Women develop pulmonary arterial hypertension (PAH) more frequently than men suggesting that female sex and/or female sex hormones i.e. estrogens play a role in disease pathogenesis. Building evidence also implicates a role for microRNAs (miRNAs) in PAH. Little is known surrounding the interplay between sex/estrogens and miRNAs in PAH. Examining the sexual dymorphism in miRNAs with regards to PAH disease may provide insight into the sex bias observed in PAH. Loss-of-function BMPR-II mutations underlie heritable PAH. Here, we showed that in pulmonary artery smooth muscle cells (PASMCs) explanted from a pulmonary hypertensive mouse model with a knock-in BMPR-II mutation (BMPR-IIR899X+/-) there were differences in miRNA expression between sexes. Among the 20 miRNAs examined, 9 miRNAs exhibited significant change in expression within female BMPR-IIR899X+/- compared to female wild-type (WT) PASMCs but remained unchanged in male PASMCs. Of interest miRNA-96 demonstrated significant down-regulation in female BMPR-IIR899X+/- but remained unchanged in male BMPR-IIR899X+/-. In silico prediction software demonstrates the 5-HT1B receptor as a putative target of miRNA-96. The 5-HT1B receptor has previously been implicated in PAH development as it is thought to play a role in pulmonary artery vasoconstriction and pulmonary artery remodelling, two major hallmarks of PAH. To verify if 5-HT1B was a true target of miRNA-96 we carried out a 3’UTR luciferase reporter assay. Indeed we found over-expression of miRNA-96 could down-regulate the luciferase output of the luciferase reporter construct containing the 3’UTR of the 5-HT1B receptor. The down-regulation of miRNA-96 within the female BMPR-IIR899X+/- mouse PASMCs was associated with a concomitant increase in 5-HT1B mRNA and protein. This expression pattern was re-iterated in PASMCs explanted from female PAH patients. Here, we found female PAH patients had a decrease in miRNA-96 expression and an increase in 5-HT1B mRNA and protein but again expression remained unchanged in male patients compared to non-PAH controls.Interestingly we also found only female PAH patient PASMCs were proliferative to the mitogen serotonin (5-HT). This could be explained by the expression pattern of 5-HT1B observed. Next we examined the effect of miRNA-96 over-expression in human PASMCs. Here, we demonstrated that over-expressing miRNA-96 had the ability to reduce 5-HT1B protein expression; however, mRNA expression remained unchanged. In addition, we found that over-expressing miRNA-96 prevented serotonin-induced proliferation in PASMCs from female PAH patients. We have previously shown a relationship between estrogen and 5-HT1B as 17β-estradiol, the main pre-menopausal circulating estrogen, increases the protein expression of 5-HT1B in human PASMCs. Here, we examined the effect of 17β-estradiol on miRNA-96 expression. We found that 17β-estradiol decreased miRNA-96 expression suggesting 17β-estradiol’s effect on 5-HT1B expression could be mediated through a decrease in miRNA-96. To determine if endogenous 17β-estradiol also influences miRNA-96 expression we assessed the expression of miRNA-96 and 5-HT1B expression within whole lung homogenates from female and male mice that had been dosed with an aromatase (estrogen synthesising enzyme) inhibitor anastrozole. These mice have depleted circulating and local lung synthesis of estrogen, and elevated BMPR-II signalling. MiRNA-96 was elevated in the lungs from the estrogen-depleted female mice and this was accompanied by a decrease in 5-HT1B mRNA expression. No changes in miRNA-96 and 5-HT1B mRNA expression were observed within male lung tissue. These results further implicated a role for estrogen in regulating miRNA-96 and subsequently 5-HT1B.As over-expression of miRNA-96 in cell culture prevented a proliferative phenotype in human PASMCs we sought to assess the effect of over-expressing miRNA-96 in vivo. We utilised both the BMPR-IIR899X+/- and hypoxic mouse model of pulmonary hypertension (PH) to examine whether a miRNA-96 mimic could both reverse and prevent a PH phenotype. The miRNA-96 mimic was administered intravenously via the tail vein once a week for 2 weeks using the MaxSuppressor™ In Vivo RNA-LANCEr II delivery method. We first confirmed that in both of these models miRNA-96 was significantly depleted in the lungs of diseased mice. Secondly we showed that intravenous injection delivered miRNA-96 mimic to the pulmonary arteries. Our in vivo results demonstrated that dosing with miRNA-96 mimic reduced the right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and % of remodelled vessels in hypoxic and BMPR-IIR899X+/- female mice. Mice dosed with a negative control mimic showed no effect on PAH indices. Interestingly we also showed that hypoxic and BMPR-IIR899X+/- mice dosed with miRNA-96 mimic had a reduction in 5-HT1B protein expression compared to those dosed with negative control mimic. This is the first study to observe sexual dimorphism in miRNA expression with regards to PAH. We have provided novel data demonstrating how miRNA-96, under the potential influence of estrogen, plays a role in the development of PH in a sex-dependent manner, by regulating 5-HT1B expression and serotonin-induced proliferation. Restoring depleted miRNA-96 levels may present a novel therapeutic approach in PAH.
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A sex-specific microRNA-96/5HT1B axis influences development of pulmonary hypertension
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