The serotonin transporter, gender and 17 beta estradiol in pulmonary arterial hypertension.
PhD thesis, University of Glasgow.
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Pulmonary arterial hypertension (PAH) is a progressive disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The consequence of this is increased pulmonary arterial pressure, right heart failure and eventual death if left untreated. Even in those patients receiving advanced PAH therapy, mortality rates remain high. Therefore, the development of novel therapeutic approaches in the treatment of PAH is urgently required. A better understanding of PAH pathogenesis is critical to achieving this. Gender differences exist in human PAH, with females up to three-fold more likely to present with disease than males. Limited evidence suggests that estrogens may be accountable for these differences. For example, the use of oral contraceptives has been associated with the development of PAH. Paradoxical to this however, experimental models of PAH exhibit male susceptibility which has limited research into the role of estrogens in the pathogenesis of PAH.
Serotonin is implicated in both experimental and human PAH. Activity of the serotonin transporter (SERT) modulates the development of PAH and mice over-expressing SERT (SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH. In the central nervous system, estrogens regulate expression of several serotonin signalling components including tryptophan hydroxylase (TPH), the 5-HT receptors and SERT. One hypothesis is that similar estrogen effects on serotonin signalling may also be apparent in the pulmonary vasculature, and this is one hypothesis for the increased female susceptibility observed in PAH. To examine this, the influence of gender and estrogen on the development of PAH in SERT+ mice was investigated. This was repeated following exposure to chronic hypoxia. Pulmonary vascular reactivity was determined using small vessel myography. The genotypic differences in SERT+ mice were also assessed via microarray analysis. Genes of interest were validated by qRT-PCR analysis and immunoblotting. To translate clinical relevance to any findings, the effects of female hormones were also investigated in human pulmonary artery smooth muscle cells (PASMCs) derived from both non-PAH (control) and idiopathic pulmonary arterial hypertension (IPAH) patients.
PAH was assessed via measurement of right ventricular systolic pressure (RVSP), pulmonary vascular remodelling and right ventricular hypertrophy (RVH). Male SERT+ mice do not develop PAH. Female SERT+ mice exhibited increased RVSP and pulmonary vascular remodelling. This increased RVSP and pulmonary vascular remodelling were completely prevented following ovariectomy in SERT+ mice. The chronic administration of 17β estradiol (1.5mg/kg/day), which is the pre-dominant circulating female hormone in pre-menopausal women, fully re-established PAH as assessed by increased RVSP and pulmonary vascular remodelling. Pulmonary vascular reactivity to serotonin was unaffected in these mice. In chronic hypoxia, female SERT+ mice exhibited exaggerated hypoxia-induced PAH whereas male SERT+ mice remained unchanged compared to their respective wildtype (WT) controls. This exaggerated hypoxia-induced PAH phenotype was attenuated in SERT+ mice following ovariectomy, as assessed by a reduction in RVSP and pulmonary vascular remodelling. The chronic administration of 17β estradiol completely re-established exaggerated hypoxia-induced PAH in ovariectomized SERT+ mice. Similar to normoxia, serotonin-induced pulmonary vascular contraction was also unaffected in these mice. The stimulation of PASMCs with 17β estradiol increased tryptophan hydroxylase-1, SERT and 5-HT1B receptor expression. Consistent with our in vivo findings, physiological concentrations of 17β estradiol (1nmol/L) stimulated PASMC proliferation whereas estrone, estriol and progesterone had no effect. This proliferation was successfully blocked by both the tryptophan hydroxylase inhibitor para-chlorophenylalanine and the 5-HT1B receptor antagonist SB224289. Microarray analysis in the pulmonary arteries of female SERT+ mice confirmed the dysregulation of multiple pathways with relevance to PAH including those associated with metabolism, cell differentiation and contraction. A large number of PAH-related pathways were also dysregulated in female SERT+ mice exposed to chronic hypoxia. In contrast, pathways were altered in normoxic and chronically hypoxic male SERT+ mice to a much lesser extent. For microarray validation, qRT-PCR analysis was performed in ten selected genes (FOS, CEBPB, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1 and C1S) and 100% concordance was reported. Three genes were selected for further investigation (FOS, CEBPB and CYP1B1). Immunoblotting confirmed that protein expression of c-FOS, C/EBPβ and CYP1B1 was increased in the pulmonary arteries of female SERT+ mice compared against female WT mice. With relevance to human PAH, we also confirmed that mRNA expression of FOS, CEBPB and CYP1B1 was increased in PASMCs derived from IPAH patients. Protein validation with immunoblotting confirmed that c-FOS, C/EBPβ and CYP1B1 expression was also increased in IPAH PASMCs.
In summary of these findings, we have identified that females may be at an increased risk to the development of PAH via the effects of 17β estradiol. Specifically, 17β estradiol appears to increase expression of several key serotonin pathway mediators including tryptophan hydroxylase-1 (the rate-limiting enzyme in serotonin synthesis), SERT and the 5-HT1B receptors, all of which have been previously implicated in the pathogenesis of PAH. Our results suggest that 17β estradiol stimulates PASMC proliferation via activation of the serotonin pathway. Microarray analysis in the pulmonary arteries of SERT+ mice confirmed that a large number of genes associated with PAH-related pathways were differentially expressed in females. RNA and protein validation further confirmed these differences in selected genes (CEBPB, CYP1B1 and FOS). With relevance to human PAH, we also confirmed the dysregulation of several key genes at both mRNA and protein level in PASMCs derived from IPAH patients. Taken together, these findings describe the critical role of 17β estradiol in PAH, and this may offer an explanation for the increased susceptibility observed in females.
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