ET-1 release in response to BMPs and ET-1 mRNA expression by BMP-9 stimulation.

<p>HPAECs were grown to confluence on 96-well plates or 6-well plates and starved for 16 hrs. Cells were then stimulated with various concentrations of BMP-2, n = 4 (A), BMP-4, n = 7 (B), BMP-6, n = 5 (C) or BMP-9, n = 6 (D). Supernatants were collected at 24 hrs of treatment and ET-1 level assayed by ELISA. RNA was extracted from cells treated with increasing concentration of BMP-9 for 24 hrs. Id-1 gene expression was determined by qRT-PCR and normalised to the average of 2 housekeeping genes and shown relative to expression of non-stimulated controls, n = 3 (E). RNA was extracted from cells treated with 1 ng/ml BMP-9 at 2 hrs and 24 hrs time points and ET-1 gene expression determined by qRT-PCR. ET-1 was normalised to the average of 2 housekeeping genes and shown relative to expression of non-stimulated controls at 2 hr and 24 hs, n = 4 (F). Data are presented as mean ± SEM. *p<0.05, **p<0.01, *** p<0.001.</p

Effects of Smad signalling pathway on BMP-9 induced prepro ET-1 mRNA transcription and ET-1 release.

<p>HPAECs were grown to confluence in 6-cm dishes, starved for 16 hrs and then stimulated with BMP-9 (1 ng/ml). Phosphorylation of Smad1/5 and Smad2 were determined by western blot (A). After transfected with Smad4 specific or non-targeting siRNA and starved for 16 hs, HPAECs were stimulated with BMP-9 (1 ng/ml). The knockdown efficiency was determined by western blotting (B). After siRNA treatment and BMP-9 stimulation, supernatants and cells were collected at 24 hrs (6-well plates) for determination of ET-1 peptides by ELISA, n = 4 (C) and mRNA levels by qRT-PCR, n = 4 (D). HPAECs were transfected with Smad1, Smad5 or non-targeting siRNA. Specific Smad1 and Smad5 knockdown were confirmed by qRT-PCR (not shown) and western blotting (E). After siRNA treatment, HPAECs were starved and stimulated with BMP-9 (1 ng/ml) for 24 hrs. RNA was extracted and gene expression determined by qRT-PCR. ET-1 was normalised to β-actin and is expressed relative to cells exposed to DH1 only, n = 5 (F). Data are presented as mean ± SEM. **p<0.01.</p

Effects of p38 MAPK and ERK1/2 inhibition on BMP-9 stimulated prepro ET-1 mRNA transcription and ET-1 release.

<p>HPAECs were grown to confluence, starved for 16 hrs and then pre-incubated with 1–10 µM SB203580, 1–10 µM UO126, 0.5 µM (5Z)-7-Oxozeanol or 1 µM BIRB796 for 60 min before adding BMP-9 (1 ng/ml) for another 24 hrs. Supernatants and cells were collected for determination of ET-1 by ELISA n = 4 (A, C, D, E), and prepro ET-1 mRNA levels by qRT-PCR, n = 3 (B). Data are presented as mean ± SEM. #p>0.05; * p<0.05; **p<0.01; ***p<0.001.</p

Effect of BMP-9 induced ET-1 on HPAEC migration.

<p>HPAECs were seeded onto transwell inserts and migration determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030075#s4" target="_blank">materials and methods</a> section. Representitive images of 3 independent experiments of cells (nuclei identified with DAPI staining) migrated to the lower chamber are shown in (A). Migrated cells, under the conditions described shown, were counted and expressed graphically, as the percentage of the control in (B). Data are presented as mean ± SEM, n = 3. **p<0.01.</p

Effect of BMP-9 induced ET-1 release on HPAEC tubule formation.

<p>HPAECs were seeded onto matrigel and tubule formation capacity analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030075#s4" target="_blank">materials and methods</a> section. Representitive images of 3 independent experiments of tubule formation are shown in (A). The average length of the tublule networks in the various conditions shown was quantified using Image J (NIH) and represented graphically in (B). Data are presented as mean ± SEM, n = 3. **p<0.01.</p

ERK1/2, JNK and p38 MAPK phosphorylation in HPAECs stimulated with BMP-9.

<p>HPAECs were grown to confluence in 6-cm dishes, starved for 16 hrs and then stimulated with BMP-9 (1 ng/ml). Phosphorylation of ERK1/2, JNK and p38 MAPK were determined by western blot (A), and quantified by densitometry (B, C and D). Data are presented as mean ± SEM. n = 3, #p>0.05; *p<0.05, one -way ANOVA, control vs. BMP-9 stimulation.</p

Effect of p38 MAPK inhibition and Smad1/Smad4 knock-down on BMP-9 induced HPAEC tubule formation.

<p>HPAECs or HPAECs transfected with negative control (CP), Smad1 and Smad4 siRNA were seeded onto matrigel and tubule formation capacity analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030075#s4" target="_blank">materials and methods</a> section. Representitive images of 3 independent experiments of tubule formation are shown in (A&B). The average length of the tublule networks in the various conditions shown was quantified using Image J (NIH) and represented graphically in (C). Data are presented as mean ± SEM, n = 3. ***p<0.001.</p

HOX expression validation by qPCR.

<p>a-e) HOXD1, D3, D4, D8 and D9 mRNA expression in BOECs, pulmonary artery ECs (PAECs), Aortic ECs (AECs), umbilical vein ECs (HUVECs), microvascular lymphatic ECs (HMECs), lung microvascular ECs (HMLECs). Expressed in relative arbitrary units. f) MicroRNA10 expression in BOECs and PAECs. Data was analyzed by ANOVA *p<0.05 **p<0.01.</p

The BOEC is functionally a mature endothelial cell.

<p>a) Phase microscopy of BOECs in culture demonstrates typical endothelial “cobblestone” morphology. Confocal microscopy of BOECs with nuclear counterstaining. b) CD31 (red), c) CD146 (green), d) fibronectin (green). e) Network formation in matrix gel, f) vacoulised BOECs in fibronectin/collagen matrix. g) Electron microscopy (EM) of whole BOEC showing extensive vesicles (Ves), h) EM close up of W-P bodies, i) EM of W-P bodies with immunogold staining for vWF. j-l) Neutrophil rolling adhesion and transmigration after stimulation with TNF in a flow-based assay.</p

BOECs have a near identical mRNA microarray profile to PAECs but differ significantly in HOX expression.

<p>a) Microarray expression of BOECs vs PAECs (n = 4) demonstrates high concordance of gene expression b) Heat map of HOX genes in BOECs vs PAECs. c) Venn diagram showing convergence of gene expression, d) Heat maps of differentially expressed HOXs. Additional lineage associated transcripts showing endothelial expression pattern, e) PCA analysis of BOEC vs 131 tissue arrays. Principal components analysis; Graph 1-blood derived cells (red), bowel (blue), brain (green); Graph 2 PAECs (blue), BOECs (red) demonstrating similarity of BOECs and PAECs when compared to other mature cell types.</p