The influence of sex on the in vivo and in vitro effects of treprostinil in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and fatal vascular disease that is more prevalent in women than men. The underlying pathology of the disease involves various factors, including genetic risk (i.e. bone morphogenetic protein receptor type II (BMPR-II) mutations) as well as the influence of hormones such as estrogen. Among the frontline treatments for PAH is prostacyclin therapy; however, the short half-life and associated problems with the need for continuous intravenous administration of synthetic prostacyclin have led to the development of newer analogues such as treprostinil. These have the advantages of a longer half-life and the possibility of subcutaneous and inhaled administration. Mortality rates for PAH are still high despite advancements in treatment, with male survival rates remaining lower than females. The BMPR-II signalling pathway may underlie some of the sex disparity that exists in incidence of PAH. However, patient responses to treatments for PAH have also demonstrated sex-specific effects. A key aim of this study was to identify the influence that sex may have on the actions of treprostinil with in-vivo and in-vitro models of PH. The ability to target treatment to specific sub-cohorts of PH is important to maximise the therapeutic effect of treprostinil. A greater understanding of how the effects of treprostinil are mediated could assist this objective. To examine any potential influence of sex on the effects of treprostinil, we examined the chronic hypoxic model of pulmonary hypertension (PH). Female and male rats were dosed with sub-cutaneously implanted pellets releasing treprostinil at 100ng/kg/min or 400ng/kg/min. Under hypoxic conditions both male and female rats had increases in right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary artery remodelling. 100ng/kg/min of treprostinil partially reversed RVSP, RVH and pulmonary artery remodelling in female hypoxic rats but not in male rats. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that a possible mechanism of treprostinil was increasing BMPR-II signalling, specifically Id1 and Id3 (inhibitor of DNA binding protein 1/3). This was only observed in female hypoxic rats. Despite no difference in terminal plasma levels of treprostinil, hypoxic males remained unaffected by treprostinil at 100ng/kg/min. However, 400ng/kg/min of treprostinil led to slightly greater decreases in RVSP, RVH and remodelling indices in female rats, also it partially reversed RVSP, RVH and remodelling in male rats. Taqman qRT-PCR of the prostaglandin receptors demonstrated an increase in prostaglandin E2 receptor 2 (EP2) under hypoxic conditions with 100ng/kg/min and 400ng/kg/min treprostinil treatment, specific to female rats. Also under hypoxic conditions female and male rats had significant increases in mRNA expression of potassium two pore domain channel subfamily K (KCNK3). To translate clinical relevance from the in-vivo findings, the influence of sex on treprostinil was investigated in human pulmonary artery smooth muscle cells (hPASMCs) taken from non-PAH (control) and PAH patients. In female control hPASMCs FBS (fetal bovine serum) induced proliferation was partially ablated by treprostinil; this effect was only observed in male control hPASMCs at the highest treprostinil concentration (10µM). In patient hPASMCs, treprostinil had a similar effect in reducing FBS-induced proliferation in both female and males. The addition of a low dose (30nM) of endothelin-1 (ET-1) increased the anti-proliferative effect of treprostinil, specifically, in female control hPASMCs. The addition of a dual endothelin receptor antagonist (SB-217242) partially reduced the anti-proliferative effect of treprostinil in combination with ET-1. Taqman qRT-PCR and western blot analysis demonstrated no difference between sexes or hPASMC groups in the expression of the prostaglandin receptors. Using receptor specific antagonists, it was determined that the anti-proliferative actions of treprostinil in PAH patient hPASMCs were partially mediated via the EP2 receptor. However, in female control hPASMCs, the IP receptor was primarily responsible for this effect. BMPR-II signalling was investigated to ascertain its role in the anti-proliferative effects of treprostinil. Taqman qRT-PCR indicated treprostinil (100nM and 1µM) induced increases in Id1 and Id3 mRNA in female control hPASMCs, this did not occur in male control hPASMCs. Treprostinil (100nM and 1µM) led to Id3 mRNA increases in female PAH patient hPASMCs, whereas in male PAH patient hPASMCs treprostinil (1µM) led to a significant Id3 mRNA increase. Western blots indicated that Id3 was upregulated by treprostinil (1µM) stimulation in female control and female PAH hPASMCs vs non-stimulated hPASMCs; this effect was not observed in males. The combination of ET-1 and treprostinil did not influence BMPR-II signalling. After 24 hours of treprostinil stimulation increased Id3 mRNA expression was observed in all hPASMCs groups. Treprostinil only increased Id1 mRNA in PAH patient hPASMCs. Although western blots confirmed treprostinil (100nM and 1µM) mediated increases in Id1 protein expression in female control hPASMCs. Treprostinil (100nM and 1µM) increased Id3 protein expression in female control and female PAH patient hPASMCs. Treprostinil (1µM) in combination with ET-1 led to a significant increase in Id3 protein expression in male control hPASMCs. As with 72-hour treprostinil stimulation, BMPR-II signalling was not influenced by the combination of ET-1 and treprostinil in the other hPASMC groups. The increased BMPR-II signalling in female control and female PAH patient hPASMCs at 24 hours led to the investigation of prostaglandin receptors role in activating BMPR-II signalling. After 24-hours of stimulation with treprostinil (100nM), Id protein induction was partially blocked by dual antagonism of the IP and EP2 receptor in both female control and female PAH patient hPASMCs. To summarise these findings, we have identified sex differences in the action of treprostinil in both in-vivo and in-vitro models of PH. Treatment with a low dose (100ng/kg/min) of treprostinil led to a significant reduction in chronic hypoxic induced PH in female rats but not in males. These differences are driven partially by increases in BMPR-II signalling. Treatment with a higher dose (400ng/kg/min) of treprostinil led to significant reductions in chronic hypoxic PH in both female and male rats. In hPASMCs the results demonstrate that treprostinil can induce the Id proteins of the BMPR-II signalling pathway and that this may account for the greater anti-proliferative effect observed in female control hPASMCs. The induction of the Id proteins was found to be partially mediated by activation of the IP and EP2 prostaglandin receptors. The results suggest that sex may influence the beneficial effects of treprostinil in an in-vivo model of PH and in hPASMCs.