Orai1 as a novel target in hypoxia-induced pulmonary VSMC remodelling

Pulmonary hypertension (PH) presents with excessive vasoconstriction and vascular smooth muscle cell (VSMC) proliferation, which contribute to right ventricular (RV) failure and death. Therapeutic strategies target vasoconstriction but are only effective in a small proportion of patients. Thus, novel anti-proliferative therapeutics may be pivotal in PH management. Orai1-mediated store-operated Ca2+ entry (SOCE) and T-type Ca2+ channel-mediated voltage-dependent Ca2+ entry are associated with proliferation in multiple diseases including PH. To investigate the potential of these Ca2+ channels as anti-proliferative targets, human pulmonary artery smooth muscle cells (HPASMCs) were validated as a suitable in-vitro model to study the mechanisms of hypoxia-induced pulmonary VSMC proliferation. Widely used T-type Ca2+ channel inhibitors, mibefradil and NNC55-0396, reduced proliferation of HPASMCs (p<0.05), but demonstrated poor selectivity due to off-target modulation of Orai1-mediated SOCE with IC50 values of 10.38 and 10.21 µM, respectively. This effect was observed in the absence of T-type Ca2+ channel expression in wild type HEK293 cells. These data add to the plethora of off-target effects previously demonstrated by these inhibitors. T-type Ca2+ channels were not upregulated in hypoxia either in vitro or in vivo, pivoting the focus to Orai1. Significant upregulation of Orai1 was observed in vitro (p<0.05), which was paralleled by marked Orai1 upregulation in vivo. The novel, selective Orai1 inhibitor, JPIII, significantly reduced HPASMC proliferation in vitro (p<0.0001), an effect that was not observed in normoxia. Continuous infusion of JPIII in an in-vivo model of CH-induced PH protected from the increase in RV hypertrophy and distal muscularisation observed in the vehicle-treated CH-induced PH cohort (p<0.05). In conclusion, no hypoxia-induced upregulation in T-type Ca2+ channel expression, combined with a limited number of selective inhibitors and complete lack of inhibitors in the clinic limit the potential of T-type Ca2+ channels as anti-proliferative targets in CH-induced PH. Hypoxia-induced upregulation of Orai1 both in vitro and in vivo, the anti-proliferative effects of JPIII in vitro and the ability of JPIII to protect from RV hypertrophy and distal muscularisation in vivo support Orai1 as a viable anti-proliferative target in CH-induced PH for a translational drug development programme.