Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 1. MGO reduces VEGFR2 protein levels and impairs endothelial cell angiogenesis. A and B: MGO reduced VEGFR2 protein levels in a time and dose dependent fashion. BAEC were incubated with MGO at indicated concentrations for up to 24 h. Then cells were subjected to Western blot respectively with a rabbit derived VEGFR2 antibody and a mouse derived b-actin antibody. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). C and D: MGO reduced endothelial cell angiogenesis in a dose dependent manner. Cells incubated with MGO (10–100 mM) were subjected to angiogenesis assessment by endothelial cell tube formation (upper panel in C) and migration (bottom panel in C). *P,0.05 vs control (n = 3). doi:10.1371/journal.pone.0046720.g001

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Incubation, Western Blot, Derivative Assay, Control, Migration

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 2. Knockdown of Glo1 enhances VEGFR2 reduction induced by MGO, whereas Glo1 overexpression prevents the reduction. A–B: MGO did not affect cell viability or apoptotic markers. BAEC were incubated with MGO at the indicated concentrations (for 24 h) or time course (up to 24 h) followed either by viability measurement (MTT assay) or Western blot staining for markers of apoptosis with respective antibodies against Bcl-2, Bax and Caspase 3. C: Knockdown of Glo1 by siRNA partly mimicked the MGO effect and enhanced MGO-induced VEGFR2 reduction. Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). D: MGO increased levels of MGO-protein adduct which were further enhanced by siRNA knockdown of Glo1. E: Overexpression of Glo1 blocked MGO-induced VEGFR2 reduction. Infection of either control or Glo1 containing plasmid DNA was performed according to the protocol provided by OriGene (Rockville, MD). F: Overexpression of Glo1 blocked MGO-increased MGO-protein adducts. G: Knockdown of RAGE by siRNA prevented MGO-induced VEGFR2 reduction. Transfection of control or RAGE siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). Western blot was performed with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control (at 0 mM of MGO). Casp-3: Caspase-3; c-Casp-3: cleaved Caspase-3. doi:10.1371/journal.pone.0046720.g002

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Knockdown, Over Expression, Incubation, MTT Assay, Western Blot, Staining, Transfection, Control, Infection, Plasmid Preparation

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 3. Inhibition of autophagy, but not proteasome or caspase, abolishes the reduction of VEGFR2 protein levels and angiogenesis by MGO. A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 mM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 mM), pepstatin A (Pep A: 10 mM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 mM), epoxomicin (Epo: 0.5 mM), lactacystin (Lact: 1 mM), and z-VAD-fmk (z-VAD: 20 mM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all cell lysates were subjected to Western blot with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 mM), endothelial cells were incubated respectively with CQ (100 mM), Pep A (10 mM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g003

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Inhibition, Incubation, Transfection, Control, Western Blot, Tube Formation Assay, Knockdown

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 4. MGO induces autophagy markers and autophagic flux, whereas genetic or pharmacological induction of autophagy mimics MGO-elicited effects. A: MGO increased autophagy markers Beclin 1 and LC3B. B: MGO induced LC3 punctae formation (autophagic flux) (Confocal microscopy imaging). C: MGO increased immunoprecipitation of VEGFR2 with LC3B, an autophagy marker and a component of the autophagosome. D: overexpression of Beclin 1 decreased VEGFR2 protein levels, and the reduction was accelerated when MGO was present. E: Rapamycin (1 mM for 1 h) decreased VEGFR2 protein levels. F: Rapamycin (1 mM) reduced endothelial tube formation. BAEC were incubated with MGO (25 mM) for 1 h (A–C) and 16 h (D). Cells were collected for Western blotting with a rabbit derived Beclin-1 or LC 3B antibody and a mouse derived b-actin antibody. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). doi:10.1371/journal.pone.0046720.g004

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Confocal Microscopy, Imaging, Immunoprecipitation, Marker, Over Expression, Incubation, Western Blot, Derivative Assay, Control

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 6. Suppression of ONOO2 generation prevents reduction of VEGFR2 protein and endothelial cell angiogenesis by MGO. A–E: Suppression of ONOO2 generation prevented reduction of VEGFR2 protein. BAEC were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 mM for 1 h), adenoviral infection of GFP (control), or SOD1 and SOD2, prior to MGO incubation (25 mM, 16 h). The treated cells were subjected to Western blot for VEGFR2 protein. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). F: Inhibition of ONOO2 generation normalized the tube formation. Endothelial cells were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 mM for 1 h) prior to MGO challenge (25 mM). The treated cells were subjected to tube formation assay. The images shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g006

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Incubation, Infection, Control, Western Blot, Inhibition, Tube Formation Assay

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 7. MGO induces endothelial VEGFR2 reduction predominantly in cytosol, which can be prevented by administration of O2 –. scavenger or autophagy inhibitor. A–B (low and high-power fields of the same slides): MGO reduced VEGFR2 immune-staining in cytosol or cytoplasm membrane, without affecting staining in the nucleus. BAEC were incubated with MGO (25 mM, 16 h) and subjected to cell immunofluorescent staining with a commercial immune-staining kit including ProLongH Gold and SlowFadeH Gold Antifade, by using a rabbit derived VEGFR2 antibody or stained with DAPI, and a goat anti-rabbit IgG conjugate labeled with fluorescent dyes. All images shown are representative of three independent experiments. C: The quantification results of B. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g007

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Staining, Membrane, Incubation, Derivative Assay, Labeling, Control

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 8. VEGFR2 protein levels and the aortic endothelial angiogenesis are reduced in diabetic mice, which can be rescued either by autophagy suppression or O2 –. scavenging. A: Aortic angiogenic response was reduced in MGO-challenged aortas of C57BL/6J mice, which could be restored by autophagy inhibitors. Aortic rings removed from normoglycemic C57BL/6J mice were incubated with MGO (25 mM) with or without the presence of autophagy inhibitors (Pep A: 10 mM, Baf A1: 5 mM) and subjected to aortic ring assay; *P,0.05 vs the vehicle treated (n = 5/ group). B: Aortic endothelial outgrowth was impaired in db/db vs genetic control mice that could be rescued by autophagy inhibitors. Aortic rings prepared from the genetic control and db/db mice were treated with vehicle or autophagy inhibitors (Pep A: 10 mM, Baf A1: 5 mM) and followed by aortic ring assay. *P,0.05 vs the vehicle treated (n = 5/group). C–D: Aortic angiogenesis and VEGFR2 protein were reduced in diabetic Akita vs genetic control mice, which could be improved by mTempol administration in vivo. Aortas prepared from the genetic control and the treated Akita mice (mTempol: 0.1 mM in the drinking water, vehicle: normal drinking water, for 4 weeks) were subjected to aortic ring assay and aortic VEGFR2 protein detection by immune-precipitation (VEGFR2 enrichment)-combined Western blot. # indicates antibody recognizing different epitope of the VEGFR2 molecule. The Western blot of b-actin in aortic tissue homogenates with a mouse derived antibody is used as an Input for the loading control. *P,0.05 vs the vehicle treated (n = 5/group). E: the proposed mechanism of diabetic angiogenesis impairment: MGO, a glycolytic metabolite which is found to be increased in patients with diabetes, impairs endothelial angiogenesis through ONOO–dependent autophagy-mediated VEGFR2 degradation. NS: not significant vs control. doi:10.1371/journal.pone.0046720.g008

Article Snippet: Materials The antibodies used in the present study included: VEGFR2 (55B11), b-actin, Beclin-1, LC3B, SOD1, and peroxidase conjugated secondary antibodies (Cell Signaling, Danvers, MA); VEGFR2 (Flk-1:A-3), Bcl-2, Bax, Caspase 3, and Glo1 (Santa Cruz Biotechnology, Santa Cruz, CA); SOD2 (Fisher scientific, Pittsburgh, PA).

Techniques: Incubation, Aortic Ring Assay, Control, In Vivo, Western Blot, Derivative Assay

Journal: Physiological Reports

Article Title: Physical activity differentially regulates VEGF, PlGF, and their receptors in the human placenta

doi: 10.14814/phy2.14710

Figure Lengend Snippet: Expression of VEGF, PlGF, VEGFR1 (Flt1) and VEGFR2 (Flk1) protein by western immunoblotting in term placenta (2:1 ratio of tissue from the central and peripheral regions of the placenta, respectively) from physically active ( n = 23) and inactive ( n = 22) women. Representative immunoblots for VEGF (a, upper panel), PlGF (b, upper panel), VEGFR1 (Flt1) (c, upper panel), and VEGFR2 (Flk1) (d, upper panel) are shown. The corresponding semi‐quantitative densitometric analysis is shown for each protein in the lower panel of a–d, respectively. All data are represented as mean ± SD. ** p < 0.01.

Article Snippet: VEGFR2 , Flk‐1 (A‐3) , Santa Cruz (Cat no. sc‐6251, Lot no. C1119, RRID: AB_628431) , Mouse monoclonal , 1:500 in Western blot (Bersini et al., ; Francis & Wei, ); 1:50 in Immunohistochemistry (Möller et al., ) .

Techniques: Expressing, Western Blot

Journal: Physiological Reports

Article Title: Physical activity differentially regulates VEGF, PlGF, and their receptors in the human placenta

doi: 10.14814/phy2.14710

Figure Lengend Snippet: Expression of VEGF, PlGF, VEGFR1 (Flt1), and VEGFR2 (Flk1) mRNA by real‐time PCR in term placenta (2:1 ratio of tissue from the central and peripheral regions of the placenta, respectively) from physically active ( n = 23) and inactive ( n = 22) women. Relative quantification of mRNA VEGF (a), PlGF (b), VEGFR1 (Flt1) (c) and VEGFR2 (Flk1) (d) expression. Relative expression was normalized to the expression of GAPDH in all samples. All data are represented as mean ± SD. * p < 0.05.

Article Snippet: VEGFR2 , Flk‐1 (A‐3) , Santa Cruz (Cat no. sc‐6251, Lot no. C1119, RRID: AB_628431) , Mouse monoclonal , 1:500 in Western blot (Bersini et al., ; Francis & Wei, ); 1:50 in Immunohistochemistry (Möller et al., ) .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Physiological Reports

Article Title: Physical activity differentially regulates VEGF, PlGF, and their receptors in the human placenta

doi: 10.14814/phy2.14710

Figure Lengend Snippet: Immunolocalization VEGF (a, b), VEGFR1 (Flt1) (d, e), and VEGFR2 (Flk1) (g, h) in term placenta from physically active and inactive women. c, f, i represent negative controls, performed in absence of primary antibodies (diluent only). red asterisk , lumen of the blood vessels; orange arrows , endothelial cells; triangles , stromal cells; arrow heads , syncytiotrophoblast border, red drafting point arrows , cytotrophoblast cells. All sections were counterstained using hematoxylin. Scale bars, 20 µm.

Article Snippet: VEGFR2 , Flk‐1 (A‐3) , Santa Cruz (Cat no. sc‐6251, Lot no. C1119, RRID: AB_628431) , Mouse monoclonal , 1:500 in Western blot (Bersini et al., ; Francis & Wei, ); 1:50 in Immunohistochemistry (Möller et al., ) .

Techniques: