, inflammation, and oxidative stress, with reduction of apelin, elabela, ACE2, and GDF15 levels, which were rescued by miR-122 inhibitor. In summary, miR-122-5p exacerbates myocardial fibrosis and dysfunction in hypertensive rats by modulating the elabela/apelin-ACE2-GDF15 signaling. MiR-122-5p has potential therapeutic significance for hypertension and hypertensive cardiac injury.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s12265-022-10214-3.

MEHP promotes liver fibrosis by down-regulating STAT5A in BRL-3A hepatocytes

ObjectiveAs an environmental endocrine disruptor, mono-2-ethylhexyl phthalate (MEHP) can interfere with liver metabolism and lead to liver diseases. We aimed to investigate the role of MEHP in liver fibrosis and its molecular mechanism. MethodsBRL-3A hepatocytes were exposed to MEHP (0, 10, 50, 100 and 200 μM) for 24 h. STAT5A gene was overexpressed by lentivirus transfection. The reactive oxygen species (ROS) was tested by the flow cytometer. The malondialdehyde (MDA), glutathione peroxidase (GSH-PX), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were detected by commercial kits. Real-Time PCR and Western blot were performed to test the relative mRNA and proteins levels, respectively. ResultsMEHP exposure significantly induced oxidative damage in BRL-3A cells, which inhibited the expression of STAT5A and promoted the expression of fibrosis related proteins MMP2, MMP9, TIMP2 and CTGF. After over-expression of STAT5A gene in BRL-3A cells, the elevated expression levels of CTGF, MMP2, MMP9 and TIMP2 induced by MEHP exposure were significantly reversed. ConclusionThis study demonstrated that MEHP exposure inhibited the expression of STAT5A by causing oxidative damage in BRL-3A hepatocytes, thus accelerating the expression of key molecules in fibrosis and promoting the occurrence of liver fibrosis.