Development of pulmonary arterial hypertension in mice over-expressing S100A4/Mts1 is specific to females

Abstract

BackgroundIdiopathic and familial forms of pulmonary arterial hypertension (PAH) occur more frequently in women than men. However, the reason for this remains unknown. Both the calcium binding protein S100A4/Mts1 (Mts1) and its endogenous receptor (receptor for advanced glycosylation end products; RAGE) have been implicated in the development of PAH. We wished to investigate if the Mts1/RAGE pathway may play a role in the gender bias associated with PAH.MethodsWe investigated the effects of gender on development of PAH in mice over-expressing Mts1 (Mts1+ mice) via measurement of pulmonary arterial remodeling, systolic right ventricular pressure (sRVP) and right ventricular hypertrophy (RVH). Gender differences in pulmonary arterial Mts1 and RAGE expression were assessed by qRT-PCR and immunohistochemistry. Western blotting and cell counts were used to investigate interactions between 17β-estradiol, Mts1 and RAGE on proliferation of human pulmonary artery smooth muscle cells (hPASMCs). Statistical analysis was by one-way analysis of variance with Dunnetts post test or two-way analysis of variance with Bonferronis post test, as appropriate.ResultsFemale Mts1+ mice developed increased sRVP and pulmonary vascular remodeling, whereas male Mts1+ mice remained unaffected. The development of plexiform-like lesions in Mts1+ mice was specific to females. These lesions stained positive for both Mts1 and RAGE in the endothelial and adventitial layers. Expression of pulmonary arterial Mts1 was greater in female than male Mts1+ mice, and was localised to the medial and adventitial layers in non plexiform-like pulmonary arteries. RAGE gene expression and immunoreactivity were similar between male and female Mts1+ mice and RAGE staining was localised to the endothelial layer in non plexiform-like pulmonary arteries adjacent to airways. In non-plexiform like pulmonary arteries not associated with airways RAGE staining was present in the medial and adventitial layers. Physiological concentrations of 17β-estradiol increased Mts1 expression in hPASMCs. 17β-estradiol-induced hPASMC proliferation was inhibited by soluble RAGE, which antagonises the membrane bound form of RAGE.ConclusionsMts1 over-expression combined with female gender is permissive to the development of experimental PAH in mice. Up-regulation of Mts1 and subsequent activation of RAGE may contribute to 17β-estradiol-induced proliferation of hPASMCs.

Hypothesis: Neuroendocrine Mechanisms (Hypothalamus-Growth Hormone-STAT5 Axis) Contribute to Sex Bias in Pulmonary Hypertension

“…We note that all previous work on sex bias in rodent-based PH models, including after administration of steroid sex hormones (for example, E2, 2-ME or testosterone) and/or gonadectomy, have focused on direct effects of steroid hormones at the level of peripheral vascular tissues in the lung (5,6,8,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). (However, the terminology used can often be confusing and, indeed, misleading.…”

“…Thus, in the context of a "neuroendocrine hypothesis," the term "central" refers to the hypothalamus and pituitary [the "neuro-" part], while the terms "distal" or "peripheral" refer to all locations in the body where a hormone can circulate [the "-endocrine" part]). Thus, there have been extensive studies of steroid hormone effects (E2, 2-ME, testosterone) in lung tissues in rodent models, in isolated human and rodent pulmonary vascular cells (smooth muscle cells [SMCs] and endothelial cells [ECs]) and on vascular cell proliferation, transcriptional regulation of BMPR2 gene expression and BMPR2-initiated cell signaling (5,6,8,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This exclusive focus on peripheral tissue effects of steroid hormones is somewhat at odds with insights gleaned over the last 30-40 years in a sister field (sex-biased expres- Figure 1.…”

“…Seventh, why do female mice overexpressing the serotonin transporter or the S100 calcium-binding protein S100A4/mts1 or the dexfenfluramineadministered mice, but not male mice, develop modest PH by 5 months (15,(19)(20)(21)(22)? Although increased serotonin (5-HT) has been implicated in the pathogenesis of PH (10,15,26,100), especially in the femalespecific mouse models developed by MacLean et al (19)(20)(21), and after administration of an anorectic drug dexfenfluramine (22,100), the mechanistic focus has largely been on effects of 5-HT on distal vascular tissues (10,15,19-22,26,100).…”

“…Although increased serotonin (5-HT) has been implicated in the pathogenesis of PH (10,15,26,100), especially in the femalespecific mouse models developed by MacLean et al (19)(20)(21), and after administration of an anorectic drug dexfenfluramine (22,100), the mechanistic focus has largely been on effects of 5-HT on distal vascular tissues (10,15,19-22,26,100). We note that it is already known that the PHcausing anorexigens fenfluramine, aminorex, phentermine and fluoxetine increase 5-HT in the hypothalamus (101)(102)(103)(104)(105) and that fenfluramine blunted GH responsiveness to GHRH (105).…”

“…In contrast, the phrase "sex biased" refers to entities that show a quantitative phenotypic difference between the two sexes.) In converse examples, female mice overexpressing the serotonin transporter or the S100 calcium-binding protein S100A4/mts1, or administered the anorexigen dexfenfluramine (but not male mice), spontaneously develop modest PH by 5 months (15,(19)(20)(21)(22). Although only up to one-third of male mice expressing the BMPR2 R899X transgene spontaneously developed right ventricular systolic pressure >30 mmHg, this proportion increased to two-thirds when the mice were fed a high-fat diet (23); in this model, 2-ME was not protective (24).…”