Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Derivation of Endothelial Cells From Human Embryonic Stem Cells by Directed Differentiation

doi: 10.1161/atvbaha.110.204800

Figure Lengend Snippet: Figure 1. Taqman low-density array (TLDA) analysis of mRNA levels in pluri- potent hESCs and cells subjected to directed differentiation to vascular endo- thelial cells. hESCs were either main- tained under pluripotency conditions or under feeder- and serum-free monolayer-directed differentiation to vas- cular endothelial cells over 21 days. TLDA analysis was performed on extracted mRNA at different time points. A, Markers shown: pluripotency- associated mRNAs: Nanog, Oct 4, and Sox 2, with significant reduction in expression observed with progression of differentiation. B, early mesoderm mark- ers (brachyury, NKX2.5, Mesp1, and Mixl1) vascular endothelial cells (FLT-1, KDR, CD31, VE-cadherin, and CD34). Data are given as the meanSEM. * P0.05, **P0.01, and ***P0.001 vs the time-matched pluripotent sample. Supplemental Table I provides the data for complete TLDA analysis.

Article Snippet: Cells were incubated at 4°C overnight with the following antibodies: mouse IgG monoclonal anti–human OCT4 (1:200; Santa Cruz Biotechnology, Inc, Heidelberg, Germany); mouse IgG monoclonal anti–human Nanog (1:200; Abcam plc, Cambridge, England); mouse IgG monoclonal anti–human CD31 (1:100; Dako UK Ltd, Ely, England); and goat IgG polyclonal VE-cadherin (1:100; R&D Systems).

Techniques: TLDA Assay, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Derivation of Endothelial Cells From Human Embryonic Stem Cells by Directed Differentiation

doi: 10.1161/atvbaha.110.204800

Figure Lengend Snippet: Figure 2. Expression of endothelial and angiogenesis-related proteins. A, FACS analysis and immunocytofluorescent staining of endothelial marker proteins. Increasing positive expression of CD31 (FL1 and far panel) and VE-cadherin (FL2 and middle panel) observed in a time-dependent manner to 21-day differen- tiation, using 4,6-diamidino-2-phenylindole (DAPI) (nuclear counterstain [blue]) (scale bar indicates 20 m). Representative scans and images from 3 independent experiments. B, Mem- brane scans of angiogenic markers in differentiated hESC-ECs (days 0, 10, 14, and 21 of differentiation). Array images are from 10-minute exposure to x-ray film. Supplemental Figure III pro- vides the corresponding densometric analysis.

Article Snippet: Cells were incubated at 4°C overnight with the following antibodies: mouse IgG monoclonal anti–human OCT4 (1:200; Santa Cruz Biotechnology, Inc, Heidelberg, Germany); mouse IgG monoclonal anti–human Nanog (1:200; Abcam plc, Cambridge, England); mouse IgG monoclonal anti–human CD31 (1:100; Dako UK Ltd, Ely, England); and goat IgG polyclonal VE-cadherin (1:100; R&D Systems).

Techniques: Expressing, Staining, Marker

Journal: Stem Cell Research & Therapy

Article Title: Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway

doi: 10.1186/s13287-015-0257-0

Figure Lengend Snippet: MSC-secreted paracrine HGF upregulated endothelial VE-cadherin protein expression and decreased caveolin-1 protein expression. The results showed that LPS stimulation of HPMECs reduced the expression of VE-cadherin and occludin protein ( p < 0.01; a , b , c ) but increased the expression of caveolin-1 protein ( p < 0.05; a , d ) and that these effects were inhibited by MSCs. However, the effect of MSCs was significantly blocked by anti-HGF antibody ( p < 0.05). Furthermore, the role of MSCs in reducing caveolin-1 protein expression was clearly inhibited by anti-HGF and anti-VEGF antibodies. Adding MSC-CM in all groups except control group and LPS group. n = 3, * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05 vs. LPS group; & p < 0.05 vs. MSC-CM group. CM Conditioned medium, HGF Hepatocyte growth factor, LPS Lipopolysaccharide, MSC Mesenchymal stem cell, VEGF Vascular endothelial growth factor

Article Snippet: Then the membranes were blocked in phosphate-buffered saline-Tween (PBS-T) containing 5 % milk for 2 h at room temperature and incubated at 4 °C overnight with primary antibodies against VE-cadherin (1:1000; Cell Signaling), occludin (1:250; Abcam) or caveolin-1 (1:1000; Epitomics).

Techniques: Expressing, Control

Journal: Stem Cell Research & Therapy

Article Title: Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway

doi: 10.1186/s13287-015-0257-0

Figure Lengend Snippet: MSC-secreted HGF restored endothelial VE-cadherin remodelling. LPS causes the remodelling of the junctional localisation of VE-cadherin, which causes HPMEC to contract, increasing paracellular permeability. After 24 h of MSC-CM and HPMEC co-culture, the remodelling of the junctional localisation of VE-cadherin was partially restored. However, neutralising HGF from the MSC-CM with anti-HGF antibody caused VE-cadherin to be disrupted again. Adding MSC-CM in all groups except control group and LPS group. CM Conditioned medium, HGF Hepatocyte growth factor, LPS Lipopolysaccharide, MSC Mesenchymal stem cell, VEGF Vascular endothelial growth factor

Article Snippet: Then the membranes were blocked in phosphate-buffered saline-Tween (PBS-T) containing 5 % milk for 2 h at room temperature and incubated at 4 °C overnight with primary antibodies against VE-cadherin (1:1000; Cell Signaling), occludin (1:250; Abcam) or caveolin-1 (1:1000; Epitomics).

Techniques: Permeability, Co-Culture Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway

doi: 10.1186/s13287-015-0257-0

Figure Lengend Snippet: VEGF/HGF and MSC treatments enhanced VE-cadherin and occludin protein expression and reduced caveolin-1 protein expression in LPS-stimulated HPMECs via the RhoA/Rac1 pathway. The results showed that the effects of MSCs and VEGF/HGF on enhancing VE-cadherin (Fig. 10a and b) and occludin protein expression (Fig. 10a and c) were weakened when injured HPMECs were pretreated with the Rac1 inhibitor NSC23766. However, caveolin-1 protein expression (Fig. 10a and d) increased in HPMECs pretreated with the Rac1 inhibitor NSC23766 or with the RhoA inhibitor C3 transferase. n = 3, * p < 0.05; ** p < 0.01 vs. MSC group; # p < 0.05 vs. VEGF/HGF group. CM Conditioned medium, HGF Hepatocyte growth factor, LPS Lipopolysaccharide, MSC Mesenchymal stem cell, VEGF Vascular endothelial growth factor

Article Snippet: Then the membranes were blocked in phosphate-buffered saline-Tween (PBS-T) containing 5 % milk for 2 h at room temperature and incubated at 4 °C overnight with primary antibodies against VE-cadherin (1:1000; Cell Signaling), occludin (1:250; Abcam) or caveolin-1 (1:1000; Epitomics).

Techniques: Expressing

Journal: Acta physiologica (Oxford, England)

Article Title: Increased R-spondin 3 contributes to aerobic exercise-induced protection against renal vascular endothelial hyperpermeability and acute kidney injury.

doi: 10.1111/apha.14036

Figure Lengend Snippet: FIGURE 5 RSPO3 ameliorates LPS-induced hyperpermeability of renal endothelial cells by suppression of MMPs expression. (A) Mice were intraperitoneally injected with LPS (12 mg/kg) with or without recombinant RSPO3 protein (0.25 mg/kg). The control group was injected with saline. Kidney samples were collected 24 h after LPS injection. The mRNA levels of MMP family members including MMP-3, MMP-7, MMP-9, MMP-13, and MMP-14 were determined by qRT-PCR (n = 7, two-way ANOVA with Bonferroni's post-tests). (B) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of RSPO3 (100 ng/mL) for 24 h. The mRNA levels of MMP-3, MMP-7, MMP-9, MMP-13, and MMP-14 were determined by qRT-PCR (n = 4, one-way ANOVA with Bonferroni's post-tests). (C,D) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of BB2516 (10 μM) for 24 h (n = 4, one-way ANOVA with Bonferroni's post-tests). (C) Protein levels of ZO-1, VE-cadherin, SDC-1, and SDC-4 were examined by Western blots. Corresponding histograms were shown on the right panel of the representative protein bands. (D) Permeability of confluent HMVEC monolayer was determined by 10-kDa FITC-dextran flux. (E,F) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of RSPO3 (100 ng/mL) for 24 h (n = 4, one-way ANOVA with Bonferroni's post-tests). (E) Protein levels of ZO-1, VE-cadherin, SDC-1, and SDC-4 were examined by Western blots. Corresponding histograms were shown on the right panel of the representative protein bands. (F) Permeability of confluent HMVEC monolayer was determined by 10-kDa FITC-dextran flux. Data were expressed as mean ± SEM. *p < 0.05, †p < 0.01, ‡p < 0.001.

Article Snippet: Membranes were incubated with primary antibody against β- actin (1:5000; Sigma Aldrich, MO, USA), ZO- 1 (1:500; Proteintech, Chicago, USA), VE- cadherin (1:500; Proteintech), SDC- 1 (1:500; Proteintech), SDC- 4 (1:1000; Abcam, Cambridge, MA, USA), and RSPO3 (1:500; ABclonal, Wuhan, Chain) at 4°C overnight.

Techniques: Expressing, Injection, Recombinant, Control, Saline, Quantitative RT-PCR, Western Blot, Permeability

Journal: Theranostics

Article Title: Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects

doi: 10.7150/thno.21966

Figure Lengend Snippet: Laminar flow specifically reduces repressive epigenetic mark H3K27me3 in endothelial cells in vitro and in vivo . (A) Diagram of PRC2. PRC2 is composed of EED, SUZ12, RbAp46/48, and EZH2, which imposes the H3K27me3 epigenetic mark to the target gene promoter, leading to gene silencing. (B) Laminar flow decreases global H3K27me3 levels. HUVECs were exposed to laminar flow for 48 h, and then total histones were purified as described in the methods section. GSK126, a specific inhibitor of EZH2 activity was used as positive control (n=4-5, **P<0.01, ***P<0.001 vs. static control or DMSO). (C) Laminar flow does not affect the level of H3K9me3 or H3K9ac. HUVECs were exposed to laminar flow. Cell alignment in response to flow was observed to ensure successful induction of laminar flow. Then, epigenetic marks H3K9me3 and H3K9ac were determined by Western blot. The same membranes were stripped and incubated with histone 3 (H3) as loading controls (n=3). (D) En face immunofluorescent staining of H3K27me3 in mouse aorta. The aortic arch and thoracic aorta were collected from 3-month-old ApoE -/- mice fed a normal chow diet for en face staining. Red, H3K27me3; green, VE-cadherin; blue, DAPI; scale bar=20 µm (n=5).

Article Snippet: Next, aorta segments were incubated with rat anti-VE-Cadherin (1:100; #555289, BD Bioscicence), rabbit anti-EZH2 (#6263, ProSci) or rabbit anti-H3K27me3 (#39155, Active Motif) antibody overnight at 4 °C.

Techniques: In Vitro, In Vivo, Purification, Activity Assay, Positive Control, Western Blot, Incubation, Staining

Journal: Theranostics

Article Title: Flow-dependent epigenetic regulation of IGFBP5 expression by H3K27me3 contributes to endothelial anti-inflammatory effects

doi: 10.7150/thno.21966

Figure Lengend Snippet: Laminar flow decreases the expression of chromatin modifier EZH2 in vitro and in vivo . (A) HUVECs were exposed to laminar flow for the indicated time points, and then EZH2 protein expression was determined by Western blot (*P<0.05, ***P<0.001 vs. static control, n=6). (B) HCAECs were exposed to laminar flow for 24 h, and then EZH2 protein expression was determined by Western blot (**P<0.01, n=3). ( C ) Illustration of atheroprone (inner curvature of aortic arch, #1) and atheroprotective (thoracic aorta, #2) regions of mouse aorta. The picture was manually drawn by powerpoint. ( D ) EZH2 mRNA expression in aortic endothelium from atheroprone and atheroprotective regions of mouse aorta. Intima-enriched RNA was isolated from the aortic arch and thoracic aorta of ApoE -/- mice fed a normal chow diet for 3 months. RNA was reverse transcribed and cDNA was used for real-time PCR quantification of EZH2 using GAPDH as internal control (**P<0.01 vs. aortic arch, n=8). (E) En face immunofluorescent staining of EZH2 in mouse aorta. The aortic arch and thoracic aorta were collected from 3-month-old ApoE -/- mice fed a normal chow diet for en face staining. Red, EZH2; green, VE-cadherin; blue, DAPI; scale bar=20 µm (n=5).

Article Snippet: Next, aorta segments were incubated with rat anti-VE-Cadherin (1:100; #555289, BD Bioscicence), rabbit anti-EZH2 (#6263, ProSci) or rabbit anti-H3K27me3 (#39155, Active Motif) antibody overnight at 4 °C.

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Staining