Journal: Bone Reports

Article Title: Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

doi: 10.1016/j.bonr.2018.06.006

Figure Lengend Snippet: Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 μm. Results are presented as mean ± SD. Student's t -test; p < 0.05 *, p < 0.01 ** and p < 0.001 *** compared to control.

Article Snippet: Human dermal microvascular endothelial cells (HDMEC; 3H Biomedical, Uppsala, Sweden) were cultured on gelatin-coated tissue-culture dishes in complete endothelial cell growth medium (EBM-MV2; PromoCell, Heidelberg, Germany).

Techniques: Expressing, Activity Assay, Staining, Control

Journal: The FASEB Journal

Article Title: CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE‐cadherin

doi: 10.1096/fj.202201623rr

Figure Lengend Snippet: FIGURE 3 Junctions in CD93 downregulated endothelial cells are restored upon ROCK inhibition. (A) VE-cadherin (VEC) immunofluorescent staining (green) and F-actin (red) in control (Mock, siCtrl) and CD93 siRNA-transfected HDBEC (siCD93_1, siCD93_2) in the presence or absence of the ROCK inhibitor Y27632. Scale bars: 20 μm. (B) Immunofluorescent staining for claudin-5 (Cldn5, green) and F-actin (red) in the presence or absence of Y27632. Scale bars: 20 μm. High magnification images indicate VEC and Cldn5 signal al the cell–cell junctions (A and B respectively). (C, D) Quantification of VEC and Cldn5-positive area normalized to cell number (n = 3). ***p < .005, **p < .01, *p < .05, ns p > .05; One-way ANOVA with Tukey's multiple comparisons test. (E) Intercellular gaps formation (light gray) in control HDBEC monolayer (Mock and siCtrl) or silenced for CD93 (siCD93_1 and siCD93_2) in the presence or absence of Y27632. (F) Quantification of the intercellular gaps area normalized to cell number in the field of view (n = 3). ****p < .0001, ***p < .005. ns p > .05. One-way ANOVA with Tukey's multiple comparisons test.

Article Snippet: VE- cadherin internalization was assessed by antibodyfeeding assay.19 Briefly, control or CD93 silenced confluent HDBECs were incubated with an antibody against VE- cadherin (Cat# MABT134; RRID: AB_10845943; Clone BV6; Merck, New Jersey, USA; 10 μg/mL) at 4°C for 1 h in EBM- MV2 containing 3% BSA without supplements.

Techniques: Inhibition, Staining, Control, Transfection

Journal: The FASEB Journal

Article Title: CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE‐cadherin

doi: 10.1096/fj.202201623rr

Figure Lengend Snippet: FIGURE 4 CD93 knockdown induces VE-cadherin (VEC) phosphorylation in a ROCK-dependent manner, causing its internalization. (A) Immunofluorescent staining of phosphorylated VEC tyrosine-658 (pY658-VEC; green) in control (Mock, siCtrl) or siCD93- (siCD93_1, siCD93_2) transfected HDBECs in the presence or absence of the inhibitor Y27632. Actin cytoskeleton is visualized by F-actin (red). Scale bars: 25 μm. (B) Quantification analysis of the pY568-VEC levels (n = 3 independent experiments). ****p < .0001, ***p < .005, ns p > .05, One- way ANOVA with Tukey's multiple comparisons test. (C) Antibody feeding assay showing internalized VEC (red, arrowheads) in control (Mock and siCtrl) and siCD93 (siCD93_1 and siCD93_2) HDBECs co-stained with total VEC (green) and nuclei (blue). Scale bars: 25 μm. (D) Quantification of internalized/total VEC in control and siCD93 cells (n = 3 independent experiments). ***p < .005, **p < .01, ns p > .05; One- way ANOVA with Tukey's multiple comparisons test.

Article Snippet: VE- cadherin internalization was assessed by antibodyfeeding assay.19 Briefly, control or CD93 silenced confluent HDBECs were incubated with an antibody against VE- cadherin (Cat# MABT134; RRID: AB_10845943; Clone BV6; Merck, New Jersey, USA; 10 μg/mL) at 4°C for 1 h in EBM- MV2 containing 3% BSA without supplements.

Techniques: Knockdown, Phospho-proteomics, Staining, Control, Transfection, Feeding Assay

Journal: The FASEB Journal

Article Title: CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE‐cadherin

doi: 10.1096/fj.202201623rr

Figure Lengend Snippet: FIGURE 5 Loss of CD93 destabilizes brain endothelial cell–cell junctions. (A) Vascular immunofluorescent staining of VE-cadherin (VEC, green) in the brain cortex of wild-type and CD93−/−. High magnification pictures show VEC expression pattern. Arrowheads in the CD93−/− group indicate irregular staining pattern. Scale bars: 20 μm. (B) Quantification of the VEC-positive signal in brain vessels of wild- type (n = 3) and CD93−/− (n = 4) mice. **p < .01, two-tailed t test. (C) Claudin-5 immunofluorescent staining (Cldn5, green) in wild-type and CD93−/− brain vessels. Arrowheads in the high magnification image indicate loss of Cldn5 signal in the CD93−/− vessels. Scale bars: 20 μm. (D) Quantification of Cldn5-positive signal in the vessels of wild-type (n = 3) and CD93−/− (n = 3) mice. *p < .05, two-tailed t test. Vessels are visualized by CD31 (red) and nuclei by Hoechst (blue).

Article Snippet: VE- cadherin internalization was assessed by antibodyfeeding assay.19 Briefly, control or CD93 silenced confluent HDBECs were incubated with an antibody against VE- cadherin (Cat# MABT134; RRID: AB_10845943; Clone BV6; Merck, New Jersey, USA; 10 μg/mL) at 4°C for 1 h in EBM- MV2 containing 3% BSA without supplements.

Techniques: Staining, Expressing, Two Tailed Test

Journal: The FASEB Journal

Article Title: CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE‐cadherin

doi: 10.1096/fj.202201623rr

Figure Lengend Snippet: FIGURE 7 CD93 deficiency induces VE-cadherin phosphorylation at tyrosines Y658 and Y685 in vivo and impairs BBB function. (A–D) VE-cadherin (VEC) phosphorylation in brain vessels of wild-type and CD93−/− littermates. Immunostaining of pY658 VE-cadherin (green) (A), pY685 VE-cadherin (green) (B). Total VEC (VEC, red) and nuclei (Hoechst; blue). Scale bars: 20 μm. (C) Quantification of pY658-VEC relative to total-VEC in wild-type (n = 3) and CD93−/− (n = 3) mice. (D) Quantification of pY685-VEC relative to total-VEC in wild-type (n = 3) and CD93−/− (n = 3) mice. **p < .01; two-tailed t test. (E–I) Analysis of blood–brain barrier permeability in mice injected intravenously with 1-kDa AlexaFluor-555 cadaverine. (E) Representative coronal sections of rostral, middle, and caudal brain regions assessed for extravasated cadaverine (red signal). Scale bars: 500 μm. (F) Schematic of a brain coronal section illustrating the areas where the cadaverine extravasation was observed (1 = cortex; 2 = midline between hemispheres; 3 = ventral region). Quantification of the cadaverine- positive area (G) and mean fluorescent intensity (H) in CD93−/− mice (n = 3) and wild-type mice (n = 3) after 2 h of circulation. ***p < .005, **p < .01, ns p > .05; Two-way ANOVA with Sidak's multi-comparisons test. (I) Confocal images of brain vessels (CD31, green) and cadaverine tracer (red). Arrowheads indicate the extravasated tracer detected in CD93−/− group. Scale bars: 25 μm. Schematics were created with BioRender.com.

Article Snippet: VE- cadherin internalization was assessed by antibodyfeeding assay.19 Briefly, control or CD93 silenced confluent HDBECs were incubated with an antibody against VE- cadherin (Cat# MABT134; RRID: AB_10845943; Clone BV6; Merck, New Jersey, USA; 10 μg/mL) at 4°C for 1 h in EBM- MV2 containing 3% BSA without supplements.

Techniques: Phospho-proteomics, In Vivo, Immunostaining, Two Tailed Test, Permeability, Injection