Journal:

Article Title: Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis

doi: 10.1172/JCI13055

Figure Lengend Snippet: Impacts of the implantation of WT mouse– or AT1a–/– mouse–derived MNCs into the ischemic hindlimbs of AT1a–/– mice on angiogenesis. The impaired LDBF ratio in AT1a–/– mice was partially but significantly restored after implantation of WT mouse–derived MNCs into the ischemic hindlimb of AT1a–/– mice (P < 0.01 vs. AT1a–/–). In contrast, implantation of AT1a–/– mouse–derived MNCs slightly improved the LDBF ratio in AT1a–/– mice, but the difference was not significant (vs. AT1a–/–). Neutralizing anti-VEGF mAb treatment abolished the WT-MNC–mediated rescue of impaired angiogenesis in AT1a–/– mice (P = NS vs. AT1a–/–).

Article Snippet: In five additional mice, neutralizing anti-mouse VEGF mAb (R&D Systems Inc., Minneapolis, Minnesota, USA) was continuously administered by a subcutaneously implanted osmotic pump (ALZA Corp.), and ischemia-induced angiogenesis was examined in AT1a –/– mice that had been subjected to WT-derived MNC transplantation.

Techniques: Derivative Assay

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 7. Maslinic acid reduces the activation of Akt and extracellular signal-related kinase (ERK) in DU145 cells. (A) Serum-deprived cells were incubated with 0– 25 mM-maslinic acid for 6 h, and cell lysates were prepared with or without a 15 min epidermal growth factor (EGF) stimulation. Serum-deprived cells were incu- bated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 for 6 h and lysed without stimulation or after 15 min of EGF stimulation to determine phospho (p)-Akt or p-ERK1/2 levels. To identify hypoxia-inducible factor-1a (HIF-1a), serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in the absence or presence of EGF for 6 h. Total cell lysates were subjected to Western blotting. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was estimated by densitometric scanning of the exposed films, and the expression levels were normalised to those of b-actin. To determine vascular endothelial growth factor (VEGF) concentrations, serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in serum-free media with or without 10 mg/l EGF for 18 h. The 18 h conditioned media were concentrated and subjected to Western blotting with VEGF antibody. The volumes of media loaded onto the gel were adjusted for equivalent protein concen- trations. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was quantified by densitometric scanning of the exposed films. The adjusted means (n 3) of each band with their standard errors are shown above each blot. a,b,c,d Mean values with unlike letters were significantly different (P , 0·05). Mr, molecular weight.

Article Snippet: The reagents used were as follows: maslinic acid (Cayman Chemical); antibodies against uPAR, TIMP-1, TIMP-2, intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and VEGF (Santa Cruz Biotechnology); antibodies against Akt, phospho-Akt, ERK1/2 and phosphoERK1/2 (Cell Signaling Technology); anti-uPA antibody (Calbiochem); anti-HIF-1a, anti-E-cadherin and Matrigele Matrix (BD Biosciences); epidermal growth factor (EGF; R&D Systems); an adhesion assay kit (Chemicon International); transwell filters (Costar).

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Molecular Weight

Journal: Journal of molecular histology

Article Title: tsRNA-3043a intensifies apoptosis and senescence of ovarian granulosa cells to drive premature ovarian failure by targeting FLT1.

doi: 10.1007/s10735-024-10256-8

Figure Lengend Snippet: Fig. 10 Diagram of the hypothesis mechanism of tsRNA-3043a facilitates POF by inhibiting FLT1

Article Snippet: The sections were blocked with 5% BSA and incubated with the anti-FLT1 antibody (1:200, 13687-1-AP, Proteintech) overnight at 4°C.

Techniques:

Journal:

Article Title: Confluent Monolayers of Cultured Human Fetal Retinal Pigment Epithelium Exhibit Morphology and Physiology of Native Tissue

doi: 10.1167/iovs.05-1622

Figure Lengend Snippet: Polarized secretion of VEGF and PEDF in cultured human fetal RPE. Media from apical and basal baths were collected separately at 8 weeks after the cells were seeded onto cell culture inserts. ELISA for VEGF and PEDF were performed in triplicate for each of the 12 wells (24 samples). VEGF protein in the basal bath was 1.7-fold higher than in the apical bath (n = 9; P < 0.005), whereas PEDF protein in the apical bath was 2.3-fold higher than in the basal bath (n = 9; P < 0.05).

Article Snippet: A 96-well EIA plate (Corning Costar) was coated with 100 μL/well of 0.4 μg/mL polyclonal goat anti-human VEGF antibody (R&D Systems, Minneapolis, MN) overnight in PBS (pH 7.4).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Viral gene expression in vitro and in vivo. (A) Comparison of Ad-VEGF-C156S and AdVEGF-C recombinant protein production in vitro. Top panels: the infected cells were metabolically labeled and cell culture media were subjected to precipitation with VEGFR-3-Ig or VEGFR-2-Ig fusion proteins. Medium from AdLacZ-infected cells was used as a negative control. Bottom panel: culture media of Ad-VEGF-C, AdVEGF-C156S, and AdLacZ infected cells were separated in 15% PAGE gel followed by Western blotting using antibodies against VEGF-C. (B) Viral transgene expression in vivo. Northern blot analysis of total RNA from mouse ears infected with recombinant adenoviruses or AAVs. The infecting virus and the duration of infection are indicated. (C) β-galactosidase staining of the ear three weeks after infection with AdLacZ. (D and E) EGFP expression in the ear 6 wk and 8 mo after AAV-EGFP infection.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Gene Expression, In Vitro, In Vivo, Comparison, Recombinant, Infection, Metabolic Labelling, Labeling, Cell Culture, Negative Control, Western Blot, Expressing, Northern Blot, Virus, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogenesis in the skin of K14-VEGF-C156S and K14-VEGF-C transgenic embryos. (A–C) Superficial lymphatic vessels of VEGFR-3+/LacZ heterozygous E14.5 embryos visualized by β-galactosidase staining. Note the increase in the number of lymphatic capillaries in B and lymphatic capillary hyperplasia in A (arrows). (D–F) Dorsal views of the same embryos. (G–I) Cross sections of the superficial lymphatic vessels of E14.5 K14-VEGF-C156S and K14-VEGF-C transgenic embryos stained with antibodies against VEGFR-3. (J–L) Cutaneous lymphatic vessels of adult (age 8 wk) VEGFR-3+/LacZ mice. The lymphatic phenotype established during embryonic development is retained. Scale bars: A–C, 650 μm; D–F, 250 μm; G–I, 35 μm; J–L, 150 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Transgenic Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogen-esis in AAV-VEGF-C156S and AAV-VEGF-C infected adult skin. (A–F) Lymphatic vessels visualized with VEGFR-3 whole mount staining 6 wk after infection with the recombinant AAVs. Note the enlargement, sprouting (arrowheads), and splitting (arrows) of the lymphatic vessels in response to VEGF-C156S and VEGF-C. Lymphatic sprouting is more obvious in the AAV-VEGF-C infected ear (B and E) than in the AAV-VEGF-C156S infected ear (A and D). (G–I) Podoplanin staining of histological sections at the same time point. Note the hyperplastic lymphatic vessel network within the subcutis and muscle cell layers of the skin in the AAV-VEGF-C156S (G) and AAV-VEGF-C (H) infected samples. Scale bars: A–C, 150 μm; D–F, 60 μm; G–I, 70 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Adenoviral VEGF-C156S does not affect blood vessel morphology. (A–C) Ears photographed 1 wk after adenoviral infection. Note the dilation and tortuosity of blood vessels in the AdVEGF-C infected skin (B) compared with the AdVEGF-C156S (A) and AdLacZ (C) infected skin. (D–F) Whole mount PECAM-1 staining of the ear skin blood vessels at the same time point. Compared with the AdLacZ control (F), the veins (V) and arteries (A) in the AdVEGF-C156S infected skin (D) appear morphologically normal, whereas in the AdVEGF-C infected ear (E) the veins are large and tortuous. Note weak staining of lymphatic vessels (L) in F. Scale bars: D–F, 150 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Adenoviral VEGF-C156S has a minimal effect on vascular permeability. (A) The difference in permeability between the treated versus the control ear measured by Evans Blue concentration ratio (ng/mg). (B and C) Mouse thoracic cavities photographed after systemic administration of AdVEGF-C156S or AdVEGF-C (1 × 10 9 pfu). Note the accumulation of pleural fluid in the AdVEGF-C infected mouse (C) but not in the AdVEGF-C156S infected mouse (B).

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Permeability, Control, Concentration Assay, Infection

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: VEGF-C156S gene therapy in the Chy lymphedema mice. (A and B) AAV-mediated overexpression of VEGF-C156S (2 mo) and VEGF-C (8 mo) induces the formation and maintenance of a functional lymphatic vessel network, as analyzed by fluorescent microlymphangiography. C and D show comparison with noninfected Chy mouse ear and wild-type control ear, respectively. (E–H) VEGFR-3 whole mount staining of the ears 2 mo after AAV-infection. Note the formation of a tree-like network of lymphatic vessels in the skin of Chy mice in response to VEGF-C156S (E) and VEGF-C (F). Inset in E shows lymphatic sprouting and splitting (arrowheads) in the AAV-VEGF-C156S infected Chy mouse skin 5 wk after infection. The control Chy mouse skin (G) contains few abnormal VEGFR-3–positive vessel structures. (H) VEGFR-3 staining of wild-type mouse skin. Scale bars: A–D, 250 μm; E–H, 200 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Over Expression, Functional Assay, Comparison, Control, Staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Molecular markers of adult lymphatic vessels. (A and B) Visualization of cutaneous lymphatic vessels in VEGFR-3+/LacZ and VEGFR-2+/LacZ mice. Blood vessels are stained brown for biotinylated lectin while the blue staining marks the expression of the β galactosidase gene. Note that whereas VEGFR-2 is found in the collecting lymphatic vessels with valves (arrows), VEGFR-3 expression is strong in the initial lymphatic capillaries (arrowheads). (C and D) Lymphatic vessels on the membranous part and (E and F) on the muscular part of the diaphragm. (G and H) The collecting lymphatic vessels of a K14-VEGF-C × VEGFR-2+/LacZ mouse and a VEGFR-2+/LacZ littermate control. Scale bars: A and B, 320 μm; C and D, 250 μm; E and F, 120 μm; G and H, 180 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Staining, Expressing, Control