A sex-specific microRNA-96/5-hydroxytryptamine 1B axis influences development of pulmonary hypertension

Rationale: Females are predisposed to pulmonary arterial hypertension (PAH); evidence suggests that serotonin, mutations in the bone morphogenetic protein receptor (BMPR) II gene, and estrogens influence development of PAH. The 5-hydroxytryptamine 1B receptor (5-HT_1BR) mediates human pulmonary artery smooth muscle cell (hPASMC) proliferation. Objectives: We aimed to determine whether selected microRNAs (miRNAs) expressed in PASMCs are influenced by sex, BMPR-II mutations, and estrogens, and contribute to PASMC proliferation in PAH. Methods: Expression levels of miRNAs targeting genes related to PAH, estrogen, and serotonin were determined by quantitative RT-PCR in hPASMCs and mouse PASMCs harboring a heterozygous mutation in BMPR-II (BMPR-II^R899X+/- PASMCs). miRNA-96 targets 5-HT_1BR and was selected for further investigation. miRNA target validation was confirmed by luciferase reporter assay. Precursor miRNA-96 was transfected into hPASMCs to examine effects on proliferation and 5-HT_1BR expression. The effect of a miRNA-96 mimic on the development of hypoxic pulmonary hypertension in mice was also assessed. Measurements and Main Results: miRNA-96 expression was reduced in BMPR-II^R899X+/- PASMCs from female mice and hPASMCs from female patients with PAH; this was associated with increased 5-HT_1BR expression and serotonin-mediated proliferation. 5-HT_1BR was validated as a target for miRNA-96. Transfection of precursor miRNA-96 into hPASMCs reduced 5-HT_1BR expression and inhibited serotonin-induced proliferation. Restoration of miRNA-96 expression in pulmonary arteries in vivo via administration of an miRNA-96 mimic reduced the development of hypoxia-induced pulmonary hypertension in the mouse. Conclusions: Increased 5-HT_1BR expression may be a consequence of decreased miRNA-96 expression in female patient PASMCs, and this may contribute to the development of PAH.