Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Mexo-P regulates vascular calcification in a let-7b-5p-dependent way. ( A ) Volcano plots show the result of miRNA sequencing between Mexo- and Mexo-P-treated MOVAS. ( B ) qRT-PCR validation of potential target miRNAs between Mexo- and Mexo-P-treated HASMCs (unpaired t -test, n = 5). ( C ) Expression levels of let-7b-5p in the artery tissue of mildly, moderately, or severely calcified CKD patients (unpaired t -test, n > 3). ARS staining ( D ) and calcium contents (( E ), one-way ANOVA, n = 4) of isolated mouse thoracic aortas, cultured in a calcifying medium (3 mM Pi) for 7 days, pretreated with let-7b-5p agomir or antagomir for 6 h. Representative images of ARS staining ( F ) and ARS quantification ( G ) of MOVAS treated with let-7b-5p-mimic or control (mimic-nc) and cultured in the calcifying medium for 7 days (Mann–Whitney test, n = 4). Representative images of ARS staining ( H ) and ARS quantification ( I ) of MOVAS treated with let-7b-5p-inhibitor or control (inhibitor-nc) and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( J ) and ARS quantification ( K ) of HASMCs treated with let-7b-5p-mimic or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( L ) and ARS quantification ( M ) of MOVAS treated with let-7b-5p-inhibitor or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Calcium content levels of calcifying-medium-cultured VSMCs (MOVAS, ( N ), HASMC, ( O )), stimulated with Mexo or Mexo-P in the presence or absence of let-7b-5p-mimic (one-way ANOVA, n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Staining, Isolation, Cell Culture, Control, MANN-WHITNEY

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Let-7b-5p mitigates vascular calcification by regulating TGFBR1. ( A ) Volcano plots showing differentially regulated genes (DEGs) in RNA-seq of Mexo- and Mexo-P-treated MOVAS. ( B ) Venn diagram showing the intersection of miRBD-predicted potential let-7b-5p targets and DEGs. ( C ) mRNA levels of 10 potential target genes were detected by qRT-PCR in let-7b-5p-mimic- or mimic-nc-treated HASMCs (unpaired t -test, n > 3). ( D ) Transcription levels of four significantly regulated genes in ( C ) were detected in Mexo- or Mexo-P-treated HASMC (unpaired t -test, n > 3). ( E ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1 overexpression plasmid (TGFBR1-OE) or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). ( F ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1-si or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). Calcium content of HASMCs with altered TGFBR1 levels with TGFBR1-OE ( G ) or TGFBR1-si ( H ), with or without the treatment of let-7b-5p-mimic or let-7b-5p-inhibitor in calcifying medium (one-way ANOVA, n = 4). ( I ) Tergetscan database predicted binding sequences of let-7b-5p on TGFBR1-3′UTR. ( J ) Wildtype (wt) and two mutations (mut1 and mut2) of TGFBR1-3′UTR were inserted into luciferase vectors. Luciferase activity assay of HASMCs co-transfected with vectors carrying wt or mutated TGFBR1-3′UTR (mut1, ( K ), mut2, ( L )), renilla vectors, mimic-nc, or let-7b-5p-mimic for 6 h, medium change, and cultured for another 48 h before assay (two-way ANOVA, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: RNA Sequencing, Quantitative RT-PCR, Staining, Over Expression, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Cell Culture

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Manipulating TGFBR1 levels regulates vascular calcification in CKD. Transcription levels of RUNX2, ALPL, and SOX9 in MOVAS ( A ) and HASMCs ( C ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 siRNA for 6 h (one-way ANOVA, n > 3). mRNA levels of RUNX2, ALPL, and SOX9 in MOVAS ( B ) and HASMCs ( D ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 overexpression plasmid (TGFBR1-OE) for 48 h (one-way ANOVA, n > 3). Representative WB images ( E ) and semi-quantification ( F ) showing RUNX2 levels in aortas from CKD mice injected with either Tgfbr1-adv or control (GFP-adv, unpaired t -test, n = 3). Representative images of ARS staining ( G ) and calcium content ( H ) of aortas from CKD mice injected with either Tgfbr1-adv or control (unpaired t -test, n > 3). ( I ) HASMCs were treated with SB525334 (1 μM), RepSOX (25 μM), and SB431542 (1 μM) for 6 h, after being transfected with SMAD3-luciferase plasmids for 24 h, and luciferase activity was detected (unpaired t -test, n = 3). ( J ) SMAD3-luciferase plasmid-transfected HASMCs were stimulated with 10 ng/mL TGFβ1 and different doses of SB525334 for 6 h, and luciferase activity was detected (one-way ANOVA, n > 3). ( K ) HASMCs were stimulated with 10 ng/mL TGFβ1 plus 1 μM SB525334 for 0, 3, 6, 12, 24, or 48 h, and RUNX2 mRNA levels were detected by qRT-PCR (one-way ANOVA, n = 5). In vitro calcification model showing the SB525334 (1 μM) inhibition of 10 ng/mL TGFβ1-induced HASMC calcification (( L ), ARS staining, ( M ), calcium content, one-way ANOVA, n = 6). Mice were orally administrated with 30 mg/kg SB525334 (n = 5) or vehicle (n = 5) daily; simultaneously with CKD modeling, aortas were collected for ARS staining ( N ), calcium content assay (( O ), unpaired t -test, n > 3), and Runx2, Alpl, and Sox9 mRNA levels detection (( P ), unpaired t -test, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Transfection, Over Expression, Plasmid Preparation, Injection, Control, Staining, Luciferase, Activity Assay, Quantitative RT-PCR, In Vitro, Inhibition

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: TGFBR1 promotes vascular calcification through the SMAD3/RUNX2 pathway. qRT-PCR results showing that SIS-3 reversed TGFβ1-induced upregulation of RUNX2 levels in HASMCs ( A ) and MOVAS (( B ), one-way ANOVA, n = 5). Western blot images showing that different doses of SMAD3 inhibitor SIS-3 alleviated TGFβ1-induced RUNX2 level increase and SMAD2/3 phosphorylation in HAMSCs ( C ) and MOVAS ( D ). ( E ) The JASPAR database predicted the binding site of SMAD3 on RUNX2 promoter, and the mutation was generated. ( F ) HASMCs were transfected with Renilla plasmid, wildtype RUNX2-promoter luciferase plasmid, and different doses of SMAD3-OE plasmid for 24 h, and luciferase activity was detected (one-way ANOVA, n = 5). ( G ) HASMC was transfected with Renilla plasmid, SMAD3-OE plasmid (3 ng per well of 12-well plate), and wildtype (wt) or mutated (mut) RUNX2-promoter luciferase plasmid, and luciferase activity was detected (Mann–Whitney test, n = 6). ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, Binding Assay, Mutagenesis, Generated, Transfection, Plasmid Preparation, Luciferase, Activity Assay, MANN-WHITNEY