Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: miR-29a-3p regulates THBS2 expression in vitro and in vivo. ( A , B ) MCT-induced expression of miR-29a-3p and exosomal THBS2 mRNA, determined by real-time PCR. ( C ) THBS2 expression in MCT-treated HCM-secreted exosomes determined by western blotting. HSP70 was used as the internal control for exosomal proteins. ( D ) Alignment of the miR-29a-3p with the THBS2 3′-untranslated region, showing the putative binding sites. ( E ) Real-time PCR analysis of miR-29a-3p expression in HCMs after transfection with an miR-29a-3p mimic or inhibitor. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05). ( F , G ) After transfection, THBS2 expression was analyzed by real-time PCR and immunocytochemical staining. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Kruskal–Wallis test was used to compare two independent groups (* p < 0.05). ( H , I ) MCT-induced expression of miR-29a-3p and THBS2 mRNA, determined by real-time PCR. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). ( J ) In situ hybridization (ISH) of miR-29a-3p expression (pink color) in myocardial tissues of MCT-induced PAH mice. U6, miRNA internal control. ( K , L ) THBS2 expression in myocardial tissues and circulating serum of MCT-induced PAH mice were examined by immunohistochemistry and western blotting, respectively. Transferrin was used as the internal control. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05 and ** p < 0.01).

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Control, Binding Assay, Transfection, MANN-WHITNEY, Staining, In Situ Hybridization, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: Cardiomyocyte-derived exosomes regulate cardiac fibroblast to myofibroblast transformation. ( A ) Schematic diagram for studying the effects of human cardiomyocyte (HCM)-derived exosome uptake by human cardiac fibroblasts (HCF) using early endosome antigen 1(EEA1) staining. ( B ) Immunocytochemistry and confocal microscopy identified the time-dependent intake and regulation of HCM-derived exosomes by EEA1 in HCFs. Nuclear DNA, exosomes, and EEA1 were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue), Dil dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine, red), and GFP fluorescence dye (green), respectively. Data are representative of three independent experiments ( n = 3). ( C ) HCFs were treated with or without HCM-derived exosomes, HCM+MCT-derived exosomes, or rTHBS2 (THBS2 recombinant protein, 1 μg/mL) for 48 h to assess cardiomyocyte-derived exosomal THBS2-mediated fibroblast to myofibroblast transformation. Expression of the myofibroblast marker proteins, α-SMA, β-catenin, fibronectin, and vimentin was examined by immunocytochemistry and confocal microscopy with Imaris software for confocal image 3D reconstruction. ( D , E ) Myofibroblast marker protein expression in myocardial tissues of MCT-induced PAH mice was examined by western blotting and immunohistochemistry. Data are representative of three independent experiments ( n = 3), and values are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (* p < 0.05).

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Transformation Assay, Staining, Immunocytochemistry, Confocal Microscopy, Labeling, Fluorescence, Recombinant, Expressing, Marker, Software, Western Blot, Immunohistochemistry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis

doi: 10.3390/ijms221910574

Figure Lengend Snippet: MiR-29a-3p/THBS2 axis regulates the progression of pulmonary artery hypertension (PAH). ( A ) Exosomal miR-29a-3p level in the serum of healthy controls (NR) and patients with PAH was determined by real-time PCR. ( B ) THBS2 expression in the serum was determined by ELISA. Data are expressed as median and interquartile range. Mann–Whitney U-test was used to compare two independent groups (** p < 0.01). ( C ) Correlation coefficient between the miR-29a-3p and THBS2 expression levels. R, Pearson’s correlation coefficient. ( D – F ) The correlation of miR-29a-3p with the mean pulmonary artery pressure (mPAP), tricuspid annular plane systolic excursion (TAPSE), and pulmonary vascular resistance (PVR) in patients with PAH. ( G – I ) The correlation of the serum THBS2 level with mPAP, TAPSE, and PVR in patients with PAH. ( J ) Suggested mechanism underlying PAH-induced cardiac fibrosis mediated by exosomal miR-29a-3p and THBS2. Serum miR-29a-3p and THBS2 (secreted by cardiomyocytes) might increase cardiomyocyte hypertrophy and promote cardiac fibroblast to myofibroblast transformation, enhance collagen production/deposition, and augment cardiac fibrosis.

Article Snippet: Plasma THBS2 levels were determined using human THBS2 ELISA Kit PicoKineTM (Catalog# EK0642; Boster Biological Technology, Pleasanton CA, USA) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Transformation Assay

Journal: Frontiers in endocrinology

Article Title: miR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

doi: 10.3389/fendo.2022.842587

Figure Lengend Snippet: FIGURE 1 | miR-218-5p induces IL1b expression and secretion. Stable transfection of mir-218-1 (left) in HTR-8/SVneo cells, transient transfection of miR-218-5p mimic in HTR-8/SVneo (middle) and Swan 71 (right) cells significantly increased IL1B mRNA (A) and IL1b protein levels in cell lysates (B) and conditioned media (C). Data are shown as mean ± SEM (n=3). **p < 0.01; ****p < 0.0001. EV, empty vector; NC, non-targeting control.

Article Snippet: The level of IL1b in cell lysates or conditioned media was determined with a human IL1b PicoKineTM ELISA kit (Boster Bio, Pleasanton, CA, USA) and analyzed using a BioTek Synergy H4 hybrid multi-mode plate reader.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control

Journal: Frontiers in endocrinology

Article Title: miR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

doi: 10.3389/fendo.2022.842587

Figure Lengend Snippet: FIGURE 3 | TGFb2 inhibits IL1b via SMAD2 signaling. (A) ELISA for IL1b in cell lysates harvested from HTR-8/SVneo and Swan 71 cells treated with recombinant TGFb2 at various concentrations for 24 hr. (B) qPCR for TGFB2 mRNA in cells transfected with siTGFB2. (C) ELISA for IL1b in cell lysates harvested from cells treated with siTGFB2. (D) qPCR for IL1B mRNA in control, siSMAD2, and siSMAD3-treated cells, in the presence of recombinant TGFb2 (1 ng/ml). Data are shown as mean ± SEM (n=3). **p < 0.01, ***p < 0.001, ****p < 0.0001. For (A, D), different letters above bars denote statistical significance.

Article Snippet: The level of IL1b in cell lysates or conditioned media was determined with a human IL1b PicoKineTM ELISA kit (Boster Bio, Pleasanton, CA, USA) and analyzed using a BioTek Synergy H4 hybrid multi-mode plate reader.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Transfection, Control

Journal: Frontiers in endocrinology

Article Title: miR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

doi: 10.3389/fendo.2022.842587

Figure Lengend Snippet: FIGURE 4 | SMAD2 blocks miR-218-5p-induced IL1B mRNA. (A) Western blotting for SMAD2 and SMAD3 in control and mir-218-1-overexpressing HTR-8/SVneo cells transiently transfected with Flag-tagged SMAD2 and/or SMAD3 expression constructs. Note that mir-218-1 overexpression reduced both endogenous and exogenous SMAD2 and SMAD3 protein levels. Data are representative of three independent experiments. (B) qPCR for IL1B mRNA in control and mir-218-1- overexpressing HTR-8/SVneo cells transfected with SMAD2 and/or SMAD3 vectors, showing that SMAD2 and SMAD3 exerted opposing effects on IL1B mRNA. Data are shown as mean ± SEM (n=3). Different letters above bars denote statistical significance.

Article Snippet: The level of IL1b in cell lysates or conditioned media was determined with a human IL1b PicoKineTM ELISA kit (Boster Bio, Pleasanton, CA, USA) and analyzed using a BioTek Synergy H4 hybrid multi-mode plate reader.

Techniques: Western Blot, Control, Transfection, Expressing, Construct, Over Expression

Journal: Frontiers in endocrinology

Article Title: miR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

doi: 10.3389/fendo.2022.842587

Figure Lengend Snippet: FIGURE 6 | IL1b promotes the acquisition of an enEVT-like phenotype. (A) Tube formation assay showing that IL1b enhanced the ability of HTR-8/SVneo and Swan 71 cells to form endothelial-like networks (n=6); scale bar = 800 µm. (B) qPCR for the expression of enEVT markers in cells treated with IL1b (n=3). Note that IL1b at low concentrations increased ITGA1, ITGA5, CDH5, and PECAM1 mRNA in a dose-dependent manner. Data are shown as mean ± SEM. Different letters above bars denote statistical significance.

Article Snippet: The level of IL1b in cell lysates or conditioned media was determined with a human IL1b PicoKineTM ELISA kit (Boster Bio, Pleasanton, CA, USA) and analyzed using a BioTek Synergy H4 hybrid multi-mode plate reader.

Techniques: Tube Formation Assay, Expressing

Journal: Frontiers in endocrinology

Article Title: miR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

doi: 10.3389/fendo.2022.842587

Figure Lengend Snippet: FIGURE 7 | Regulation of miR-218-5p by IL1b. (A) HTR-8/SVneo and Swan 71 cells were treated with different concentrations of IL1b for 24 hr. qPCR revealed that lower doses of IL1b reduced miR-218-5p level. Data are shown as mean ± SEM (n=3). Different letters above bars denote statistical significance. (B) A schematic outlining the interactions among miR-218-5p, IL1b, and the TGFb/SMAD2 pathway and their potential roles in enEVT differentiation.

Article Snippet: The level of IL1b in cell lysates or conditioned media was determined with a human IL1b PicoKineTM ELISA kit (Boster Bio, Pleasanton, CA, USA) and analyzed using a BioTek Synergy H4 hybrid multi-mode plate reader.

Techniques:

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 plasma levels rise after running and XCL1 treatment increases the number of neurospheres. ( a ) XCL1 plasma levels measured by an ELISA in standard-housed mice (STD, n = 5 mice) and mice housed for 4 days with a running wheel (RUN, n = 6 mice). * p < 0.05, Student’s t -test. ( b ) qPCR gene expression analysis of lymphotactin receptors reveals that neural precursor cells express Itga9 but not Xcr1 (left), although both Itga9 and Xcr1 are detected in splenic control tissue (right). Uncropped gels are presented in Supplementary Fig. . ( c ) Representative images of a SVZ neurosphere (top) and a DG neurosphere (bottom). Scale bars: 100 μm. ( d ) Neurosphere assays with DG-derived primary cells cultured with XCL1. n = 9 to 10 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Neurosphere assays with SVZ-derived primary cells cultured with XCL1. n = 6 to 9 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Neurosphere assays with XCL1-neutralizing antibodies. n = 3 to 6 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( g ) Size distribution of DG-derived neurospheres cultured with XCL1. n = 7 to 8 independent experiments. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression, Control, Derivative Assay, Cell Culture

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 promotes neuronal differentiation in adherent monolayer and neurosphere cultures. ( a ) Viability assay in adherent NPC cultures with XCL1. n = 5 to 6 independent experiments. ( b ) CFSE proliferation assay in adherent NPC cultures with XCL1. n = 3 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( c ) Motility of adherent monolayer-cultured NPCs determined by semi-automated tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. XCL1: n = 92 cells, Control: n = 104 cells, *** p < 0.001, Student’s t -test. ( d ) Quantifica t ion of β-tubulin + cells in proliferating NPC cultures two days after the addition of XCL1. n = 4 independent experiments, * p < 0.05, one-way ANOVA with Dunnett test. ( e ) Representative image of differentiated NPCs in adherent monolayer cultures showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( f ) Quantification of GFAP + and β-tubulin + cells in differentiated adherent monolayer cultures treated with XCL1. n = 4 to 5 independent experiments, * p < 0.05, *** p < 0.001, one-way ANOVA with Dunnett test. ( g ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( h ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures treated with XCL1. n = 5 independent experiments, *** p < 0.001, one-way ANOVA with Dunnett test. Dashed lines represent control cultures normalized to 100%. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Viability Assay, Proliferation Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: XCL1 influences the cell cycle progression of NPCs in vitro . ( a ) Representative images of a dividing NPC followed by time-lapse microscopy. Images are 5 min apart. Yellow arrows mark the process of cell division. Scale bar: 10 μm. ( b ) Example of a generation tree of a re-dividing cell obtained from semi-automated cell tracking of NPCs to calculate the mean generation time. ( c ) Generation time of NPCs cultured with and without XCL1 determined by semi-automated cell tracking. Data are plotted as the 5 th /95 th percentile with outliers represented as circles. Control: n = 23 cells, XCL1: n = 26 cells. ( d ) Representative flow cytometry plots of the click-iT EdU proliferation assay. Viable cells were first defined using forward scatter and side scatter (left). Doublets were then excluded from single cell signals by plotting Hoechst-width against Hoechst-area (middle). Finally, to determine the cell cycle phase, the DNA content (Hoechst label) was plotted against the EdU signal (right). ( e ) Percentage of NPCs in S phase. n = 4 independent experiments, ** p < 0.01, one-way ANOVA with Dunnett test. ( f ) Percentage of NPCs in G2/M phases. n = 4 independent experiments. ( g ) Percentage of NPCs in G1/G0 phases. n = 4 independent experiments. All data represent the mean ± SEM.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: In Vitro, Time-lapse Microscopy, Cell Tracking Assay, Cell Culture, Control, Flow Cytometry, Proliferation Assay

Journal: Scientific Reports

Article Title: The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

doi: 10.1038/s41598-019-48360-5

Figure Lengend Snippet: Neurogenesis in XCL1 KO mice is reduced ex vivo . ( a ) Neurosphere assays with primary DG cells from XCL1 KO mice (−/−) and WT littermates (+/+). n = 6 mice per group, * p < 0.05, paired Student’s t -test. ( b ) Representative image of differentiated neurospheres showing GFAP + astrocytes in green and β-tubulin + neurons in red. Scale bar: 50 μm. ( c ) Quantification of GFAP + and β-tubulin + cells in differentiated neurosphere cultures from XCL1 −/− and +/+ mice. n = 4 mice per group, * p < 0.05, Student’s t -test. ( d ) and ( e ) Neurosphere assays with primary DG cells from XCL1 −/− and +/+ mice in the presence of ( d ) potassium chloride (n = 4 to 5 independent experiments) and ( e ) norepinephrine (n = 6 independent experiments). *** p < 0.001, **** p < 0.0001, Student’s t -test.

Article Snippet: Protein levels were also measured using the mouse XCL1 PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Ex Vivo