Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis

doi: 10.3389/fcvm.2022.941946

Figure Lengend Snippet: Generation of endothelia specific-Gsα knockout mice. (A) Schematic diagram of generation of endothelial-specific Gsα knockout mice. (B) Representative immunofluorescence staining for Gsα, CD31 for endothelial cells, and DAPI for nuclei in aortas from control (CTR) and Gsα ECKO mice. Scale bar, 100 μm. (C,D) Western blot analysis and quantification of Gsα protein in lung tissues of CTR and Gsα ECKO mice. n = 8/group. (E) The cAMP level was measured in lung tissue of CTR and Gsα ECKO mice. n = 5/group. ** P < 0.01, *** P < 0.001 vs. CTR (unpaired Student’s t -test). Data are mean ± SEM.

Article Snippet: Eight-week-old male CTR and Gsα ECKO mice were anesthetized as described above and injected subcutaneously into the flank with 300 μL growth factor-reduced Matrigel (Corning, NY, United States), mixed with vascular endothelial growth factor (VEGF-100 ng/mL; PeproTech, Cranbury, NJ, United States) and heparin sodium (40 U/mL; Bio Vision, Milpitas, CA, United States).

Techniques: Knock-Out, Immunofluorescence, Staining, Control, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis

doi: 10.3389/fcvm.2022.941946

Figure Lengend Snippet: Endothelial Gsα deficiency impairs blood flow recovery after ischemic injury. (A) Representative images of laser doppler blood flow before or on days 1, 3, 7, and 14 after femoral artery resection. (B) Blood flow in ischemic hind limbs. Data are expressed as a ratio of ischemic to non-ischemic limb perfusion. n = 6/group. (C) Representative immunofluorescence staining at day 14 of CD31 for endothelial cells and DAPI for nuclei in gastrocnemius muscle from ischemic hind limbs of CTR and Gsα ECKO mice. Scale bar, 100 μm. (D) Quantification of CD31-positive area in (C) , n = 5/group, * P < 0.05, ** P < 0.01 vs. CTR (unpaired Student’s t -test). Data are mean ± SEM.

Article Snippet: Eight-week-old male CTR and Gsα ECKO mice were anesthetized as described above and injected subcutaneously into the flank with 300 μL growth factor-reduced Matrigel (Corning, NY, United States), mixed with vascular endothelial growth factor (VEGF-100 ng/mL; PeproTech, Cranbury, NJ, United States) and heparin sodium (40 U/mL; Bio Vision, Milpitas, CA, United States).

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis

doi: 10.3389/fcvm.2022.941946

Figure Lengend Snippet: Loss of endothelial Gsα inhibits neovascularization and tumor growth in vivo . (A) Representative matrigel plugs retrieved from CTR and Gsα ECKO mice. (B) Representative immunofluorescence staining of CD31 and DAPI in matrigel plugs. Scale bar, 50 μm. (C) Quantification of the ratio of CD31-positive to DAPI staining. n = 5 for CTR; n = 6 for Gsα ECKO . (D) Images of LLCs harvested from CTR and Gsα ECKO mice. (E) Quantification of tumor volume. n = 5/group. (F) Representative immunofluorescence staining of CD31 and DAPI from LLC tumors. Scale bar, 100 μm. (G) Quantification of the ratio of CD31-positive area. n = 4 for CTR, n = 5 for Gsα ECKO , * P < 0.05, *** P < 0.001 vs. CTR (unpaired Student’s t -test). Data are mean ± SEM. (H) Transcriptome sequencing of genes with > 1.5- fold upregulation or > 1.5-fold down-regulation in Gsα virus transfected HUVEC compared with GFP.

Article Snippet: Eight-week-old male CTR and Gsα ECKO mice were anesthetized as described above and injected subcutaneously into the flank with 300 μL growth factor-reduced Matrigel (Corning, NY, United States), mixed with vascular endothelial growth factor (VEGF-100 ng/mL; PeproTech, Cranbury, NJ, United States) and heparin sodium (40 U/mL; Bio Vision, Milpitas, CA, United States).

Techniques: In Vivo, Immunofluorescence, Staining, Sequencing, Virus, Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis

doi: 10.3389/fcvm.2022.941946

Figure Lengend Snippet: Gsα regulates AGGF1 expression via CREB in endothelial cells. (A,B) Western blot analysis and quantification of AGGF1 in HUVECs infected with adenovirus-expressing GFP or Gsα. n = 5 for GFP; n = 4 for Gsα, * P < 0.05 vs. GFP (unpaired Student’s t -test). (C) RT-qPCR analysis of AGGF1 mRNA in HUVECs infected with adenovirus-expressing GFP or Gsα. n = 5/group, ** P < 0.01 vs. GFP (unpaired Student’s t -test). (D,E) Western blot analysis and quantification of AGGF1 in HUVECs treated with forskolin (10 μM) for 24 h. n = 4/group, * P < 0.05 vs. DMSO (unpaired Student’s t -test). (F) Predicted CRE site in the human AGGF1 promoter. (G) Binding of CREB to the CRE of the AGGF1 promoter was shown by Chromosome immunoprecipitation assay. (H) Luciferase activity in HEK-293T cells transfected with wild-type or a mutant AGGF1 promoter-luciferase construct and infected with adenovirus-expressing GFP or Gsα for 24 h. Results of luciferase promoter assay were firefly/Renilla luciferase activity, n = 6/group, ** P < 0.01 vs. GFP (One-Way ANOVA and Bonferroni post-tests). Data are mean ± SEM.

Article Snippet: Eight-week-old male CTR and Gsα ECKO mice were anesthetized as described above and injected subcutaneously into the flank with 300 μL growth factor-reduced Matrigel (Corning, NY, United States), mixed with vascular endothelial growth factor (VEGF-100 ng/mL; PeproTech, Cranbury, NJ, United States) and heparin sodium (40 U/mL; Bio Vision, Milpitas, CA, United States).

Techniques: Expressing, Western Blot, Infection, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Luciferase, Activity Assay, Transfection, Mutagenesis, Construct, Promoter Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis

doi: 10.3389/fcvm.2022.941946

Figure Lengend Snippet: Knockdown of AGGF1 attenuates the proangiogenic effect of Gsα in endothelial cells. (A) Representative scratch wound assays of HUVECs transfected with CTR or AGGF1 siRNA and infected with GFP or Gsα virus. Scale bar, 100 μm. (B) Quantification of scratch wound closure of HUVECs shown in (A) . n = 3/group. (C,D) Western blot analysis and quantification of CyclinD1 protein level in HUVECs. n = 5/group. (E) Cell proliferation was measured in HUVECs by using a CCK8 ELISA kit. n = 5/group. (F) The spheroid assay of HUVECs transfected with CTR or AGGF1 siRNA and infected with GFP or Gsα virus. Scale bar, 100 μm. (G,H) Quantification of the average sprout number and tube length of HUVECs transfected with CTR or AGGF1 siRNA and infected with GFP or Gsα virus. n = 5/group. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. GFP + CTR siRNA, ## P < 0.01, ### P < 0.001 vs. Gsα + CTR siRNA (One-Way ANOVA and Bonferroni post-tests). Data are mean ± SEM.

Article Snippet: Eight-week-old male CTR and Gsα ECKO mice were anesthetized as described above and injected subcutaneously into the flank with 300 μL growth factor-reduced Matrigel (Corning, NY, United States), mixed with vascular endothelial growth factor (VEGF-100 ng/mL; PeproTech, Cranbury, NJ, United States) and heparin sodium (40 U/mL; Bio Vision, Milpitas, CA, United States).

Techniques: Knockdown, Transfection, Infection, Virus, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Reproduction and Development

Article Title: Increased supply from blood vessels promotes the activation of dormant primordial follicles in mouse ovaries

doi: 10.1262/jrd.2019-091

Figure Lengend Snippet: Effect of transplantation of biodegradable gels containing recombinant vascular endothelial growth factor (VEGF) in 10-week-old ICR mouse ovaries. (A) Number of blood vessels in the ovaries transplanted with a biodegradable gel containing recombinant VEGF. (B) Ratio of activated primordial follicles in the ovaries in which a biodegradable gel containing recombinant VEGF was transplanted. Mean ± SD (n = 3 for 10 μg/ml VEGF, n = 4 for 100 ng/ml and 1 μg/ml VEGF, and n = 5 for control). * P < 0.05.

Article Snippet: To evaluate the effect of recombinant VEGF (R&D System) on the activation of dormant primordial follicles, the left ovaries were cultured as controls and the right ovaries were cultured in medium containing recombinant VEGF (50, 100, and 200 ng/ml).

Techniques: Transplantation Assay, Recombinant, Control

Journal: The Journal of Reproduction and Development

Article Title: Increased supply from blood vessels promotes the activation of dormant primordial follicles in mouse ovaries

doi: 10.1262/jrd.2019-091

Figure Lengend Snippet: Effect of recombinant vascular endothelial growth factor (VEGF) on the activation of primordial follicles in cultured P0 Oog1pro3.9/R26-H2B-mCherry mouse ovaries. Mean ± SD (n = 3 for 200 ng/ml, n = 4 for 50 ng /ml, n = 5 for 100 ng /ml, and n = 12 for control.

Article Snippet: To evaluate the effect of recombinant VEGF (R&D System) on the activation of dormant primordial follicles, the left ovaries were cultured as controls and the right ovaries were cultured in medium containing recombinant VEGF (50, 100, and 200 ng/ml).

Techniques: Recombinant, Activation Assay, Cell Culture, Control

Journal: The Journal of Reproduction and Development

Article Title: Increased supply from blood vessels promotes the activation of dormant primordial follicles in mouse ovaries

doi: 10.1262/jrd.2019-091

Figure Lengend Snippet: Expression of KIT ligand (KITL) in 10-week-old ICR mouse ovaries in which a biodegradable gel containing vascular endothelial growth factor (VEGF) was transplanted and in cultured P0 mouse ovaries. (A) A primordial follicle that expressed KITL (KITL-positive primordial follicles) in a 10-week-old ICR mouse ovary. (B) Primordial follicles that did not express KITL in a 10-week-old ICR mouse ovary. (C) Ratio of KITL-positive primordial follicles in the ovaries in which a biodegradable gel containing 10 μg/ml recombinant VEGF was transplanted. (D) Ratio of KITL-positive primordial follicles in the cultured P0 mouse ovaries. Arrows indicate primordial follicles. Mean ± SD (n = 3 for 10 μg/ml recombinant VEGF, n = 5 for control; n = 3 for 10% fetal bovine serum (FBS), n = 4 for 5% FBS and n = 7 for 0% FBS). Scale bar: 50 μm. * P < 0.05, *** P < 0.005.

Article Snippet: To evaluate the effect of recombinant VEGF (R&D System) on the activation of dormant primordial follicles, the left ovaries were cultured as controls and the right ovaries were cultured in medium containing recombinant VEGF (50, 100, and 200 ng/ml).

Techniques: Expressing, Cell Culture, Recombinant, Control

Journal: Communications Biology

Article Title: Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

doi: 10.1038/s42003-019-0609-x

Figure Lengend Snippet: sEV-VEGF is signaling competent. a Immunoblot of phosphorylated VEGFR2 (p-VEGFR2) and total VEGFR2 in HUVEC at 5 min following stimulation with sEVs of ES2, HCT116, and 786-0 cells or with recombinant VEGF 165 (rVEGF 165 ). b – e HUVEC were pretreated with inhibitors of VEGFR tyrosine kinase activity ( b , c ) and with neutralizing Ab to VEGFR2 ( d , e ), stimulated with sEVs or rVEGF 165 , and then assayed for tube formation at 4 h thereafter. In b and d , mean ± SD of n = 4 independent experiments. In c and e , representative images of tube formation. Scale bar = 100 μm. f VEGF levels in conditioned media (CM) of ES2 and HCT116 cells that were depleted of sEVs (sEV-dep) or left non-depleted (whole). Shown are mean ± SD of n = 3 independent experiments. g – i HUVEC were stimulated with whole and sEV-depleted conditioned media, and then assayed for VEGFR2 phosphorylation ( g ) and tube formation ( h , i ). In h , mean ± SD of n = 4 independent experiments. In i , representative images of tube formation. Scale bar = 100 μm. ** P < 0.01, *** P < 0.001, **** P < 0.0001, by two-way ANOVA with Bonferroni’s corrections in b , d , and h , and by a two-sided unpaired t -test in f . Source data used for graphs in b , d , f , and h can be found in Supplementary Data

Article Snippet: Wells of 96-well High Bind microplates were coated with recombinant VEGF (100 ng) at RT for 16 h. After washing three times with wash buffer (R&D Systems), wells were blocked with 10% goat serum for 1 h and washed again.

Techniques: Western Blot, Recombinant, Activity Assay

Journal: Communications Biology

Article Title: Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

doi: 10.1038/s42003-019-0609-x

Figure Lengend Snippet: sEV-VEGF predominantly comprises dimeric VEGF 189 . a Immunoblot of cellular VEGF in lysates of cells of parental cancer cell lines that were treated with brefeldin A to block protein secretion. Recombinant VEGF proteins were included as controls. Overexposure shows VEGF 121 and VEGF 165 dimers. b Immunoblot of VEGF in sEVs isolated from the same cell lines as in a but without brefeldin A treatment. c Immunoblot of VEGF in tumor tissues (T) of three patients with ovarian cancer and in sEVs isolated from ascites of the same patients. d Immunoblot of VEGF in sEVs isolated from serum or plasma of five patients with colorectal carcinoma (CRC) and two patients with renal cell carcinoma (RCC). TSG101 was assayed as a control in c , d

Article Snippet: Wells of 96-well High Bind microplates were coated with recombinant VEGF (100 ng) at RT for 16 h. After washing three times with wash buffer (R&D Systems), wells were blocked with 10% goat serum for 1 h and washed again.

Techniques: Western Blot, Blocking Assay, Recombinant, Isolation, Control

Journal: Communications Biology

Article Title: Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

doi: 10.1038/s42003-019-0609-x

Figure Lengend Snippet: Selective localization of VEGF 189 in sEVs is mediated by heparin-binding and increases ligand stability. a VEGF levels in conditioned media and sEVs of ES2 VEGF −/− and HCT116 VEGF −/− cells transfected with VEGF 121 , VEGF 165 , or VEGF 189 . TSG101 was assayed in sEVs as a control. Mean ± SD of n = 3 independent experiments are shown. b , c VEGF levels in whole and sEV-depleted conditioned media of VEGF −/− cells transfected with VEGF 189 ( b ) and VEGF 121 ( c ). Mean ± SD of n = 3 independent experiments are shown. d Conditioned media of non-transfected VEGF −/− cells was incubated with addition of recombinant VEGF 189 (rVEGF 189 ) at a concentration equivalent to the VEGF concentration in conditioned media of VEGF 189 -transfected VEGF −/− cells (2500 pg/mL for ES2; 1500 pg/mL for HCT116; see data in a ). Thereafter, sEVs were isolated. Amounts of VEGF 189 detected in these sEVs were compared with VEGF content in sEVs secreted by VEGF 189 -transfected VEGF −/− cells. Mean ± SD of n = 3 independent experiments are shown. e Levels of human VEGF 189 in conditioned media and sEVs of CHO-K1 and pgsD-677 cells transfected with human VEGF 189 . Mean ± SD of n = 3 independent experiments are shown. f , g PKH67-labeled sEVs of parental ES2 cells were pretreated with heparinase, chondroitinase, or no enzyme, and then incubated with VEGF Ab coupled to microbeads. VEGF on sEVs was detected by flow cytometric analysis of PKH67 fluorescence in Ab-coupled microbeads (solid histograms). Dotted histograms show background fluorescence when sEVs were incubated with control Ig-coupled beads. As a negative control for enzymatic digestion, the same approach was used to detect the transmembrane protein CD63. In f , representative histogram plots. In g , MFI values of n = 3 independent experiments (mean ± SD). Contour plots are shown in Supplementary Fig. . h rVEGF 189 and sEVs with an equivalent content of VEGF were added to healthy donor plasma. Following incubation at 37 °C for the indicated times, VEGF levels in plasma were assayed. Shown are mean of n = 2 independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001, by one-way ANOVA with Bonferroni’s corrections in a and g , by two-sided unpaired t -test in b – e . Source data used for graphs in a – e , g , and h can be found in Supplementary Data

Article Snippet: Wells of 96-well High Bind microplates were coated with recombinant VEGF (100 ng) at RT for 16 h. After washing three times with wash buffer (R&D Systems), wells were blocked with 10% goat serum for 1 h and washed again.

Techniques: Binding Assay, Transfection, Control, Incubation, Recombinant, Concentration Assay, Isolation, Labeling, Fluorescence, Negative Control

Journal: Communications Biology

Article Title: Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

doi: 10.1038/s42003-019-0609-x

Figure Lengend Snippet: Heparin-bound sEV-VEGF is not neutralized by bevacizumab in vitro. a , b rVEGF 189 was captured by HMW heparin or by VEGF capture Ab (positive control), and then incubated with bevacizumab or VEGFR1/R2-Fc. Bevacizumab bound to VEGF 189 and VEGFR1/R2-Fc bound to VEGF 189 were detected by anti-human IgG. In a , experimental scheme. In b , relative levels of bevacizumab bound to VEGF 189 and VEGFR1/R2 bound to VEGF 189 . Shown are mean ± SD of n = 6 independent experiments. c , d Microbeads were coupled to bevacizumab, incubated with sEVs of VEGF +/+ cells or sEVs of VEGF −/− cells, and then stained with exo-FITC dye to label sEV membrane. The same procedure was performed using microbeads coupled to VEGFR1/R2-Fc (positive control). Binding of bevacizumab and VEGFR1/R2-Fc to VEGF on the surface of sEVs was evaluated by flow cytometric analysis of exo-FITC fluorescence in the gated population of microbeads. In c , representative histogram plots. In d , MFI values of n = 3 independent experiments (mean ± SD). Gating strategy is shown in Supplementary Fig. . Contour plots are shown in Supplementary Fig. . e Bevacizumab and VEGFR1/R2-Fc were incubated with recombinant VEGF and with sEVs that have a VEGF content equivalent to the range of amounts of recombinant VEGF. Following incubation, levels of unbound bevacizumab and unbound VEGFR1/R2-Fc were assayed. Shown are mean ± SD of n = 3 independent experiments. f – h HUVEC were stimulated with sEVs or rVEGF 189 that were pre-incubated with control Ig, bevacizumab, or VEGFR1/R2-Fc, and then assayed for phosphorylated and total VEGFR2 ( f ) and tube formation ( g , h ). In g , mean ± SD of n = 3 independent experiments. In h , representative images of tube formation. Scale bar = 100 μm. * P < 0.05, *** P < 0.001, **** P < 0.0001 by ANOVA with Bonferroni’s corrections; one-way in d ; two-way in b and g . Source data used for graphs in b , d , e , and g can be found in Supplementary Data

Article Snippet: Wells of 96-well High Bind microplates were coated with recombinant VEGF (100 ng) at RT for 16 h. After washing three times with wash buffer (R&D Systems), wells were blocked with 10% goat serum for 1 h and washed again.

Techniques: In Vitro, Positive Control, Incubation, Staining, Membrane, Binding Assay, Fluorescence, Recombinant, Control