Journal: Blood

Article Title: VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

doi: 10.1182/blood-2011-09-378174

Figure Lengend Snippet: Figure 1. VEGFR1 stimulation, but not VEGFR2 stimu- lation, is responsible VEGF-A165–induced thrombocy- tosis. (A-B) Mice were administered either VEGF-A165 (100 g/kg intraperitoneally), PlGF-2, VEGF-E (100 g/kg intraperitoneally), or vehicle (PBS) each day for 4 con- secutive days. (A) Some groups of mice receiving the 4-day VEGF-A165 regimen were also administered block- ing Abs to either VEGFR1, VEGFR2, VEGFR1&2, or control IgG 20 minutes before VEGF-A165 administration on days 1 and 3. Enumeration of circulating platelets was then conducted 24 hours after last growth factor adminis- tration. Bone marrow or spleens were also harvested, and MKs were identified by flow cytometry via forward and side scatter and CD41 expression. (C) Percentage of MKs expressing VEGFR1 and VEGFR2 in vehicle con- trol–treated mice. (D) The number of MKs per million cells from bone marrow or spleen; n 5-6 animals per group. Data are expressed as mean SEM; *P .05; ***P .001 compared with control or as indicated.

Article Snippet: In some experiments, VEGF-A165–treated mice were administered previously defined neutralizing doses of either anti-VEGFR1 Ab (50 g/mouse IP; R&D Systems), anti-VEGFR2 Ab (50 g/mouse IP; R&D Systems), or both, 2 hours before VEGF-A165 administration on days 0 and 2.9,19 Chromopure goat IgG (Jackson ImmunoResearch Laboratories) was administered to control groups.

Techniques: Blocking Assay, Control, Cytometry, Expressing

Journal: Blood

Article Title: VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

doi: 10.1182/blood-2011-09-378174

Figure Lengend Snippet: Figure 2. Effect of VEGFR1 and VEGFR2 stimulation on MK maturation. Mice were administered either VEGF-A165 (100 g/kg intraperitoneally), PlGF-2, VEGF-E (100 g/kg intraperitoneally), or vehicle (PBS) each day for 4 consecutive days. (A-B) Representative histograms of CD41 MKs stained with TOPRO-3 revealing respective peaks of increasing DNA ploidy number. (A) CD41 MKs with 2n, and (B) 2n peak subtracted to reveal 4n to 128n. (C) Percentage of bone marrow and splenic MKs with a ploidy number of 16n or greater in mice administered either VEGF-A165, PlGF-2, or VEGF-E. (D) Histogram revealing spread of bone marrow MK ploidy above 16n in mice administered either VEGF-A165, PlGF-2, or VEGF-E; n 5-6 animals per group. Data are expressed as mean SEM; *P .05 com- pared with control.

Article Snippet: In some experiments, VEGF-A165–treated mice were administered previously defined neutralizing doses of either anti-VEGFR1 Ab (50 g/mouse IP; R&D Systems), anti-VEGFR2 Ab (50 g/mouse IP; R&D Systems), or both, 2 hours before VEGF-A165 administration on days 0 and 2.9,19 Chromopure goat IgG (Jackson ImmunoResearch Laboratories) was administered to control groups.

Techniques: Staining, Control

Journal: Blood

Article Title: VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

doi: 10.1182/blood-2011-09-378174

Figure Lengend Snippet: Figure 5. VEGFR1 increases MK migration to vascu- lar niche via a CXCR4-dependent process. Mice were administered either VEGF-A165, PlGF-2, VEGF-E (100 g/kg intraperitoneally), or vehicle (PBS) each day for 4 consecutive days before bone marrow and spleens were harvested 24 hours later. (A) Percentage of CD41

Article Snippet: In some experiments, VEGF-A165–treated mice were administered previously defined neutralizing doses of either anti-VEGFR1 Ab (50 g/mouse IP; R&D Systems), anti-VEGFR2 Ab (50 g/mouse IP; R&D Systems), or both, 2 hours before VEGF-A165 administration on days 0 and 2.9,19 Chromopure goat IgG (Jackson ImmunoResearch Laboratories) was administered to control groups.

Techniques: Migration

Journal: Blood

Article Title: VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

doi: 10.1182/blood-2011-09-378174

Figure Lengend Snippet: Figure 4. VEGFR1 stimulation redistributes MKs to the vascular niche. Mice were administered either VEGF-A165, PlGF-2, VEGF-E (100 g/kg intraperitoneally), or vehicle (PBS) each day for 4 consecutive days before femurs were excised for bone marrow histology. (A) Quantification of MKs per field of view, and (B) quantification of the number of MKs adjacent to sinusoidal vessels; n 5-6 animals per group. Data are expressed as mean SEM; ***P .001 compared with control.

Article Snippet: In some experiments, VEGF-A165–treated mice were administered previously defined neutralizing doses of either anti-VEGFR1 Ab (50 g/mouse IP; R&D Systems), anti-VEGFR2 Ab (50 g/mouse IP; R&D Systems), or both, 2 hours before VEGF-A165 administration on days 0 and 2.9,19 Chromopure goat IgG (Jackson ImmunoResearch Laboratories) was administered to control groups.

Techniques: Control

Journal: PLoS ONE

Article Title: Glycolaldehyde induces synergistic effects on vascular inflammation in TNF-α-stimulated vascular smooth muscle cells

doi: 10.1371/journal.pone.0270249

Figure Lengend Snippet: (A) MOVAS-1 cells were incubated with TNF-α (10 ng/mL) in the presence or absence of GA (25 and 50 μM) for 2 h. The level of ROS was determined as described in materials and methods. (B) The production of ROS in the VSMCs was evaluated using the immunofluorescence microscopy. (C and D) MOVAS-1 cells were activated with TNF-α (10 ng/mL) in the presence or absence of GA (25 and 50 μM) for 4 h. Protein level of PI3K and AKT was determined by Western blot assay. The level of β-actin was measured as an internal control. Results are shown as means ± SEM from a representative experiment (n = 5). *p<0.05 significantly different from the group treated with TNF-α.

Article Snippet: The following antibodies were used: AGEs (LS-C664030) from LSBio (Seattle, WA, USA), VCAM-1 (#39036), RAGE (#42544), phospho-p65 (#3033), p65 (#8242), phospho-IκB (#2859), IκB (#9242); all from Cell Signaling Technologies (Danvers, MA, USA), ICAM-1 (sc8439), phospho-ERK (sc81492), ERK (sc7383), phospho-JNK (sc81502), JNK (sc6254), phospho-p38 (sc166182), p38 (sc271120), phospho-PI3K (sc166365), phospho-AKT (sc514032), TNF-α (sc52746), IL-6 (ab57315); all from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Incubation, Immunofluorescence, Microscopy, Western Blot, Control

Journal: Experimental Neurobiology

Article Title: Glutamine Supplementation Ameliorates Chronic Stress-induced Reductions in Glutamate and Glutamine Transporters in the Mouse Prefrontal Cortex

doi: 10.5607/en.2019.28.2.270

Figure Lengend Snippet: The glutamate/glutamine cycle. High-affinity excitatory amino acid transporters (EAATs) of astrocytes primarily clear Glu in the synaptic cleft that is released from presynaptic neurons. GLT1 (glutamate transporter 1/EAAT2/SLC1A2) is the major regulator that transports Glu to astrocytes. In astrocytes, Glu is converted into Gln by glutamine synthetase (GS). Synthesized Gln is released from the astrocytes through sodium-coupled neutral amino acid transporters (SNAT3/SLC38A3 and SNAT5/SLC38A5). Released Gln is transported into neurons via SNAT1 and SNAT2 (SLC38A1 and SLC38A2) and converted back to Glu by phosphate-activated glutaminase (PAG or GLS1/2, mainly GLS2 in brain) for synaptic transmission of glutamatergic neurons. Glutamate is further transformed to γ-aminobutyric acid (GABA) by glutamic acid decarboxylases (GAD1 and GAD2) in the GABAergic neurons. Glu can be synthesized in the astrocytes from glucose via the tricarboxylic acid (TCA) cycle, followed by transamination of α-ketoglutarate (α-KG). Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate that is converted to α-KG via the TCA cycle. Glutamate dehydrogenase (GDH) connects the TCA and Glu-Gln cycles by reversibly converting Glu into α-KG and vice versa. Expressional changes by chronic immobilization stress (CIS) and Gln supplementation are marked by red and green arrows, respectively.

Article Snippet: The membranes were blocked with 5% skim milk or 3% bovine serum albumin in Tris-buffered saline with Tween (50 mM Tris, 150 mM NaCl, 0.1% or 1% Tween-20, pH 7.4) for 2 h and incubated overnight at 4°C with the following primary antibodies: PC (1:2,000, SC373937; Santa Cruz Biotechnology, Dallas, TX, USA), GDH [1:5,000, SC160383 (GDH1/2); Santa Cruz Biotechnology], GLT1 (1:1,000, SC7760; Santa Cruz Biotechnology), GS (1:10,000, MAB302; EMD Millipore, Temecula, CA, USA), SNAT3 (1:1,000, ab211516; Abcam, Cambridge, UK), SNAT5 (1:2,000, ab72717; Abcam), SNAT1 (1:1,000, SC67080; Santa Cruz Biotechnology) SNAT2 (1:1,000, SC166366; Santa Cruz Biotechnology), GLS2 (1:2,000, AB113509; Abcam), GAD1 (1:2,000, 5305; Cell Signaling, Danvers, MA, USA), GAD2 (1:2,000, 3988; Cell Signaling), α-tubulin (1:10,000, T5168; Sigma-Aldrich, St. Louis, MO, USA), and β-actin (1:10,000, A5441; Sigma-Aldrich).

Techniques: Synthesized, Transmission Assay, Transformation Assay