MonOrail to cure? Targeting Orai1 to Reverse Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rapidly fatal disease with no cure. In PAH, increased pulmonary vascular resistance and mean pulmonary arterial (PA) pressure (mPAP) (≥ 20 mmHg at rest) due to vasoconstriction and remodeling of small pulmonary arteries (PAs) leads to elevation of right ventricle (RV) afterload and premature death of right heart failure (1). Over the last decades, development of new therapeutic options significantly improved patients’ outcomes. However, available therapies, predominantly vasodilators, do not reverse pulmonary vascular remodeling or stop disease progression, and PAH remains a life-limiting disease with high mortality rates and no curative therapeutic options (2).

Pulmonary vascular remodeling is a dynamic and multi-factorial process. The combination of pro-remodeling factors (genetic predisposition, epigenetic factors, environmental exposure, inflammation, etc.) trigger injury and apoptosis of PA endothelial cells (PAECs), excessive production and release of growth factors, pro-inflammatory mediators, stiffening of small PAs, and hyper-proliferation and apoptosis resistance of resident pulmonary vascular cells, including PAECs, PA smooth muscle cells (PASMCs), and adventitial fibroblasts (PAAFs) (3). As disease progresses, PASMCs acquire proliferative, apoptosis-resistant phenotype, which is supported by complex rewiring of intracellular signaling pathways. Similar to cancer cells, molecular basis for PAH PASMC hyper-proliferation and apoptosis resistance is supported by up-regulation of pro-proliferative/pro-survival cascades (e.g. Ras/Raf/MEK/MAPK, PI3K/Akt/mTOR), Notch, HIF1, cMyc, cyclin D1, transcriptional coactivators YAP/TAZ, and pro-inflammatory NFAT and NFκB. This is accompanied by deficiency or dysfunction of growth suppressors, including BMPR2, HIPPO, FoxO transcription factors, p27, p21, and outward rectifier K⁺ channel KCNK3 (3).

Calcium (Ca²⁺) is a master regulator of mitochondrial respiration, contraction, proliferation, and migration of vascular smooth muscle cells (4), Not surprisingly, alterations in intracellular Ca²⁺ homeostasis are linked with PASMC dysfunction in PAH. An increase in cytosolic free Ca²⁺ and a decrease in mitochondrial Ca²⁺ concentrations promote pulmonary vasoconstriction and PASMC proliferation through activation of PI3K/Akt/mTOR and MAPK pathways (5, 6). One of the important regulators of Ca²⁺ homeostasis is a store-operated Ca²⁺ channel (SOC), which mediates store-operated Ca²⁺ entry (SOCE). SOC consists of the Ca²⁺ channels Orai1–3 and, in part, transient receptor potential canonical channels (TRPCs), which are activated by stromal interacting molecules (STIM) in response to G-protein coupled receptors/phospholipase C (PLC)-induced endoplasmic reticulum (ER)-sarcoplasmic reticulum (SR) Ca²⁺ store depletion (7). Orai promotes growth and metastasis of various human cancers (8) and cellular remodeling in cardiorespiratory diseases, including systemic hypertension, restenosis, and atherosclerosis (9), making Orai an attractive target molecule for developing new anti-proliferative therapies. Accumulating evidence suggests importance of Orai in pulmonary vascular remodeling (7, 9, 10), but the mechanisms of regulation, function, and potential therapeutic attractiveness of Orai channels in PAH remain elusive.

In the current issue of the Journal, Masson and colleagues (11) demonstrate that expression and function of Orai1 are increased in PASMCs from small remodeled PAs from human PAH lungs, which was required for the elevated SOCE amplitude, PASMC hyper-proliferation and survival (Figure 1). Intriguingly, the authors found that depletion or inactivation of Orai1 reduced mitochondrial Ca²⁺ uptake in PAH PASMCs. This observation is intriguing in view of previous reports showing a strong association between a decrease in mitochondrial Ca²⁺ and PAH pathogenesis. Indeed, deficiency of the mitochondrial calcium uniporter or uncoupling protein 2 caused a decrease in mitochondrial Ca² and induced the hyper-proliferative pro-survival PAH PASMC phenotype, pulmonary vascular remodeling, and experimental PH by influencing mitochondrial dynamics and suppressing glucose oxidation (6, 12). Although further studies are needed to determine connections among major players in mitochondrial Ca²⁺ regulation, it is possible that the “Goldilocks principle” may apply to the mitochondrial Ca²⁺ homeostasis, where either too much or too little mitochondrial Ca²⁺ triggers PASMCs hyper-proliferation and remodeling via different signaling mechanisms.

Figure 1. Potential mechanism of Orai1 signaling in pulmonary arterial hypertension.

Left panel: Orai1 is up-regulated in PASMCs in small PAs in human PAH and rat experimental PH. Middle panel: Orai1 up-regulation in PAH PASMCs is triggered by PDGF-Akt-mTOR and hypoxia, and supported by MEK1/2, NFκB, deficiency of KCNK3, and calcineurin-NFAT loop. Orai1 induces SOCE and cytosolic Ca²⁺ increase, mitochondrial Ca²⁺ uptake, reduces p21, p38, and cleaved Caspase 9, promotes PASMC proliferation, migration, and apoptosis resistance, pulmonary vascular remodeling, and PH. Right panel: Down-regulation of Orai1 reduces SOCE, mitochondrial Ca²⁺ uptake, proliferation, migration, and apoptosis resistance of PAH PASMCs. Pharmacological inhibition of Orai1 reduces PH and improves RV in rats with experimental PH. MCT – monocrotaline; mTOR – mechanistic target of rapamycin; PAs – pulmonary arteries; PAH – pulmonary arterial hypertension; PASMCs – pulmonary arterial smooth muscle cells; PDGF – platelet-derived growth factor; PH – pulmonary hypertension; RV – right ventricle; RVSP – RV systolic pressure.

Little is known about the mechanisms driving Orai1 overexpression in PAH pulmonary vasculature. Suggestive of the multi-factorial nature of Orai1 regulation, in PASMCs Orai1 is up-regulated by platelet-derived growth factor (PDGF)/Akt/mTOR axis and hypoxia, well-described pro-remodeling players in PAH (10, 11, 13). Further expanding our knowledge, Masson et al. report that Orai1 over-accumulation in PASMCs from patients with advanced PAH is supported by pro-proliferative-pro-survival MEK1/2, pro-inflammatory NFAT and NFkB, and by a deficiency of anti-proliferative KCNK3 independently of BMPR2 and Akt/mTOR (11). Continuing to unravel Orai1-dependent signaling, the authors show that Orai1 supports the pro-proliferative/pro-migratory PAH PASMC phenotype by down-regulating cell cycle inhibitor p21, p38 signaling, caspase 9 activity, and through activation of serine/threonine phosphatase calcineurin. This leads us to hypothesize that in PAH PASMCs Orai1 could act as a signaling hub that coordinates growth factor and inflammatory signals to enable cell cycle progression, enhance migratory potential, and protect cells from apoptosis. Furthermore, the calcineurin/NFAT pathway appeared to be acting both upstream and downstream of Orai1. This raises the intriguing possibility that up-regulation of Orai1 in proliferative PAH PASMCs is self-supported via the calcineurin/NFAT loop, explaining the molecular basis of unstimulated PAH PASMC proliferation.

In addition to describing the novel mechanism of Orai1 regulation and function in PAH PASMCs, Masson et al. (11) provide preclinical evidence highlighting the potential attractiveness of targeting Orai1 to reduce established PAH. The authors demonstrate that pharmacological inhibition of Orai1 reduced hyper-proliferation and migration of human PAH PASMCs in vitro, decreased constriction of human isolated PAs, and prevented or attenuated pulmonary vascular remodeling, PH, RV hypertrophy and fibrosis in vivo in rats with chronic hypoxia, monocrotaline, and SU5416/hypoxia-induced PH (Figure 1). Overall, the study by Masson et al. provides compelling evidence of the important role of Orai1 in PASMC remodeling and suggests potential benefits of pharmacological targeting of Orai1 to reduce both, pulmonary vascular remodeling and vasoconstriction.

There are, however, some important questions that remain. Uncovered by the authors, the importance of Orai1 in the PAH PASMC signalome calls for broader investigation of Orai-dependent molecular mechanisms in PAH. Orai channels play a role in endothelial proliferation, apoptosis, permeability, and endothelial-to-mesenchymal transition, immune cells behavior, and platelet activation and thrombosis (8–10), all of which are involved in PAH pathogenesis. Although the role of Orai1 in PASMCs has been evaluated by the authors in depth, the involvement of Orai1 in PAEC and PAAF pathobiology needs to be further investigated, and its role in the inflammatory response in PAH remains to be determined. Further, in addition to Orai1, the roles of pro-oncogenic Orai2 and 3 isoforms in PAH also remain to be established.

In vivo experiments performed by Masson et al. (11) show multiple benefits of single-agent treatment by Orai1 inhibitors to attenuate pulmonary vascular remodeling and PH in male rats. However, additional preclinical studies are needed to assure a safe transition to the clinical trials. PAH is a predominantly female disease, and the role of estrogen in PASMC proliferation, survival, and remodeling is well documented (1). Importantly, estrogen acts as an upstream regulator of Orai1 and Ca²⁺ influx in modulating cell proliferation (14), and affects p38, MAPK, and Akt signaling in a sex-dependent manner. Thus, it is important to test for potential differences in Orai1 inhibitor responses in males and females, and to determine whether combining Orai1 inhibition with aromatase inhibitors benefits patients with PAH. Another potential challenge is that the standard of care for patients with PAH is a double or triple combination therapy that includes vasodilators. Orai1 promotes arterial vasoconstriction (15). Careful preclinical analysis of potential systemic side effects of Orai1 inhibitors administered in combination with current standard of care vasodilators would be beneficial.

In conclusion, the article by Masson et al. (11) provides important information about the role and translational significance of Orai1 in PASMC remodeling in PAH and opens several important avenues for further investigation to expand our understanding of the role of Orai channels beyond PASMCs and to further explore the potential attractiveness of targeting Orai1 to treat this devastating disease.