We have presented evidence that the Mts1/RAGE pathway may play a role in the gender bias associated with PAH. Female Mts1+ mice develop increased pulmonary vascular remodeling and elevated right ventricular pressure compared to male Mts1+ mice. A subset of Mts1+ female mice (25%) also exhibit neointimal formation and lumen obliteration in a small number of pulmonary resistance arteries, consistent with the formation of plexiform-like lesions. These plexiform-like lesions are not apparent in male Mts1+ mice. Development of PAH and plexiform lesions have previously been described in Mts1+ mice, however in these studies there was no documentation of gender distribution and cohorts were likely mixed [6, 19]. This, therefore, is the first study to show that the PAH phenotype in Mts1+ mice is specific to females. In accordance with Greenway et al.  we show that plexiform-like lesions are observed only in a sub-group of Mts1+ mice, and that these lesions are predominantly comprised of smooth muscle cells in the neo-intimal layer with a thin layer of endothelial cells lining the obliterated lumen. Also in agreement with Greenway et al, we show Mts1 expression to be in the endothelial and adventitial layers of the plexiform-like lesions. We further show that RAGE is also expressed in the endothelial and adventitial layers of the lesions in a similar pattern to Mts1. However, there are also some discrepancies between the current study and a previous study describing PAH in Mts1+ mice . While we describe increased pulmonary vascular remodeling in female Mts1+ mice, the Mts1+ mice studied by Merklinger et al showed no pulmonary vascular remodeling . In addition, we did not observe any differences in RVH in either male or female Mts1+ mice compared to controls. The original Mts1+ mice studied by Merklinger et al. showed increased RVH , but a later study by the same group showed Mts1+ mice to have similar RVH to control mice . There are three notable differences between our study and that of Merklinger et al  which may contribute to the different results obtained. Our study used mice at 5 months of age whilst Merklinger et al. studied younger mice (~ 2months of age). In addition, our study has separated data from male and female mice whilst gender distribution was not described in the Merklinger et al. study and cohorts were likely mixed. Finally, Merklinger et al used C57BL6 mice as their wildtype controls while the current study employed C57BL6 x CBA mice as wildtype controls (Mts1+ mice are bred on a C57BL6 x CBA background) . The reasons we studied mice at 5 months of age were two-fold. Firstly, we have demonstrated that the PAH phenotype does not present until 5 months of age in some transgenic mice, for example in mice over-expressing the serotonin transporter . Secondly, female mice reach sexual maturity at 6-8 weeks of age, and can continue reproducing until around 9-12 months old, although litter sizes will drop around this time . Thus at 5 months of age, female mice are mid-late reproductive age which approximates the age (average 35) in humans at which PAH presents itself.
We show female Mts1+ mice to develop increased sRVP despite a relatively small increase in pulmonary vascular remodeling. This is in line with previous reports describing increased sRVP in mice despite only 5-15% of vessels being remodeled [18, 23]. Indeed, as discussed above, Merklinger et al showed increased sRVP and RVH in Mts1+ mice despite these mice having similar levels of pulmonary vascular remodeling to wildtype controls. Thus it is likely that factors additional to pulmonary vascular remodeling contribute to increased pulmonary pressures in mice. Indeed Mts1+ mice show decreased lumen diameter of the left pulmonary artery, and decreased attenuation of sRVP in response to nitric oxide, suggesting elevated pulmonary vascular resistance .
The dissociation between RVP and RVH observed in female Mts1+ mice is consistent with previous observations using female mice at this age. For example, dexfenfluramine-dosed female mice develop increased RVP in the absence of RVH, as do female mice over-expressing the human SERT gene [18, 21, 23]. We and others have shown that estrogen may have a protective effect against RVH [18, 24], and this may explain the dissociation between RVH and RVP we observe in the present study.
Corresponding with the increased PAH phenotype observed in female Mts1+ mice, Mts1 gene expression was increased in the lungs of female Mts1+ mice compared to male Mts1+ mice. Immunohistochemical analysis showed increased Mts1 protein expression in the medial layer of small, resistance pulmonary arteries in female compared to male Mts1+ mice. These gender differences in Mts1 expression were not attributable to gender influences on the expression of the HMGCoA reductase promoter. Thus our results suggest an influence of sex hormones on Mts1 expression and subsequent PAH phenotype and we wished to assess the effects of 17β-estradiol on Mts1 expression. To determine if our in vivo results in mice could be translated to a human cell model, we chose to study hPASMCs. In line with female Mts1+ mice showing increased expression of Mts1 in the medial layer of pulmonary arteries, physiologically relevant concentrations of 17β-estradiol up-regulated Mts1 expression in hPASMCs. Mts1 has previously been shown to act in an autocrine fashion in hPASMCs; it is released from and then acts upon these cells to mediate proliferation and migration . Thus increased expression of Mts1 within the medial layer of small resistance pulmonary arteries may contribute to the increased remodeling observed in female Mts1+ mice. Mts1 exerts its effects on proliferation and migration in hPASMCs via RAGE . Interestingly, we show sRAGE to inhibit 17β-estradiol-induced proliferation of hPASMCs. As Mts1 is the ligand for RAGE, collectively, the results suggest that 17β-estradiol induces increased expression of Mts1 that then activates RAGE and stimulates hPASMC proliferation.
The mechanism by which Mts1 is released from hPASMCs by serotonin involves co-operation between the SERT and the 5-HT1B receptor . In line with this, Mts1 mRNA is up-regulated in the lungs of female mice over-expressing SERT (SERT+ mice) . We have recently shown that female gender is permissive in the development of PAH in SERT+ mice . In addition we have shown a critical role for 17β-estradiol in the development of PAH in female SERT+ mice as ovariectomy reduces the PAH phenotype which can then be re-established using subcutaneous 17β-estradiol implants .
Previous studies have demonstrated that female gender and/or estrogens can be protective in experimental models of PAH, such as the hypoxic and monocrotaline models and mice lacking the vasoactive intestinal peptide gene [4, 5, 25]. This suggests that, in certain circumstances, female sex hormones may actually be protective against PAH. Indeed, the estradiol metabolite 2-methoxyestradiol (2-ME) has been shown to mediate protective effects in monocrotaline induced PAH . Estradiol is metabolized to 2-hydroxyestradiol (2-OHE) mainly via the estrogen metabolizing enzymes CYP1A1/2 and to a lesser extent via CYP1B1. 2-OHE is then converted to 2-ME via catechol O-methyltransferase. Estradiol can also be metabolized to 16α-hydroxyestrone (16-OHE1) via various CYP enzymes including CYPs 1A1, 1A2 and 1B1 . 2-OHE/2-ME may have anti-proliferative effects on cells , while 16α-OHE1 stimulates cellular proliferation by constitutively activating the estrogen receptor . Hence, both pro- and anti-proliferative effects of estrogens may be observed, depending on their metabolism. Consistent with this, both gene and protein expression of CYP1B1 is increased in PASMCs from PAH patients . In addition, higher penetrance of PAH is observed among BMPRII mutation carriers with a polymorphism in the CYP1B1 genotype . Interestingly, the 2-OHE/16α-OHE1 ratio was also decreased in PAH patients with a BMPRII mutation compared to unaffected BMPRII mutation carriers . Disruption in the balance of estrogen metabolites may therefore account for the differential effects of female sex hormones in different models of PAH.