Compromised BMPR2 Signalling Results in a Sex Specific Heightened Serotonin System

E Wallace1, K. M. Mair1, X. D. Yang2, L Long2, N. W Morrell2, M. R. MacLean1. 1University of Glasgow, Glasgow, UK, 2University of Cambridge, Cambridge, UK

Pulmonary arterial hypertension (PAH) is ~ 4 times more frequent in women than men1 suggesting the influence of sex hormones. Heritable PAH (HPAH) is associated with mutations within the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) and one mutation is the R899X. In BMPR2 haploinsufficient mice exogenous serotonin uncovers a pulmonary hypertensive phenotype2. Serotonin and its predominant pulmonary receptor 5-HT1B have a well-defined role in pulmonary artery smooth muscle cell (PASMC) vasoconstriction and proliferation, two hallmarks of PAH pathology3. Yet the contribution of gender in this model is unclear. Thus we hypothesised that gender contributes to the PH phenotype in mice with the BMPR2R899X+/- mutation via a serotonin-dependent mechanism.

5-HT1B expression was determined in female and male BMPR2R899X+/- mouse PASMCs (n=6) aged 5-6 months that exhibit a PH phenotype and human PASMCs (n=3) by qRT-PCR and immunoblotting. Wild type (WT) and non-diseased human PASMCs were studied as controls. To determine the contribution of oestrogen on 5-HT1B expression, whole lung tissue was harvested from female and male WT mice dosed with an aromatase (oestrogen-synthesizing enzyme) inhibitor, Anastrozole (Tocris; 3mg/kg/day s.c.) or vehicle (1% carboxymethyl cellulose) for 14 days (n=5). Animals were sacrificed by anaesthetic overdose (isoflourane supplemented with 5% oxygen). mRNA expression was normalised to GAPDH and delta CT value obtained for statistical analysis. Protein expression was normalised to alpha-tubulin. Statistical analysis was performed by a t-test or one-way ANOVA followed by a Bonferronis post-hoc test.

5-HT1B mRNA and protein expression were increased in female BMPR2R899X+/- mouse PASMCs compared to their WT counterparts (4.82 ± 1.474 vs. 1.00 ± 0.677, p<0.05, and 0.644 ± 0.148 vs. 0.274 ± 0.035, p<0.05 respectively). 5-HT1B expression was unchanged in male mice (mRNA, 0.52 ± 0.151 vs. 0.43 ± 0.319; and protein, 0.328 ± 0.137 vs. 0.358 ± 0.081). Similarly, this gender difference in 5-HT1B expression also observed in human PASMCs from female PAH patients, with an increase in 5-HT1B expression compared to control (9.54 ± 3.355 vs. 1.00 ± 0.591, p<0.01) whereas no changes were observed in the male PASMCs (0.705 ± 0.06 vs. 1.00 ± 0.396). In the absence of circulating oestrogen via inhibition of aromatase, 5-HT1B mRNA expression was decreased in female mice compared to vehicle treated animals (0.16 ± 0.035 vs. 1.00 ± 0.365, p<0.01). Interestingly, there was no difference in 5-HT1B expression amongst male mice (0.98 ± 0.058 vs. 1.00 ± 0.249).

In summary, only female BMPR2R899X+/- mice and hPASMCs from PAH patients with a BMPR2R899X+/- mutation exhibit an increased expression of 5-HT1B receptor compared to control tissues. In addition, 5-HT1B receptor expression appears in part to be controlled by the presence of circulating oestrogens, providing a mechanistic sex-specific regulation of its expression profile. This may explain the disproportionate prevalence of PAH amongst females and suggests novel therapeutic strategies targeting the 5HT1B receptor.

1. Walker, A.M. et al, American Heart Journal, 152: 521-526, 2006

2. Long, L. et al, Circulation Research, 98: 818-827, 2006

3. Morecroft, I. et al, The Journal of Pharmacology and Experimental Therapeutics, 313: 539-548, 2005