Bastien Masson1,2, Hélène Le Ribeuz1,2, Jessica Sabourin3, Loann Laubry1,2,4, Emily Woodhouse4, Richard Foster4, Yann Ruchon1,2,5, Mary Dutheil1,2,5, Angèle Boët1,2,5, Maria-Rosa Ghigna1,2, Vincent Thomas De Montpreville6, Olaf Mercier7, David J Beech4, Jean-Pierre Benitah3, Marc A Bailey4, Marc Humbert1,2,8, David Montani1,2,8, Véronique Capuano1,2,5, Fabrice Antigny1,2. 1. Université Paris-Saclay, Faculté de Médecine, Le Kremlin-Bicêtre, France (B.M., H.L.R., L.L.., Y.R, M.D, A.B., M.-R.G., M.H., D.M., V.C., F.A.). 2. INSERM UMR_S 999 « Hypertension pulmonaire: Physiopathologie et Innovation Thérapeutique », Hôpital Marie Lannelongue, Le Plessis-Robinson, France. B.M., H.L.R., L.L.., Y.R, M.D, A.B., M.-R.G., M.H., D.M., V.C., F.A.). 3. Inserm, UMR-S 1180, Signalisation et Physiopathologie Cardiovasculaire, Université Paris-Saclay, Châtenay-Malabry, France (J.S., J.-P.B.). 4. Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, United Kingdom (E.W., R.F., L.C., D.J.B., M.A.B.). 5. Hôptal Marie Lannelongue, Groupe Hospitalier Paris Saint-Joseph, Le Plessis Robinson, France (Y.R., M.D., A.B., V.C.). 6. Department of Pathology, Groupe Hospitalier-Marie Lannelongue, 92350 Le Plessis-Robinson, France (V.T.D.M.). 7. Service de Chirurgie Thoracique, Vasculaire et Transplantation Cardio-Pulmonaire, Hôpital Marie Lannelongue, Groupe Hospitalier Paris Saint Joseph, Le Plessis Robinson, France (O.M.). 8. Assistance Publique - Hôpitaux de Paris (AP-HP), Service de Pneumologie et Soins Intensifs Respiratoires, Centre de Référence de l'Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin-Bicêtre, France (M.H., D.M.).
Abstract
BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.
BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.