Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: scRNA-seq analyses revealed Sca1 + cells repopulate ECs in mouse femoral artery after injury (A) Normal femoral arteries (NC) and injured femoral arteries were digested and analyzed by scRNA-seq after 2 (FAI2W) or 4 (FAI4W) weeks. Umap plot showed visualization of unsupervised clustering of all groups. (B) Dot plot of canonical cell markers identified EC cluster ( Kdr , Flt1 , Cdh5 , Pecam1 , and Nos3 ) and SPC cluster ( Cd34 , Ly6a , and Pdgfra ). (C) Bar plot showed the constitution of Ly6a (Sca1) + or Ly6a − cells among ECs of each group. (D) Differentially expressed genes between Ly6a + and Ly6a − cells were identified and analyzed using GO enrichment. The top 10 enriched biological process terms were presented. (E) Trajectory analysis of the differentiation process from SPCs to ECs; the color change from green to blue indicates the cell’s progression from a primitive to a more developed state. (F) The expression levels of canonical EC markers and SPC markers are shown alongside the differentiation trajectory. See also Figure S1 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Expressing

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Vascular Sca1 + cells exhibit endothelial differentiation potential in artery injury and repair (A) Immunofluorescence staining of healthy femoral artery for CD31 and Sca1 + lineage marker tdTomato (tdT) using Sca1-CreER T2 ;Rosa26-tdTomato mouse model. The right panel showed magnification of the boxed region. (B) Immunofluorescence staining of injured femoral artery for CD31 and tdTomato (Sca1 + lineage). The right panel and the panel below showed magnification of the boxed regions. For merge images, scale bars, 100 μm. For magnification images, scale bars, 20 μm. (C) Quantification of the percentage of CD31 + tdT + cells in CD31 + cells of healthy femoral artery (HFA) and injured femoral artery (IFA). Data represent mean ± SEM, n = 6 in each group. p value was specified in the graph. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values. See also Figure S2 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Marker, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Vascular Sca1 + cells differentiate into ECs in vein graft (A) Immunofluorescence staining of normal vena cava for CD31 and tdTomato (tdT, Sca1 + lineage). The right panel showed magnification of the boxed region. (B) Immunofluorescence staining of vein graft for CD31 and tdTomato. The right panel and the panel below showed magnification of the boxed regions. (C) Quantification of the percentage of CD31 + tdT + cells in CD31 + cells of both groups. (D) Sca1-CreER T2 ; Rosa26-tdTomato mice were bred with Rosa26-iDTR mice. Tamoxifen was given to label Sca1 + cells and diphtheria toxin (DT) injection was performed to ablate certain cells. Immunostaining for tdTomato and CD31 on vein graft sections after DT administration. For merge images, scale bars, 100 μm. For magnification images, scale bars, 20 μm. Data represent mean ± SEM, n = 5 in each group. p value was specified in the graph. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Injection, Immunostaining, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: Isolated Sca1 + cells could differentiate into ECs with VEGF treatment in vitro (A) The morphological changes in Sca1 + cells after treatment with 50 ng/ml VEGF for 0–5 days. (B) mRNA expression level of EC markers Kdr , Flt1 , and Pecam1 as determined by RT-PCR at different time points after culture with VEGF. (C) Protein expression level of PECAM1, CDH5, and KDR as determined by western blotting assay. (D) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (E) Immunofluorescence staining of CD31 in Sca1 + cells treated with VEGF at different time points. Scale bars, 25 μm. Data represent mean ± SEM. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns p ≥ 0.05. n = 3 in each group. Statistical differences between groups were determined by one-way ANOVA analysis of variance with method of multiple comparisons.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Isolation, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: The effect of miR-145-5p on the differentiation of Sca1 + cells to ECs with VEGF treatment (A) RT-PCR analysis for the expression of miR-145-5p in Sca1 + cells treated with VEGF at different time points. (B) RT-PCR showed expression of miR-145-5p in Sca1 + cells after transfection with mimic control/miR-145-5p mimic and inhibitor control/miR-145-5p inhibitor. (C) and (D) RT-PCR analysis for the expression of Pecam1 , Flt1 , and Cdh5 in Sca1 + cells transfected with mimic control/miR-145 mimic or inhibitor control/miR-145 inhibitor and cultured in VEGF. (E) the protein expression level of PECAM1, CDH5 and KDR as determined by western blotting assay in each group. (F) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (G) Immunofluorescence staining of CD31 ( Pecam1 ) in Sca1 + cells and (H) analysis for fluorescence intensity of CD31 in each group, n = 3. scale bars, 25 μm. Data represent mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns p ≥ 0.05. Statistical differences between groups were determined by one-way analysis of variance with method of multiple comparisons. See also Figure S3 .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, Cell Culture, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: The effects of miR-145-5p on the proliferation and migration of Sca1 + cells (A) CCK8 assay for the effect of miR-145-5p overexpression on the proliferation of Sca1 + cells. Transwell migration assay (B) and wound healing assay (C) for the effect of miR-145-5p overexpression on the migration of Sca1 + cells. n = 3 in each group. Scale bars, 25 μm. Data represent mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ns p ≥ 0.05. Statistical differences between groups were determined by two-tailed Student’s t test for normally distributed values.

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Migration, CCK-8 Assay, Over Expression, Transwell Migration Assay, Wound Healing Assay, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet: MiR-145-5p targets ERG to regulate differentiation of Sca1 + cells into ECs (A) The database TargetScan shows the combining site of miR-145-5p on ERG’s 3′-UTR region. (B) Luciferase activity of 293T cells co-transfected with wild type ERG-3′UTR and mimic control/miR-145-5p mimic or inhibitor control/miR-145-5p inhibitor. (C) Luciferase activity of Sca1 + cells co-transfected with mutant ERG-3′UTR and mimic control/miR-145-5p mimic or inhibitor control/miR-145-5p inhibitor. (D) Erg mRNA expression in Sca1 + cells transfected with mimic control or miR-145-5p mimic as measured by RT-PCR. (E) ERG expression of Sca1 + cells transfected with control plasmid vector (pcDNA-control) or plasmid to overexpress ERG (pcDNA-ERG) as measured by RT-PCR. (F) mRNA expression of Kdr , Pecam1 and Flt1 in Sca1 + cells transfected with pcDNA-control or pcDNA-ERG after treated with VEGF. (G) Protein expression level of KDR, PECAM1, and CDH5 in Sca1 + cells transfected with pcDNA-control or pcDNA-ERG after treated with VEGF. (H) mRNA expression of Kdr , Pecam1 and Flt1 in Sca1 + cells of each group as indicated. (I) Protein expression level of KDR, PECAM1, and CDH5 in Sca1 + cells of each group as indicated. (J) Protein bands were quantified by densitometry and normalized to the density of Tubulin. (K) Expression level of miR-145-5p in healthy cephalic vein and arteriovenous fistula (AVF) as determined by RT-PCR. p value between the 2 groups were specified in the graph. n = 12 in the healthy group and n = 8 in the AVF group. Data represent mean ± SEM. Statistical differences between groups were determined by one-way ANOVA analysis of variance with method of multiple comparisons or (D,E,K) two-tailed Student’s t test for normally distributed values.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns p ≥ 0.05. See also Figure S4 - .

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Transfection, Control, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Resident vascular Sca1 + progenitors differentiate into endothelial cells in vascular remodeling via miR-145-5p/ERG signaling pathway

doi: 10.1016/j.isci.2024.110080

Figure Lengend Snippet:

Article Snippet: Cells isolated from the aortic arch of C57 wild-type mice were then sorted with Sca1 (Miltenyi Biotec, 130-122-615) microbeads according to manufacturer’s instructions.

Techniques: Recombinant, Lysis, Purification, Luciferase, Reporter Assay, CCK-8 Assay, Plasmid Preparation, Software