Journal: Biomedicines

Article Title: Neutralization of Marinobufagenin Demonstrates Efficacy In Vitro and In Vivo in Models of Pre-Eclampsia.

doi: 10.3390/biomedicines13040782

Figure Lengend Snippet: Figure 4. (A) MBG-induced decreased expression of VEGF was attenuated by pretreatment with H3L2 (4 µg/mL) (* p < 0.05). (B) MBG-induced decreased expression of PlGF was attenuated by pretreatment with H3L2 (4 µg/mL) (* p < 0.05). (C) MBG-induced increased expression of sFlt-1 was attenuated by pretreatment with H3L2 (4 µg/mL) (* p < 0.05). (D) MBG-induced increased expression of sEng was attenuated by pretreatment with H3L2 (4 µg/mL) (* p < 0.05).

Article Snippet: Measurement of angiogenic factors: Levels of the pro-angiogenic factors VEGF and PlGF, and the anti-angiogenic factors sFlt-1 and sEng, were measured with commercially available kits (Human VEGF Quantikine ELISA Kit (DVE00); Human PlGF Quantikine ELISA Kit (DPG00); Human sVEGF R1/Flt-1 Quantikine ELISA Kit (DVR100B); Human Endoglin/CD105 Quantikine ELISA Kit (DNDG00); R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Signal transduction and biological function of placenta growth factor in primary human trophoblast.

doi: 10.1095/biolreprod60.4.887

Figure Lengend Snippet: FIG. 1. PlGF induced JNK and p38 kinase but not ERK-1 and -2 activities in term trophoblast. Trophoblast was rendered quiescent by serum dep- rivation and treated with rhPlGF, rhEGF, or UV irradiation in serum-free media for the indicated times. Cell lysates were subjected to in vitro ki- nase assays for JNK (A) and ERK-1/2 (C) using GST-c-jun and MBP as target substrates, respectively. B) p38 Kinase activity was assessed in the same trophoblast lysates by Western blot using anti-active p38 kinase antibody (top panel). The immunoblot was stripped and reprobed with anti-p38 antibodies to confirm that equal amounts of protein lysates were present in each lane (B, bottom). Results are from representative experi- ments that were repeated on three separate trophoblast preparations with comparable results (p 5 0.0007, ANOVA). pAb, polyclonal antibody.

Article Snippet: Materials Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Irradiation, In Vitro, Activity Assay, Western Blot

Journal: Biology of reproduction

Article Title: Signal transduction and biological function of placenta growth factor in primary human trophoblast.

doi: 10.1095/biolreprod60.4.887

Figure Lengend Snippet: FIG. 2. PlGF induced ERK-1 and -2 but not JNK or p38 kinase activities in HUVE cells. HUVE cells were serum-starved for 18 h and treated with rhPlGF, rhEGF, or UV irradiation in serum-free media for the indicated times. Cell lysates were analyzed for ERK-1 and -2 activity by Western blot using anti-active MAPK antibodies (A, top) and total ERK-1 and -2 protein with anti-ERK-2 antibodies (A, bottom). HUVE cell lysates were also subjected to an in vitro kinase assay for JNK using GST-c-jun fusion protein as a target substrate (B). These experiments were repeated on three different trophoblast preparations with similar results. pAB, polyclonal an- tibody.

Article Snippet: Materials Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Irradiation, Activity Assay, Western Blot, In Vitro, Kinase Assay

Journal: Biology of reproduction

Article Title: Signal transduction and biological function of placenta growth factor in primary human trophoblast.

doi: 10.1095/biolreprod60.4.887

Figure Lengend Snippet: FIG. 3. PlGF protected trophoblast from growth factor withdrawal-in- duced apoptosis: DNA fragmentation analyses. Term trophoblast was iso- lated and cultured as described. After attachment (4 h), complete medium was replaced with serum-free medium containing no supplements (lane 3), 10 ng/ml rhPlGF (lane 4), 10 ng/ml rhEGF (lane 5), or 10% FCS (lane 2). After 72 h, total DNA from each culture was isolated and analyzed for DNA fragmentation by electrophoresis in 1.6% agarose. Molecular weight markers (lane 1) are in basepairs (bp). This experiment was re- peated on four independent trophoblast preparations with similar DNA fragmentation patterns. SF, Serum-free.

Article Snippet: Materials Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Cell Culture, Isolation, Electrophoresis, Molecular Weight

Journal: Biology of reproduction

Article Title: Signal transduction and biological function of placenta growth factor in primary human trophoblast.

doi: 10.1095/biolreprod60.4.887

Figure Lengend Snippet: FIG. 4. PlGF protected trophoblast from growth factor withdrawal-in- duced apoptosis: MTT reduction analyses. Trophoblast was isolated and treated as described in Figure 3. MTT reductive capacities were deter- mined after 48 h and normalized to control cultures (10% FCS) within each experiment. Results shown are mean percentage 6 SEM of quadru- plicate wells of four independent experiments. SF, Serum-free.

Article Snippet: Materials Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Isolation, Control

Journal: Biology of reproduction

Article Title: Signal transduction and biological function of placenta growth factor in primary human trophoblast.

doi: 10.1095/biolreprod60.4.887

Figure Lengend Snippet: FIG. 5. PlGF did not inhibit pro-inflammatory cytokine-induced apopto- sis. Term trophoblast was isolated and cultured as described. After attach- ment (4 h), complete medium was replaced with medium containing ei- ther 10% FCS, 10 ng/ml TNFa and 100 U/ml IFN-g, TNFa/IFN-g (T/I) plus 10 ng/ml rhPlGF, or TNFa/IFN-g (T/I) plus 10 ng/ml rhEGF. The trophoblast was cultured an additional 48 h, and MTT reductive capacity was as- sessed as described in Material and Methods. The MTT results were nor- malized to control cultures (10% FCS), and results shown are means 6 SEM of quadruplicate wells of four independent experiments. ANOVA of the treatment groups showed significant results (p , 0.005).

Article Snippet: Materials Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Isolation, Cell Culture, Control

Journal: Hypertension

Article Title: VEGF (Vascular Endothelial Growth Factor) Functionalized Magnetic Beads in a Microfluidic Device to Improve the Angiogenic Balance in Preeclampsia

doi: 10.1161/hypertensionaha.118.12380

Figure Lengend Snippet: Figure 2. Impact of the grafting rates of VEGF (vascular endothelial growth factor)-coated beads (V-beads) on their capture/release performances in conditioned media (CM) of human trophoblastic cells. The amount of bt-VEGF (biotinylated VEGF) coated on streptavidin Dynabeads was evaluated from 30 to 259 pmol/mg of beads corresponding to a surface ligand density ranging from 11.7·103 to 102·103 VEGF/µm2. Grafting of V-beads is done following the protocol in Bouzas-Ramos et al.13 A, sFlt-1 capture and (B) PlGF (placental growth factor) release after 2.5-h incubation at 37ºC with stirring. Note that the marked excess is the molar excess of VEGF compared with the initial concentration of sFlt-1.

Article Snippet: Human recombinant sFlt-1 with an antibody constant fraction (rhVEGFR1/ Fc Chimera, Ser27-His687) and human recombinant PlGF (rh PlGF) were from R&D Systems.

Techniques: Incubation, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 1. A, structure-based sequence alignment of PlGF-1 and VEGF-A receptor binding domain. The secondary structural elements are shown as black arrows (-helices) and colored bars (-strands). The residues of VEGF-A involved in Flt-1 binding are underlined. The PlGF-1 putative residues involved in Flt-1 binding (based on the modeling studies) are underlined, and the PlGF-1 residues that have been mutated are in bold. B, schematic representation of part of the PlGF-1 structure that is involved in the binding to Flt-1. The residues chosen for mutation are ball-and-stick representations. The residues from monomer A are shown in blue and those from monomer B in green. Glu-73 and Asn-74 were modeled as alanines in the structure of PlGF-1 (25).

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 2. Western blot analysis of PlGF-1 variants. Conditioned media from cells transfected with wt or mutated PlGF-1 cDNAs were loaded onto a 12% nonreducing SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The filter was probed using purified rabbit polyclonal antibody against h-PlGF-1. A band co-migrating with wt PlGF-1 dimer (46 kDa) was detected in all lanes except for the two mutants of the glycosylation sites, N16A and N84A, and the mutant that prevents dimer assembly, C60A.

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Western Blot, Transfection, SDS Page, Membrane, Purification, Glycoproteomics, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 3. ELISA-based binding of PlGF-1 variants to the soluble portion of Flt-1. A, binding of PlGF-1 variants to human Flt-1, coated at 0.5 g/ml on a 96-well plate, was performed using increasing concentrations of soluble proteins ranging between 1 and 16 ng/ml. Wild type PlGF-1 was used as a positive control. B, percentage of binding of PlGF-1 variants at a concentration of 8 ng/ml calculated with respect to the binding of wt PlGF-1. The results shown represent the average of three independent experiments.

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 4. ELISA-based binding of PlGF-1 variants to Flt-1 at saturating concentrations. A, linear binding of the Q27A, D72A, E73A, and D72A/E73A PlGF-1 variants to Flt-1 coated at 0.5 g/ml, performed using saturating concentrations of soluble proteins. B, percentage of binding of PlGF-1 variants at a concentration of 50 ng/ml calculated with respect to the binding of wt PlGF-1. The results shown represent the average of three independent experiments.

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 5. Analysis of Flt-1 activation induced by wt PlGF-1 and PlGF-1 variants. Starved 293-hFlt1 cells were stimulated with PlGF-1 and Q27A- PlGF-1, D72A-PlGF-1, and D72A/E73A PlGF-1 variants at a concentration of 10 ng/ml for 10 min. A, 1 mg of cell lysate was immunoprecipitated (IP) with anti- Flt1 antibodies and analyzed by Western blot, probed first with anti-phospho- tyrosine antibodies (Anti p-Tyr), and sub- sequently normalized with anti-Flt1 anti- bodies (Anti Flt-1). B, 100 g of the cell protein extracts was analyzed by Western blot (WB) to reveal the phosphorylation state of the signaling protein, Erk. The blots were first immunodetected with an- ti-phospho-Erk antibodies (Anti p-Erk) and subsequently with anti-Erk1 antibod- ies (Anti Erk1). The values of densitome- try analyses performed using Image- Quant 5.2 software are reported. The values of 0 and 100 have been assigned arbitrarily to non-induced and wt PlGF- 1-induced samples, respectively.

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Activation Assay, Concentration Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Software

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 6. D72A/E73A-PlGF-1 variant fails to induce HUVEC cap- illary-like tube formation. A, negative control performed with an amount of conditioned medium from 293 pcDNA3 comparable with that utilized in C and E, diluted in HUVEC EBM-2 medium. B, positive control performed with complete HUVEC EGM-2 medium. C, condi- tioned medium containing wt PlGF-1 at a concentration of 100 ng/ml. D, conditioned medium containing wt PlGF-1 at a concentration of 100 ng/ml preincubated with a cross-reacting neutralizing monoclonal an- tibody against mPlGF at a concentration of 2.5 g/ml. E, conditioned medium containing D72A/E73A-PlGF-1 at a concentration of 100 ng/ml. F, conditioned medium containing D72A/E73A-PlGF-1 at a concen- tration of 100 ng/ml preincubated with a cross-reacting neutralizing monoclonal antibody anti-mPlGF at a concentration of 2.5 g/ml. Magnification 10.

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Variant Assay, Negative Control, Positive Control, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

doi: 10.1074/jbc.m401418200

Figure Lengend Snippet: FIG. 7. Effect of PlGF-1 variants on CAM neovascularization. Nine-day-old chicken embryos were implanted with 3 106 of 293-pcDNA3 (A), 293-PlGF-1 (B), 293-Q27A-PlGF-1 (C), 293-D72A-PlGF-1 (D), or 293-D72A/E73A-PlGF-1 cells (E). All of the stable cell lines produced comparable amounts of the protein (30–40 ng/ml). After 72 h of implantation, the new capillaries around the graft were evaluated optically by stereoscopic microscopy and photographed using a digital camera. F, vessel number at the time of the implant (green bars) and 72 h post- implantation (blue bars).

Article Snippet: Materials—Anti-human PlGF antibody, biotinylated anti-human PlGF antibody, recombinant human PlGF, and recombinant human VEGFR-1 (Flt-1/Fc chimera) were purchased from R&D Systems.

Techniques: Stable Transfection, Produced, Microscopy

Journal: European journal of pharmacology

Article Title: PlGF signaling and macrophage repolarization contribute to the anti-neoplastic effect of metformin.

doi: 10.1016/j.ejphar.2019.172696

Figure Lengend Snippet: Fig. 5. VEGFR-1 signaling mediated autocrine and exogenous PlGF-induced proliferation. (A and B) In vitro proliferative curves of (A) 4T1 and MCF-7 cells, treated with PBS (Con), recombinant PlGF, or in combination of siRNA against VEGFR-1 or VEGFR-2 (n = 6). (C) Immunoblotting for VEGFR-2 of endothelial cell (EC), 4T1 and MCF-7 breast cancer cells. EC is used as the control. (D) Immunostaining for VEGFR-1 (Left) and VEGFR-2 (Right) in 4T1 cells from untreated mouse. (E and F) In vitro proliferation curves of (E) MCF-7 and (F) 4T1 breast cells, treated with PBS (Con), recombinant PlGF or in combination with GDC-0994 (n = 6). Measurement data are expressed as mean ± S.D. **P < 0.01; ***P < 0.001; ns indicates not statistically significant (P > 0.05).

Article Snippet: Recombinant human and mouse PlGF proteins were obtained from R&D SYSTEMs.

Techniques: In Vitro, Recombinant, Western Blot, Control, Immunostaining