Targeting L‐arginine‐nitric oxide‐cGMP pathway in pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) remains a devastating and deadly disease with a poor long-term prognosis. The median survival for untreated patients is only 2.8 years, with pitiful three-year, five-year, and sevenyear survival rates of 68%, 57%, and 49%, respectively. The goal of a breakthrough discovery leading to a meaningfully life-extending treatment protocol has not yet been achieved, as the exact pathogenic mechanisms of pulmonary arterial hypertension (PAH) are too complex for current understanding. Research has, on the bright side, yielded a promising, increasingly detailed map of disease processes. Endothelium-derived nitric oxide (NO) is a major vasodilator, while its downstream effectors, cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG), have been demonstrated to cause vasodilative, anti-proliferative, anti-coagulant, and anti-inflammatory effects on pulmonary vasculature. Therefore, the L-arginine-NO-cGMP-PKG signaling cascade is an important pathway for developing therapies for PAH (Fig. 1). L-arginine is a semi-essential basic amino acid that contains four nitrogen molecules and serves as a substrate for NO synthase (NOS). Activated NOS produces NO by converting L-arginine to L-citrulline in the presence of O2, Ca2þ, heat shock protein 90 (Hsp 90), and tetrahydrobiopterin (BH4). There are three known NOS isoforms: neuronal constitutive NOS (nNOS or NOS1); inducible NOS (iNOS or NOS2); and endothelial constitutive NOS (eNOS or NOS3). NOS1/nNOS and NOS3/eNOS are constitutive NOS (cNOS). Under normal conditions, continual NO production by cNOS is Ca2þ/calmodulin-dependent. An increase in cytosolic Ca2þ concentration ([Ca]cyt) in endothelial cells (ECs), induced by shear force (flowdependent NO formation) or EC membrane receptors (receptor-stimulated NO formation), can activate cNOS, increase NO synthesis and release, and result in vasodilation. The activity of iNOS, which is also expressed in ECs, is very low under normal and basal conditions, and its activation is independent of Ca2þ. During inflammation, iNOS can be activated by bacterial endotoxins (e.g. lipopolysaccharide) and cytokines (e.g. tumor necrosis factor) and produce NO at about 1000-fold greater levels than that produced by cNOS. NO is a potent vasodilator and a modulator of pulmonary hemodynamics. NO rapidly diffuses from the endothelium to the vascular smooth muscle cells (SMCs). Once in the SMC, NO activates the soluble guanylate cyclase (sGC) by binding to the heme-NO/O2-binding domain on the b1 subunit of sGC. Activated sGC catalyzes guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) which is then catalyzed by phosphodiesterase 5 (PDE5) to become 5-GMP (Fig. 1). cGMP is an important intracellular second messenger that activates PKG, cGMP-gated, or cGMP/PKG-sensitive ion channels and Ca2þ-activated Kþ (BK) channels in SMCs. Increased cytoplasmic cGMP and activated PKG exert relaxant and anti-proliferative effects on pulmonary arterial smooth muscle cells (PASMCs) through activation of cGMP-gated Kþ channels and BK channels, blockade of Ca2þ-permeable channels (e.g. VDCC and TRP channels), and inhibition of specific intracellular signaling cascades that are related to cell proliferation, growth, and migration. Upregulation and maintenance of the L-arginineNO-cGMP signaling pathway is an attractive therapeutic strategy in PAH because of the extensive evidence that altered NO levels play a pivotal role in the pathogenesis of sustained vasoconstriction and excessive vascular remodeling. Intermittent administration of inhaled NO effectively lowers PA pressure in patients with PAH and a patient’s vasoreactive response to NO is a strong predictor of longterm survival. Augmenting the activity of sGC, a downstream target of NO signaling, also provides a reasonable strategy to ameliorate the development of pulmonary hypertension (PH). Riociguat, a sGC stimulator, is the recent drug approved by Food and Drug Administration (FDA) for the treatment of PAH and chronic thromboembolic pulmonary hypertension (CTEPH). Two of PDE5 inhibitors, sildenafil and tadalafil, which inhibit cGMP hydrolysis, have also been approved for the treatment of PAH. Another therapeutic strategy is to improve and restore the activity of endothelial NOS by increasing the availability of the substrates (L-arginine) or stabilizing NOS to improve