In this study, we report that NAC reduces TPR and improves right ventricular function in rats with MCT-induced PH, partly through its immune-modulatory and cardioprotective properties. Due to the strong rationale of inflammation in PAH pathophysiology, a lot of studies have already evaluated the benefit of immune-modulatory drugs in PH animal models. However, most of them explored the drug before installation of pulmonary vascular lesions reflecting their capacity to prevent the disease and not to cure it. Despite improvement in the field of PAH, diagnosis of PAH patients is still delayed with an advanced progression of the disease, while breathlessness appears when 80% of small pulmonary arteries are affected. It is therefore essential that drugs predicted for PH improvement be studied in advanced conditions of the disease. In this study, NAC was introduced 2 weeks after MCT exposure when pulmonary vascular lesions were already developed
, as it has been previously performed in a study using dexamethasone in the same model
. We choose the MCT-induced PH model for its technical simplicity and reproducibility. Although criticized by some authors, MCT remains a great model regarding inflammation process in PH
. It is noteworthy that other models of PH have been developed: Sugen + hypoxia- and MCT + pneumonectomy-induced PH. These models display intimal remodeling and some degree of angioproliferative lesions resembling plexiform lesions. Even if these lesions are pathognomonic of PAH (group 1 of the updated clinical classification of PH
), it is not in fact known whether these lesions participate to the elevated pulmonary vascular resistance responsible for PAH, or if they are just a surrogate marker of disturbed blood flow within the PAH-affected pulmonary vasculature. In contrast, the progressive neomuscularization and obstruction of precapillary resistance arteries occuring in the MCT-induced PH is a robust mechanism of TPR elevation. However it remains interesting to test NAC efficacy in other models of PAH representative of different etiologies and of potential different pathomechanisms, like chronic hypoxia or hypoxia + sugen-induced PH.
In our current study, two weeks treatment with NAC 14 days after MCT exposition led to a significant decrease of pulmonary vascular remodeling characterized hemodynamically by a reduced level of TPR and an unmodified mPAP and RVSP. In a previous study, we reported that severity of pulmonary vascular remodeling in a permanent high-flow challenged animal model associated with MCT correlated with lung expression of pro-inflammatory cytokines and recruitment of both monocyte/macrophages and dendritic cells in the pulmonary vasculature
. The reduction of the lung inflammatory status observed in this study with the reduced lung cytokine expression and inflammatory cells recruitment could partly explain these results. Potential disease-relevant mechanistic factors of NAC include NF-κB and Angiotensin II (Ang II) signaling. Firstly, NF-κB is the key inflammatory transcription factor well known to be activated by oxidative stress
 which is implicated in human
 and experimental PAH
; its activation leads to the upregulation of chemokines and inflammatory cytokines implicated in human IPAH
 and in the MCT-induced PH model where its inhibition ameliorates PH - both preventatively and therapeutically
[35, 36]. Secondly, involvement of Ang II through its receptor 1 (AT(1)) activation was reported in the pathophysiology of PAH
. Interestingly, NAC decreases Ang II binding to the AT(1) receptor in vascular smooth muscle cells (VSMC) in a concentration-dependent manner, leading to a reduction of VSMC proliferation
. The beneficial effect of NAC could therefore be in part depends on this property. Although it has been reported evidence of NAC-induced vasodilation and hypotension
[39, 40], we didn’t found any systemic effect of NAC in our study.
PAH is characterized by remodeling of the pulmonary arterial vessels, which involves progressive distal vessel obliteration leading to an increase in the TPR. This increase in TPR leads to an increase in the RV afterload leading to RV dysfunction and CO decrease. In our study, mPAP was not statistically changed but TPR were decreased, the degree of distal obliteration (%) was reduced, and CO was increased in the NAC treated group. We might explain the decrease in TPR by the significant decrease in the distal artery occlusion. The simplified classical relationship between mPAP, CO and TRP is the following: mPAP = CO*TPR. In other terms, CO = mPAP/TPR. So even if mPAP remains not statistically changed, the increase in CO might be the consequence of reduced TPR and therefore of reduced distal pulmonary vascular occlusion. CO depending on proper calibration of RV contractility and impedance to blood flow through the lungs, NAC may also have a direct beneficial impact on CO though improvement of RV contractility. However, this parameter has not been analyzed in our study but deserve further investigation in order to ascertain a direct RV protective effect in PH. Another argument in favor of a direct impact of NAC on right ventricle is provided in a recent review by Voelkel on oxidative stress and PH
. In this review, influence of oxidative stress on pulmonary vasculature and cardiac cells was extensively analyzed, particularly in the RV failure mechanism. The authors propose a kinetic model of oxidative stress with an impact on pulmonary vasculature at an early stage and on RV at a later stage and throughout the development of the RV disease. In order to confirm their hypothesis on cardiac dysfunction, protandim treatment (having antioxidant properties) of the Su/Hx rats prevented the development of RV failure and fibrosis. Analysis of mitochondrial and metabolic gene remodeling in the RV as it was recently reported by Gomez et al., would probably also afford significant information on cardio protective pathway of NAC and need to be performed in next experiments
Pressure overload usually increases pulmonary vascular resistance and cardiomyocyte stress leading to cardiomyocyte hypertrophy and extracellular matrix changes with fibrosis. Endomyocardial biopsy specimens from patients with PAH show increased levels of fibrosis affecting myocardial systolic and diastolic function
[43, 44]. In a recent review analyzing the RV under pressure, maladaptative neurohormonal signaling, oxidative stress and inflammation in the heart were reported as processes possibly accelerating the development of right-heart failure in PAH
. In vitro and in vivo studies have shown that reactive oxygen species (ROS) induce cardiomyocyte hypertrophy as well as fibrosis
 and in the MCT-induced model of PH, right ventricular failure was associated with oxidative stress
. Moreover, in conditions of ischemia/reperfusion, macrophages recruit neutrophils through the secretion of IL-6, which are an important source of ROS
. Interestingly, Ang II induces cardiomyocyte hypertrophy, inflammation, fibrosis and contractile dysfunction through in part by the formation of ROS
. Due to its antioxidant, anti-inflammatory and cardioprotective properties, NAC improved right ventricular function (CO) with inhibition of cardiomyocyte hypertrophy and fibrosis.
Although specific mechanisms of NAC on RV preservation still remain elusive, improvement of right ventricular function with NAC is a particularly relevant issue since despite PAH specific therapeutics, pulmonary microvascular obstruction usually progresses and imposes an increasing load on the RV
. The patient outcome is therefore predominantly determined by the response of the RV to the increased afterload and RV function is therefore a strong marker of prognosis and disease severity
. Advance in new therapies acting on right ventricular function is therefore relevant in PAH management. However, current available or experimental PAH treatments are used to induce pulmonary vasodilation and reverse pulmonary vascular remodeling, and little is known about their effect on the heart. An ideal PAH treatment strategy would therefore both reduce pulmonary vascular resistance and improve right ventricular function. Here, we report that NAC, a well-known safe drug in current clinical use, has beneficial effect on these parameters in an experimental model of PH.