Obstructive sleep apnea (OSA) characterized by cyclic episodes of intermittent hypoxia is a health problem that affect a large worldwide adult population (Somers et al., 2008; Dempsey et al., 2010; Floras, 2018). Obstructive sleep apnea is associated with somnolence, sleep fragmentation and cognitive dysfunction, but it is also considered as an independent risk factor for systemic diurnal hypertension (Somers et al., 2008; Dempsey et al., 2010; Iturriaga et al., 2021). Furthermore, OSA is associated with mild pulmonary hypertension with an incidence ranging from 20-50% (Sajkov and McEvoy, 2009; Floras, 2018). Chronic intermittent hypoxia (CIH) is the main factor for develop systemic hypertension in OSA patients (Somers et al., 2008; Dempsey et al., 2010) and animal exposed to CIH (Del Rio et al., 2010, 2014; Iturriaga et al., 2014, 2021; Iturriaga, 2018; Harki et al., 2021). In addition, CIH produces vascular pulmonary remodeling and increases right ventricular systolic pressure (RVSP) in rodents (Nisbet et al., 2009; Jin et al., 2014, 2016; Iturriaga and Castillo-Galán, 2019; Castillo-Galán et al., 2020). Recently, we found that CIH progressively increased the right ventricular systolic pressure (RVSP) over 35 mm Hg in the rat, enhanced the vasoconstrictor response of small pulmonary arteries to KCl and endothelin-1 (ET-1), increased the medial layer thickness, and increased the right-ventricular wall thickness and the right-ventricular end systolic volume related to the control rats (Castillo-Galán et al., 2020). However, even though the link between OSA and pulmonary hypertension is well recognized, the underlying pathogenic mechanisms are not completely known. The pathological pulmonary vascular alterations induced by CIH has been attributed to Ca^2+–dependent enhanced contractility and proliferation of pulmonary arterial smooth muscle cells (PASMC) induced by the activation of hypoxic inducible factors (HIFs) and oxidative stress (Nisbet et al., 2009: Jin et al., 2014, 2016; Iturriaga and Castillo-Galán, 2019). Thus, it is likely that the activation of HIFs and reactive oxygen species (ROS)-dependent signaling pathways may increase intracellular PASMC calcium levels contributing to the development of the CIH-induced enhanced vasoconstriction, cell proliferation and pulmonary hypertension (Waypa et al., 2002; Sylvester et al., 2012).

Among channels and transporters involved in Ca²⁺ intracellular homeostasis, it has been proposed that store operated calcium entry contributes to enhance the PASMC contractility in sustained hypoxia (Kuhr et al., 2012; Wang et al., 2012, 2017; Reyes et al., 2018). The binding of endothelin-1 (ET-1) or serotonin (5-HT) to their receptors coupled to G-protein evokes the release of the second messenger IP₃, which in turn activates intracellular calcium channels (IP3R) depleting the Ca²⁺ stores of the sarcoplasmic reticulum (Zhang et al., 2003; Wang et al., 2012). STOC are activated by a depletion of Ca²⁺ stores in the sarcoplasmic reticulum, which allow the entry of calcium from the extracellular space. A growing body of evidence supports the contribution of STOC in the contraction of pulmonary arteries in response to acute and sustained hypoxia (Weigand et al., 2005; Lu et al., 2008, 2009; Ng et al., 2008; Guibert et al., 2011). Furthermore, STOC-mediated calcium entry participates in pathological pulmonary vascular remodeling process, such as hypercontractility, proliferation and differentiation of PASMC (Golovina et al., 2001; Hou et al., 2013; Reyes et al., 2018). From a structural point of view, STOC are composed by homo or heterotetramer pore-forming subunits from the TRPC family (TRPC 1 to 7), or to the ORAI family (ORAI 1 to 3) (Reyes et al., 2018). On the other hand, an auxiliary subunit of the family of stromal interaction molecules [stromal interaction molecule (STIM) 1 and 2] acts as a calcium sensor activating pore-forming subunits in response to calcium depletion from sarcoplasmic reticulum deposits (Fuchs et al., 2010; Johnstone et al., 2010; Wray and Burdyga, 2010; Yang et al., 2010). Indeed, a growing body of evidence suggests that STIM-activated TRPC-ORAI Ca²⁺ channels (STOC) play a key role in PASMC remodeling and hypercontractility, which led to pulmonary hypertension in animals exposed to sustained hypoxia (Weigand et al., 2005; Ng et al., 2008; Castillo-Galán et al., 2016, 2022)

Recently, we found that CIH produced the upregulation of mRNA and protein levels of TRPC1, TRPC4, TRPC6, ORAI1, and STIM1 subunits in the rat lung tissue, which paralleled the vascular remodeling and pulmonary hypertension (Castillo-Galán et al., 2020), suggesting that STOC participate in the vascular alterations induced by CIH. Accordingly, to assess the contribution of STOC on the CIH-induced pulmonary vascular alterations, we study the effects of the STOC non-selective inhibitor 2-aminoethyldiphenylborinate (2-APB) (Bootman et al., 2002; Smith et al., 2015; Castillo-Galán et al., 2016, 2022; Jain et al., 2021) on the development of pulmonary hypertension and vascular remodeling induced by CIH in a well stablished preclinical model of OSA (Del Rio et al., 2010, 2012, 2014; Iturriaga et al., 2014, Iturriaga and Castillo-Galán, 2019). We also determined the effects of 2-APB on systemic and lung oxidative stress induced by CIH, because 2-APB ameliorates the oxidative stress in a heart ischemia-reperfusion model in mice (Morihara et al., 2017) and rat (Shen et al., 2021).

We studied the effects of 2-APB, a drug with inhibitory actions on STOC (Bootman et al., 2002, Castillo-Galán et al., 2016, 2022), in a rat model of CIH-induced pulmonary hypertension (Del Rio et al., 2010, 2012, 2014; Iturriaga et al., 2014; Castillo-Galán et al., 2020). Consistently with previous studies, rats exposed to CIH for 28 days, develop vascular remodeling characterized by an increased thickness of the medial layer in pulmonary arteries and an elevated right ventricular systolic pressure (Nisbet et al., 2009; Jin et al., 2014, 2016; Iturriaga and Castillo-Galán, 2019; Castillo-Galán et al., 2020). The main findings of this study are that 2-APB treatment prevented the raise of RVSP, the pulmonary vascular remodeling characterized by an increased medial layer thickness, the increased α-actin-it and Ki-67 immunoreactive levels, and the lumen vascular surface reduction. In addition, we found that 2-APB did not reduce the lung and plasma oxidative stress, suggesting that the effects of 2-APB on vascular remodeling and pulmonary hypertension are independent of the oxidative stress action. Thus, present results strongly suggest that STOC contribute to the pulmonary hypertension and vascular remodeling in a pre-clinical model of OSA.

Present results showing that 2-APB infusion prevented the increase of RVSP induced by CIH, agree and extended previous studies, which found that 2-APB treatment attenuated the pulmonary vascular remodeling, the increased RVSP and the Fulton index in mice exposed for 4 weeks to sustained hypoxia, as compared to hypoxic animals treated with Vehicle (Smith et al., 2015). Similarly, the daily infusion of 2-APB reduced the vascular remodeling and pulmonary hypertension induced by pregestational hypoxia in newborn lambs in high altitude (Castillo-Galán et al., 2016). Interestingly, our results showed that 2-APB prevented the over-expression of TRPC subunits 1, 4 and 6 induced by CIH in the lung tissue. On the other hand, CIH activates the ET-1 – IP3R pathway in PASMC, releasing Ca²⁺ from the reticulum, which in turn activate STOC (Wang et al., 2012; Castillo-Galán et al., 2020) and raise the intracellular Ca²⁺ concentration (Reyes et al., 2018). In addition, Ca²⁺ activates or stabilized transcription factors that upregulated STOC subunits such as HIFs, NFAT, AP-1, NF-KB and the Platelet Derived Growth Factor (Shibasaki et al., 1996; Timmerman et al., 1996; Dolmetsch et al., 1997; Chen et al., 1998; Salnikow et al., 2002; Mottet et al., 2003; Ogawa et al., 2012; Jiang et al., 2014; Domínguez-Rodríguez et al., 2015; Wang et al., 2015, 2017). Taken together, the evidence suggests that 2-APB would induce a decrease in intracellular calcium (Parrau et al., 2013), which may inhibit the upregulation of TRPC1, TRPC4 and TRPC6. However, more studies are required to determine if all of these factors that regulated STOC changed in the lung tissue following CIH.

In this study, 2-APB was administered with osmotic pumps at a delivery rate of 10 mg/kg per day, which is similar to the daily dose range used by Smith et al. (2015), Castillo-Galán et al. (2016, 2022) and Jain et al. (2021) to block STOC in vivo. Indeed, Smith et al. (2015) treated chronic hypoxic mice with 2-APB (1 mg/kg/day i.p.) to reduce pulmonary hypertension and right ventricular enlargement. Castillo-Galán et al. (2016, 2022), administered 2-APB (10 mg/kg/day i.v.) to neonatal lambs with a partial or full gestation in high altitude to ameliorate the pulmonary hypertension and vascular remodeling, and Jain et al. (2021) administrated 2-APB (1 mg/kg per day i.p.) to attenuate pulmonary hypertension in hypoxic mice. We did not measure the plasma concentrations of 2-APB after its administration, but Parrau et al. (2013) estimated that a single dose of 2-APB (8 mg/kg i.v.) would reach a concentration in the extracellular space in the range of 10–100 μM, assuming a distribution volume of 40% of the body weight. The same order of 2-APB concentration blocks STOC in isolated pulmonary arteries from hypoxic lambs (Parrau et al., 2013) and in HEK293 cells transfected with Orai/Stim or TRPC subunits (Lievremont et al., 2005; Peinelt et al., 2008).

Since 2-APB is a non-selective STOC blocker, we cannot preclude effects of 2-APB at the IP3R level. This make it uncertain to determine to what extent the effects of 2-APB on pulmonary vascular remodeling and hypertension are due to its effects on any of these targets. The participation of IP3R on the response of pulmonary arteries to hypoxia is controversial and depend on the species and type of vessel studied. While in rat distal pulmonary artery PASMC, both Ca²⁺ release from intracellular stores and Ca²⁺ entry from the extracellular space induced by hypoxia are abolished by IP3R blockade with xestonpongin-C (Wang et al., 2012), in canine pulmonary artery PASMC, Ca²⁺ entry mediated by STOC in response to hypoxia is unaffected by IP3R blockade (Ng et al., 2008). On the other hand, in rat main pulmonary arteries, both cytosolic Ca²⁺ increase and contraction in response to hypoxia are biphasic, with a rapid transient fast response followed by a slowly developing and sustained response. In these arteries, the contraction in response to hypoxia develop without changes in IP3 content (Jin et al., 1993), Indeed, it has been proposed that STOC account for the sustained Ca²⁺ increase in response to hypoxia (Sylvester et al., 2012). Moreover, in isolated pulmonary arteries of newborn lambs, the relaxation elicited by SKF-96365, a specific STOC blocker without effect on IP3R is similar to the relaxation induced by 2-APB, and both of them increased in pulmonary arteries from hypoxic lambs (Parrau et al., 2013). Taken together these observations suggest that 2-APB may act on IP3R and STOC, but the final effect is an indirect or a direct reduction of Ca²⁺ entry mediated by STOC in pulmonary PASMC from animals exposed to hypoxia. Considering that 2-APB is not a selective STOC blocker, new drugs with a selective effects on ORAI1/STIM1 interaction are needed to evaluate the role of the STOC in OSA patients with pulmonary hypertension (Reyes et al., 2018; Waldron et al., 2019).

Chronic intermittent hypoxia induces oxidative stress at the pulmonary and systemic level, which may affect calcium homeostasis and contribute to the lung vascular remodeling (Del Rio et al., 2010, 2012; Jin et al., 2014, 2016; Iturriaga and Castillo-Galán, 2019; Castillo-Galán et al., 2020). Antioxidants such as procyanidin and melatonin reduced the elevated RVSP and the vascular remodeling induced by CIH (Jin et al., 2014, 2016). The activation of TRPC1 and TRPC6 by the transcription factor BMP4 in rat PASMC depended on the increased ROS levels mediated by NOX4 (Jiang et al., 2014). In addition, it has been proposed that the increase in ROS levels induced by hypoxia, activates the ryanodine receptors (RyR), which in turn induces the release of the reticular Ca²⁺, activating STIM1, and allowing the interaction with ORAI in the plasma membrane and subsequent activation of the STOC (Suresh and Shimoda, 2016). These results suggest that the effects of antioxidants may be related with the inactivation of STOC and subsequently decrease in intracellular calcium. Although an antioxidant action of 2-APB has been shown in ischemia/reperfusion damage in mice and rat cardiomyocytes (Morihara et al., 2017; Shen et al., 2021), our results showed that 2-APB did not prevent the systemic and lung oxidative stress, suggesting that the effects of 2-APB on PASMC in the lung are not related by a reduction in oxidative stress. Thus, it is likely that the effects of 2-APB were related to its inhibitory action on the increased intracellular Ca²⁺ concentrations induced by STOC (Bootman et al., 2002). Thus, the treatment of hypoxic pulmonary hypertensive rats with 2-APB, has a strong effect preventing the increase of RVSP and the pulmonary vascular remodeling without any significant action on the oxidative stress markers.

Present results indicated that 2-APB treatment to rats exposed to CIH prevented the increased RVPS and the pulmonary vascular remodeling, without effects on both systemic and pulmonary oxidative stress markers, suggest that STOC are and attractive therapeutic target to prevent pulmonary hypertension in OSA. In summary, present results showed that 2-APB strongly ameliorated the pulmonary vascular alterations induced by CIH, indicates that the STOC inhibition may potentially be used to treat the pulmonary hypertension induced by OSA.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Scientific Ethical Committee for the Animal and Environment Care Pontificia Universidad Católica de Chile, Santiago, Chile (ID:180803006).

SC-G and BR performed the experiments. SC-G and RI contributed to the conception and design of research, analyzed the data, interpreted the results, and drafted the manuscript. All authors revised the manuscript and approved final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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