Journal: International Journal of Molecular Medicine

Article Title: Lack of endogenous parathyroid hormone delays fracture healing by inhibiting vascular endothelial growth factor-mediated angiogenesis

doi: 10.3892/ijmm.2018.3614

Figure Lengend Snippet: (A) Immunohistochemical staining of VEGF-A (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, VEGF-A expression was significantly reduced in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, VEGF-A expression was increased in PTHKO mice, but remained significantly lower than that in WT mice. (B) Immunohistochemical staining of pVEGFR2 (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, a significantly smaller number of pVEGFR2-positive cells was detected in the cartilaginous callus in PTHKO mice compared with in WT mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, a large number of pVEGFR2-positive cells was observed in the cartilaginous callus in WT mice, whereas a much lower level of angiogenesis was detected in PTHKO mice. (C) Immunohistochemical staining for HIF1α (magnification, ×400) in fractures. (a) In PTHKO and (b) WT mice 1 week after fracture, the expression levels of cytoplasmic HIF1α were significantly lower in PTHKO mice. (c) In PTHKO and (d) WT mice 2 weeks after fracture, HIF1α expression was increased in both groups; however, the expression remained lower in PTHKO mice compared with in WT mice. Black arrows indicate positive areas. (D) Protein expression levels of VEGF, pVEGFR2 and HIF1α were detected by western blot analysis. HIF1α, hypoxia inducible factor-1α; PTH, parathroid hormone; PTHKO, PTH knockout; pVEGFR, phosphorylated-VEGF receptor 2; VEGF, vascular endothelial growth factor; WT, wild-type.

Article Snippet: RUNX2 (ab76956; 1:600), HIF1α (ab8366; 1:600), pVEGF receptor 2 (pVEGFR2; ab131241; 1:200), proliferating cell nuclear antigen (PCNA; ab92552; 1:400), VEGF (ab46154; 1:200), PKA (ab75991) and pAKT monoclonal antibodies (ab81283) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Immunohistochemistry, Staining, Mouse Assay, Expressing, Western Blot, Knock-Out

Journal: eLife

Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

doi: 10.7554/eLife.48095

Figure Lengend Snippet: Assessment of VEGF-A-induced cell proliferation and migration upon modulation of miR-424–5p or miR-29a-3p. ( A ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA in control spheroids (SPHC) or VEGF-A-stimulated spheroids (SPHV) generated with ECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors or their respective controls. Representative plots for n = 3 experiments. ( B ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication in 2D cultured HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls. The assay was performed after 18 hr of VEGF stimulation. Data are represented as mean ± SEM from n = 3 experiments. ( C ) VEGF-induced cell migration of HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls, assessed by xCELLigence and represented as the slope of the migration curve over a 24 hr experiment. Data are represented as mean ± SEM from n = 3 experiments. ***, p

Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

Techniques: Migration, Generated, Transfection, Cell Culture

Journal: eLife

Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

doi: 10.7554/eLife.48095

Figure Lengend Snippet: DICER knock-down. ( A ) Sprouting assay performed with spheroids generated with cells that had been transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4), and the corresponding quantification of the sprouted area. Data are represented as mean ± SEM from n = 6 experiments. Scale bars, 200 µm. ( B ) DICER expression measured by real-time PCR assay in cells transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4). Data are represented as mean ± SEM from n = 3 experiments. ( C ) GSEA study performed against the canonical pathways gene sets collection, comparing control spheroids (SPHC) generated with DICER KD or DICER WT cells. FDR q value > 0.05, not significant. ( D ) VEGF-A-induced cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication, in 2D-cultured HUVECs. Cells were transduced either with a scrambled shRNA ( DICER WT ) or with a shRNA targeting DICER ( DICER KD ). Plots are representative of n = 3 experiments. Data represent mean percentage of proliferating cells ± SEM from n = 3 experiments. p

Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

Techniques: Generated, Transduction, shRNA, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

Journal: eLife

Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

doi: 10.7554/eLife.48095

Figure Lengend Snippet: Assessment of the effective concentrations of ERK and p38 inhibitors in ECs. ( A ) Western blot showing ERK phosphorylation, detected with an anti-phospho-ERK antibody (P-ERK), upon VEGF-A stimulus in the presence of an increasing concentration (10, 100, 300 nM) of the ERK inhibitor SHC 772984, or vehicle (DMSO). Total ERK was used as protein loading control. ( B ) Western blot showing p38 phosphorylation, detected with an anti-phospho-p38 antibody (P–p38), upon VEGF-A stimulus in the presence of increasing concentration (10, 100, 300 μM) of the p38 inhibitor SB 202190, or vehicle (DMSO). Total p38 was used as protein loading control. Data are representative of three independent experiments.

Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

Techniques: Western Blot, Concentration Assay

Journal: PeerJ

Article Title: Cellular prion protein and γ-synuclein overexpression in LS 174T colorectal cancer cell drives endothelial proliferation-to-differentiation switch

doi: 10.7717/peerj.4506

Figure Lengend Snippet: Overexpression of γ-Syn and PrPC inhibits LS 174T cells from adhering onto EA. (A) Cell attachment analysis of the adhesiveness of unstimulated and stimulated (VEGF, 20 ng/mL) LS 174T cell lines on EA. Images were taken at 100× magnification using the Eclipse TS100 inverted microscope (Nikon, New York, NY, USA). (B) Fluorescence intensity was quantified. Data were expressed as fluorescence intensity and represent the mean ± SEM (error bars) of three independent experiments. Mean values were compared using one-way ANOVA followed by LSD’s post hoc test. Asterisk indicates p

Article Snippet: Briefly, LS 174T cell lines (1 × 106 cells/T25 flask) unstimulated or stimulated with vascular endothelial growth factor (VEGF) (Sigma-Aldrich, St. Louis, MO, USA) at 20 μg/mL for 4 h were trypsinized, centrifuged, and cell pellets were mixed with Calcein AM (Trevigen, Gaithersburg, MD, USA).

Techniques: Over Expression, Cell Attachment Assay, Inverted Microscopy, Fluorescence

Journal: PeerJ

Article Title: Cellular prion protein and γ-synuclein overexpression in LS 174T colorectal cancer cell drives endothelial proliferation-to-differentiation switch

doi: 10.7717/peerj.4506

Figure Lengend Snippet: Partial secretome analysis of conditioned media from LS 174T cell lines. (A) Angiogenesis antibody array proteome profiling of CM of LS 174T cell lines. Red boxes indicate reference spots whereas green boxes indicate negative control. Numbered yellow boxes indicate the ten angiogenic factors in the CM that yielded distinct signals, which are CXCL16 (1), DPPIV (2), TIMP-1 (3), TIMP-4 (4), angiogenin (5), IGFBP-2 (6), uPA (7), IL-8 (8), amphiregulin (9) and VEGF (10). (B) Secretion levels of different angiogenesis-related proteins in LS 174T CM, LS 174T-γ-Syn CM and LS 174T-PrP CM were analyzed with ImageJ software and presented as densitometry values. (C) The ten angiogenic factors that yielded distinct signals were relatively quantified. Data of densitometry value were expressed as relative intensity compared to LS 174T CM (set as 1) and represent the mean ± SEM (error bars) of two independent experiments. Mean values were compared using one-way ANOVA followed by LSD’s post hoc test. Asterisk indicates p

Article Snippet: Briefly, LS 174T cell lines (1 × 106 cells/T25 flask) unstimulated or stimulated with vascular endothelial growth factor (VEGF) (Sigma-Aldrich, St. Louis, MO, USA) at 20 μg/mL for 4 h were trypsinized, centrifuged, and cell pellets were mixed with Calcein AM (Trevigen, Gaithersburg, MD, USA).

Techniques: Ab Array, Negative Control, Software

Journal: Cell Death & Disease

Article Title: FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells

doi: 10.1038/s41419-017-0162-9

Figure Lengend Snippet: FLI1 and PKC co-activation mediated hESCs differentiation into iECs a Schematic illustration of the EC differentiation strategy from hESCs. b Typical morphological images of hESC-EC differentiation on days of 0, 1, 2, and 3. Scale bar, 100 μm. c The ratio of CD31+/CD144+ cells gradually increased during induction. Columns represent the mean ± SD; n = 5 independent differentiation experiments. d Representative results of the percentage of CD31+/CD144+ cells during the induction process detected by FCM. e Overexpression of FLI1 and activation of PKC in different hESC lines (hESC-254 or hESC-137) induced iECs. f Overexpressing FLI1 with different PKC activators (PMA or prostratin) yielded iECs. g Expression levels of VEGF , GATA2 , CD31 and CD144 genes in hESCs, human fibroblasts (HFs), iECs and EPCs. Columns represent the mean ± SD; n = 5 independent differentiation experiments

Article Snippet: After 3 days, supplements were changed with 50 ng ml−1 VEGF-A (96-100-20-10, PEPROTECH).

Techniques: Activation Assay, Over Expression, Expressing

Journal: PLoS ONE

Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

doi: 10.1371/journal.pone.0134308

Figure Lengend Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Quantitative analyses of the number of large lipid droplets ( > 50 μm 2 ) per field at x400 magnification ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (B) Quantitative analyses of vascularity of interscapular BAT demonstrated by isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantitatively analyzed by comparison to the group treated with intravitreal PBS injection as 100%. (C) The changes in body weight from P14 to P56. Anti-VEGF, anti-VEGF antibody. NS , not significant (two-tailed, unpaired T-test).

Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

Techniques: Injection, Staining, Two Tailed Test

Journal: PLoS ONE

Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

doi: 10.1371/journal.pone.0134308

Figure Lengend Snippet: Ocular and systemic consequences of intravitreally injected anti-VEGF antibody. (A) Effects of intravitreally injected anti-VEGF antibody (1 μg/eye) on retinal neovascularization in OIR mice ( n = 6). Neovascular tufts were highlighted with yellow pseudocolor on representative images of isolectin B4-stained retina. The area of neovascular tufts was normalized to total retinal area; then, the effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). Scale bar, 200 μm. (B) Retinal VEGF concentrations at P17 with intravitreal injection of PBS or anti-VEGF antibody ( n = 3). The level of VEGF was normalized to total amounts of proteins in the retina. (C) Serum concentrations of anti-VEGF antibody after intravitreal injection at P14, P17, and P21 ( n = 3–6). (D) Serum VEGF concentrations after intravitreal injection of anti-VEGF antibody at P17, P21, and P28 ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. NS , not significant; **, P

Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

Techniques: Injection, Mouse Assay, Staining

Journal: PLoS ONE

Article Title: Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

doi: 10.1371/journal.pone.0134308

Figure Lengend Snippet: Effects of intravitreally injected anti-VEGF antibody on BAT of neonatal mice. (A) Concentrations of VEGF in interscapular BAT at P21 and P28. The level of VEGF was normalized to total amounts of proteins in BAT ( n = 3–6). (B) Representative images of H E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody show enlarged lipid droplets. Scale bar, 20 μm. (C) Quantitative analyses of vascularity of interscapular BAT based on isolectin B4 staining ( n = 3–6). The effects of anti-VEGF antibody were quantified and normalized to the control (intravitreal PBS injection). (D) Relative expression of Ucp1 and Ppargc1a in interscapular BAT ( n = 3–6). Data are presented as mean ± SEM in graphs. Anti-VEGF, anti-VEGF antibody. *, P

Article Snippet: Long-term effects of intravitreally injected anti-VEGF antibody on BAT. (A) Representative images of H & E staining of interscapular BAT after intravitreal injection of PBS or anti-VEGF antibody.

Techniques: Injection, Mouse Assay, Staining, Expressing

Journal: EMBO Reports

Article Title: EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

doi: 10.15252/embr.201745054

Figure Lengend Snippet: Expression of endogenous Fgf2 and endogenous and total Vegfa A–D Gene expression of Fgf2 (A) and total (B) or endogenous Vegfa (C) was quantified in skeletal muscles 3 days after myoblast implantation and expressed as fold‐change vs. control muscles. The relative magnitude of gene expression changes can be better appreciated by plotting the data on the same scale (D). Mean ± SEM; n = 4–6 independent samples/group; ** P

Article Snippet: Expression of genes of interest was determined using the following TaqMan Gene Expression assays (Applied Biosystems) according to manufacturer's instructions: mouse Tnfa (Mm00443258_m1); mouse Gapdh (Mm03302249_g1); mouse Pdgfb (Mm00440678_m1); mouse Pdgfrb (Mm00435545_m1); mouse Fgf2 (Mm01285715_m1); mouse Vegfa (Mm00437306_m1); human Igfbp3 (Hs00365742_g1); human Esm1 (Hs00199831_m1); and human Gapdh (Hs02758991_g1).

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Oxidative stress, autophagy and pyroptosis in the neovascularization of oxygen-induced retinopathy in mice

doi: 10.3892/mmr.2018.9759

Figure Lengend Snippet: VEGF-A expression is activated in the retinas of OIR mice. (A) Immunoblot analysis of the protein expression levels of VEGF-A and HIF-1α in the retinas. (B) Quantification revealed an increase in the expression levels of VEGF-A and HIF-1α in the retinas of the OIR mice compared with WT mice. The relative protein expression level was normalized to GAPDH (n=3 mice per group). Data are presented as the mean ± standard deviation of the mean. *P

Article Snippet: Following electrophoresis and wet transfer to a polyvinylidene difluoride (PVDF) membrane, the membrane was blocked in 5% skim milk in TBS with Tween-20 (TBST) at room temperature for 1 h. Immunostaining was conducted using antibodies against VEGF-A (cat. no. ab52917; 1:500), caspase-1 (cat. no. ab1872; 1:1,000), pro-caspase-1 (cat. no. ab179515; 1:1,000), interleukin (IL)-1β (cat. no. ab2105; 1:500), pro-IL-1β (cat. no. ab2105; 1:500), microtubule associated protein 1 light chain 3α (LC3; cat. no. ab48394; 1:500) (Abcam, Cambridge, MA, USA), HIF-1α (cat. no. 36169; 1:1,000), autophagy protein (Atg)5 (cat. no. 12994; 1:1,000), Atg7 (cat. no. 8558; 1:1,000), Atg12 (cat. no. 4180; 1:1,000), Beclin1 (cat. no. 3495; 1:1,000), p62 (cat. no. 23214; 1:1,000), NOD-like receptor family pyrin domain-containing 3 (NLRP3; cat. no. 15101; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc, Danvers, MA, USA) at 4°C overnight.

Techniques: Expressing, Mouse Assay, Standard Deviation