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: 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

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Effects of passages, starvation, cytokines and growth factors on ADAM15 expression in cultured cells. The mRNA expression of ADAM15 was examined by RT-PCR at 25 cycles as described in Materials and methods. (a) Effect of passages on the mRNA expression of ADAM15 in rheumatoid arthritis (RA) synovial fibroblasts (SFs). Lanes 1 to 5 indicate passages 5, 6, 7, 8 and 9 of RA SFs. (b) Effect of starvation on the mRNA expression of ADAM15 in RA SFs. (c) Effect of tumor necrosis factor (TNF)-α (0, 0.1, 1 and 10 ng/ml), IL-1α (0, 0.1, 1 and 10 ng/ml) or transforming growth factor (TGF)-β (0, 0.1, 1 and 10 ng/ml) on the mRNA expression of ADAM15 in RA SFs after stimulation with these factors for 24 h. (d) Regulation of the mRNA expression of ADAM15 by vascular endothelial growth factor (VEGF) 165 in RA SFs and human umbilical vein endothelial cells (HUVECs). Cells were stimulated with VEGF 165 (0, 1, 10 and 50 ng/ml) for 24 h. Note that VEGF 165 enhances the expression of ADAM15 only in HUVECs.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Effects of cytokines and growth factors on expression of vascular endothelial growth factor receptors (VEGFRs). The mRNA expression of the VEGFRs was examined by RT-PCR at 30 cycles as described in Materials and methods. (a) The expression of VEGFR-1, VEGFR-2 and neuropilin-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) of different passages and human umbilical vein endothelial cells (HUVECs). Lanes 1 to 5 correspond to RA SFs of passages 5, 6, 7, 8 and 9, respectively. (b) The mRNA expression of VEGFR-1, VEGFR-2 and neuropilin-1 in RA SFs after 24 h stimulation with tumor necrosis factor (TNF)-α (0, 0.1, 1, 10 and 50 ng/ml), IL-1α (0, 0.1, 1, 10 and 50 ng/ml) or transforming growth factor (TGF)-β (0, 0.01, 0.1, 1 and 10 ng/ml). Note that TNF-α selectively induces the mRNA expression of VEGFR-2 in RA SFs.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Immunohistochemistry of ADAM15, vascular endothelial growth factor receptor (VEGFR)-2 and von Willebrand factor (vWF). (a-d) Rheumatoid arthritis (RA) synovial fibroblasts (SFs) and (e-h) human umbilical vein endothelial cells (HUVEC) were cultured on Lab-Tek II chamber slides and immunostained with antibodies against (a,e) ADAM15, (b,f) VEGFR-2 or (c,g) vWF or (d,h) non-immune mouse IgG as described in Materials and methods. Immunostaining of VEGFR-2 in RA SFs (b) was performed with RA SFs that were treated with 10 ng/ml TNF-α for 24 h prior to immunohistochemistry. Note that vWF is not immunostained in RA SFs (c) , but VEGFR-2 is expressed in those stimulated with TNF-α (b) . Scale bar, 25 μm.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Immunohistochemistry, Cell Culture, Immunostaining