1. Ca²⁺ Channels

1.1. Direct Pharmacological Action

The L-type Ca²⁺ channel blockers, nifedipine, diltiazem, and amlodipine, are currently the only therapy used in PAH acting on ion channels ^[1][2]. The use of these inhibitors cause significant clinical and hemodynamic improvement in patients with PAH. However, this therapy approach is not possible for all patients and, therefore, they should undergo an acute trial with a pulmonary vasodilator, NO, beforehand. Patients with iPAH underwent oral treatment with Ca²⁺ channel blockers (CCBs), but only 54% responded positively. Patients who responded positively to treatment demonstrated clear improvement including less severe disease, a higher proportion of patients in NYHA functional class II, better 6MWD test and less severe hemodynamic parameters, as well as improvement after one year of treatment ^[2]. Although they are already used in PAH therapy, there are no known advantages in terms of their action at the endothelial level.

1.2. Indirect Pharmacological Action

Blocking T-type Ca²⁺ channels has a greater impact on inhibiting cell proliferation than inhibiting L-type Ca²⁺ channels. Mibefradil, a T-type Ca²⁺ channel blocker, completely inhibits cell proliferation and prevents entry into the cell cycle. On the other hand, diltiazem, an L-type Ca²⁺ channel blocker, did not show marked effects. Selective blocking of Cav3.1 expression with siRNA completely inhibited proliferation and prevented entry into the cell cycle ^[3]. In animal models of CH-induced PH, blockade of T-type Ca²⁺ channels by TTA-A2 prevented induced PH, reduced right cardiac hypertrophy, and induced pulmonary artery remodeling. In addition, TTA-A2 decreased PASMCs proliferation and prevented vascular hyperreactivity ^[4]. Verapamil and SKF 525A, known antagonists of Ca²⁺ channels, were also responsible for inhibiting hypoxic pulmonary vasoconstriction, suggesting that this was essentially mediated by the transmembrane influx of extracellular Ca^{2+ [5]}.

There are several pharmacological compounds with a preventive effect on PAH, however, they do not specifically affect ECs. Sildenafil, for example, an inhibitor of phosphodiesterase type 5, has a positive impact on therapy in patients with PAH ^[6][7][8] as it has an inhibitory effect on the proliferation of hPASMCs. The antiproliferative effect of sildenafil may be related to the downregulation of TRPC1 gene expression ^[9]. Pyrazol2, a blocker of TRPC channels, has been shown to significantly attenuate RV hypertrophy and PAH in mice with MCT-induced PAH ^[10]. Furthermore, iloprost, the synthetic analog of prostacyclins, substantially decreased the expression of TRPC3 in iPAH-PASMCs, consequently inducing a decrease in the proliferation of PASMCs; suggesting that the vasodilatory and antiproliferative effects of prostacyclin and its analogs may be involved in the inhibition of TRPC expression in PASMCs ^[11]. Therefore, targeting PAH therapies to TRPC channels may be a useful therapeutic strategy. At the EC level, further studies should be carried out to understand the interactions of these channels in ECs and, consequently, in PAH.

2. Na⁺ Channels

The regulation of NHE is essential to maintain intracellular pH, however, it only plays a permissive role in the proliferation of PASMCs, and little is known about its action at the level of PAECs. However, NHE inhibitors appear to be attractive therapeutic targets for PAH.

2.1 Indirect Pharmacological Action

The use of NHE inhibitors, such as dimethyl amiloride and ethylisopropylamiloride, have been shown to significantly reduce pulmonary vascular remodeling in a hypoxia-induced PH animal model ^[12]. Sabiporide, a member of the NHE1 inhibitor family, also inhibits the proliferation and migration of hPASMCs, blocking cell progression ^[13]. Cariporide, another NHE inhibitor, attenuates the development of right heart failure. The use of this inhibitor in mice with MCT-induced PH promoted a decrease in RVSP and RV hypertrophy. In addition, cariporide attenuated necrosis fibrosis and induced RV myocardium mononuclear cell infiltration ^[14].

Another mechanism to inhibit NHE is the upstream inhibition of hypoxia-inducible factor 1 (HIF-1). HIF-1 is a transcription factor that upregulates the NHE1 gene in response to hypoxia ^[15]. Studies show that digoxin, a cardiac glycoside, inhibits the transcriptional activity of HIF-1. Digoxin treatment in mice with hypoxia-induced PH has been shown to attenuate increase in RVSP, RV hypertrophy, and pulmonary vascular remodeling and to delay the progression of established PH. Furthermore, digoxin treatment prevented increase in NHE1 expression in PASMCs. Treatment with acriflavine, another HIF inhibitor, but by a different path, also prevented the development of PH ^[16]. However, to date, at the level of PAECs, little is known about the inhibition of these channels and their possible involvement in the pathophysiology of PAH.

3. Cl-Channels

TMEM16A expression and function are significantly increased in PAH. Studies on iPAH-PASMCs have shown that TMEM16A is upregulated, increasing the Cl^- current. Inhibition of TMEM16A by benzbromarone (BBR) reversed membrane depolarization in iPAH-PASMCs to healthy levels. Chronic treatment with BBR in animal models of PH promoted a considerable reduction in RVSP and pulmonary artery muscularization, demonstrating a potent attenuation of vascular remodeling. BBR has been approved for the treatment of gout in humans, with a maximum oral dose of 200 mg. Therefore, more studies should be undertaken to understand the possible effects of BBR in the treatment of iPAH and to determine recommended doses ^[17].

Treatment with T16Ainh-A01, an aminophenylthiazole inhibitor of TMEM16A, was shown to be beneficial in MCT-induced PAH mice. Administration of T16Ainh-A01 significantly alleviated pulmonary arteriole remodeling and RV hypertrophy, decreased pulmonary artery pressure, and decreased upregulation of proliferating nuclear antigen (PCNA) in pulmonary arteries. T16Ainh-A01, in addition to inhibiting the activity of the TMEM16A channel, also suppressed its effect on cell proliferation. Administration of T16Ainh-A01 significantly improved PAH. However, it did not lead to full recovery. Thus, T16Ainh-A01 appears to be a promising drug in improving vascular remodeling ^[18]. Increase in TMEM16A expression in healthy PAECs has functional consequences in PAECs, such as changes in Ca²⁺ dynamics and eNOS activity, decreasing NO production, promoting PAECs proliferation, wound healing, tube formation and relaxation of pulmonary artery mediated by ACh ^[19]. Studies should be carried out to understand whether silencing TMEM16A can reverse the PAH-induced phenotype in healthy PAECs and whether it will be a therapeutic candidate for PAH.

4. K⁺ Channels

4.1. Direct Pharmacological Action

PAH is associated with a loss of K⁺ channel expression and activity. Pozeg et al. demonstrated that in vivo gene transfer of the Kv1.5 channel in CH-induced PH rats promoted a significant improvement in PH as well as hypoxic pulmonary vasoconstriction ^[20]. Furthermore, studies have demonstrated that KCNA5 gene transfer, in addition to increasing K⁺ currents, increased caspase-3 activity and accelerated apoptosis. Thus, the induction of apoptosis through gene therapy can be a fundamental strategy to prevent the progression of pulmonary vascular wall thickening and for treating iPAH patients ^[21]. However, the authors only focused on KCNA5 re-expression in PASMCs. The researchers did not find any information on the consequences of KCNA5 re-expression in PAECs.

Nicorandil, a nicotinamide ester, is a K_ATP channel opener with a NO release vasodilator function. In an animal model of MCT-induced PAH, nicorandil protected the pulmonary endothelium from damage, reduced apoptosis, and attenuated PAH development through upregulation of eNOS expression anti-apoptotic factors, mediated by PI3K/Akt and ERK1/2 signaling pathways ^[22]. In this way, nicorandil could be an attractive therapeutic target at the endothelial level for the treatment of PAH. In addition, iptakalim (2,3-dimethyl-n-(1-methylethyl)-2-butanamine hydrochloride), a compound responsible for opening K_ATP channels, is involved in inhibiting PASMCs proliferation and pulmonary vascular remodeling by downregulation of PKC-alpha ^[23]. In several animal models of PH, treatment with iptakalim attenuated induced PH and pulmonary arterial wall remodeling. In addition, it attenuated the inflammatory response and prevented endothelial damage ^[24][25]. However, at PAEC level, little is known about its therapeutic effect.

4.2. Indirect Pharmacological Action

Levosimendan is a calcium-sensitizing medication used clinically to treat right heart failure in PH. Studies in an MCT model of PH demonstrated that levosimendan attenuated increase in pulmonary vascular medial wall thickness and significantly decreased the proliferation of PASMCs in vitro and in vivo. This effect appeared to be K_ATP-dependent. In addition, levosimendan increased endothelial NO generation and decreased the expression of inflammatory genes in ECs. Thus, levosimendan reduces pulmonary vascular remodeling, probably due to an antiproliferative and anti-inflammatory effect, making it an interesting target for treating PAH ^[26].

In 2013, six new heterozygous missense variants were identified in KCNK3. Electrophysiological studies found that all variants resulted in the loss of function. The administration of the phospholipase A2 inhibitor ONO-RS-082 reactivated the current of some KCNK3 mutants. In this sense, ONO-RS-082 could be a new therapeutic approach for PAH ^[27]. Several studies were carried out after these discoveries. Antigny et al. demonstrated that KCNK3 expression and function were reduced in patients with PAH and rats with MCT-induced PH. In vitro, using the patch-clamp technique in PASMCs and PAECs, it was found that ONO-RS-082 promoted an improvement in the endogenous KCNK3 current in rats. Furthermore, it was found that, although treatment with ONO-RS-082 was ineffective in reducing PH symptoms, preventive therapy with ONO-RS-082 improved hemodynamically induced PH symptoms in RV hypertrophy and pulmonary vascular remodeling ^[28]. Thus, since the loss of KCNK3 may be a key event in the pathogenesis of PAH, there is interest in furthering this therapeutic approach.

4.3. Untested Pharmacological Compounds

K_v channels are downregulated in patients with PAH. Morecroft et al. studied the influence that flupirtine, a K_v7 channel activator, had in two models of induced PAH. In both animal models, an increase in mean RV pressure (mRVP) was associated with remodeling of the pulmonary vasculature and RV hypertrophy. In the presence of flupirtine, these effects were attenuated as a specific result of increase in mRVP through the activation of the K_v7 channel. Thus, therapies that involve the activation of K_v7 channels appear to be beneficial in treating PAH of different etiologies ^[29]. In addition, flupirtine reduced vascular resistance in the lung and inhibited hypoxic PH ^[30].