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S141 BMP9 is required for LPS-mediated neutrophil recruitment to PAH-patient derived blood outgrowth endothelial cells with BMPR-II mutations
  1. SL Appleby1,
  2. J Deighton1,
  3. CM Yates2,
  4. MR Toshner1,
  5. GB Nash2,
  6. ER Chilvers1,
  7. NW Morrell1
  1. 1University of Cambridge, Cambridge, united Kingdom
  2. 2University of Birmingham, Birmingham, United Kingdom

Abstract

Introduction Heterozygous mutations in the gene encoding bone morphogenetic protein (BMP) receptor II (BMPR-II) are present in >70% of patients with heritable pulmonary arterial hypertension (hPAH) and 15–26% of idiopathic PAH (iPAH) cases. The genetic data strongly implicates the endothelium as the initiating cell type in PAH, and BMP9 is the major circulating ligand for BMPR-II on endothelial cells (EC). Since inflammation is emerging as a potential trigger for disease in BMPR-II mutation carriers, we hypothesised that BMP9 and BMPR-II mutation might impact on endothelial:leukocyte interaction.

Methods We employed blood outgrowth endothelial cells (BOEC) isolated from healthy donors (control-BOEC) and BOEC derived from PAH patients with BMPR-II mutations (PAH-BOEC) in this study.BOEC can be generated from venous blood and are recognised as a non-invasive source of patient-derived EC. Endothelial:neutrophil interaction was studied using an in vitro flow-based model. Neutrophil recruitment to LPS (100 ng/ml; 4h)-stimulated BOECs was quantified in the presence or absence of BMP9 (5 ng/ml; 16h) under physiologically relevant sheer stress (0.1 Pa). Surface expression of E-selectin was quantified using flow cytometry, ELISA was used to assess the secretion of IL-8 and mRNA levels were quantified by qPCR.

Results LPS stimulation induced robust neutrophil recruitment to control-BOEC, whereas BMP9 had no effect.Co-stimulation with LPS and BMP9 resulted in a synergistic increase in neutrophil recruitment and control-BOEC E-selectin and IL-8 expression, compared to stimulation with LPS alone. Moreover, BMP9 stimulated the upregulation of TLR4 on control-BOEC (the receptor for LPS), thus providing a potential mechanism for the enhanced inflammatory response seen with LPS and BMP9 co-stimulation. In contrast to the findings in control-BOEC, LPS stimulation failed to induce neutrophil recruitment in PAH-BOEC. However, co-stimulation with LPS and BMP9 restored neutrophil recruitment in PAH-BOEC.

Conclusions BMP9 markedly enhances neutrophil recruitment to the endothelium in response to LPS. Deficiency in BMP9 signalling (as a consequence of BMPR-II mutations in PAH-BOEC) impairs this process, and BMP9 restores normal endothelial:neutrophil interactions in PAH-BOECs. These findings identify a hitherto unknown role for BMP9 in endothelial:neutrophil interaction and implicate this process in the pathobiology of PAH.

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