Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG activates pro-proliferative signalling and a disease-relevant transcriptome. Panel ( a ) Signalling Pathway Impact Analysis (SPIA) with each pathway represented by one dot. The pathways to the right of the red diagonal line are significant after Bonferroni correction of the global p -values obtained using Fisher’s methods from the combination of pPERT and pNDE values, the pathways to the right of the blue line are significant after FDR correction. ( b ) shows a heat map of significant differentially regulated genes after gene enrichment against PAH-associated genes in OPG stimulated PASMCs, ( c ) TaqMan validation of gene expression microarray, TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Panel ( d ) shows a heat map of cell cycle/CDK proteins significantly regulated by OPG at 10 and 60 min expressed as a ratio to unstimulated controls from the same time point from Kinex phospho-arrays identified, with ( e ) showing those specifically related to NF-κβ. f Western blot validation of Kinex array data in unstimulated (0.2% FCS, Un) or OPG-stimulated (50 ng ml −1 ) PASMCs at 10 min (10) and 60 min (60) with relative band densities of phospho-ERK1/2, phospho-HSP27, phospho-mTOR, phospho-CDK4 and total CDK5 are shown by the bar graphs and representative western blot images shown above the graph. Heat maps show Z-ratio gene or protein expression. Bars represent mean with error bars showing the standard error of the mean, n = 3 for pooled triplicate samples ( a , b ), n = 12 ( c ), n = 4 ( d , e ), n = 5 ( f ) from three donors of PASMCs, dots represent experimental repeats. Bars from unstimulated cells are white, OPG stimulated blue. * p < 0.05, ** p < 0.01, *** p < 0.001 compared OPG-stimulated to unstimulated PASMCs using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Biomarker Discovery, Gene Expression, Microarray, Expressing, Control, Western Blot

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG binds to Fas, which is increased in IPAH lung and right ventricle. Panel ( a ) demonstrates confirmed protein binding between OPG and syndecan-1 (SDC-1), RANKL (TNFSF11), Growth Associated Protein 43 (GAP43), Fas, IL1-receptor accessory protein (IL-1RAcP) and transmembrane protease, serine 11D. b TaqMan expression of Fas, IL-1RAcP and GAP43 in control (white bars, 0.2% FCS) and OPG-stimulated (blue bars, 50 ng ml −1 ) purchased PASMCs, and ( c ) PASMCs from patients with IPAH (grey bars) and healthy controls (white bars). d Anti-Fas co-immunoprecipitation of OPG in endogenous primary human PASMC lysates or recombinant protein replicated 3 times. e OPG and Fas are expressed within remodelled pulmonary arteries and the right ventricle of patients with IPAH. TaqMan expression of Fas in whole lung RNA (f) and protein expression in lung sections ( g ) isolated from control (saline), monocrotaline (d28), control (normoxia) and SuHx (wk9) rats. TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Bars represent the mean with error bars showing the standard error of the mean. Panel ( c ) n = 4 and panel ( d ) n = 3 from three individual donors, dots represent experimental repeats. * p < 0.05, ** p < 0.01, *** p < 0.001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used. Scale bar represents 25 µm

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Protein Binding, Expressing, Control, Immunoprecipitation, Recombinant, Isolation, Saline

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF & OPG stimulations) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Expressing, Inhibition, Migration

Journal: The Journal of Experimental Medicine

Article Title: The angiopietin-1–Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice

doi: 10.1084/jem.20090389

Figure Lengend Snippet: Effect of 5-HT or IL-6 on Ang1 protein levels in vivo and in vitro. (A) Decreased lung Ang1 protein levels in WT and Tie2 +/− mice after exposure to 5-HT (A; n = 5 mice per group) or IL-6 (B; n = 5 mice per group) for 1 wk. (C and D) Decreased Ang1 protein secretion measured by ELISA in pulmonary artery SMCs serum starved overnight and stimulated with 10 −8 –10 −5 M 5-HT (C; n = 4 per group) or equal concentrations of a combination of IL-6 and sIL-6R (10, 50, and 100 ng/ml; D; n = 4 per group) for 24 h. Results in A and B are from two independent experiments; results in C and D are from four independent experiments. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01 versus saline (in vivo) or control (in vitro).

Article Snippet: Human pulmonary artery SMCs (Lonza, Ltd.) at 70–80% confluency were serum starved in basal medium with 0.1% BSA for 24 h and exposed to 5-HT (Sigma-Aldrich) at concentrations of 10 −8 –10 −5 M for 24 h. In a separate experiment, serum-starved cells were exposed to a combination of human IL-6 and human sIL-6R (IL-6 + sIL-6R; Sigma-Aldrich), each at concentrations of 10, 50, and 100 ng/ml for 24 h. At the end of the experiments, supernatants were collected for ELISA and cells were harvested for protein quantification.

Techniques: In Vivo, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG activates pro-proliferative signalling and a disease-relevant transcriptome. Panel ( a ) Signalling Pathway Impact Analysis (SPIA) with each pathway represented by one dot. The pathways to the right of the red diagonal line are significant after Bonferroni correction of the global p -values obtained using Fisher’s methods from the combination of pPERT and pNDE values, the pathways to the right of the blue line are significant after FDR correction. ( b ) shows a heat map of significant differentially regulated genes after gene enrichment against PAH-associated genes in OPG stimulated PASMCs, ( c ) TaqMan validation of gene expression microarray, TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Panel ( d ) shows a heat map of cell cycle/CDK proteins significantly regulated by OPG at 10 and 60 min expressed as a ratio to unstimulated controls from the same time point from Kinex phospho-arrays identified, with ( e ) showing those specifically related to NF-κβ. f Western blot validation of Kinex array data in unstimulated (0.2% FCS, Un) or OPG-stimulated (50 ng ml −1 ) PASMCs at 10 min (10) and 60 min (60) with relative band densities of phospho-ERK1/2, phospho-HSP27, phospho-mTOR, phospho-CDK4 and total CDK5 are shown by the bar graphs and representative western blot images shown above the graph. Heat maps show Z-ratio gene or protein expression. Bars represent mean with error bars showing the standard error of the mean, n = 3 for pooled triplicate samples ( a , b ), n = 12 ( c ), n = 4 ( d , e ), n = 5 ( f ) from three donors of PASMCs, dots represent experimental repeats. Bars from unstimulated cells are white, OPG stimulated blue. * p < 0.05, ** p < 0.01, *** p < 0.001 compared OPG-stimulated to unstimulated PASMCs using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Expressing, Microarray, Western Blot

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG binds to Fas, which is increased in IPAH lung and right ventricle. Panel ( a ) demonstrates confirmed protein binding between OPG and syndecan-1 (SDC-1), RANKL (TNFSF11), Growth Associated Protein 43 (GAP43), Fas, IL1-receptor accessory protein (IL-1RAcP) and transmembrane protease, serine 11D. b TaqMan expression of Fas, IL-1RAcP and GAP43 in control (white bars, 0.2% FCS) and OPG-stimulated (blue bars, 50 ng ml −1 ) purchased PASMCs, and ( c ) PASMCs from patients with IPAH (grey bars) and healthy controls (white bars). d Anti-Fas co-immunoprecipitation of OPG in endogenous primary human PASMC lysates or recombinant protein replicated 3 times. e OPG and Fas are expressed within remodelled pulmonary arteries and the right ventricle of patients with IPAH. TaqMan expression of Fas in whole lung RNA (f) and protein expression in lung sections ( g ) isolated from control (saline), monocrotaline (d28), control (normoxia) and SuHx (wk9) rats. TaqMan expression data normalised using ΔΔCT with 18 s rRNA as the endogenous control gene. Bars represent the mean with error bars showing the standard error of the mean. Panel ( c ) n = 4 and panel ( d ) n = 3 from three individual donors, dots represent experimental repeats. * p < 0.05, ** p < 0.01, *** p < 0.001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test. When there were only two groups, unpaired t-tests were used. Scale bar represents 25 µm

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Protein Binding, Expressing, Immunoprecipitation, Recombinant, Isolation

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF & OPG stimulations) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 following one-way ANOVA with Bonferroni’s multiple comparisons post hoc test

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Expressing, Inhibition, Migration

Journal: Nature Communications

Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

doi: 10.1038/s41467-019-13139-9

Figure Lengend Snippet: Ky3 blocks OPG-induced proliferation, migration and NF-κβ activation. Box and whisker plots shows the inhibition of OPG-induced proliferation ( a ) and migration ( b ) in PASMC stimulated with serum-free media (SFM), PDGF or OPG in the presence of either IgG4 (grey) or Ky3 antibody (green), Fas siRNA (yellow) or non-targeting siRNA (NTsi) (white). Bar graph shows the mean with the error bars showing the standard error o the mean with ( c ) showing the activation of NF-κβ in response to OPG (blue) in the presence of either IgG4 (grey) or Ky3 antibody (green). Box and Whisker plots represent the interquartile range (box) with the line representing the median and whisker the full range of the data, each dot represents an experimental repeat, n = 6 ( a ), n = 5 ( b ) and n = 4 ( c ), * p < 0.05 following two-way ANOVA followed by Sidak’s multiple comparisons test ( a ), or one-way ANOVA with Bonferroni’s multiple comparisons post hoc test (b&c)

Article Snippet: Human PASMCs (Lonza) were stimulated in triplicate with 0.2% FCS (control) or 50 ng ml −1 OPG (Peprotech).

Techniques: Migration, Activation Assay, Whisker Assay, Inhibition