Dexfenfluramine associated pulmonary arterial hypertension in mice is dependent on serotonin synthesis

Neil MacRitchie1, Ian Morecroft1, Lynn Loughlin1, Michael Bader2, Diego Walther2, Margaret MacLean1
1University of Glasgow, Glasgow, United Kingdom, 2Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

Dexfenfluramine (Dfen) is an appetite suppressant associated with an increased risk of pulmonary arterial hypertension (PAH), a disease characterised by vasoconstriction and vascular remodelling of pulmonary resistance arteries (PRAs; Abenhaim et al., 1996). Only a small percentage of people exposed to Dfen develop PAH and underlying genetic modifying factors are thought to increase susceptibility to the disease. One candidate is as increase in serotonin (5-HT) resulting from overexpression of the 5-HT transporter (5-HTT) or an increase in the rate-limiting enzyme for 5-HT synthesis in the periphery, tryptophan hydroxylase 1 (TPH1), that has recently been found to be increased in lungs and pulmonary endothelial cells in patients with idiopathic PAH. In the present study we have investigated whether changes in the serotonergic system might influence the effects of Dfen in mice.

Mice (C57BL/6J, 2-3 months, 15-25g, n=4-9), either wild-type (WT) or lacking peripheral 5-HT, due to deficiency in tryptophan hydroxylase 1 (TPH1-/-) were treated with Dfen (5 mg/kg/day) or vehicle for 28 days via oral-gavage dosing. Mice were then maintained under anaesthesia (1.25% halothane, 6:1 O2:N20) and pulmonary haemodynamics assessed. Right ventricular pressure (RVP) was measured by transdiaphragmatic cardiac puncture using a 25-gauge needle attached to a pressure transducer.

Three sagittal sections were obtained from left lungs. Sections were stained with ElasticaVanGieson and microscopically analysed for muscularization of airway associated small pulmonary arteries (<80μM external diameter). The percentage of remodelled vessels was assessed by measuring vessels with a double elastic lamina and expressing this as a percentage of the vessels examined. Statistical comparisons were made by one-way ANOVA followed by Tukey’s multiple comparison test. All data expressed as mean ± s.e.m.

Dfen significantly increased (p<0.001) systolic RVP in WT (31.97 ± 0.48 mmHg) mice compared with the vehicle treated WT (17.34 ± 1.53 mmHg) group. However, Dfen failed to increase systolic RVP in TPH1-/- mice with vehicle treated and Dfen treated groups having similar pressures (15.50 ± 1.01 mmHg and 14.09 ± 0.97 mmHg respectively). The percentage of remodelled vessels was also significantly higher in WT (12.10 ± 1.17%) mice treated with Dfen compared with vehicle dosed WT (3.26 ± 0.33%) mice (p<0.001). No such difference was seen between TPH1-/- Dfen and vehicle treated mice (3.63 ± 0.36% and 3.08 ± 0.43% respectively).

This study demonstrates the requirement for peripheral 5-HT synthesis in Dfen induced PAH in mice. There is considerable evidence implicating a role for 5-HT in PAH (De Caestecker, 2006) and Dfen can induce release of 5-HT from pulmonary arterial smooth muscle cells. People carrying genetic polymorphisms that result in increased 5-HT signalling may be predisposed to PAH if exposed to other risk factors.

Abenhaim, A. et al. (1996) N Engl J Med 335:609-616
De Caestecker, M. (2006) Circulation Research 98:1229-1231