Journal: The Journal of Neuroscience

Article Title: Dural Calcitonin Gene-Related Peptide Produces Female-Specific Responses in Rodent Migraine Models

doi: 10.1523/JNEUROSCI.0364-19.2019

Figure Lengend Snippet: Drugs administered in these experiments, sources, doses/volumes, and routes of administration

Article Snippet: Human recombinant BDNF (R&D Systems) stock solution was made in sterile PBS containing 1% BSA.

Techniques: Recombinant

Journal: The Journal of Neuroscience

Article Title: Dural Calcitonin Gene-Related Peptide Produces Female-Specific Responses in Rodent Migraine Models

doi: 10.1523/JNEUROSCI.0364-19.2019

Figure Lengend Snippet: Intracisternal administration of BDNF induces priming to dural CGRP in females, but not males. Facial withdrawal thresholds were measured in female (A) and male (B) rats before and after either cisternal injection of 1 pg BDNF (n = 6 females, n = 6 males) or vehicle (aCSF, pH 7.4) (n = 6 females, n = 6 males). At 72 h after dural IL-6, when animals had baselined, 0.1 pg CGRP was administered to the dura. Two-way ANOVA indicated a significant effect of CGRP in the BDNF-treated group of females. The significant differences of the means for each group were determined by ANOVA followed by Bonferroni post hoc test (F(9,100) = 7.467). These data are represented as means ± SEM; *p < .05, **p < .01, ****p < 0.0001).

Article Snippet: Human recombinant BDNF (R&D Systems) stock solution was made in sterile PBS containing 1% BSA.

Techniques: Injection

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: (a) Representative Western blot of proteins confirming PASMC phenotype including smooth muscle myosin, smooth muscle actin, acetylcholine receptor, and transgelin. Hypoxia enhances PASMC brain derived neurotrophic factor (BDNF) secretion. (b) Western blot analysis of human pulmonary artery smooth muscle cells (PASMCs) demonstrated presence of BDNF, with increased expression following 48h of 1% hypoxia. (c) Human PASMCs secrete BDNF, and such secretion is increased with hypoxia, as demonstrated by ELISA of supernatant in PASMC cultures. (d) Representative Western analysis showed increased TrkB expression (particularly full-length; FL) in PASMCs following hypoxia. (e) Immunocytochemistry demonstrating enhanced BDNF and TrkB expression in hypoxia-exposed human PASMCs compared to normoxia (green color represents BDNF or TrkB, red color represents α-smooth muscle actin, blue color (DAPI) represents nucleus staining. Values are means ± SE (n = 5 patients without pulmonary vascular disease). *Significant difference from normoxia (p<0.05).

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Staining

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: (a) Representative Western blots of endothelium-intact human PA show presence of both full length (FL) and truncated (T1) isoforms of TrkB. Exposure to 1% hypoxia increased TrkB-FL expression (a, b). Expression of TrkB-FL was also higher in PA of patients with moderate PH (a, b). BDNF expression in endothelium-intact human PA was also increased by hypoxia (c) albeit not to the extent as for TrkB. Importantly, ELISA of supernatants showed that human PA secretes BDNF and such secretion is enhanced by hypoxia and in PH (d). *Significant difference from normoxia (p<0.05). Values are means ± SE (n = 5 unique patients for normoxia and hypoxia and 3 unique PH patients in (b) and (c)).

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: (a) In human PASMCs, 48h of 1% hypoxia enhanced expression of hypoxia-inducible factor (HIF-1α). Expression of β-actin or transgelin was not substantially altered showing maintenance of smooth muscle phenotype. (b) Hypoxia enhancement of TrkB was suppressed by pharmacological inhibition of HIF1α. Values are means ± SE (n = 7 patients). *Significant difference from normoxia, # significant effect of HIF-1α inhibition (p<0.05). (c) Western blot demonstrating phosphorylated TrkB expression in normoxia or hypoxia with exposure to BDNF for indicated times.

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: (a) Exposure of PASMCs to 1 μM serotonin (5HT; arrow) resulted in a transient [Ca 2+ ] i response. Under normoxic conditions, 24h exposure to 100 pM BDNF enhanced the subsequent response to 1 μM 5HT, as reflected by increased amplitude (bar graph). (b) Hypoxia substantially enhanced [Ca 2+ ] i responses of PASMCs to 5HT. Under conditions of hypoxia, BDNF did not produce any additional augmentation of these responses. However, neutralization of extracellular BDNF using the chimeric TrkB-Fc (1 μg/ml) protein blunted the effect of hypoxia. Similarly inhibition of HIF1α reduced the effect of hypoxia on the responses to 5HT. Values are means ± SE (n = 7 patients). *Significant difference from normoxia control, #significant effect of inhibitors from hypoxia control (p<0.05).

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: Neutralization, Inhibition, Control

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: Western blots for pPI3k, pAkt, and pERK in denuded human PA (aand b). After 24 hr hypoxia exposure, PI3 kinase (c) and pAkt/Akt (d) levels were both increased: effects blunted by BDNF neutralization or HIF1α inhibition. In normoxia, 100 pM BDNF enhanced PI3 kinase expression and Akt phosphorylation. 24h hypoxia also enhanced phosphorylation of ERK1/2 (e). In normoxia, 100 pM BDNF enhanced ERK1/2 phosphorylation. Extracellular neutralization of BDNF using TrkB-Fc (1 μg/ml) or inhibition of HIF1α blunted hypoxia effects on ERK phosphorylation. Values are means ± SE (n = 3 patients). *Significant difference from normoxia control; # significant inhibitor effect from hypoxia control (p<0.05).

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Neutralization, Inhibition, Expressing, Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Role of Hypoxia-Induced Brain Derived Neurotrophic Factor in Human Pulmonary Artery Smooth Muscle

doi: 10.1371/journal.pone.0129489

Figure Lengend Snippet: PASMC proliferation was measured using a fluorescent CyQuant assay (a). Compared to baseline proliferation over 48h in normoxia (~6%), proliferation in BDNF exposed cells were substantially higher. Hypoxia by itself substantially increased proliferation, with no additional effect of BDNF. In contrast BDNF neutralization or HIF1 inhibition substantially blunted proliferation. The fluorescent proliferation assay results were matched by changes in proliferating cell nuclear antigen (PCNA) Western analysis (b). In contrast to the increase in proliferation, BDNF had only minimal effects on anti-apoptotic proteins MCL1 and BCL2 (c, d) but did reduce levels of the apoptotic proteins caspase 3 and BAX. Hypoxia effects were overall more pronounced for anti-apoptotic proteins, but less for apoptotic proteins. These protein changes were consistent with small changes in apoptotic activity as measured for caspase 3 vs. 7 (f). Values are means ± SE (n = 7 unique patients). *Significant BDNF (black band) or hypoxia (gray band) effect, #significant inhibitor effect (p<0.05).

Article Snippet: For BDNF exposure, intact PA or isolated PASMCs were incubated for 24 or 48 h (see protocols) at 37°C in regular growth media (vehicle control) or 100 pM recombinant human BDNF (#248BD, R&D Systems, Minneapolis, MN).

Techniques: CyQUANT Assay, Neutralization, Inhibition, Proliferation Assay, Western Blot, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

doi: 10.1074/jbc.m800668200

Figure Lengend Snippet: FIGURE 2. BDNF enhances bone/cementum-related protein mRNA expression in HCEM cells via TrkB. A, time course effect. HCEM cells were exposed to BDNF (20 ng/ml) for the periods indicated before the end of incubation on day 7. B, dose-dependent effect. HCEM cells were exposed to increasing concentrations of BDNF for 12 h before the end of incubation on day 7. Graphs show the ratio of OPN, ALP, or BMP-2 mRNA to GAPDH mRNA. Values represent means S.D. of three cultures. *, p 0.01; value differs significantly from the control (analysis of variance). C and E, effectiveness of siRNA of TRKB and p75NTR. HCEM cells, having been transfected withnegativecontrol(Neg),TRKB,orp75NTRsiRNA,wereculturedfor2days.TheeffectivenessofthesiRNAswas confirmed by real time PCR. Graphs show the ratio of TRKB or p75NTR mRNA to GAPDH mRNA. Values represent means S.D. of three cultures. *, p 0.01; value differs significantly (t test). D and F, role of TrkB and p75NTR in the enhancement of bone/cementum-related protein mRNA expression by BDNF. HCEM cells, having been transfected with negative control (Neg), TRKB (D), or p75NTR (F) siRNA, were cultured for 2 days and then exposed to 20 ng/ml BDNF for 12 h. Total RNA was isolated from the cells, and mRNA levels of OPN, ALP, and BMP-2 were determined by real time PCR. Values represent means S.D. for three cultures. *, p 0.01; value differs significantly (t test).

Article Snippet: The HCEM cells were pretreated with or without PD98059 (50 M, Calbiochem), SB203580 (10 M, Calbiochem), SP600125 (10 M, Calbiochem), or PDTC (10 M, Cal- biochem) for 30 min and then exposed to various concentrations of human recombinant BDNF (R & D Systems, Minneapolis, MN) for specified periods before the end of incubation in -MEMsupplementedwith penicillinG solution (100 units/ml) and streptomycin (100 g/ml) (mediumC).

Techniques: Expressing, Incubation, Control, Transfection, Real-time Polymerase Chain Reaction, Negative Control, Cell Culture, Isolation

Journal: Journal of Biological Chemistry

Article Title: Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

doi: 10.1074/jbc.m800668200

Figure Lengend Snippet: FIGURE 3. Involvement of the ERK1/2 signaling pathway in the BDNF-induced enhancement in bone/ cementum-related protein mRNA levels and calcification in HCEM cells. A and B, effect of an ERK inhibitor and an NF-B inhibitor on BDNF-induced bone/cementum-related protein mRNA expression. HCEM cells, having been pretreated with PD98059, an ERK inhibitor (50 M) (A), or PDTC, an NF-B inhibitor (10 M) (B), for 30 min, were exposed to 20 ng/ml BDNF for 12 h before the end of incubation on day 7. mRNA expression of ALP, OPN, and BMP-2 was assayed by real time PCR. The graphs show the ratio of OPN, ALP, and BMP-2 mRNA to GAPDH mRNA. Values represent means S.D. for three cultures. *, p 0.01; value differs significantly (t test). C, calcification induced by BDNF. HCEM cells were cultured in mineralizing media containing BDNF (20 ng/ml), PD98059 (50 M), or PDTC (10 M). Media were changed every other day. Alizarin red S staining was performed on day 14 to visualize mineral deposition. D, activation of ERK1/2 by BDNF. HCEM cells were exposed to BDNF (20 ng/ml) for the periods indicated before the end of incubation on day 7. E, effect of an ERK inhibitor on the activation of ERK1/2 by BDNF. HCEM cells, having been pretreated with PD98059 (50 M) or PDTC (10 M) for 30 min, were cultured in the presence or absence of BDNF for 10 min. F, role of TrkB in the activation of ERK1/2 by BDNF. HCEM cells, having been transfected with negative control (Neg) and TRKB siRNA, were cultured for 2 days and exposed to BDNF (20 ng/ml) for 10 min. The activity of phosphorylated ERK1/2 and the total ERK1/2 levels in the cell lysates were analyzed by immunoblotting.

Article Snippet: The HCEM cells were pretreated with or without PD98059 (50 M, Calbiochem), SB203580 (10 M, Calbiochem), SP600125 (10 M, Calbiochem), or PDTC (10 M, Cal- biochem) for 30 min and then exposed to various concentrations of human recombinant BDNF (R & D Systems, Minneapolis, MN) for specified periods before the end of incubation in -MEMsupplementedwith penicillinG solution (100 units/ml) and streptomycin (100 g/ml) (mediumC).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Staining, Activation Assay, Transfection, Negative Control, Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

doi: 10.1074/jbc.m800668200

Figure Lengend Snippet: FIGURE4.NucleartranslocationofERK1/2byBDNFinHCEMcells.A–E,immunofluorescenceoftotalERK1/2 in HCEM cells. Total ERK is visualized toward the z axis direction. Each panel represents serial images (1 m) through the thickness of the cell. A–C, HCEM cells, having been pretreated with PD98059 (50 M) for 30 min, were exposed to 20 ng/ml BDNF for 12 h before the end of incubation on day 7. A, control; B, BDNF; C, PD98059 and BDNF. D and E, HCEM cells, having been transfected with negative control or TRKB siRNA, were cultured for 2 days and then exposed to 20 ng/ml BDNF for 10 min. D, TRKB siRNA and BDNF. E, negative control siRNA and BDNF. Scale bars, 10 M. F, immunoblotting of ERK1/2 in HCEM cells. HCEM cells were cultured as described above. Cytoplasmic and nuclear proteins were separately extracted with NE-PER nuclear and cytoplasmic extraction kit as described under “Experimental Procedures.” Neg, negative control.

Article Snippet: The HCEM cells were pretreated with or without PD98059 (50 M, Calbiochem), SB203580 (10 M, Calbiochem), SP600125 (10 M, Calbiochem), or PDTC (10 M, Cal- biochem) for 30 min and then exposed to various concentrations of human recombinant BDNF (R & D Systems, Minneapolis, MN) for specified periods before the end of incubation in -MEMsupplementedwith penicillinG solution (100 units/ml) and streptomycin (100 g/ml) (mediumC).

Techniques: Incubation, Control, Transfection, Negative Control, Cell Culture, Western Blot, Extraction

Journal: Journal of Biological Chemistry

Article Title: Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

doi: 10.1074/jbc.m800668200

Figure Lengend Snippet: FIGURE 5. Involvement of Elk-1 in the BDNF-induced increase in bone/cementum-related mRNA levels in HCEM cells. A–C, HCEM cells, having been pretreated with PD98059 (50 M) for 30 min, were exposed to 20 ng/ml BDNF for 12 h before the end of incubation on day 7. Phosphorylated Elk-1 was observed using immu- nofluorescence microscopy in HCEM cells. A, control; B, BDNF; C, PD98059 and BDNF. D and E, HCEM cells, havingbeentransfectedwithnegativecontrolorTRKBsiRNA,wereculturedfor2daysandexposedto20ng/ml BDNF for 10 min. D, TRKB siRNA and BDNF. E, negative control siRNA and BDNF. Scale bars, 10 M. F, effective- ness of ELK-1 siRNA. HCEM cells, having been transfected with negative control (Neg) or ELK-1 siRNA, were cultured for 2 days. The effects of siRNAs were confirmed by real time PCR. Values represent means S.D. for three cultures. *, p 0.01; value differs significantly (t test). G, role of Elk-1 in the elevation of bone/cementum- related protein mRNA levels by BDNF. HCEM cells, having been transfected with negative control (Neg) or Elk-1 siRNA, were cultured for 2 days, and exposed to 20 ng/ml BDNF for 12 h. Total RNA was isolated from HCEM cells,andmRNAexpressionofOPN,ALP,andBMP-2wasdeterminedbyrealtimePCR.Thegraphsshowtheratio of OPN, ALP, or BMP-2 mRNA to GAPDH mRNA. Values represent means S.D. for three cultures. *, p 0.01; value differs significantly (t test).

Article Snippet: The HCEM cells were pretreated with or without PD98059 (50 M, Calbiochem), SB203580 (10 M, Calbiochem), SP600125 (10 M, Calbiochem), or PDTC (10 M, Cal- biochem) for 30 min and then exposed to various concentrations of human recombinant BDNF (R & D Systems, Minneapolis, MN) for specified periods before the end of incubation in -MEMsupplementedwith penicillinG solution (100 units/ml) and streptomycin (100 g/ml) (mediumC).

Techniques: Incubation, Microscopy, Control, Negative Control, Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Isolation

Journal: Journal of Biological Chemistry

Article Title: Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

doi: 10.1074/jbc.m800668200

Figure Lengend Snippet: FIGURE6.EffectofBDNFonc-Raf,anupstreamtargetofERK1/2.A,phospho- rylationofc-RafatSer-338byBDNF.HCEMcellswereexposedtoBDNF(20ng/ml) for the periods indicated before the end of incubation on day 7. B and C, role of TrkBinthephosphorylationofc-RafatSer-338byBDNF.HCEMcells,havingbeen transfected with negative control (Neg), TRKB (B), and p75NTR (C) siRNA were cultured for 2 days and exposed to BDNF for 10 min. The phosphorylated c-Raf and total Raf levels in the cell lysates were analyzed by immunoblotting.

Article Snippet: The HCEM cells were pretreated with or without PD98059 (50 M, Calbiochem), SB203580 (10 M, Calbiochem), SP600125 (10 M, Calbiochem), or PDTC (10 M, Cal- biochem) for 30 min and then exposed to various concentrations of human recombinant BDNF (R & D Systems, Minneapolis, MN) for specified periods before the end of incubation in -MEMsupplementedwith penicillinG solution (100 units/ml) and streptomycin (100 g/ml) (mediumC).

Techniques: Incubation, Transfection, Negative Control, Cell Culture, Western Blot