antibodies against vegf a  (Abcam)

Journal: Arthritis Research & Therapy

Article Title: Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

doi: 10.1186/s13075-018-1592-1

Figure Lengend Snippet: Synovial tissue (ST) angiogenesis, oxidative stress and cellular bioenergetics. To support the concept that oxidative stress, angiogenesis and energy metabolism are interconnected processes that co-exist during the inflammation milieu, double-immunofluorescence staining was performed. ST slides were co-incubated with primary mouse antibody against human 4-hydroxy-2-nonenal (4-HNE) and with primary rabbit antibodies against angiogenic factors (vascular endothelial growth factor [VEGF], angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]), glycolytic proteins (glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase isozyme 2 [PKM2], glucose transporter 1 [GLUT1]) and a mitochondrial marker (adenosine triphosphate synthase subunit β [ATP5B]). Representative merged immunofluorescence images demonstrate examples of co-localisation ( yellow ) of 4-HNE with VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Cells stained green are positive for 4-HNE only; cells stained red are positive only for VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Arrows indicate examples of co-localisation. Magnification of photomicrographs × 20, insets : Figure S4

Article Snippet: ST sections were fixed with acetone for 10 minutes and co-incubated with primary mouse antibody against human 4-HNE (GENTAUR, Kampenhout, Belgium) and with primary rabbit antibodies against VEGF, Ang2, Tie2, ATP5B and glucose transporter 1 (GLUT1) (all from Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA) and pyruvate kinase isozyme 2 (PKM2) (Abgent, San Diego, CA, USA).

Techniques: Double Immunofluorescence Staining, Incubation, Marker, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

doi: 10.1186/s13075-018-1592-1

Figure Lengend Snippet: 4-Hydroxy-2-nonenal (4-HNE) induces pro-angiogenic and pro-inflammatory mechanisms in primary rheumatoid arthritis synovial fibroblast cells (RASFC). Increased vascular endothelial growth factor (VEGF) immunofluorescence in RASFC subjected to 4-HNE compared to the basal cells and quantification of VEGF, angiopoietin 2 (Ang2), basic fibroblast growth factor (bFGF), interleukin (IL)-8, platelet-derived growth factor subunit B (PDGF-B), regulated on activation, normal T cell expressed and secreted (RANTES), intercellular adhesion molecule (ICAM) in RASFC supernatants ( n = 7) following cell culture with 4-HNE. Data are presented as mean ± SEM. * p

Article Snippet: ST sections were fixed with acetone for 10 minutes and co-incubated with primary mouse antibody against human 4-HNE (GENTAUR, Kampenhout, Belgium) and with primary rabbit antibodies against VEGF, Ang2, Tie2, ATP5B and glucose transporter 1 (GLUT1) (all from Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA) and pyruvate kinase isozyme 2 (PKM2) (Abgent, San Diego, CA, USA).

Techniques: Immunofluorescence, Derivative Assay, Activation Assay, Cell Culture

Journal: CNS Neuroscience & Therapeutics

Article Title: DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway, et al. DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway

doi: 10.1111/cns.13042

Figure Lengend Snippet: SIRT1 inhibitor nicotinamide partially counteracts the effects of DPP‐4 inhibitors on the cell proliferation, migration, production of VEGF, and mRNA expression of SIRT1, HIF‐1α, eNOS, and VEGF in rBMVECs under hypoxic/high‐glucose conditions. A, MTT experiment results for different groups showing nicotinamide partially counteracted the effects of DPP‐4 inhibitors, for example, linagliptin and berberine, on the reduced cell proliferation of rBMVECs by the induction of hypoxic/high‐glucose treatment (model control). B, Number of migratory cells in different groups showing nicotinamide partially counteracted the effects of DPP‐4 inhibitors, for example, linagliptin and berberine, on the reduced cell migration of rBMVECs by the induction of hypoxic/high‐glucose treatment (model control). C, ELISA experiment results showing nicotinamide partially counteracted the effects of DPP‐4 inhibitors, for example, linagliptin and berberine, on the VEGF production in rBMVECs by the induction of hypoxic/high‐glucose treatment (model control). D, qRT‐PCR experiment results showing nicotinamide partially counteracted the effects of DPP‐4 inhibitors, for example, linagliptin and berberine, on the mRNA expression of SIRT1, HIF‐1α, eNOS, and VEGF in rBMVECs by the induction of hypoxic/high‐glucose treatment (model control). The difference between groups was confirmed with statistical significance (*** P

Article Snippet: Antibodies against VEGF and DPP‐4 were purchased from Abcam (Cambridge, MA, USA), while HIF‐1α, eNOS, SIRT1, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Migration, Expressing, MTT Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: CNS Neuroscience & Therapeutics

Article Title: DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway, et al. DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway

doi: 10.1111/cns.13042

Figure Lengend Snippet: ELISA experiment results showing that DPP‐4 inhibitors linagliptin and berberine attenuated the reduction of VEGF production in rBMVECs by the induction of hypoxic/high‐glucose conditions (model control). The difference between groups was confirmed with statistical significance (*** P

Article Snippet: Antibodies against VEGF and DPP‐4 were purchased from Abcam (Cambridge, MA, USA), while HIF‐1α, eNOS, SIRT1, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway, et al. DPP‐4 inhibitors promote proliferation and migration of rat brain microvascular endothelial cells under hypoxic/high‐glucose conditions, potentially through the SIRT1/HIF‐1/VEGF pathway

doi: 10.1111/cns.13042

Figure Lengend Snippet: DPP‐4 inhibitors linagliptin and berberine attenuated the reduction in mRNA and protein expressions of VEGF and eNOS in rBMVECs by the induction of hypoxic/high‐glucose conditions. A, Results of qRT‐PCR experiment showing the mRNA expression of VEGF and eNOS was reduced by the induction of hypoxic/high‐glucose treatment in rBMVECs (model control), which was attenuated to a large extent by the administration of DPP‐4 inhibitors, for example, linagliptin and berberine. B, Western blot results showing the protein expression of VEGF and eNOS was decreased by the induction of hypoxic/high‐glucose treatment in rBMVECs (model control), which was attenuated to a large extent by the administration of DPP‐4 inhibitors, for example, linagliptin and berberine. C, Quantitative results of Western blot analysis. The difference between groups was confirmed with statistical significance (*** P

Article Snippet: Antibodies against VEGF and DPP‐4 were purchased from Abcam (Cambridge, MA, USA), while HIF‐1α, eNOS, SIRT1, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Morphological Plasticity of Emerging Purkinje Cells in Response to Exogenous VEGF

doi: 10.3389/fnmol.2017.00002

Figure Lengend Snippet: Vascular endothelial growth factor receptor 2 (VEGFR-2) expression in cerebellar p6, p17 and p30 rats. (A-I) Immunostaining of calbindin positive PCs (red), VEGFR-2 (green) and cell nuclei (blue) in cryosections of rat cerebella at p6, p17, p30. VEGFR-2 receptors are localized within the soma (simple white arrowheads; A–I ) of PCs and in Bergmann glia fibers (white arrows; B ), reaching up to the MCL; exemplarily few cell nuclei are labeled in different cell layers (curved white arrowheads; B,C,E,F,H,I ). Scale bars: (A–I) 20 μm.

Article Snippet: p6, p17 and p30 Cerebellar Rat Cryosections After blocking and permeabilization, cryosections were incubated with primary antibodies against VEGFR-2 (rabbit, polyclonal, 1:100, ab39256; Abcam) and placed in a fridge (4°C) overnight.

Techniques: Expressing, Immunostaining, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Morphological Plasticity of Emerging Purkinje Cells in Response to Exogenous VEGF

doi: 10.3389/fnmol.2017.00002

Figure Lengend Snippet: mRNA expression levels in PCs. (A,B) In situ hybridization: expression of VEGFR-2 in rat cerebella at the age of p6 (A) and p30 (B) . Cryosections (15 μm) of each stage were incubated with 80 nM double-DIG-LNA TM probe (Exiqon). In p6 nearly every PC showed a positive VEGFR-2 signal. During development the expression of VEGFR-2 extremely decreases. In p30 VEGFR-2 positive PCs were hardly detectable. P6 and (C) p30 PCs were laser microdissected at 200× magnification and subjected to the method of quantitative polymerase chain reaction (qPCR) (D) . For relative quantification of KDR expression, the 2 −ΔΔCt method was conducted using the housekeeping gene GAPDH for normalization; data are provided as means ± SEM. Data were tested for significance using Student’s t -test. Significant differences are indicated by *** p

Article Snippet: p6, p17 and p30 Cerebellar Rat Cryosections After blocking and permeabilization, cryosections were incubated with primary antibodies against VEGFR-2 (rabbit, polyclonal, 1:100, ab39256; Abcam) and placed in a fridge (4°C) overnight.

Techniques: Expressing, In Situ Hybridization, Incubation, Real-time Polymerase Chain Reaction

Journal: Diabetes

Article Title: Vascular Endothelial Growth Factor-Mediated Islet Hypervascularization and Inflammation Contribute to Progressive Reduction of ?-Cell Mass

doi: 10.2337/db12-0134

Figure Lengend Snippet: Increased VEGF expression in VEGF low and VEGF high transgenic β-cells leads to islet hypervascularization. A : Representative Western blot showing higher expression of VEGF in both VEGF high and VEGF low . B : A 2.7- and 17-fold increase in VEGF protein levels were found in islets from 2-month-old VEGF low and VEGF high mice measured by ELISA, wild-type (WT) mice (white bar), transgenic VEGF low mice (gray bar), and transgenic VEGF high mice (black bar). * P

Article Snippet: Proteins (50–100 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with primary antibodies against VEGF (Abcam), E-cadherin (Santa Cruz Biotechnology), and β-actin (Abcam) overnight at 4°C.

Techniques: Expressing, Transgenic Assay, Western Blot, Mouse Assay, Enzyme-linked Immunosorbent Assay

Journal: Diabetes

Article Title: Vascular Endothelial Growth Factor-Mediated Islet Hypervascularization and Inflammation Contribute to Progressive Reduction of ?-Cell Mass

doi: 10.2337/db12-0134

Figure Lengend Snippet: Two-month-old VEGF low and VEGF high mice showed disorganization of islet architecture, but normal β-cell mass. A : Top panel : immunohistochemical analysis of insulin (green) and glucagon (red). In transgenic mice, islets appeared disorganized with α-cells in the core. Bottom panel : insulin immunostaining used to visualize islet architecture. B : β-Cell mass was measured in 2-month-old mice ( n = 4–7/group): wild-type (WT) mice (white bar), transgenic VEGF low mice (gray bar), and transgenic VEGF high mice (black bar). C : Ki67 (green) and insulin (red) immunostaining were used to label proliferating β-cells (arrow). D : Percentage of Ki67-positive (replicative) β-cells. E : TUNEL (green) and insulin (red) immunostaining showed apoptotic nuclei (arrow). F : Quantification of TUNEL-positive (apoptotic) β-cells. G and H : Western blot analysis of E-cadherin using islet homogenates from 2-month-old mice. G : A representative immunoblot is shown. H : Densitometric analysis of three different immunoblots: WT mice (white bar), transgenic VEGF low mice (gray bar), and transgenic VEGF high mice (black bar). Scale bars, 100 µm. * P

Article Snippet: Proteins (50–100 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with primary antibodies against VEGF (Abcam), E-cadherin (Santa Cruz Biotechnology), and β-actin (Abcam) overnight at 4°C.

Techniques: Mouse Assay, Immunohistochemistry, Transgenic Assay, Immunostaining, TUNEL Assay, Western Blot

Journal: Diabetes

Article Title: Vascular Endothelial Growth Factor-Mediated Islet Hypervascularization and Inflammation Contribute to Progressive Reduction of ?-Cell Mass

doi: 10.2337/db12-0134

Figure Lengend Snippet: AAV-mediated VEGF overexpression in β-cells increased islet vascularization and inflammation. Two-month-old wild-type (WT) mice were injected with VEGF-expressing (AAV9-VEGF) or nonexpressing (null) AAV9 vectors (10 12 vector genomes/mouse). A : Ten days after AAV injection vasculature structure was revealed by immunostaining for collagen IV (red) and insulin (green) ( top panel ). VEGF-treated islets showed increased basement membrane compared with AAV-null. FITC-dextran (green) together with insulin (red) immunostaining was used to label functional blood vessels ( top middle panel ). Insulin (green) and glucagon (red) expression showed islet disorganization ( bottom middle panel ). Macrophage infiltration in AAV-VEGF–treated animals was determined by Mac-2 immunostaining 10 days after AAV injection ( bottom panel ). Scale bars, 100 µm. B : AAV-VEGF–injected animals showed increased Mac-2–positive area/islet area when compared with AAV-null–treated mice as early as 10 days after injection: AAV-null–treated mice (white bar) and AAV-VEGF–treated mice (black bar) ( n = 4 mice/group). C : Fed blood glucose levels were determined before AAV injection (day 0) and at several time points thereafter: AAV-null-treated mice (white circle) and AAV-VEGF–treated mice (black square) ( n = 10 mice/group). D : Glucose tolerance was measured 20 days after AAV administration (2 g/kg body weight) ( n = 10 animals/group). * P

Article Snippet: Proteins (50–100 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with primary antibodies against VEGF (Abcam), E-cadherin (Santa Cruz Biotechnology), and β-actin (Abcam) overnight at 4°C.

Techniques: Over Expression, Mouse Assay, Injection, Expressing, Plasmid Preparation, Immunostaining, Functional Assay