BMPR2 gene delivery shifts intracellular Smad activation profile

PAH is a fatal disease associated with decreased BMPR2 and increased TGF-β mediated cellular signalling. We have shown that BMPR2 upregulation in pulmonary endothelium reduces PAH in vivo, but the downstream signalling effects are unknown. BMPR2 is thought to signal primarily via Smad 1/5/8 and TGF-β via Smad 2/3, thus we hypothesised that BMPR2 upregulation may alter the relative activity of these pathways. Aims and Objectives: To assess the effect of BMPR2 upregulation on downstream Smad signalling in endothelial cells, and the response to BMP ligands. Methods: Human Microvascular Endothelial Cells (HMVEC) were transduced with an Adenoviral vector carrying the BMPR2 gene, and then stimulated with BMP7 or 9. Results: Efficient upregulation of BMPR2 was confirmed by western blot and persisted beyond 48 hours. Incubation with BMP7 and 9 showed similar increases in p-Smad1/5/8 and Smad 5 at 24 hours following transduction, but by 48 hours after transduction, BMP7 effects were significantly greater than BMP9. Interestingly, BMP9 stimulated cells had increased Endoglin compared to BMP7 stimulated cells at 48hrs. Both ligands lead to reduced p-Smad 3 with a similar profile. Conclusion: BMPR2 upregulation shifts the intracellular signalling profile toward Smad 1/5/8, and away from Smad 3 consistent with an alteration in downstream TGF-β/BMPR2 pathways, which may in part explain the effects on PAH seen in vivo. There may be differential effects depending on the BMP ligand involved, which requires further analysis. Supported by NHMRC & NHF.