Endothelial cell-specific knockout of the ETB receptor exaggerates hypoxia induced pulmonary arterial hypertension

Kelland, N.F., Bagnall, A.J., Morecroft, I.*, Gulliver-Sloan, F.H., Dempsie, Y.*, Nilson, M.*, MacLean, M.R.*, Kotelevtsev, Y., & Webb, D.J. Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, UK. *Division of Neuroscience and Biomedical Systems, IBLS, University of Glasgow, UK.

Pulmonary ETB receptors elicit both protective (vasodilator, anti-inflammatory and clearance) and deleterious (vasoconstrictor and pro-mitogenic) effects that may contribute to the pathogenesis of pulmonary arterial hypertension (PAH). We hypothesised that by decreasing vascular tone and limiting ETA receptor activation through clearance of ET-1, endothelial cell (EC) ETB receptors protect against the development of hypoxia-induced PAH.

Mice with loxP sites flanking ETB receptor coding regions (FF/--) were crossed with Tie2-Cre (Kisanuki et al., 2001) mice to produce EC-specific ETB receptor knockout (KO) mice (FF/Tie2-Cre). Male mice (8-12 weeks; 30-40g) were subjected to 2 weeks of either hypoxic (10% FiO 2) or normoxic conditions. Under halothane anaesthesia right ventricular pressure (RVP) was measured by direct cardiac puncture, right ventricular (RV) / left ventricle+septum (LV+S) ratio and percentage of remodeled pulmonary arteries (PAs) were determined as described previously (MacLean et al., 2004) . PAs (~300 μm ID) were removed and cumulative concentration responses to ET-1 (1fM-0.1μM) constructed using a wire myograph. Statistical analysis was by one-way ANOVA using the Newman-Keuls multiple comparison test.

Under normoxic conditions, there was no difference between systolic RVP in FF/Tie2-Cre mice (23.7 ± 1.7 mmHg, n=9) and FF/-- controls (20.2 ± 1.5 mmHg, n=10). Hypoxia induced an exaggerated increase in systolic RVP in FF/Tie2-Cre mice (34.4 ± 1.2 mmHg, n=10) compared with FF/-- mice (24.6 ± 1.4mmHg, n=10; p<0.05). Hypoxia resulted in increased RV/LV+S ratio in FF/Tie2-Cre mice (normoxia: 0.224 ± 0.009; hypoxia: 0.285 ± 0.017; p<0.01), but not in FF/-- mice. Whilst ET-1 induced a dose dependent constriction of PAs from both FF/Tie2-Cr

e and FF/-- mice, maximum contractile responses were similar, irrespective of FiO2. Hypoxia increased the percentage of remodeled vessels in FF/-- mice (normoxia: 5.6 ± 0.6% ; hypoxia: 11.4 ± 0.6 %; n=6; p<0.001), and this was augmented in FF/Tie2-Cre mice (normoxia: 7.1 ± 0.5% ; hypoxia: 18.5 ± 1.2 %; n=6; p<0.001) .

EC ETB KO mice develop hypoxia induced PAH and RV hypertrophy due to an increase in pulmonary vascular remodeling rather than an increase in pulmonary vascular responsiveness to ET-1 .

Kisanuki et al.,(2001) Dev Biol , 230, 230-42
MacLean et al., (2004). Circulation, 109, 2150-2155.