Fig 2: Prodomain-bound BMP10 is active in endothelial cells. A and B, phosphorylation of Smad1/5 in HPAECs (A) and HAECs (B) treated with 0.1 ng/ml, 0.33 ng/ml, and 1 ng/ml of BMP10 GFD or three independent preparations of pBMP10 for 1 h, detected by Smad1/5 phosphorylation in immunoblotting analysis; total Smad1 was used as a loading control. The concentrations of pBMP10 in all the cell assays refer to the concentrations of mature GFD in the pBMP10 complex. Relative phosphorylation of Smad1/5 upon treatment was measured using densitometry, corrected to total Smad1 and normalized to 0.1 ng/ml BMP10 GFD treatment condition and plotted on the right. C, induction of ID1 and BMPR2 mRNA expression by recombinant pBMP10, compared with BMP9 and BMP10 GFD. HPAECs were treated with the ligands at indicated concentrations for 8 h before samples were harvested for RNA extraction and qPCR analysis as described in “Experimental Procedures.” n = 3; *, p = 0.05; n.s., not significant; paired t test.

Fig 3: Generation of recombinant human pBMP10. A, schematic diagram of BMP10 production and processing. B, FPLC chromatography gel filtration trace of purified pBMP10. C, purified pBMP10 shown as the peak fraction from the gel filtration in B on a non-reducing SDS-PAGE. A single asterisk denotes a nonspecific protein. D, both D- and M-forms of BMP10 GFD could be detected by monoclonal anti-BMP10 antibody. E, prodomain can be detected by anti-BMP10 prodomain antibody. BMP10 GFD from R&D Systems was used as a positive control in D and negative control in E.

Fig 4: BMP10 derived from atrium or plasma is fully active. A, BMP10 mRNA expression in human heart tissues. n = 3. **, p = 0.01; one-way ANOVA, Tukey's post test. B, BMP10 mRNA expression in mouse heart tissues. BMP10 expression is significantly higher in RA than LA in mouse. n = 4, paired t test; **, p = 0.01; C, BMP activity could be detected in the conditioned medium of cultured mouse RA. Conditioned medium from LA or RA was applied to serum-starved HPAECs (both at 5% v/v), and the BMP activity was measured by the induction of ID1 gene expression. No BMP activity can be detected from LA-conditioned medium (LA CM), while significant level of ID1 gene induction activity can be detected in the RA-conditioned medium (RA CM). n = 3, * p = 0.05; n.s., not significant. One-way ANOVA, Tukey's post test; D, identification of BMP activity in RA CM. The ID1-induction activity from RA CM (0.4% v/v) could not be inhibited by anti-BMP9 antibody (at 20 µg/ml), but can be partially inhibited by anti-BMP10 antibody (at 20 µg/ml), and very effectively inhibited by ALK1-Fc (at 2.5 µg/ml). n = 3, *, p = 0.05; n.s., not significant. One-way ANOVA, Dunnett's post test; E, control experiments showed that anti-BMP9 antibody (at 10 µg/ml) could specifically neutralize BMP9 activity very effectively, but not the activity of BMP10 or pBMP10, whereas ALK1-Fc (at 2.5 µg/ml) can inhibit both BMP9 and BMP10 activity very effectively. The concentrations of BMP9, BMP10, and pBMP10 used in this assay were all 1 ng/ml. n = 3, one-way ANOVA for each BMP ligand group, Dunnett's post test, **, p = 0.01; *, p = 0.05; n.s., not significant; F, BMP10 activity can be detected in human plasma. Freshly frozen human plasma was used to treat serum-starved HPAECs (1% v/v final concentration), and BMP activity was measured by ID1 gene induction. All the ID1-induction activity from 1% plasma can be completely inhibited by ALK1-Fc (at 2.5 µg/ml) alone, suggesting that all the ID1-gene induction activity in 1% plasma was due to BMP9 and BMP10. While most of this activity can be inhibited by anti-BMP9 antibody (at 15 µg/ml), the residual ID1-induction activity cannot be inhibited by additional amounts of anti-BMP9 antibody (at 20 µg/ml). It can be only inhibited by either anti-BMP10 antibody (at 15 µg/ml) or ALK1-Fc (at 2.5 µg/ml), suggesting the residual ID1-induction activity is due to BMP10. n = 3, one-way ANOVA, Dunnett's post test. *, p = 0.05; #, p = 0.05; ##, p = 0.01; n.s., not significant.

Fig 5: BMP10 prodomain does not inhibit BMP10 activity in endothelial cell lines. A, titration of BMP10 GFD activities in HPAECs (left) and HAECs (right). Increasing concentrations of BMP10 GFD as indicated were used to treat the serum-starved cells. After 1 h treatment, cells were harvested and the phosphorylation of Smad1/5 and the induction of ID1 and ID3 proteins were analyzed using immunoblotting. B, BMP10 GFD was pre-incubated with increasing amounts of BMP10 prodomain in same molar ratio as in Fig. 1B before applying to serum-starved endothelial cells in (B) HPAECs, (C) HAECs, and (D) HMEC-1. Remaining activity of BMP10 was measured by phosphorylation of Smad1/5 with total Smad1 as a loading control. One representative blot from four repeats is shown for all experiments. Prodomain inhibitions were quantified and analyzed as in Fig. 1B and shown below.