Smad1 deficient mice display female susceptibility to pulmonary hypertension.

KM Mair1, L Long2, XD Yang2, E Wallace1, NW Morrell2, MR MacLean1. 1University of Glasgow, Institute of Cardiovascular and Medical Science, Glasgow, UK, 2University of Cambridge, School of Clinical Medicine, Cambridge, UK

Pulmonary hypertension (PH) occurs more frequently in females than males (1). The primary genetic defects associated with PH are mutations in the gene encoding bone morphogenetic protein receptor 2 (BMPR2) (2). Smad1 signals downstream of BMPR2 and dysfunctional Smad signalling is associated with the abnormal smooth muscle cell proliferation observed in PH (3). Here we characterise the pulmonary haemodynamics of Smad1 heterozygous knockout (Smad1+/-) mice to determine the role of Smad1 in the development of PH and assess the contribution of gender to this disease model.

Smad1 conditional knockout mice were generated on a C57BL/6 background using the Cre-loxP system as previously described (4). Haemodynamic measurements were carried out using male and female Smad1+/- mice and their wildtype (WT) littermates, aged 5-6 months. Right ventricular systolic pressure (RVSP) was measured via catheterisation of the right ventricle and systemic arterial blood pressure (SAP) was measured by cannulation of the carotid artery. Pulmonary vascular remodelling was determined by staining with elastic Van Gieson and pulmonary arteries (<80 μm external diameter) microscopically assessed for degree of muscularisation. RNA extracted from pulmonary artery smooth muscle cells (PASMCs) was analysed and mRNA expression quantified using TaqMan® gene expression assays. Additionally, contractile responses of small third order pulmonary arteries (~250µm internal diameter) were examined using wire myography. Cumulative concentration response curves to serotonin (1nM-300µM in half log increments) were constructed. All data are expressed as mean ± SEM. Data were analysed using one-way ANOVA with Bonferroni post-test.

Female Smad1 +/- mice spontaneously developed PH at 5-6 months of age, whilst the males did not. Significant elevations in RVSP in female Smad1+/- mice were observed: female Smad1+/- = 30.06 ± 0.71 mmHg versus male Smad1+/- = 23.8 ± 0.71 mmHg; p<0.001, n=6. The percentage of remodelled pulmonary arteries was also significantly increased: female Smad1+/- 12.11 ± 1.72 %versus male Smad1+/- 8.92 ± 0.92 %; p<0.05, n=6. Pulmonary arteries from female Smad1+/- mice were also found to display augmented contractility to serotonin (female Smad1+/- Emax = 172.7 ± 5.67 % versus female wildtype Emax = 104.7 ± 5.67 %; p<0.05, n≥4). Furthermore, PASMCs isolated from female Smad1+/- mice express significantly elevated levels of 5HT1B receptor and tryptophan hydroxylase 1 (TPH1) (the enzyme responsible for peripheral serotonin synthesis) compared to the wildtype controls (5HT1B = 4.63 ± 0.82 fold increase, p<0.01, n≥3; TPH1 = 2.00 ± 0.33 fold increase, p<0.05, n≥3).

These findings highlight the role of dysfunctional Smad1 signalling in the development of PH and characterise a new murine model which exhibits female susceptibility. Increased expression of the 5HT1B receptor and TPH1 in PASMCs may contribute to the increased vascular reactivity in female Smad 1 +/- mice. Given the PH occurs up to four times more frequently in females than males, the Smad1 +/- mouse may prove extremely effective to further investigate this condition.

(1) Badesch DB et al, Chest 137:376, 2010

(2) Nichols WC et al, Nature Genetics 15:277,1997

(3) Yang XD et al, Circ Res 96:1053-1063, 2005

(4) Huang S. et al, Genesis 32:76, 2002