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S35 Bmp9 And Bmp10 Mediate Connexin Expression In Endothelial Cells: Implications For Pah And Hht
  1. PD Upton,
  2. JM Wilkinson,
  3. KA Wiggins,
  4. NW Morrell
  1. University of Cambridge, Cambridge, UK

Abstract

Background Germ-line mutations in the bone morphogenetic protein type-II receptor, BMPR-II, underlie 80% of heritable pulmonary arterial hypertension (PAH) cases and approximately 25% of idiopathic PAH cases. PAH may arise due to endothelial dysfunction as mice with BMPR-II deficiency exhibit increased pulmonary vascular permeability.

BMP9 is an endothelial quiescence factor and is thought to maintain the integrity of the endothelium. We previously reported that BMPR-II and ALK1 are the key receptors through which BMP9 inhibits the proliferation of human pulmonary artery endothelial cells (hPAECs). We hypothesised that BMPR-II deficiency impacts on endothelial cell connectivity and may contribute to endothelial dysfunction in PAH.

Methods Human pulmonary artery endothelial cells were obtained from Lonza and blood outgrowth endothelial cells (BOECs) were isolated from peripheral blood of unaffected controls or PAH patients with identified BMPR-II mutations. Cells were transfected with siRNAs targeting BMPR-II followed by stimulation with BMP9. RNA was extracted and the expression of candidate genes determined by quantitative PCR. Further siRNA studies were performed for ALK1 and endoglin siRNAs. The promotion of gap junction assembly by BMP9 and BMP10 were assessed by immunofluorescence, Western blotting and functionally using parachute assays.

Results Screening of candidate BMP9-induced junctional and structural proteins highlighted a subset of endothelial connexins that are BMP9 and BMP10-responsive and dependent on BMPR-II and ALK1. BMP9 and BMP10 increased the expression of the connexins, assessed by Western blotting and immunostaining. In addition, BMP9 and BMP10 significantly increased the transfer of calcein from labelled donor cells to unlabelled acceptor cells, indicating a promotion of endothelial cell connectivity.

Conclusion In addition to their roles promoting endothelial quiescence, BMP9 and BMP10 directly promote endothelial cell connectivity by increasing connexin expression and assembly. The central contributions of BMPR-II and ALK1 to this process may implicate impaired endothelial connectivity as a pathological component of PAH and HHT.

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