Journal: Nature communications

Article Title: VEGFR2 signaling drives meningeal vascular regeneration upon head injury.

doi: 10.1038/s41467-020-17545-2

Figure Lengend Snippet: Fig. 8 Rapid recovery of BV basement membrane in the injured dura mater but not in the injured brain after PTI. a, b Representative images and comparisons of distributions of collagen IV along CD31+BVs in putative injury area (white dotted-lined circle) at indicated days in the dura mater of adult mice after PTI. Empty arrowheads indicate broken alignment of collagen IV. Scale bars, 200 µm. n = 4 mice/group from two independent experiments. Mean fluorescence intensity (MFI) of collagen IV per MFI of CD31 in the putative injury area at S14 is regarded as 100%. Dots and error bars indicate mean ± SD. **P < 0.01 versus S14 and ##P < 0.01 versus D1 of collagen IV by Kruskal–Wallis test. c, d Representative images and comparisons of distributions of collagen IV and CD31+BVs in putative injury area (white dotted line) at indicated days in the brain of adult mice after PTI. Scale bars, 500 µm. Each indicated box area is magnified and arrayed in the right panels. Empty arrowheads indicate increased collagen IV in perivascular areas, whereas white arrowheads indicate collagen IV accumulation in avascular areas. n = 4 mice/group from two independent experiments. MFI of collagen IV or MFI of CD31 in the putative injury area at S14 is regarded as 100%. Dots and error bars indicate mean ± SD. **P < 0.01 versus S14 by Kruskal–Wallis test.

Article Snippet: The following antibodies were used for MACS: biotin anti-mouse CD31 (rat monoclonal, 130-119-562, Miltenyi-Biotec); anti-biotin microbeads (130-090-485).

Techniques: Membrane

Journal: Stem cells and development

Article Title: Discrimination between lung homeostatic and injury-induced epithelial progenitor subsets by cell-density properties.

doi: 10.1089/scd.2012.0468

Figure Lengend Snippet: FIG. 2. Density distribution of common lung cell lineages. (A) Fractionated cells from sham or 2-day bleomycin-treated mice were subjected to flow cytometry and average percent of cells ( – SEM) per fraction are presented. P-value represen- tations shown: normal font—differences in the proportion of protein-expressing cells isolated between fractions from the homeostatic lung; bold font—differences in the proportion of protein-expressing cells between fractions postbleomycin treatment, and italics—overall differences in the proportion of protein-expressing cells between pre- and postbleomycin treatment regardless of fraction. No statistically significant differences were observed in the proportion of CD45-, CD31-, and EpCAM-positive cells between the density fractions in the homeostatic lung. In contrast, cells positive for c-KIT and CD49f were more prevalent in the lower fractions, while SCA-1 expressing cells equilibrated largely in the intermediate fractions. At day 2 following bleomycin treatment, only CD45-positive cells showed significant differences among the fractions equili- brating at the highest density (n ‡ 7; P = 0.01). (B) Representative western blots depicting levels of protein expression per fraction (n ‡ 4). The fibroblastic, smooth muscle, a1-actin (ACTA1) protein signal is highest in fractions 3 and 4 (P < 0.01 by densitometry). Levels of the epithelial (pro-) surfactant protein-C (SFTPC) and secretoglobin family 1A member 1 (SCGB1A1) protein bands are statistically significant between the fractions (P < 0.001 for both). Levels of the AT1 epithelial protein, aquaporin-5 (AQP5) are highest in the lighter fractions (P < 0.001). Bands representing levels of the endothelial CD31 protein and b-actin are also shown.

Article Snippet: Flow cytometry and FACS analysis Fractionated cells were washed and labeled with the following antibodies: FITC-conjugated anti-CD45R (Invitrogen), R-PE anti- SCA-1 (Ly6A/E; Invitrogen), PE/Cy5 antic-KIT (CD117; eBioscience), PE-anti-CD49f (eBioscience), Alexa-488-anti-EpCAM (CD326; Biolegend, San Diego, CA), biotinylated-CD31 (Santa Cruz Biotech) followed by PE/Cy5 streptavidin (Biolegend), and mouse anti-BRDU (ebioscience) followed by anti-mouse Alexa 488 (Invitrogen).

Techniques: Cytometry, Expressing, Isolation, Western Blot

Journal: Stem cells and development

Article Title: Discrimination between lung homeostatic and injury-induced epithelial progenitor subsets by cell-density properties.

doi: 10.1089/scd.2012.0468

Figure Lengend Snippet: FIG. 3. Density properties of lung progenitor cell lineages. Sham or 2-day postbleomycin-treated mice were injected with BrdU, euthanized, and lungs harvested after 1 h. Cells were then dissociated, colabeled for BrdU and CD45, SCA-1, c-KIT, CD31, EpCAM, or CD49f, and processed for flow cytometry. (A) In untreated mice, BrdU-incorporating cells of FR3, primarily belonged to EpCAM and CD49f cell lineages. In contrast, BrdU-incorporating cells of fraction FR4 were SCA-1-positive (n ‡ 5). (B) Bleomycin treatment diminished the presence of BrdU-incorporating EpCAM and CD49f putative progenitor cells in fraction 3 introducing, instead, proliferating CD45, SCA-1, and c-KIT cell lineages to fraction 5 (n ‡ 4). Data are presented as average – SEM. (C) Re- presentative flow cytometry analysis of CD49f and EpCAM expression demonstrating the gating and percentage of sorted CD49f and EpCAMhi cells from density fractions.

Article Snippet: Flow cytometry and FACS analysis Fractionated cells were washed and labeled with the following antibodies: FITC-conjugated anti-CD45R (Invitrogen), R-PE anti- SCA-1 (Ly6A/E; Invitrogen), PE/Cy5 antic-KIT (CD117; eBioscience), PE-anti-CD49f (eBioscience), Alexa-488-anti-EpCAM (CD326; Biolegend, San Diego, CA), biotinylated-CD31 (Santa Cruz Biotech) followed by PE/Cy5 streptavidin (Biolegend), and mouse anti-BRDU (ebioscience) followed by anti-mouse Alexa 488 (Invitrogen).

Techniques: Injection, Cytometry, Expressing

Journal: PLoS ONE

Article Title: Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

doi: 10.1371/journal.pone.0067550

Figure Lengend Snippet: DiI fluorescence-labeled PEG-Lip (a–c), PRP-PEG-Lip (d–f), RGD-PEG-Lip (g–i) or Dual-PEG-Lip (j–l) were intravenously injected into Colon26 NL-17-bearing mice at day 10 after tumor implantation. At 3 h after injection, the tumors were dissected, and then frozen-sections (10-µm thickness) were prepared. Left panels (a, d, g, and j) show the distribution of endothelial cells as visualized by immunostained CD31 (green color); and middle panels (b, e, h, and k), the distribution of the liposomes (red color). Merged images are shown in the right panels (c, f, i, and l). Scale bars represent 20 µm.

Article Snippet: After these sections had been blocked with 1% BSA in PBS, they were incubated with biotinylated anti-mouse CD31 rat monoclonal antibody (Becton Dickinson Lab., Franklin Lakes, NJ, USA) for 18 h at 4°C and then visualized after incubation with streptavidin-Alexa fluor 488 conjugates (Molecular Probes Inc., Eugene, OR, USA) for 30 min at room temperature in a humid chamber.

Techniques: Fluorescence, Labeling, Injection, Tumor Implantation, Liposomes

Journal: Nature aging

Article Title: Characterization of cellular senescence in aging skeletal muscle

doi: 10.1038/s43587-022-00250-8

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD31 MicroBeads, mouse , Miltenyi , Cat# 130-097-418.

Techniques: Western Blot, Plasmid Preparation, Variant Assay, Sequencing, Recombinant, Red Blood Cell Lysis, Reverse Transcription, RNAscope, Multiplex Assay, DNA Library Preparation, Sample Prep, Immunodetection, Avidin-Biotin Assay, Blocking Assay, Protease Inhibitor, DC Protein Assay, Membrane, Lysis, Isolation, Software

Journal: European Heart Journal

Article Title: Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans

doi: 10.1093/eurheartj/ehq254

Figure Lengend Snippet: Effects of aldosterone treatment in vivo on intracellular oxidative stress and function of endothelial progenitor cells (EPCs). ( A ) Scheme of in vivo experiments: aldosterone or vehicle was delivered by implanted osmotic minipumps continuously for 2 weeks at a dose rate of 50 µg/kg/day. After 7 days matrigel plugs were implanted. After 14 days EPC function and number were determined, as well as vascularization of the implanted matrigel plug and sprouting capacity of explanted aortic rings. ( B ) Systolic and diastolic blood pressure of mice after 14 days of aldosterone- or vehicle infusion with and without concomitant oral treatment with the mineralocorticoid receptor-antagonist eplerenone (Epl, 100 mg/kg of body weight). Effects of aldosterone treatment and mineralocorticoid receptor antagonism in vivo on oxidative stress (oxidated dihydroethidium) in monocytic (early) EPCs ( C ), EPC differentiation ( D ) and EPC migratory capacity ( E ). ( F ) Number of Dil-acLDL-labelled monocytic (early) EPCs in implanted matrigel plugs after infusion of 1 × 10 6 mineralocorticoid receptor-positive or -negative EPCs to hyperaldosteronemic or control mice. ( G ) Integration (white arrow) or adhesion (gray arrow) of infused EPCs into CD31-positive vascular structures in explanted matrigel plugs. Data are mean and SEM.; *Significance against control; # significance against respective Aldo dose without Epl; * , # P ≤ 0.05, ** , ## P ≤0.01, *** , ### P ≤0.005. n = 4–6 individual experiments or animals per group.

Article Snippet: After additional 7 days matrigel plugs were explanted, shock frozen in TissueTec, sliced, stained with a mouse CD31 antibody (PECAM-1, #MCA2388, Serotec, dilution 1:50) overnight and subsequently stained with a biotinylated antimouse antibody and Texas Red Avidin DCS (Vector #A2006) and investigated by fluorescence microscopy.

Techniques: In Vivo, Control

Journal: European Heart Journal

Article Title: Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans

doi: 10.1093/eurheartj/ehq254

Figure Lengend Snippet: Effects of aldosterone-infusion and mineralocorticoid receptor antagonism on endothelial function and vascularization capacity in vivo . ( A ) Endothelium-dependent relaxation induced by acetylcholine (left) and endothelium-independent relaxation induced by 2-( N , N -diethylamino)-diazenolate-2-oxide (right) function measured in aortic ring preparations from mice treated with aldosterone (aldo) or control and/or concomitant eplerenone (Epl) treatment (see Scheme Figure A ). ( B ) Left CD31 staining of invading capillaries in matrigel plugs explanted 2 weeks after continuous aldosterone- or placebo infusion to mice. ( right ) Statistical summary of the amount of invading capillaries into matrigel plugs per section ( upper ) and haemoglobin content of matrigel plugs as a further marker of vascularization ( bottom ). ( C ) Number of sproutings from aortic ring preparations explanted from mice treated for 2 weeks with aldosterone or placebo and/or mineralocorticoid receptor antagonism. Data are mean and SEM. *Significance against control; # significance against respective Aldo dose without Epl; * , # P ≤ 0.05, ** , ## P ≤0.01. n = 5–6 individual experiments or animals per group.

Article Snippet: After additional 7 days matrigel plugs were explanted, shock frozen in TissueTec, sliced, stained with a mouse CD31 antibody (PECAM-1, #MCA2388, Serotec, dilution 1:50) overnight and subsequently stained with a biotinylated antimouse antibody and Texas Red Avidin DCS (Vector #A2006) and investigated by fluorescence microscopy.

Techniques: In Vivo, Control, Staining, Marker

Journal: Scientific Reports

Article Title: Laminin-guided highly efficient endothelial commitment from human pluripotent stem cells

doi: 10.1038/srep35680

Figure Lengend Snippet: ( a ) Schematic outline of endothelial cell differentiation. ( b ) Purity of PSC-EPCs on day 7. Data are presented as the mean ± SEM (n = 3) and were statistically analyzed using ANOVA test. ( c ) Representative flow cytometry plots of cells (day 7) on LM411 (KhES-1). ( d ) Tube formation assay of PSC-ECs induced on LM411. Scale bar: 200 μm. ( e ) Ac-LDL-uptake and CD31 expression of PSC-ECs induced on LM411. Scale bar: 10 μm.

Article Snippet: Anti-human KDR antibodies (BioLegend), anti-human CD34 antibodies (Beckman coulter), anti-human VE-cadherin antibodies (eBioscience), sheep anti-human CD31 antibodies (R&D systems), FITC-conjugated anti-sheep IgG antibodies (Jackson ImmunoResearch), mouse anti-human nuclei antibodies (Millipore), Cy3-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch), Alexa-Fluor 647-labelled rat anti-mouse TER-119 antibodies (BioLegend), rat anti-human CD49f antibodies (GoH3, BD), anti-human CD29 antibodies (AIIB2; developed by Caroline H. Damsky and obtained from Developmental Studies Hybridoma Bank) were used in this study.

Techniques: Cell Differentiation, Flow Cytometry, Tube Formation Assay, Expressing

Journal: Scientific Reports

Article Title: Laminin-guided highly efficient endothelial commitment from human pluripotent stem cells

doi: 10.1038/srep35680

Figure Lengend Snippet: ( a ) Schematic process of the modified differentiation system via activation of the canonical Wnt signaling pathway. ( b ) Representative flow cytometry plots of the day 6 cell population (KhES-1). ( c ) Efficiency of endothelial differentiation in the modified system compared to the unmodified system. The ordinates indicate the number of PSC-EPCs per starting PSC number. Data are presented as the mean ± SEM (n = 3) and were statistically analyzed using Student’s t -test. Representative results of at least three independent experiments are shown. ( d ) The cell numbers on day 3 (undefined system) and day 2 (defined system), normalized by starting PSC numbers. The data were analyzed using Student’s t -test, n = 3. ( e ) CD34 + VE-cadherin + cell numbers at day 6. Cell numbers were normalized by plated cell numbers on LM411-E8. The data were analyzed using Student’s t -test, n = 3. ( f ) A tube formation assay of PSC-ECs. Scale bars: 200 μm. ( g ) Ac-LDL-uptake and CD31 expression of PSC-ECs. Scale bar: 10 μm. ( h ) Expression of vWF, eNOS and PECAM1 in PSC-EPCs compared to undifferentiated (day 0) cells. The RQ values were calculated via the ΔΔCt-method using GAPDH on day 0 as an internal control. ( i ) ICAM-1 expression in PSC-ECs stimulated with 10 ng/mL TNF-α for 18 hours. ( j ) The proportion of CD34 + VE-Cadherin + cells on LM411-E8 or LM511-E8. Day 2 cells were plated cultured for four days. The data were analyzed using Student’s t -test, n = 3. ( k ) Immunofluorescence images of Matrigel-plug sections. PSC-EPCs (KhES-1) generated according to the above method were suspended in Matrigel and subcutaneously transplanted to NOG mice. Human endothelial cells (positive for HuNu and CD31) can be seen surrounding murine erythrocytes (positive for TER-119). Scale bars: 5 μm.

Article Snippet: Anti-human KDR antibodies (BioLegend), anti-human CD34 antibodies (Beckman coulter), anti-human VE-cadherin antibodies (eBioscience), sheep anti-human CD31 antibodies (R&D systems), FITC-conjugated anti-sheep IgG antibodies (Jackson ImmunoResearch), mouse anti-human nuclei antibodies (Millipore), Cy3-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch), Alexa-Fluor 647-labelled rat anti-mouse TER-119 antibodies (BioLegend), rat anti-human CD49f antibodies (GoH3, BD), anti-human CD29 antibodies (AIIB2; developed by Caroline H. Damsky and obtained from Developmental Studies Hybridoma Bank) were used in this study.

Techniques: Modification, Activation Assay, Flow Cytometry, Tube Formation Assay, Expressing, Control, Cell Culture, Immunofluorescence, Generated

Journal: Frontiers in Cardiovascular Medicine

Article Title: The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation

doi: 10.3389/fcvm.2023.1190460

Figure Lengend Snippet: IL-1R1 expression is associated with EndMT under disturbed blood flow areas. ( A ) Representative image of Oil O Red-stained aortic arch section of ApoE KO. Scale bars = 1 mm. Dash boxes represent the athero-resistant and atheroprone areas. ( B,C ) Immunofluorescent staining of IL-1R1 (red) and CD31 (green). Scale bars = 50–100 µm. ( D ) Shows immunofluorescence staining of CD31 (purple) Snail (red) and IL-1R1 (green) in atheroprone and athero-resistant areas in the EC-specific lineage tracing mice, YFP positive expression in ECs as stained by GFP (grey) ( n = 3). Scale bars = 500 − 100 µm. ( E,F ) Human aortic ECs (HAOECs) exposed to laminar shear stress (LSS 10 dynes/cm 2 ; mimicking in vivo athero-protective flow and oscillatory shear stress (OSS; 1 dynes/cm 2 ; mimicking in vitro atheroprone flow) for 24 h. and lysed and mRNA was quantified using qRT-PCR for IL-1R1 ( E ) and SNAI1 ( F ). Data are represented as mean ± SEM, and 1-way ANOVA and Tukey's post-test test, * p < 0.05, n = 4 independent experiments.

Article Snippet: Slides were incubated with secondary antibodies after frequent washing with 0.01% (v/v) TBST for 1 h (1:2,000 dilution each; Donkey anti-mouse Alexa Fluor 555—A32773, Donkey anti-mouse Alexa Fluor 488—A32766tr, Donkey anti-rabbit Alexa Fluor 488—A32790tr) or for the CD31 staining Donkey anti-rat IgG Biotin-SP (Jackson ImmunoResearch, 712-065-150, 1:100 dilution) at room temperature.

Techniques: Expressing, Staining, Immunofluorescence, Shear, In Vivo, In Vitro, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation

doi: 10.3389/fcvm.2023.1190460

Figure Lengend Snippet: IL-1R1 knockdown prevents OSS-induced EndMT in vitro . ( A ) Module of Endothelial phenotypic switches to EndMT in atherosclerosis. ( B ) immunofluorescent images of mock vs. IL-1R1 siRNA HAECs subjected to oscillatory shear (OSS; 1 dynes/cm 2 for 24 h), from n = 3. The cells were stained for Phalloidin (F-actin, purple), N-cadherin (red; mesenchymal marker), and CD31 (green; endothelial marker). ( C,D ) Immunofluorescent images of Snail1 (red) and DAPI (white: shows nuclei) in mock vs. IL- 1R1 siRNA HAOECs subjected to oscillatory shear (OSS; 24 h) and assessed for Snail1 nuclear translocation. Snail1 nuclear translocation was compared between IL-1R1 siRNA cells and mock controls in static and OSS conditions. Data are mean ± SEM, multiple unpaired t test, *** p < 0.001, n = 6 independent experiments. Scale bars = 400 − 100 µm.

Article Snippet: Slides were incubated with secondary antibodies after frequent washing with 0.01% (v/v) TBST for 1 h (1:2,000 dilution each; Donkey anti-mouse Alexa Fluor 555—A32773, Donkey anti-mouse Alexa Fluor 488—A32766tr, Donkey anti-rabbit Alexa Fluor 488—A32790tr) or for the CD31 staining Donkey anti-rat IgG Biotin-SP (Jackson ImmunoResearch, 712-065-150, 1:100 dilution) at room temperature.

Techniques: Knockdown, In Vitro, Shear, Staining, Marker, Translocation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation

doi: 10.3389/fcvm.2023.1190460

Figure Lengend Snippet: HAOECs were transfected with siRNA targeting IL-1R1 or mock and exposed to oscillatory shear stress (OSS) for 24 h. Cells lysates were collected for proteins of IL-1R1 ( A ), N-Cadherin ( B ), α-SMA ( C ), or mRNA ( D ). Protein or mRNA levels of endothelial markers Ve-Cadherin ( E ), and CD31 ( F ) were quantified using Western blot or qRT-PCR. Data analyzed by 2-way ANOVA and Bonferroni's post-test; * p < 0.05, ** p < 0.01, representative blots are from n = 4–5 independent experiments.

Article Snippet: Slides were incubated with secondary antibodies after frequent washing with 0.01% (v/v) TBST for 1 h (1:2,000 dilution each; Donkey anti-mouse Alexa Fluor 555—A32773, Donkey anti-mouse Alexa Fluor 488—A32766tr, Donkey anti-rabbit Alexa Fluor 488—A32790tr) or for the CD31 staining Donkey anti-rat IgG Biotin-SP (Jackson ImmunoResearch, 712-065-150, 1:100 dilution) at room temperature.

Techniques: Transfection, Shear, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation

doi: 10.3389/fcvm.2023.1190460

Figure Lengend Snippet: IL-1R1 knockout prevents d-flow-induced EndMT in vivo . ( A,B ) sc-RNA seq datasets (Bioproject #PRJNA646233) are re-analyzed. ( A ) EndMT clusters were induced by d-flow after 2 weeks of left carotid artery ligation (LCA) maximum after 2 weeks (LCA_2W). ( B ) IL-1R1 (Il1r1) expression levels in left carotid artery (LCA) compared to right carotid artery (RCA) 2 days (2D) and 2 weeks (2W) post-ligation. ( C ) Intimal mRNA levels of SNAI1 and CD31 ( D ) were compared 2 weeks after ligation between Control-iEC and IL-1R1-iEC mice. Data are mean ± SEM, analyzed by multiple unpaired t test, n = 6 mice per condition, * p < 0.05, ** p < 0.01.

Article Snippet: Slides were incubated with secondary antibodies after frequent washing with 0.01% (v/v) TBST for 1 h (1:2,000 dilution each; Donkey anti-mouse Alexa Fluor 555—A32773, Donkey anti-mouse Alexa Fluor 488—A32766tr, Donkey anti-rabbit Alexa Fluor 488—A32790tr) or for the CD31 staining Donkey anti-rat IgG Biotin-SP (Jackson ImmunoResearch, 712-065-150, 1:100 dilution) at room temperature.

Techniques: Knock-Out, In Vivo, RNA Sequencing, Ligation, Expressing, Control