Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Stromal Cell–Derived Factor-1α/C-X-C Chemokine Receptor Type 4 Axis Promotes Endothelial Cell Barrier Integrity via Phosphoinositide 3-Kinase and Rac1 Activation

doi: 10.1161/atvbaha.114.303890

Figure Lengend Snippet: Figure 4. Stromal cell–derived factor (SDF)-1α promotes endo- thelial barrier integrity by Rac1 activation and subsequent corti- cal actin rim formation. A, Effect of 30-minute pretreatment with NSC23766 (NSC; 100 μmol/L) on the SDF-1α (100 ng/mL)– induced transendothelial electrical resistance increase of bovine aortic endothelial cells (BAECs; n=5–6). **P<0.01 compared with BSA-treated cells. #P<0.05 compared with SDF-1α–treated cells. B, Effect of SDF-1α (100 ng/mL) or sphingosine-1-phosphate (1 μmol/L, 3 minutes) stimulation on Rac1 activation in BAECs. A typical example (top) and quantification of GTP-Rac and total- Rac band strength (bottom) are shown (n=3–5). Relative Rac1 activity was shown as folds of untreated cells. *P<0.05 compared with untreated cells. C, Typical pictures of immunostaining of vascular endothelial (VE)-cadherin (top, green) and F-actin (middle, red). Bottom, Merged pictures of VE-cadherin, F-actin, and DAPI (4’,6-diamidino-2-phenylindole; blue) staining (n=3–4). BAECs were stimulated with SDF-1α (100 ng/mL, 5 minutes) or thrombin (1 U/mL, 20 minutes) with or without SDF-1α (100 ng/ mL, 5 minutes) pretreatment. Arrowheads indicate intercellular space. Scale bar, 10 μm. Data are presented as a mean±SEM.

Article Snippet: The chemicals obtained were: Akt1/2 kinase inhibitor, AMD3100, ATP, bovine serum albumin (BSA), bradykinin, croton oil, 4',6-diamidino-2-phenylindole (DAPI), Evans blue, ionomycin calcium salt, mouse anti-β-actin antibody, thrombin (Sigma, USA); histamine, L-NG-Monomethylarginine (L-NMMA), L-NG-Nitroarginine Methyl Ester (L-NAME), LY294002, Triton X-100, paraformaldehyde (PFA), VEGF (Wako, Japan); CCX771 (Chemo Centryx, USA); SDF-1α, Rac1 Activation Assay Kit (Millipore, USA); fetal bovine serum (FBS) (JRH Bioscience, USA); D-erythro-sphingosine-1-phosphate (Merck, USA); pertussis toxin (KAKETUKEN, Japan); NSC23766 (Tocris Bioscience, USA); Fura-2/AM (Dojindo); cremophor EL (Nacalai Tesque, Japan); siGENOME SMARTpool against p110α or p110β, ON-TARGETplus siRNA against p110γ (Thermo Scientific, USA); lipofectamine RNAiMAX, CXCR4 and CXCR7 StealthTM Select RNAi (Bos taurus) sets, goat anti-rabbit IgG antibody Alexa Fluor 680, rabbit anti-goat IgG antibody Alexa Fluor 488 (Invitrogen, USA); rhodamine phalloidin (Molecular Probes, USA); Endothelial Cell Growth Medium-2 (EGM-2), Endothelial Cell Basal Medium-2 (EBM-2), EGM-2-MV (Lonza, Switzerland); rabbit anti-phospho-AktSer473 antibody, rabbit anti-p110α antibody, rabbit anti-p110β antibody, rabbit anti-p110γ antibody (Cell Signaling Technology, USA); rabbit anti-Akt antibody (BD, USA); mouse anti-Rac1 antibody (Merck, USA); rabbit anti-CXCR4 antibody (Abcam, UK); goat anti-vascular endothelial-cadherin (VE-cadherin) antibody (Santa Cruz); goat anti-mouse IgG antibody IRDye 800 (ROCKLAND, USA); goat anti-rabbit IgG antibody IRDye 680, goat anti-mouse IgG antibody IRDye 800CW (LI-COR Biosciences, USA).

Techniques: Derivative Assay, Activation Assay, Activity Assay, Immunostaining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

doi: 10.3390/ijms232012627

Figure Lengend Snippet: Increased vascular endothelial cell adhesion by NPR1 deficiency. ( A ) Immunofluorescent staining for NPR1 (red), endothelium (green), and nuclei (blue) in aorta frozen sections from Npr1 +/- and WT mice. n = 4. Scale bar: 20 µm. L: Lumen. Arrows indicate the endothelium. Relative expression levels were calculated according to mean fluorescent intensity. ( B ) Volcano plots of differentially expressed genes in aorta of Npr1 +/- and WT mice. n = 3. |log2 fold change| ≥ 0.5, q value < 0.05. Col6a6 : collagen type VI alpha 6 chain; Thbs4 : thrombospondin 4; Itgb4 : integrin beta 4; Col6a5 : collagen type VI alpha 5 chain; Tnr : tenascin R; Vtn : vitronectin; Parvg : parvin gamma. ( C ) GO annotation of differentially expressed genes in the aorta of Npr1 +/- and WT mice. n = 3. PMAM: plasma membrane adhesion molecules. Y-axis indicates biological processes and X-axis indicates the rich ratio. The intensity of color and bubble size denote the q value and gene number, respectively. ( D ) Immunofluorescent staining for ICAM-1 (red), endothelium (green), and nuclei (blue) in the aorta of Npr1 +/- and WT mice. n = 4. Scale bar: 20 µm. L: Lumen. Arrows indicate the endothelium. Relative expression levels were calculated according to the mean fluorescent intensity. Data are mean ± S.D. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight.

Techniques: Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

doi: 10.3390/ijms232012627

Figure Lengend Snippet: Altered adhesion of monocytes to HUVECs by knockdown or overexpression of NPR1 . ( A ) Western blotting for NPR1 expression in HUVECs transfected with siRNAs (si NPR1 -1 and si NPR1 -2). Quantitative data were analyzed by densitometry. ( B ) Levels of cGMP in HUVECs transfected with siRNAs (si NPR1 -1 and si NPR1 -2). ( C ) Monocyte (green)–endothelial cell adhesion assay for HUVECs transfected with si NPR1 -1 and si NPR1 -2. Scale bar: 200 µm. Relative adhesion was evaluated by monocyte counts. ( D ) Western blotting for ICAM-1 expression in HUVECs transfected with siRNAs (si NPR1 -1 and si NPR1 -2). Quantitative data were analyzed by densitometry. ( E ) Overexpression of NPR1 in HUVECs treated with TNF-α or GFP as a control. ( F ) Levels of cGMP in HUVECs transfected with overexpression NPR1 . ( G ) Monocyte (green)–endothelial cell adhesion assay for TNF-α-treated HUVECs with overexpressed NPR1 . Relative adhesion was evaluated by monocyte counts. Scale bar: 200 µm. ( H ) Western blotting for ICAM-1 expression in HUVECs transfected with overexpression of NPR1 . Quantitative data were analyzed by densitometry. Data are mean ± S.D. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Endothelial Cell Adhesion Assay

Journal: International Journal of Molecular Sciences

Article Title: Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

doi: 10.3390/ijms232012627

Figure Lengend Snippet: Decreased cell adhesion induced by NPR1 inhibition in ITGB4 -knockdown HUVECs. ( A ) Western blot analysis for NPR1, ITGB4, and ICAM-1 expression in HUVECs treated with si NPR1 and followed by transfection with si ITGB4 -1 and si ITGB4 -2. Analysis of endogenous NPR1 and ITGB4 levels, normalized to GAPDH, by densitometry, respectively. ( B ) Monocyte (green)–endothelial adhesion assay of HUVEC treated with si NPR1 and followed by transfection with si ITGB4 -1 and si ITGB4 -2. Scale bar: 200 µm. Data are mean ± S.D. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Cell Adhesion Assay

Journal: International Journal of Molecular Sciences

Article Title: Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

doi: 10.3390/ijms232012627

Figure Lengend Snippet: NPR1 and ITGB4 expression in the aorta from a mouse model of atherosclerosis (As). ( A ) Oil red O staining of mouse aortic root in the As model. Scale bar: 100 µm. ( B ) Immunofluorescent staining for ICAM-1 (red), endothelium (green), and nuclei (blue) in aorta frozen sections from the mouse model of As. n = 4. Scale bar: 20 µm. L: Lumen. Arrows indicate the endothelium. Relative expression levels were calculated according to the mean fluorescent intensity. ( C ) qPCR for Npr1 and Itgb4 mRNA expression in mouse aorta from the As model. ( D , E ) Immunofluorescent staining for NPR1 (red), ITGB4 (red), endothelium (green), and nuclei (blue) in aorta frozen sections from the mouse model of As. n = 4. Scale bar: 20 µm. L: Lumen. Arrows indicate the endothelium. Relative expression levels were calculated according to the mean fluorescent intensity. Data are mean ± S.D. * p < 0.05, ** p < 0.01 vs. WT.

Article Snippet: After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight.

Techniques: Expressing, Staining