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The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway. ispartof: PULMONARY CIRCULATION vol:9 issue:4 ispartof: location:United States status: published

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.

Mutations in the bone morphogenetic protein receptor 2 ( BMPR2 ) gene are associated with heritable pulmonary arterial hypertension (PAH). In addition, BMPRII signalling is impaired in idiopathic PAH. Since BMPR2 mutation carriers do not all develop PAH, inflammatory mediators could trigger the disease in BMPR2 mutation carriers. Since circulating levels and pulmonary tissue expression of interleukin-18 (IL18) are elevated in PAH, we hypothesized that IL18 could contribute to PAH pathogenesis, in addition to impaired BMPRII signalling. Lentiviral vectors encoding microRNA 30-based knockdown hairpins derived from BMPR2 siRNAs were generated and transduced in human lung microvascular endothelial cells (HLMVEC) to mimic impaired BMPR2 signalling in vitro. Effects of IL-18 on i) adhesion of HLMVEC to human monocytic cell line THP-1, endothelial integrity, adhesion molecule expression and activation of p38 MAP kinase and SMAD proteins were investigated. Knockdown of BMPR2 in HLMVEC resulted in decreased activation of SMAD proteins, increased activation of p38 MAP kinase and endothelium permeability. In BMPR2 knocked-down HLMVEC, IL18 and TNFα increased adhesion to monocytes by 35% and 77%, respectively and activated p38 MAP kinase. Interestingly, TNFα induced mRNA IL18 receptor expression in BMPR2 knocked-down HLMVEC. To conclude, in BMPR2 knocked-down HLMVEC, IL18 induced adhesion and p38 MAP kinase activation, suggesting that IL-18 could trigger PAH pathogenesis, in addition to BMPR2 impaired signalling.