Journal: Journal of Cell Science

Article Title: VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

doi: 10.1242/jcs.179960

Figure Lengend Snippet: BMP6 induces vascular permeability in vitro . (A) BMP6 increases endothelial cell monolayer solute permeability. HUVECs seeded in transwell inserts were subjected to 10 nM BMP6 or 2 nM VEGF-165 and, as indicated, pharmacological inhibitor (0.5 µM LDN-193189) treatment for 24 h and in vitro transendothelial solute permeability was measured by FITC-Dextran flux. Mean±s.e.m. normalized to untreated control cells from ten biological replicates in three independent experiments. (B) BMP6 stimulation decreases transendothelial electrical resistance (TEER). HUVECs were seeded in transwell inserts and stimulated with growth factors for 24 h. At the indicated time points, TEER was measured. Mean±s.d. normalized to untreated control cells from nine biological replicates in three independent experiments. (A,B) *** P ≤0.001; ns, not significant. (C) Immunocytochemical staining of VE-cadherin in HUVECs treated with growth factors for 4 and 24 h. Main images are representative regions from the insets. Scale bars: 5 µm.

Article Snippet: HUVECs were seeded at 200,000 cells/cm 2 in HUVEC growth medium on fibronectin/collagen-coated Costar 24-well transwell inserts (0.4 µm polyester membrane, Corning) 72 h prior to the experiment.

Techniques: Permeability, In Vitro, Staining

Journal: Journal of Cell Science

Article Title: VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

doi: 10.1242/jcs.179960

Figure Lengend Snippet: BMP6 stimulation increases internalization of VE-cadherin. (A) BMP6 increases the internalization rate of cell-surface-labeled VE-cadherin. HUVECs were incubated with the VE-cadherin extracellular domain-targeting antibody BV6 at 4°C. VE-cadherin internalization was monitored by uptake of BV6 antibody upon growth factor treatment for 60 min at 37°C. Remaining cell-surface antibodies, visible in ‘no acid wash’ conditions, were washed away with a mild acid solution and internalized VE-cadherin antibodies were visualized in fixed cells by addition of a fluorophore-coupled secondary antibody (green). Blue, DAPI. Scale bars: 20 µm. (B) Quantitation of VE-cadherin vesicles visible in A, normalized to untreated control cells. Mean±s.d. from three independent experiments. (C) BMP6 triggers internalization of cell-surface biotinylated VE-cadherin. HUVEC surface proteins were biotinylated at 4°C and growth factor-induced endocytosis was allowed to occur for 60 min at 37°C. Subsequently, remaining cell-surface biotin, which is visible in 4°C versus 4°C stripped samples, was stripped at 4°C and cells were rinsed and solubilized. Internalized biotinylated proteins were subjected to immunoprecipitation and samples were blotted as indicated. Total cell lysates (TCLs) represent samples before precipitation. Dotted line indicates merge of the same, but differentially exposed blot (left, 10 s; right, 60 s). (D) Quantitation of VE-cadherin signal intensities depicted in C and normalized to TCL VE-cadherin and untreated control cells. Mean±s.e.m. from four independent experiments. (B,D) ** P ≤0.005, *** P ≤0.001; ns, not significant.

Article Snippet: HUVECs were seeded at 200,000 cells/cm 2 in HUVEC growth medium on fibronectin/collagen-coated Costar 24-well transwell inserts (0.4 µm polyester membrane, Corning) 72 h prior to the experiment.

Techniques: Labeling, Incubation, Quantitation Assay, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

doi: 10.1242/jcs.179960

Figure Lengend Snippet: BMP6 induces phosphorylation of VE-cadherin via activation of c-Src. (A) VE-cadherin is phosphorylated at Tyr685 upon BMP6 treatment. VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody from confluent HUVECs treated with BMP6 for 30 min and samples were blotted as indicated. Normal IgG antibody served as the immunoprecipitation (IP) control. TCLs represent lysates not subjected to IP. Dotted lines indicate where samples from the same blot have been omitted. (B) Quantitation of pVE-cadherin_Tyr685 signal intensities depicted in A and normalized to TCL VE-cadherin and untreated control cells. Mean±s.d. from four independent experiments. (C) c-Src kinase is activated upon BMP6 treatment. c-Src was immunoprecipitated with a c-Src-specific antibody from confluent HUVECs treated with growth factors for 15 min. Normal IgG antibody served as an IP control. (D) Quantitation of pSrc_Tyr416 signal intensities shown in C and normalized to TCL c-Src and untreated control cells. Mean±s.d. from three independent experiments. (E) BMPRII interacts endogenously with c-Src. BMPRII was immunoprecipitated with a BMPRII-specific antibody from confluent HUVECs stimulated with BMP6 for 30 min and samples were blotted as indicated. Quantitation shows the c-Src versus BMPRII IP signal intensity ratio. Normal IgG antibody served as an IP control. (F) ALK2 associates with c-Src. HEK293T cells were transfected with ALK2-HA and c-Src, and HA-tagged ALK2 was immunoprecipitated with an HA-specific antibody. Normal IgG antibody served as IP control. (G) ALK2 and c-Src are required for BMP-induced phosphorylation of VE-cadherin. HUVECs were transfected with siRNA targeting either nonspecific sequences (si-scr), human ALK2 (si- ALK2 ) or human c-Src (si- SRC ) and treated with BMP6 for 30 min. Cells were lysed and blotted as indicated. (H) BMP6-induced permeability is mediated by ALK2 and c-Src. HUVECs were transfected with si-scr, si- ALK2 or si- SRC , seeded in transwell inserts and stimulated with BMP6 for 24 h. At the indicated time points, TEER was measured. Mean±s.d. normalized to untreated control cells from three independent experiments. *** P ≤0.001; ns, not significant. For BMP6-treated si- ALK2 and si- SRC versus untreated si-scr: ## P ≤0.005, ### P ≤0.001.

Article Snippet: HUVECs were seeded at 200,000 cells/cm 2 in HUVEC growth medium on fibronectin/collagen-coated Costar 24-well transwell inserts (0.4 µm polyester membrane, Corning) 72 h prior to the experiment.

Techniques: Activation Assay, Immunoprecipitation, Quantitation Assay, Transfection, Permeability

Journal: Journal of Cell Science

Article Title: VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

doi: 10.1242/jcs.179960

Figure Lengend Snippet: VE-cadherin regulates endothelial BMP signaling. (A) VE-cadherin is required for efficient BMP signal transduction. HUVECs were transfected with si-scr or si- CDH5 and treated with BMP6 for the indicated times. Cells were lysed and blotted as indicated. (B) Quantitation of pSMAD1/5 signal intensities shown in A and normalized to GAPDH and untreated control cells. Mean±s.d. from three independent experiments. (C) VE-cadherin is required for optimal activation of SMAD1/5 target gene expression. HUVECs were transfected with si-scr or si- CDH5 and treated with BMP6 for 60 min. ID1 mRNA expression was determined using qRT-PCR and normalized to GAPDH control mRNA expression and to untreated control cells. Mean±s.e.m. from three independent experiments. (D) CDH5 mRNA expression was determined after siRNA transfection using qRT-PCR and normalized to GAPDH control mRNA expression and to si-scr-treated control cells. Mean±s.e.m. from three independent experiments. (E) VE-cadherin clusters are needed for efficient BMP signaling. HUVECs were incubated with the VE-cadherin extracellular domain-targeting antibody BV6 or isotype control antibody and treated with BMP6 for 45 min. Cells were lysed and blotted as indicated. (F) Quantitation of pSMAD1/5 signal intensities depicted in E and normalized to GAPDH and untreated control cells. Mean±s.d. from three independent experiments. ** P ≤0.005, *** P ≤0.001. For BMP6-treated si-scr and si- CDH5 : # P ≤0.05, ### P ≤0.001.

Article Snippet: HUVECs were seeded at 200,000 cells/cm 2 in HUVEC growth medium on fibronectin/collagen-coated Costar 24-well transwell inserts (0.4 µm polyester membrane, Corning) 72 h prior to the experiment.

Techniques: Transduction, Transfection, Quantitation Assay, Activation Assay, Expressing, Quantitative RT-PCR, Incubation

Journal: Journal of Cell Science

Article Title: VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

doi: 10.1242/jcs.179960

Figure Lengend Snippet: VE-cadherin interacts with BMP receptors. (A) In situ proximity ligation assay (PLA) of VE-cadherin and the BMP receptors ALK2 and BMPRII. HUVECs were treated with BMP6 for 60 min and association of VE-cadherin and ALK2 or BMPRII was visualized using an in situ PLA (green). DAPI, blue. Scale bars: 10 µm. (B) Quantitation of VE-cadherin–ALK2 and VE-cadherin–BMPRII heteromers shown in A. Mean±s.d. from three independent experiments. (C) VE-cadherin interacts endogenously with BMPRII. VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody from confluent HUVECs stimulated with BMP6 for 60 min and samples were blotted as indicated. Recombinant protein A-Sepharose beads served as an IP control. TCLs represent lysates not subjected to immunoprecipitation. (D) VE-cadherin associates with BMPRII upon overexpression. HEK293T cells were transfected with VE-cadherin and BMPRII-HA, and VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody. Normal IgG antibody served as IP control. (E) VE-cadherin associates with ALK2 upon overexpression. HEK293T cells were transfected with VE-cadherin and ALK2-HA, and VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody. Normal IgG antibody served as IP control. (F) VE-cadherin facilitates BMP receptor complex formation. HEK293T cells were transfected with BMPRII-Myc and ALK2-HA in the absence or presence of co-expressed VE-cadherin. Cells were stimulated with BMP6 for 45 min, followed by immunoprecipitation of Myc-tagged BMPRII with a Myc-specific antibody. Mouse IgG isotype antibody served as IP control. ** P ≤0.005, *** P ≤0.001.

Article Snippet: HUVECs were seeded at 200,000 cells/cm 2 in HUVEC growth medium on fibronectin/collagen-coated Costar 24-well transwell inserts (0.4 µm polyester membrane, Corning) 72 h prior to the experiment.

Techniques: In Situ, Proximity Ligation Assay, Quantitation Assay, Immunoprecipitation, Recombinant, Over Expression, Transfection

Journal: Oral and Maxillofacial Surgery

Article Title: Geranyl-geraniol addition affects potency of bisphosphonates—a comparison in vitro promising a therapeutic approach for bisphosphonate-associated osteonecrosis of the jaw and oral wound healing

doi: 10.1007/s10006-021-00982-8

Figure Lengend Snippet: Migration assay of human umbilical cord endothelial cells (HUVEC) with substituted bisphosphonates in 50 μM concentration; # significantly enhanced migration compared to control w/o geranyl-geraniol; * significantly enhanced migration to control w geranyl-geraniol; + significantly enhanced migration w geranyl-geraniol compared to the same bisphosphonate w/o geranyl-geraniol addition. p < 0.05 indicates statistical significance

Article Snippet: For the incubation of HUVEC Endothelial Cell Growth Basal Medium (500 ml EBM, Lonza Group AG, Basel, Switzerland, plus 10% fetal calf serum, 50 μg/ml gentamicin, 50 ng/ml amphotericin B, 12 μg/ml bovine brain extract, 1 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor).

Techniques: Migration, Concentration Assay, Control

Journal: Oral and Maxillofacial Surgery

Article Title: Geranyl-geraniol addition affects potency of bisphosphonates—a comparison in vitro promising a therapeutic approach for bisphosphonate-associated osteonecrosis of the jaw and oral wound healing

doi: 10.1007/s10006-021-00982-8

Figure Lengend Snippet: MTT assay of human umbilical cord vein endothelial cells (HUVEC) with substituted bisphosphonates in 5 μM and 50 μM concentrations; # significantly enhanced cell viability compared to control w/o geranyl-geraniol; * significantly enhanced cell viability to control w geranyl-geraniol; + significantly enhanced cell viability w geranyl-geraniol compared to the same bisphosphonate w/o geranyl-geraniol addition. p < 0.05 indicates statistical significance

Article Snippet: For the incubation of HUVEC Endothelial Cell Growth Basal Medium (500 ml EBM, Lonza Group AG, Basel, Switzerland, plus 10% fetal calf serum, 50 μg/ml gentamicin, 50 ng/ml amphotericin B, 12 μg/ml bovine brain extract, 1 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor).

Techniques: MTT Assay, Control

Journal: The Journal of Cell Biology

Article Title: An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

doi: 10.1083/jcb.200503054

Figure Lengend Snippet: Perinuclear WPBs are coated. (a–d) Conventional transmission EM of HUVECs shows extensive coating of WPBs. Bars, 200 nm. (a) Arrowheads point to the coat covering a WPB. (b) Such coating is more often observed in the perinuclear region (arrowheads). (c) A transverse section of a WPB (arrowhead) and a typical CCV (arrow) have different diameters. (d) Partial coating (arrowhead) on a WPB is seen where the VWF tubules are less structured. (e) Newly emerging WPBs at the TGN (arrowheads) in two different cells partially colocalize with clathrin by immunofluorescence. Bar, 10 μm.

Article Snippet: HUVECs were purchased from TCS-Cellworks and were grown in HUVEC growth medium, which contains M199 medium with Earle's modified salts (GIBCO BRL), 20% FCS, 10 U/ml heparin (Sigma-Aldrich), 30 μg/ml endothelial growth supplement (Sigma-Aldrich), and 50 μg/ml gentamicin as previously described ( ).

Techniques: Transmission Assay, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

doi: 10.1083/jcb.200503054

Figure Lengend Snippet: AP-1 is present on WPBs. (a–e) Partial colocalization of AP-1 and VWF shown by immunofluorescence. HUVECs were fixed in 6% PFA, permeabilized, and labeled with mouse anti–AP-1 and rabbit anti-VWF, followed by FITC-conjugated anti–mouse and Texas red–conjugated anti–rabbit secondary antibodies. Bars, 10 μm. (a) A perinuclear WPB is substantially covered with AP-1. The insets show a higher magnification of the boxed area (top inset, AP-1; bottom inset, VWF). (b–e) Further examples of partial colocalization of VWF (b and d) and AP-1 (c and e). (f–h) Immuno-EM of HUVEC cryosections show the presence of AP-1 on WPBs. Bars, 200 nm. (f) A WPB labeled with rabbit anti-VWF followed by 15 nm gold particles conjugated to protein A. (g) AP-1 is observed on CCVs (arrows) and it is also found at the rim of transverse sections of WPBs (arrowheads) near the Golgi. (h) An electron-dense coat that is labeled with an AP-1 antibody (arrowhead) is associated with an elongated VWF-containing structure.

Article Snippet: HUVECs were purchased from TCS-Cellworks and were grown in HUVEC growth medium, which contains M199 medium with Earle's modified salts (GIBCO BRL), 20% FCS, 10 U/ml heparin (Sigma-Aldrich), 30 μg/ml endothelial growth supplement (Sigma-Aldrich), and 50 μg/ml gentamicin as previously described ( ).

Techniques: Immunofluorescence, Labeling

Journal: The Journal of Cell Biology

Article Title: An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

doi: 10.1083/jcb.200503054

Figure Lengend Snippet: Clathrin and AP-1 are required for WPB biogenesis. (a and b) HEK293 cells were cotransfected with AP180-C and full-length VWF constructs. WPB formation was impaired in a cell transfected with AP180-C. In contrast, elongated WPBs were clearly seen in a cell that did not contain AP180-C (*). (c–j) The depletion of AP-1, but not AP-3, impairs the formation of WPBs in HEK293 cells. Cells were transfected with a full-length VWF construct and AP-1 siRNA (e and f), AP-3 siRNA (i and j), or mock transfected (c, d, g, and h). Although elongated WPBs were clearly observed upon AP-3 RNAi (j), WPBs formation was severely impaired upon AP-1 siRNA treatment (f). (k and l) Similar results were obtained in HUVECs. Elongated WPBs are abundant in a mock-treated cell (k) but not in the AP-1–depleted cell (l). Insets show a magnified view of the boxed regions. Bars, 10 μm.

Article Snippet: HUVECs were purchased from TCS-Cellworks and were grown in HUVEC growth medium, which contains M199 medium with Earle's modified salts (GIBCO BRL), 20% FCS, 10 U/ml heparin (Sigma-Aldrich), 30 μg/ml endothelial growth supplement (Sigma-Aldrich), and 50 μg/ml gentamicin as previously described ( ).

Techniques: Construct, Transfection

Journal: The Journal of Cell Biology

Article Title: An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

doi: 10.1083/jcb.200503054

Figure Lengend Snippet: The AP-1/clathrin coat is involved in an early stage of WPB biogenesis, but not in the maintenance of the cigar shape of preformed WPBs. (a and b) A BFA-sensitive coat is not required for the maintenance of the shape of existing WPBs. HUVECs were treated with 5 μg/ml BFA for 30 min (b). Compared with untreated cells in panel a, there was no significant change to the appearance in anti-VWF staining. (c) Anti–γ-adaptin staining shows that AP-1 dissociates from WPBs and other membranes upon BFA treatment. (d–f) HUVECs were treated with 10 μM of monensin for 1 h (see Fig. S2), nucleofected with GFP-VWF, and allowed to recover overnight. The cells were labeled with rabbit anti-VWF (d), followed by Texas red–conjugated anti–rabbit antibody. Although the old rounded WPBs did not return to the original shape, newly synthesized WPBs had a classic rod shape (e). (g–l) HUVECs were microinjected with GFP-VWF together with a control vector plasmid (g–i) or AP180-C (j–l), fixed 16 h later, and labeled with anti-VWF (g and j; blue in i and l) and anti-GFP (h and k; green in i and l). Although old WPBs remained clearly visible in both cases, the cell microinjected with AP180-C failed to make new cigar-shaped WPBs. (insets) Anti-myc labeling shows the microinjected AP180-C construct. Bars, 10 μm.

Article Snippet: HUVECs were purchased from TCS-Cellworks and were grown in HUVEC growth medium, which contains M199 medium with Earle's modified salts (GIBCO BRL), 20% FCS, 10 U/ml heparin (Sigma-Aldrich), 30 μg/ml endothelial growth supplement (Sigma-Aldrich), and 50 μg/ml gentamicin as previously described ( ).

Techniques: Staining, Labeling, Synthesized, Plasmid Preparation, Construct

Journal: The Journal of Cell Biology

Article Title: An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

doi: 10.1083/jcb.200503054

Figure Lengend Snippet: VWF-positive puncta in AP-1–depleted HUVECs cannot recruit WPB components. (a–d) GFP-Rab27a was recruited onto peripheral WPBs in mock-transfected cells (b), but upon AP-1 RNAi, became cytosolic (d) and failed to associate with the VWF-positive puncta (c). (e–h) HUVECs were treated with 100 μM leupeptin and 100 μM pepstatin for 6 h before fixation. The colocalization of P-selectin and VWF (e and f) was lost upon AP-1 RNAi (g and h). P-selectin was observed only after the lysosomal inhibitor treatment (h), suggesting that it was targeted to lysosomes for degradation in the absence of WPBs in AP-1–depleted cells. Bar, 10 μm.

Article Snippet: HUVECs were purchased from TCS-Cellworks and were grown in HUVEC growth medium, which contains M199 medium with Earle's modified salts (GIBCO BRL), 20% FCS, 10 U/ml heparin (Sigma-Aldrich), 30 μg/ml endothelial growth supplement (Sigma-Aldrich), and 50 μg/ml gentamicin as previously described ( ).

Techniques: Transfection