Effect of BMPRII on endothelial function in human lung microvascular endothelial cells

Abstract

Bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension (PAH) and mutations in the BMPR2 gene are associated with both heritable and idiopathic PAH. Endothelial dysfunction is a key process in initiation and progression of PAH pathogenesis. We aimed to investigate whether loss of BMPRII could affect endothelial function, including endothelial integrity and angiogenesis of pulmonary lung microvascular endothelial cells (HLMVEC). BMPR2 knockdown was performed in HLMVEC using lentiviral vectors encoding microRNA-based hairpins against BMPR2. HLMVEC lines were established and phenotyped. BMPRII protein expression and activation of BMPRII downstream effectors, SMAD and p38MAPK, were investigated by Western blotting. Effects of BMPR2 knockdown on endothelial integrity using a permeability assay and angiogenic capacities were further evaluated in vitro using migration and tube formation assays. Stable BMPR2 silencing did not affect HLMVEC phenotype, resulted in i) a 87% decrease in BMPRII protein expression, ii) a 50% decrease in SMAD protein activation and iii) a 48% increase in p38 MAPK phosphorylation. Interestingly, loss of BMPRII resulted in a 24% significant decrease in barrier function and a 29% decrease in migration capacity and impaired 3D-angiogenesis in vitro. Stable BMPR2 silencing in HLMVECs results in a shift in attenuated activation of the canonical SMAD signaling into enhanced activation of the non-canonical p38 MAPK signaling. In addition, impaired BMPRII signaling induced a loss of barrier function and decreased angiogenic capacities indicating the involvement of BMPRII in endothelial dysfunction in PAH.