Interestingly systemic circulating levels of NOx

Interestingly, systemic circulating levels of NOx were not dramatically increased by treatment with isosorbide mononitrate or sodium nitrite at a dose which exerted beneficial effects on the progression of PH. In line with our results, Mathew et al. [13] demonstrated that the long-acting NO donor molsidomine has therapeutic potential against MCT-induced PH, without a significant increase in plasma nitrate levels. On the other hand, Maruyama et al. [16] showed that continuous NO inhalation fails to improve MCT-induced PH, though it markedly increases urine nitrate levels. These findings suggest that a significant systemic increase in NO should be avoided to treat PH where the cause is in the pulmonary circulatory system. Unfortunately, this study did not measure the local NOx levels, but Cilengitide converting isosorbide mononitrate and sodium nitrite to NO have been proven to be upregulated in the lungs of MCT-injected rats [11,25]. Enough NO may have been supplied to the lungs by treatment with low-dose isosorbide mononitrate or sodium nitrite. RV hypertrophy induced by MCT injection was not inhibited in rats treated with isosorbide mononitrate or sodium nitrite regardless of the dose. Several signaling pathways are intricately related to the progression of PH [4], and it is not always the case that activation or inhibition of only one signaling pathway is enough to completely suppress the progression of disease. For example, the inhibitory effect of the sGC stimulator BAY 41‐8543 on MCT-induced cardiac remodeling is somewhat inferior to its effect on RVSP elevation or pulmonary arterial medial thickening [26]. Furthermore, Paffett et al. [27] and Xie et al. [28] separately reported that chronic administration of the phosphodiesterase 5 inhibitor sildenafil reverses MCT-induced RV systolic dysfunction and pulmonary vascular remodeling but not RV hypertrophy. These are in line with the present data and therefore suggest that MCT-induced RV hypertrophy might not be prevented by the stimulation of the NO/sGC/cGMP pathway. Further studies are needed to investigate this hypothesis. It is difficult at present to identify why NO-enhancing drugs given at a low dose were superior to those given at a high dose in this study. Under stress conditions, excess NO reacts with superoxide to form peroxynitrite, inducing further oxidative and nitrosative stress [29]. There was no difference in the ratio of nitrite/nitrate levels, a marker of oxidative stress, between doses, but peroxynitrite formation was not examined in this study. Since many earlier studies have found that excess exogenous NO produces peroxynitrite in the cardiopulmonary system [[30], [31], [32]], high doses of NO-enhancing drugs may have increased peroxynitrite levels, leading to less effective results. Alternatively, systemic blood pressure was unchanged even in rats subjected to high-dose isosorbide mononitrate or sodium nitrite supplementation, but that might be as a result of baroreflex-mediated sympathetic activation. There is no question that the sympathetic transmitter noradrenaline is capable of enhancing RV systolic function [33,34]. In addition, this transmitter induces left ventricular, but not RV, hypertrophy [35,36]. Furthermore, a blockade of the adrenergic receptors improves RV function and prevents vascular remodeling in MCT-induced PH rats [37]. From this evidence, baroreflex-mediated sympathetic activation can also be considered as one possible reason for the inferiority of the high-dose treatment. As described above, the balance between NO-sensitive and -insensitive sGC is normal in the early stage of PH, but there is a possibility that the redox equilibrium is disrupted in the late stage. We tried to confirm this based on vascular reactivity, but as has been reported previously [[38], [39], [40]], extralobar pulmonary arteries no longer contracted sufficiently in response to spasmogens, including KCl, phenylephrine, U-46619, and endothelin-1, at 28 days after MCT injection (Supplementary Fig. S1). Therefore, it was impossible to check the relaxant responses to BAY 41-2272 and BAY 60-2770. However, in this regard, Pankey et al. [41] reported that changes in pulmonary arterial pressure in response to intravenous injections of sodium nitroprusside and BAY 60–2770 did not decrease and increase, respectively, in rats 28 days after MCT injection. Based on this finding, they concluded that sGC in the pulmonary vascular bed is not oxidized in rats with MCT-induced PH. The sGC redox state might be normal even in the late stage of MCT-induced PH.