Journal: Frontiers in Physiology

Article Title: Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia

doi: 10.3389/fphys.2018.00083

Figure Lengend Snippet: Role of VEGF in mitochondrial bioenergetics in preeclampsia. ECs and HTR-8/SVneo cells were treated with 2% serum from NOR, CTL, and PE women with and without exogenous VEGF (20 ng/mL) for 24 h. (A) Maximal respiration (OCR after FCCP administration) and spare respiratory capacity (Difference between basal and maximal OCR) in endothelial cells, (B) Basal and maximal (Max) RCR, (C,D) are the same experiments as in (A,B) , respectively, but evaluated in HTR-8/SVneo cells. Data are presented as means ± SEM. ( n = 5). In (A,C) : * P < 0.05, vs. NOR, # P < 0.05, vs. cells exposed to PE serum alone. In (B,D) : * P < 0.05, vs. NOR maximal RCR, # P < 0.05, vs. PE maximal RCR. ANOVA ( Bonferroni's post hoc test ).

Article Snippet: Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia

doi: 10.3389/fphys.2018.00083

Figure Lengend Snippet: sFlt-1 induces a metabolic phenotype switch to glycolysis in ECs but not in trophoblasts. ECs and HTR-8/SVneo cells were exposed to 0, 10, 25, and 50 ng/mL of rh-sFlt-1 for 24 h. (A) Oxygen consumption rates (OCR), maximal respiration (OCR after FCCP administration) and spare respiratory capacity (Difference between basal and maximal OCR), (B) Energetic phenotype map, basal and maximal OCR and extracellular acidification rates (ECAR), (C) ECAR determinations of glycolysis rate and glycolytic reserve, evaluated in endothelial cells. (D-F) , are the same experiments as in (A–C) , respectively, but evaluated in HTR-8/SVneo cells. Data is presented as means ± SEM. ( n = 5), * P < 0.05, ** P < 0.01, *** P < 0.001 vs. untreated controls, In (A,D) : Student T -test. In (C,F) : ANOVA ( Bonferroni's post hoc test ).

Article Snippet: Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia

doi: 10.3389/fphys.2018.00083

Figure Lengend Snippet: sFlt-1 acts as a mitochondrial bioenergetics disruptor in preeclampsia. (A) Morphological changes in endothelial cells (ECs) and trophoblasts cultured in glucose and galactose media (40X). (B) Cell viability of ECs and (C) trophoblasts cultured in glucose and galactose media and exposed to exogenous 50 ng/mL of sFlt-1 during 24 h. Scale: 100 μm. Data are presented as means ± SEM. ( n = 3), ** P < 0.01, *** P < 0.001, vs. galactose exposed cells. # P < 0.05, vs. glucose-exposed cells. Student T -test.

Article Snippet: Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA).

Techniques: Cell Culture

Journal: Frontiers in Physiology

Article Title: Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia

doi: 10.3389/fphys.2018.00083

Figure Lengend Snippet: sFlt-1 induces mitochondrial dysfunction in vitro . (A) Mitochondrial ROS (mtROS) determinations by fluorescent microscopy using MitoSOX Red fluorescent probe demonstrate that sFlt-1 significantly induced mtROS formation in endothelial cells (ECs), while these effects were not observed in (B) trophoblasts. (C) sFlt-1 dissipated the mitochondrial membrane potential (Ψ m ) measured by fluorescent microscopy using JC-1 fluorescent probe in ECs, but not in (D) trophoblasts. Cells were exposed to sFlt-1 (50 ng/mL) for 24 h. Data is presented as means ± SEM. ( n = 3), * P < 0.05 vs. untreated controls. Student T -test.

Article Snippet: Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA).

Techniques: In Vitro, Microscopy, Membrane

Journal: Frontiers in Physiology

Article Title: Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia

doi: 10.3389/fphys.2018.00083

Figure Lengend Snippet: Schematic view of the effects of sFlt-1 dysregulation over cellular metabolism and bioenergetics in PE. Dysregulated VEGF signaling due to up-regulation of sFlt-1 levels in preeclampsia leads to reduced activation of VEGF receptors Flt-1 and Flk-1/KDR, respectively. Effects of dysregulated VEGF bioavailability affect mitochondrial oxygen consumption (OCR), inducing a metabolic phenotype switch enhancing glycolytic response (ECAR) in endothelial cells, but not in trophoblasts. sFlt-1 due to loss of mitochondrial bioenergetics increase oxidative stress in mitochondria (mtROS). Together, these events lead to mitochondrial dysfunction, that would result in vascular dysfunction and the onset of preeclampsia.

Article Snippet: Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA).

Techniques: Activation Assay

Journal: Journal of Immunology Research

Article Title: Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease

doi: 10.1155/2018/8972572

Figure Lengend Snippet: Flow analysis of circulating Tfh cells in GD patients. Human PBMCs from GD patients (BT: 36; AT: 21) and 20 HC were stained with anti-CD4, anti-CXCR5, anti-ICOS, anti-CD45RA, and anti-PD-1. (a) The cells were gated initially on lymphocytes and then circulating Tfh cells were analyzed by flow cytometry; (b) the numbers of circulating CD4 + CXCR5 + CD45RA − Tfh (cTfh) cells; (c) the numbers of CD4 + CXCR5 + CD45RA − ICOS + T cells; (d) CD4 + CXCR5 + CD45RA − PD-1 + Tfh cells; (e) the correlation between PD-1 + Tfh cell proportions and TPO-Ab levels in GD patients. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns: no significant difference.

Article Snippet: The peripheral blood mononuclear cells (PBMCs) were separated immediately by density gradient separation with Ficoll-Hypaque solution (CL5020, CEDARLANE, Canada).

Techniques: Staining, Flow Cytometry

Journal: Journal of Immunology Research

Article Title: Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease

doi: 10.1155/2018/8972572

Figure Lengend Snippet: Expanded frequency of circulating plasma cells in GD patients. Human PBMCs from GD patients (BT: 36; AT: 21) and 20 HC were stained with anti-CD19, anti-CD38, and anti-CD27. (a) The cells were gated initially on lymphocytes and then on CD19 + B cells; afterwards, the numbers of CD27 high CD38 high PCs were analyzed by flow cytometry. (b) Changes in PCs in GD patients. (c) Relation of PC proportions and serum TPO-Ab levels in GD patients. (d) Relation of Tfh2 subset proportions with PCs in GD patients. (e) The correlation between the proportions of PCs and ICOS + Tfh cells in GD patients. (f) The correlation between the proportions of PCs and PD-1 + Tfh cells in GD patients. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns: no significant difference.

Article Snippet: The peripheral blood mononuclear cells (PBMCs) were separated immediately by density gradient separation with Ficoll-Hypaque solution (CL5020, CEDARLANE, Canada).

Techniques: Clinical Proteomics, Staining, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Small-Molecule Activation of p53 Blocks Hypoxia-Inducible Factor 1α and Vascular Endothelial Growth Factor Expression In Vivo and Leads to Tumor Cell Apoptosis in Normoxia and Hypoxia

doi: 10.1128/mcb.00959-08

Figure Lengend Snippet: FIG. 1. RITA induces p53-dependent inhibition of HIF-1 and VEGF in normoxia and hypoxia. (A to C) Western blot analysis shows HIF-1 and p53 protein levels in MCF-7 cells (A), MDA-MB-231 (B), and p53/ HCT116 or p53/ HCT116 cells (C) treated with RITA over a concentration range for 16 h in normoxia or hypoxia (1% O2). Actin was used as a load control. (D) The graph shows VEGF (pg/ml) secreted into cell culture medium from p53/ and p53/ HCT116 cells treated with RITA (0 to 1 M) for 16 h in hypoxia (1% O2).

Article Snippet: Following treatment, conditioned medium was removed from cells and analyzed by an enzyme-linked immunosorbent assay for secreted vascular endothelial growth factor (VEGF) (QuantiGlo; R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Western Blot, Concentration Assay, Control, Cell Culture

Journal: Molecular and Cellular Biology

Article Title: Small-Molecule Activation of p53 Blocks Hypoxia-Inducible Factor 1α and Vascular Endothelial Growth Factor Expression In Vivo and Leads to Tumor Cell Apoptosis in Normoxia and Hypoxia

doi: 10.1128/mcb.00959-08

Figure Lengend Snippet: FIG. 8. RITA induces p53 and H2AX phosphorylation and blocks HIF-1 and VEGF expression in tumor xenografts. (A) Western blot analysis of p53/ HCT116 tumor xenograft lysates. Mice bearing p53/ HCT116 cell-derived tumor xenografts were administered with a single intraperitoneal dose of RITA at 10 mg/kg for 24 h. Tumor cell lysates from eight tumors were analyzed per group. Western blots show a representation of two independent tumors (tumor 1 [T1] and T2) from the DMSO- and RITA-treated groups, assessed for HIF-1, HDM2, p53, phosphorylated-S15-p53 (P-S15-p53), and phosphorylated-S139-H2AX (P-S139-H2AX) proteins. Actin was used as a load control. (B) The graph shows VEGF levels in tumor lysates. Four data points are shown for each condition. Experiments show two independent tumors (T1 and T2) in two independent experiments (diamonds and circles). Abbreviations: IP, intraperitoneally; hr, hours.

Article Snippet: Following treatment, conditioned medium was removed from cells and analyzed by an enzyme-linked immunosorbent assay for secreted vascular endothelial growth factor (VEGF) (QuantiGlo; R&D Systems, Minneapolis, MN).

Techniques: Phospho-proteomics, Expressing, Western Blot, Derivative Assay, Control