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: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 2. Gas6 and Protein S bioavailabilities peak at different times of the light–dark cycle. Analysis of the mRNA (A,C) and protein (B,D) expression profiles for Gas6 (A,B) and Protein S (C,D) in the RPE/choroid, retina, or IPM for wildtype (wt, blue) and β5−/−mice (β5 ko, pink) at different times of day as indicated. (A) qPCR experiments allowed us to show that Gas6 mRNA expression levels are slightly increased just before (retina) and after (RPE/choroid) the phagocytic peak in wt mice. Gas6 expression levels were lower in the RPE/choroid of β5−/−mice between peak phagocytosis time and 22.00 while levels were unchanged in the retina fraction. (B) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes showed a decrease at light onset followed by a marked increase at the time of the phagocytic peak in wt animals. In β5−/−mice, expression levels did not vary. (C) Pros1 mRNA expression increases just before and at the time of peak phagocytosis in wt RPE/choroid and retina, respectively. In both tissue samples, a second peak occurs at night offset (retina) of just after (RPE/choroid). The 7.00 and 22.00 RPE/choroid peaks, as well as the phagocytosis retina peak, are lost in β5−/−mice, but median levels are not changed. (D) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes follows a combination of RPE/choroid and retina gene expression profiles depicted in (C). Results are in arbitrary units (a.u.) as means ± SDs, n = 3–8 independent samples; reference: wildtype sample at 8.00. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Western Blot, Gene Expression

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 3. Gas6 is more expressed than Pros1, and ligands are more expressed in the retina than in the RPE/choroid. (A) Gas6 and Protein S (Pros1) mRNA expression profiles in RPE/choroid (green) and retina (orange) fractions of wildtype (wt) mice were compared at different times of day as indicated. Both ligands were more expressed in the retina than in the RPE/choroid. (B) Respective Gas6 (blue) and Pros1 (pink) mRNA expression profiles were compared in both the RPE/choroid and retina fractions of wt mice at different times of day as indicated. In both tissue types, Gas6 was much more expressed than Pros1. (A,B) Results are in arbitrary units (a.u.) as means ± SDs, n = 3–7 independent samples; references: RPE/choroid (A) or Pros1 (B) sample at 8.00. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak. (C) siRNA samples were used to downregulate the endogenous production of each ligand by RPE-J cells. Cells were then subjected to phagocytosis assays for 1.5 and 3 h as indicated. Decrease in Gas6 synthesis (blue bars) leads to diminished binding and internalization of POSs compared to control siRNA (Ctrl, white bars). Blocking the production of Protein S (Pros1, pink/purple bars) only slightly affects binding at 1.5 h. Adding both siRNAs has the same effect than adding the Gas6 siRNA alone. Targeting of both ligands’ production (purple bars) has the same effect as the decrease in Gas6 alone. Results of FITC/DAPI ratios are in arbitrary units (a.u.) expressed as means ± SDs, n = 5–6 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with a Tukey post-test compared to each series corresponding control; reference: total phagocytosis (binding + internalization) for the control condition.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Binding Assay, Control, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 5. Gas6 and Protein S bind to different amino acids of MerTK Ig-like domains. Mutants target- ing ligand binding sites in MerTK Ig-like domains 1 (pink bars) and 2 (blue bars) were transfected in RPE-J and tested for their influence on POS binding (top left) and internalization (bottom left) when compared to non-mutated MerTK (black bar) with or without the addition of Gas6 and Protein S— alone or in combination—as indicated. The p.Gly122Arg (G122R, light pink bars) mutant significantly increases POS binding in DMEM while addition of Gas6 diminishes binding and addition of Protein S importantly increases internalization compared to the wt construct. Among the neighbor sites p.Thr140Ala (T140A) and p.Phe142Val (F142V), only p.Phe142Val (F142V) shows a slight increase in POS binding in the presence of Gas6. The p.Lys263Ile (K263I) mutant has a negative impact on both binding and internalization of POSs alone, as well as a positive effect on the internalization of POSs with Gas6. The p.Lys269Leu (K269L) mutant has almost no effect besides slightly less binding of POSs alone. When challenged with fluorescent beads (right bar graphs), no difference was observed in this study between the different clones. Results of FITC/DAPI ratios in arbitrary units (a.u.) are expressed as means ± SDs, with n = 4–6 independent experiments (POSs, left) or n = 3–4 independent experiments (beads, right). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; one-way ANOVA with a Tukey post-test compared to each series corresponding wildtype; reference: total phagocytosis (binding + internalization) for the control condition (WT). Significance brackets compare different ligand conditions for a single mutant.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Ligand Binding Assay, Transfection, Binding Assay, Mutagenesis, Construct, Clone Assay, Control

Journal: PloS one

Article Title: Embedding synthetic microvascular networks in poly(lactic acid) substrates with rounded cross-sections for cell culture applications.

doi: 10.1371/journal.pone.0073188

Figure Lengend Snippet: Figure 3. Seeding and culture of bovine aortic endothelial cells (BAECs) throughout PLA microchannel networks. (a) Confocal microscopy shows that the interior walls of a 200 mm diameter straight circularized microchannel can be uniformly seeded with endothelial cells that subsequently are confluently cultured in a monolayer lining the channel wall. (b) Fluorescent images show BAECs survive and maintain their morphology after 5 days in the straight circularized microchannel (bar, 50 mm). (c) BAECs seeded in four generations of branched microchannel network with diameters extending below 50 mm uniformly cover all channel walls and maintain viability after 3 days of culture (bar, 50 mm). doi:10.1371/journal.pone.0073188.g003

Article Snippet: Bovine Aortic Endothelial Cells (BAECs; Cell Applications, Inc., CA) obtained as a kind gift from Dr. Mariah Hahn at Texas A&M University were resuspended at 56106 cells mL–1 medium for 500 mm diameter channels or at 26107 cells mL–1 medium for 100 mm diameter and branched channels.

Techniques: Confocal Microscopy, Cell Culture

Journal: Mediators of inflammation

Article Title: Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4 + CD25 + Regulatory T Cells Mainly through Axl Receptor.

doi: 10.1155/2017/6848430

Figure Lengend Snippet: Figure 6: Gas6 modulates the Foxp3 and CTLA4 expression mainly through Axl receptor. (a–d) CD4+CD25+Tregs were treated with anti- Axl or anti-Mertk Abs or PBS in the presence of 100 ng/ml rmGas6. After 24 h of incubation, the expression of CTLA-4 and Foxp3 was determined by flow cytometry (𝑛= 4/group). ∗𝑃< 0.05 compared with the value for the Gas6 group, and #𝑃< 0.05 compared with the value for rmGas6+anti-Mertk group. (e–h) The expression of CTLA-4 and Foxp3 in Tregs with or without Axl knockout was determined by flow cytometry. ∗𝑃< 0.05 compared with the value for the Gas6 group.

Article Snippet: To investigate the effect of Gas6 on CD4+CD25+Tregs in vivo, healthy mice were administered 1, 3, or 6 μg/mouse of rmGas6 (8310-GS, R&D Systems, Minneapolis, MN) via tail vein.

Techniques: Expressing, Incubation, Flow Cytometry, Knock-Out