Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: E10.5 (left panels) and E12.5 (right panels) placentas were dissociated to a single cell suspension and analyzed. Prom1+Sca1+CD34+ cells are displayed for (A) FCS/SSC, CD49f and (B) CD31, CD105, VE-Cadherin, Tie2, CD41 and cKit expression. Red boxes highlight the phenotype Prom1+Sca1+CD45− cells.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: Placentas from (A) E10.5, (B) E11.5 and (C) E12.5 were stained with antibodies against CD133 (Prom1, green). The left panels show composite low magnification pictures of transversal sections of placentas. The region of the labyrinth that contains Prom1+ cells is highlighted (white circles).

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Staining

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) Hemogenic PS34 cells from E10.5 and E12.5 placentas were fractioned into CD45−cKit−, CD45−cKit+ and CD45+cKit+ populations. HSPCs and mature blood cells (MBC) were also sorted for comparison.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques:

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E10.5 AGM+VU regions were isolated, dissociated to a single cell suspension and analyzed by flow cytometry. PS34CD45− and PS34CD45+ cells are gated and displayed for the expression of the 23GFP Runx1 reporter. Percentages are shown as mean ± SD, n=3.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E12.5 PS34CD45− cells were co-cultured on OP-9 in the presence of cytokines (SCF, Fltl3L, IL-3, IL-6 and TPO) for 4 days. FACS analysis shows the generation of CD45+CD34+cKit+ cells that were tested for clonogenic activity.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Cell Culture, Activity Assay

Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Isolation and characterization of mouse retinal EC. Vdr +/+ and Vdr −/− retinal EC were prepared and cultured as described in Materials and Methods. ( A ) Morphology of cells at subconfluent and confluent. ( B ) Vitamin D receptor expression levels were accessed by qPCR analysis (n = 3, *** p < 0.001). ( C ) Cells were incubated with medium alone (cells), 0.41% ethanol (vehicle), or 10 µM calcitriol for 24 h and VDR levels were measured by Western blot analysis. ( D ) Expression levels of EC markers were determined by flow cytometry. VE-cad; VE-cadherin and PECAM-1; Platelet endothelial cell adhesion molecule-1. Relative median fluorescent intensities are indicated in the top left corner of each panel. These cells were also positive for B4-lectin (a mouse EC specific lectin) and lacked the expression of PDGFRβ and smooth muscle actin (pericyte markers; not shown).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Isolation, Cell Culture, Expressing, Incubation, Western Blot, Flow Cytometry

Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Cellular localization and expression levels of ZO-1, N-cadherin, VE-cadherin, p120-catenin, and β-catenin. ( A ) Retinal EC were cultured on fibronectin-coated chamber slides to confluence and stained with specific antibodies as described in . ( B , C ) Western blot analysis of junctional proteins (n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Expressing, Cell Culture, Staining, Western Blot

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Low shear stress induced mesenchymal genes via Snail. ( a ) HUVEC were exposed to low oscillatory (+/−4 dyn/cm 2 ) or high (13 dyn/cm 2 ) wall shear stress (WSS) using a parallel plate system. ( b ) PAEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) wall shear stress. ( a , b ) After 72 h, levels of EndMT marker transcripts and VE-cadherin transcripts were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Data were pooled from six independent experiments using cells from different donors and mean levels +/− SEM are shown. ( c–e ) HUVEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) WSS for 72 h. ( c ) Expression of N-cadherin (green) and VE-cadherin (red) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. The proportion of cells that expressed N-cadherin or VE-cadherin was measured. ( d ) The expression levels of N-cadherin (left) and VE-cadherin (right) were assessed by Western blotting using specific antibodies and anti-PDHX antibodies were used to control for total protein levels. Representative blots are shown. Bands were quantified by densitometry. ( e ) Expression of Snail (green) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. Fluorescence intensity was quantified in multiple cells. ( f ) HUVEC were transfected with siRNA targeting Snail or with scrambled sequences and exposed to orbital flow for 72 h. Cells exposed to low WSS (5 dyn/cm 2 ) were collected and transcript levels of Slug, N cadherin and α-SMA were quantified by qRT-PCR. ( c – f ) Data were pooled from three independent experiments using cells from different donors and mean levels +/− SEM are shown.

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Marker, Quantitative RT-PCR, Expressing, Staining, Western Blot, Fluorescence, Transfection

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Snail was preferentially expressed at low shear atherosusceptible sites. ( a ) EC were freshly-isolated from low wall shear stress (WSS; inner curvature) and high WSS (outer curvature) regions of the aorta in six pigs. Levels of Snail, Slug, N-cadherin, α-SMA and VE-cadherin mRNA were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Mean levels +/− SEM are shown. ( b , c ) EC at low WSS (susceptible) or high WSS (protected) regions of the aorta were studied by en face staining. ( b ) C57BL/6 mice (n = 5) were stained using anti-Snail antibodies (red), co-stained using anti-CD31 antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). ( c ) TWIST1 cKO or TWIST1 fl/fl mice (n = 4 each group) were stained using anti-Snail antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). Representative images (scale bar, 10 μm) and quantitation of Snail fluorescence levels (mean +/− SEM) are shown. Differences between means were assessed using a paired t-test ( b ) or two-way ANOVA ( c ).

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Staining, Quantitation Assay, Fluorescence

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Comparison of cell dissociation protocols. For the first condition, 1.5 mL of 0.25% Trypsin-EDTA was added after collagenase digestion and the mixture was digested for an additional 15 min at 37 °C. The optimal condition (boxed) yields the highest number of viable cells in two independent experiments. NA, not available. (B) Sorting plots of an adult (>8 week-old) mouse lung with genetically labeled ECs. Left two panels: comparison of antibody staining with native fluorescence from tdT (red contour), which colocalizes mostly with CD31 and ICAM2, but also a subset of CD45 cells, perhaps due to inadvertent labeling of hematopoietic cells by Cdh5-CreER . Right two panels: for all sorting experiments, CD45 negative selection was first performed and followed by ICAM2 positive selection (red box), which labels the same ECs as CD31 but has better separation from the corresponding negative ECs.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Labeling, Staining, Fluorescence, Selection

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Representative en face view of immunostaining images from at least 5 mice. Boxed region is magnified as a section view in the first three images of the bottom row. CAR4 staining covers, whereas PLVAP staining surrounds, alveolar islands (dash). Perinuclear CAR4 and PLVAP staining allows assignment of ERG nuclei to CAR4 (cyan arrowhead) versus PLVAP (magenta arrowhead) ECs, which is automatically identified, as shown in the lower rightmost image (grey nuclei are ambiguous). Scale: 10 um. (B) Wholemount immunostaining of lungs with sparsely-labeled ECs, representative of at least 5 mice, viewed as a stack (40 um), a slab (top 20 um), or a section (1 um). Accumulation of tdT to ERG nuclei allows cell numeration (1 through 5). Cell #1 is a Car4 EC and cells #2-5 are non-Car4 ECs. Line profile analysis shows aligned versus shifted peaks for Car4 versus non-Car4 ECs, respectively. For shifted peaks, Car4 ECs are closer to the airspace than non-Car4 ECs (e.g. cell #3). Asterisk, avascular tissue surrounded by a single net-like Car4 EC. Open arrow, CDH5 junction overlapping with a single Car4 EC. Cell perimeter is measured by connecting protrusions that are visible in a projection view. Tam, 0.25 ug tamoxifen. Scale: 10 um. (C) Quantification of cell perimeter and comparison using Student’s t-test.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Staining, Labeling

Journal: Frontiers in physiology

Article Title: Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

doi: 10.3389/fphys.2017.00666

Figure Lengend Snippet: FIGURE 1 | Indoxyl sulfate (IS) induces activation in human umbilical vein endothelial cells (HUVECs). HUVECs treated with IS (256 µg/ml) for 24 h showed significant increases in markers of oxidative stress and apoptosis. The expression was quantified by changes in the mean fluorescent intensity (MFI) of (A) hydroethidine (HE) and (B) annexin V. In addition, (C) IS induced significantly elevated expression of ICAM-1, VE-cadherin, and VCAM-1, but not PECAM-1. Data are the means ± SEM of five independent experiments. *p = 0.004 vs. untreated HUVECs; **p = 0.045 vs. untreated HUVECs; and #p ≤0.001 vs. untreated HUVECs.

Article Snippet: Next, VE-cadherin (FITC rabbit anti human CD144, AbD Serotec, UK), PECAM-1 (PE-labeled monoclonal antiCD31, Caltag Laboratories, Burlingame, CA, USA), ICAM-1 (PE mouse anti human CD54, Invitrogen), and VCAM-1 (PE Mouse Anti-Human CD106, BD Pharmingen) antibodies were used to assess the expression in the experimental conditions.

Techniques: Activation Assay, Expressing

Journal: Frontiers in physiology

Article Title: Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

doi: 10.3389/fphys.2017.00666

Figure Lengend Snippet: FIGURE 3 | Indoxyl sulfate (IS) modulates the expression of adhesion molecules and annexin V in microvesicles (MV). MV (MV/µl) derived from indoxyl sulfate-treated HUVECs (IsEMV) showed a significant increase in annexin V and adhesion molecules, such as PECAM-1, VE-cadherin, and ICAM-1, but not VCAM-1. Results are the mean ± SEM of five independent experiments. *p < 0.001 vs. MV derived from untreated HUVECs (EMV); **p = 0.002 vs. EMV and #p = 0.014 vs. EMV.

Article Snippet: Next, VE-cadherin (FITC rabbit anti human CD144, AbD Serotec, UK), PECAM-1 (PE-labeled monoclonal antiCD31, Caltag Laboratories, Burlingame, CA, USA), ICAM-1 (PE mouse anti human CD54, Invitrogen), and VCAM-1 (PE Mouse Anti-Human CD106, BD Pharmingen) antibodies were used to assess the expression in the experimental conditions.

Techniques: Expressing, Derivative Assay