Investigating the role of sex and the right ventricle in the development and treatment of experimental pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterised by pathological remodelling of the pulmonary vasculature resulting in increased pulmonary vasculature resistance and pulmonary artery pressure. This eventually results in maladaptive changes within the right ventricle (RV) leading to RV dysfunction and ultimately RV failure. Investigations of heritable PAH identified mutations in BMPR2 as a major risk factor for disease development. Approximately 75% of heritable cases and 20% of idiopathic cases are associated with heterozygous mutations in this gene. However, penetrance rates are low, suggesting the involvement of other endogenous and environmental factors in triggering the development of disease. PAH patient registries have highlighted an increased female to male ratio within the patient population, in some registries the ratio is as high as 4:1. This indicated the female sex was associated with increased risk of disease and encouraged investigation into the role of sex hormones in the development of disease. Estrogen (17b-estradiol) and estrogen metabolites have been a focus of recent research, however, studies have produced conflicting results on the protective or pathological effect of estrogen in the pulmonary vasculature. This research is further complicated by the finding that although fewer males develop PAH, male PAH patients tend to have poorer clinical outcomes. This is thought to be associated with more rapid decline in RV function. These contradictions are known within the field as the “estrogen paradox”. Currently, the therapies licenced for the treatment of PAH only target the vasoconstriction of the pulmonary vasculature, therefore, there is a need to identify new therapeutic targets. The role of sex in the development of disease highlights a need to investigate the sex specific effects of potential PAH therapies. The main aim of this thesis was to investigate the sex-specific effects of two potential therapies for PAH patients – FK506 (tacrolimus) and Anastrozole (aromatase inhibitor). An additional aim was to characterise the male and female RV and identify sex-specific differences that may contribute to disease progression. Firstly, the effect of FK506 treatment (0.05 mg/kg/day) was investigated in the chronic hypoxia rat model. To our knowledge, this was the first time FK506 treatment had been investigated in this model. Administration of FK506 in male and female chronic hypoxia rats had no significant effect on right ventricular systolic pressure (RVSP), pulmonary artery remodelling or RV/LV+S. However, further investigation of the RV indicated that RV mass and RV mass/body weight were significantly decreased in the hypoxic FK506 treated males. This led to a gene expression study to investigate the effect of hypoxia and FK506 treatment in the male and female RV, as well as a direct comparison between the male and female RV. The key finding from this study was that hypoxia increased expression of Tgfb1 in the male RV only and treatment with FK506 significantly reduced expression. Furthermore, Tgfb1 expression was significantly higher in the male RV in normoxia and hypoxia compared with the female RV. Expression of genes associated with cardiac remodelling – Col1a1, Ctgf, Nppa and Myh7 – were all differentially expressed between the hypoxic male and female RV, with males displaying increased expression. These results are preliminary but could indicate a differential response to hypoxia by the male and female RV, which could be associated with disease progression. A model to isolate adult rat RV cardiomyocytes was under development during this thesis in order to investigate these findings further in male and female cells. The next study involved investigation of treatment with Anastrozole in the chronic hypoxia mouse model. This study was focused on female mice as Anastrozole had previously been shown to reverse pulmonary hypertension (PH) in female mice. However, Anastrozole had no significant effect on RVSP or RV/LV+S in female mice and had no significant effect on RV/LV+S in male mice. As the first study in this thesis had highlighted key differences in the male and female RV in adult rats, the male and female mouse RV was compared to understand if these findings were due to differences across species. These investigations showed that Id1 and Id3 proteins, downstream components of the BMPR2 signalling pathway, were expressed at significantly lower levels in the female normoxic RV. This again highlighted differences in a key signalling pathway in PAH that could contribute to the differences in susceptibility and progression of disease between males and females. Continuing to assess the role of estrogen in the development of PAH, final investigations carried out in rat pulmonary artery smooth muscle cells (PASMCs) investigated the effects of 17b-estradiol (E2) and 16a-hydroxyestrone (16a- OHE1) on cell migration. These results highlighted that although the use of charcoal stripped serum is preferred for in vitro investigations involving steroid hormones, the stripping process may also remove factors that promote cell migration. E2 and 16a-OHE1 did not stimulate migration of male or female rat PASMCs. In summary, this thesis provides preliminary evidence that there are significant differences between the male and female RV which could contribute to disease development, progression and the success of treatment. In future, it would be important to confirm these findings and the downstream effects of differential gene and protein expression utilising appropriate cell models. It is important that the sex-specific effects of potential PAH therapeutics are investigated as well as the lung and RV specific effects. PAH patients may benefit from sex- specific treatment protocols which could ultimately reduce disease burden and mortality rates