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ABSTRACT: BACKGROUND: Many cases of pulmonary arterial hypertension (PAH) are heritable and related to gene mutations in bone morphogenic receptor-2 (BMPR2). These patients consequently may have a signaling imbalance within the transforming growth factor beta (TGFβ) receptor superfamily. The causes of increased endothelin-1 (ET-1), which contributes to PAH, are unknown, and we therefore studied the contribution of various BMPs and their receptors on ET-1 production in vitro, after knockdown of BMPR2 in human pulmonary microvascular endothelial cells (HMVEC-LBl). METHODOLOGY/PRINCIPAL FINDINGS: Receptor knockdown in HMVEC-LBl was performed using siRNA to BMPR2, and activin like-kinases 1 and 2 (ALK1, ALK2). ET-1 and TGFβ levels in the medium were measured by ELISA. In some experiments, cells were exposed to TGFβ or BMP7 or FK506 (tacrolimus). Using Western blotting, levels of BMPR2, endothelin ET(B) receptor, phosphorylated Smad 2 (pSmad 2), phosphorylated Smad 1,5 (pSmad 1,5), ALK1, ALK2, ALK5, TGFβ receptor 2, plasminogen activator inhibitor-1 (PAI-1) and ID1 were measured. BMPR2 knockdown significantly increased ET-1 levels. It did not affect ET(B) receptor or TGFβ levels. TGFβ increased ET-1 levels, with or without BMPR2 knockdown. BMPR2 knockdown did not affect TGFβ (pSmad 2 and PAI-1) signaling. BMP7 increased ET-1 levels after BMPR2 knockdown but this was prevented by ALK2 knockdown as was the increase in ID1 caused by BMPR2 knockdown. FK506, which interacts with ALK2, increased ET-1 levels and ID1 levels, and this was blocked by ALK2 knockdown. CONCLUSION/SIGNIFICANCE: ALK2 may be an important receptor in ET-1 production during BMPR2 knockdown.
Microvascular Research 11/2012; · 2.83 Impact Factor
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ABSTRACT: Abnormalities of signalling for the transforming growth factor beta (TGFβ) family of peptides, including bone morphogenic proteins (BMP), have been described in heritable pulmonary arterial hypertension (PAH). TGFβ can modulate synthesis of the vasoconstrictor and mitogen, endothelin-1 (ET-1), a mediator that contributes to the pathogenesis of PAH. BMP-9 is a circulating peptide recently recognized to affect endothelial function. The stimuli for increased microvascular endothelial production of ET-1 in PAH are unknown. We therefore studied the effects of BMP-9 on ET-1 production by human lung blood microvascular endothelial cells (HMVEC-LBl) in vitro. In vitro, BMP-9 increased ET-1 production by HMVEC-LBl. The effect was identical to TGFβ-1, but BMP-9 and TGFβ-1 combined further increased ET-1 levels by 29%. As compared to TGFβ-1, BMP-9 induced more potent and rapid phosphorylation of Smad 1/5, the downstream signalling molecules of the activin-like kinase 1 (ALK-1) receptor. Moreover, as has been previously shown for endothelial cells of other origin, BMP-9 also induced Smad 2 phosphorylation in HMVEC-LBl. In conclusion, BMP-9 stimulates ET-1 production by HMVEC-LBl in vitro. BMP-9 signals via several Smad pathways. These studies provide novel mechanisms for the potentiation of PAH.
Microvascular Research 12/2010; 80(3):349-54. · 2.83 Impact Factor
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ABSTRACT: Altered endothelial cell (EC)-derived mediator levels, including increased endothelin-1 (ET-1), are hallmarks of human pulmonary arterial hypertension (PAH). Gene mutations for receptors for bone morphogenic proteins (BMP), or transforming growth factor-beta (TGF-beta) cause heritable PAH. The effects of BMPs and TGF-beta on ET-1 production by human pulmonary microvascular EC (HMVEC-LBl) are unknown.
HMVEC-LBl were exposed in-vitro to BMPs 2, 4, and 7 or TGF-beta1 in basal or complete medium. ET production was measured, as well as total cellular protein. Levels of Smad 5 and phosphorylated Smads 1/5 were also measured.
BMP-4 did not increase ET-1 while BMP-2 increased it minimally in basal medium. BMP-7 increased ET-1, but only at 100 ng/ml. By contrast, TGF-beta increased ET-1 throughout most of the studied dose range. All BMPs and TGF-beta increased levels of phosphorylated Smads 1/5 without depleting levels of Smad 5.
With the exception of BMP-7 at high-concentrations, the BMPs that interact with BMP receptor 2, the receptor implicated in heritable PAH, do not or minimally modulate in-vitro constitutive ET-1 production by HMVEC-LBl. TGF-beta increases ET-1 synthesis, and this may have clinical relevance in PAH.
Vascular Pharmacology 10/2008; 50(1-2):45-50. · 1.99 Impact Factor
