**1. Introduction**

In 1980, Furchgott noted that the endothelium produces and releases a vasodilatory substance named endothelium-derived relaxing factor (EDRF), which diffuses into adjacent vascular smooth muscle cells and results in vascular relaxation [1]. In 1987, EDRF was identified as nitric oxide (NO) by Ignarro [2] and Moncada [3]. Murad reported the vasodilatory effect of nitroglycerin and NO formation from nitroglycerin in 1977 [4]. However, at the time, it was not known that endogenous NO is produced and released as a physiological substance in the body, especially in the vascular endothelium.

High-temperature combustion accelerates the reaction of oxygen and nitrogen in air to generate nitrogen oxides (NOx), such as NO, NO2, and N2O3. A common source of NOx is car engines, among which diesel engines have particularly high production. NO reacts with O2 to produce NO2, which is more toxic than NO. Thus, NOx, including NO, is considered an air pollutant. Accordingly, measuring instruments and NO gas standards with known concentrations are needed to assess NO concentrations in air. In addition, NO gas has various industrial applications, including uses in the production of chemicals, semiconductors, integrated circuits, and memory storage elements and devices. Therefore, measuring instruments for NO and the delivery of NO from gas cylinders were developed long before the discovery of NO as a physiological substance in the body. NO is now recognized as a gas and a physiological substance. Pioneering clinicians determined that "as a gas, NO can be administered to the body through the lung." It was fortunate for these clinicians who first conducted NO inhalation in humans that measuring instruments for NO and NO cylinders were available.

The present chapter discusses endogenous NO production in normal and hypertensive pulmonary vasculature, the history of NO inhalation for therapeutic use, the fate of inhaled NO (iNO), effects of iNO in remote organs other than the lung, and iNO as a therapeutic strategy in pediatrics.

## **2. Endogenous NO and its role in the pathogenesis and pathophysiology of pulmonary hypertension**

NO is primarily synthesized by endothelial NO synthase (eNOS, NOSIII) in pulmonary vascular endothelial cells. NO reacts with a receptor, soluble guanylate cyclase (sGC), in adjacent smooth muscle cells. Activated sGC produces cGMP, which stimulates protein kinase G (PKG) and exerts many physiological effects, including pulmonary vascular relaxation. The inhibition of NO production by l-NMMA (*N*-omega-monomethyl-l-arginine, a NOS inhibitor) decreases pulmonary flow in conscious healthy adults [5]. A deficiency in eNOS, but not iNOS or neuronal NOS, induces augmented hypoxic pulmonary vasoconstriction and a lack of endothelium-dependent vasodilation [6]. These findings support the important roles of the eNOS-NO-cyclic guanosine monophosphate (GMP) pathway in maintaining pulmonary circulation. Alteration of eNOS expression and/or function may contribute to decreased NO synthesis in pulmonary hypertension (PH). Human PH has many different etiologies. Depending on the pathological state, patients may exhibit alterations in the eNOS-NO-cGMP pathway.

#### **2.1 Effects of NO in isolated pulmonary arteries**

The effects of NO differ among cell types. NO induces relaxation in vascular smooth muscle cells, prevents aggregation and adhesion in platelets, prevents adhesion in leucocytes, and acts as a neurotransmitter in synapses. Thus, NO regulates various cell functions. In the vasculature, NO is released from endothelial cells, reaches adjacent smooth muscle cells, and causes vascular relaxation, indicating that it functions in intercellular signaling. Among the physiological roles of endogenous NO, vascular relaxation was discovered first.

In isolated rat main pulmonary arterial rings precontracted with prostaglandin F2α (PGF2a), acetylcholine (ACh) induces relaxation in endothelium-preserved pulmonary arteries, but not in endothelium-denuded pulmonary arteries, suggesting that the endothelium in pulmonary arteries produces a relaxation-inducing substance in response to acetylcholine (**Figure 1(a)** and (**b**)).

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**Figure 1.**

*8, 10<sup>−</sup><sup>8</sup>*

endothelium-independent relaxation [7].

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension*

In pulmonary arteries isolated from experimental PH models (chronic hypoxic PH in rat), the relaxation response to acetylcholine (ACh) is depressed, as observed in endothelium-denuded arteries, suggesting that endothelial function is impaired in PH arteries. Both ACh- and sodium nitroprusside (SNP, an NO donor)-induced relaxations were impaired in pulmonary arteries from rats with chronic hypoxic PH, suggesting that NO-induced relaxation is depressed in hypertensive pulmonary arteries [7, 8] (**Figure 2**). However, the magnitude of impairment seems to be higher in ACh-induced endothelium-dependent relaxation than in SNP-induced

 *mol/L, the same for 7, 6, 5, and 4. (B) Scanning electron micrograph of the endothelium-preserved pulmonary artery and endothelium-denuded pulmonary artery. Endothelium was removed by gently rubbing luminal surface by fine stainless wire. Left: luminal surface of the endothelium-preserved pulmonary artery.* 

*(a) Acetylcholine induces relaxation in endothelium-preserved pulmonary arterial rings. Pulmonary artery rings were obtained from normal control air rats. Rings were suspended in an 20 ml organ bath, and isometric tension was measured. Relaxation responses to acetylcholine (Ach) in endothelium-preserved (END+) and endothelium-denuded (END-) rings of the extrapulmonary artery were obtained. Endothelium was removed by gently rubbing luminal surface by fine stainless wire in endothelium-denuded rings. Rings were* 

*as 100%. Bars mean standard error. Relaxation responses to ACh were abolished in the endothelium-denuded pulmonary arterial rings, showing that pulmonary vascular endothelium releases vasorelaxation substance named endothelium-derived relaxing factor (EDRF). The absence of the endothelium was confirmed by scanning electron micrography (b). END−, endothelium-denuded rings; END+, endothelium-preserved rings;* 

 *M papaverine (Pap 4) was taken* 

*precontracted with prostaglandin F2a (PGF2a). Relaxation induced by 10<sup>−</sup><sup>4</sup>*

*Right: luminal surface of the endothelium-denuded pulmonary artery.*

*DOI: http://dx.doi.org/10.5772/intechopen.89381*

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.89381*

#### **Figure 1.**

*(a) Acetylcholine induces relaxation in endothelium-preserved pulmonary arterial rings. Pulmonary artery rings were obtained from normal control air rats. Rings were suspended in an 20 ml organ bath, and isometric tension was measured. Relaxation responses to acetylcholine (Ach) in endothelium-preserved (END+) and endothelium-denuded (END-) rings of the extrapulmonary artery were obtained. Endothelium was removed by gently rubbing luminal surface by fine stainless wire in endothelium-denuded rings. Rings were precontracted with prostaglandin F2a (PGF2a). Relaxation induced by 10<sup>−</sup><sup>4</sup> M papaverine (Pap 4) was taken as 100%. Bars mean standard error. Relaxation responses to ACh were abolished in the endothelium-denuded pulmonary arterial rings, showing that pulmonary vascular endothelium releases vasorelaxation substance named endothelium-derived relaxing factor (EDRF). The absence of the endothelium was confirmed by scanning electron micrography (b). END−, endothelium-denuded rings; END+, endothelium-preserved rings; 8, 10<sup>−</sup><sup>8</sup> mol/L, the same for 7, 6, 5, and 4. (B) Scanning electron micrograph of the endothelium-preserved pulmonary artery and endothelium-denuded pulmonary artery. Endothelium was removed by gently rubbing luminal surface by fine stainless wire. Left: luminal surface of the endothelium-preserved pulmonary artery. Right: luminal surface of the endothelium-denuded pulmonary artery.*

In pulmonary arteries isolated from experimental PH models (chronic hypoxic PH in rat), the relaxation response to acetylcholine (ACh) is depressed, as observed in endothelium-denuded arteries, suggesting that endothelial function is impaired in PH arteries. Both ACh- and sodium nitroprusside (SNP, an NO donor)-induced relaxations were impaired in pulmonary arteries from rats with chronic hypoxic PH, suggesting that NO-induced relaxation is depressed in hypertensive pulmonary arteries [7, 8] (**Figure 2**). However, the magnitude of impairment seems to be higher in ACh-induced endothelium-dependent relaxation than in SNP-induced endothelium-independent relaxation [7].

#### **Figure 2.**

*The relaxation responses are depressed in isolated pulmonary arterial rings from chronic hypoxic pulmonary hypertensive rat. Pulmonary artery rings were obtained from normal control air rats and rats exposed to 10 days of hypoxia with chronic hypoxic pulmonary hypertension (PH). Isometric tension was measured. Relaxation responses to acetylcholine (ACh) in prostaglandin F2a (PGF2a)-precontracted rings of extrapulmonary were recorded. Relaxation induced by 10<sup>−</sup><sup>4</sup> M papaverine (Pap 4) was taken as 100%. Relaxation responses to ACh were depressed in rings from rats with chronic hypoxic PH, showing that the release of vasorelaxation substance is impaired in PH rings. Although the relaxation responses to sodium nitroprusside (SNP) are impaired in pH rings compared with control, this means that there was a room where SNP could cause relaxation in PH rings from chronic hypoxic PH.*

The relaxation responses to SNP are caused by the liberation of NO from SNP. To determine the vasodilatory effects of NO directly, a NO solution was made by bubbling 10% NO in pure N2 into deoxygenated distilled water. Although depressed, the relaxation responses were indeed induced by NO in hypertensive pulmonary arteries [7] (**Figure 3**). Importantly, iNO exhibits selectivity, resulting in vasodilation in pulmonary arteries (**Figure 4**) when administered by inhalation through the trachea. The intravenous injection of NO donors simultaneously decreases both pulmonary and systemic arterial pressure.

#### **2.2 Pulmonary arterial hypertension**

#### *2.2.1 eNOS in pulmonary arterial hypertension in humans*

Although the role of the eNOS-derived NO-related pathway in pulmonary arterial hypertension (PAH) has been determined, its pathophysiology remains unclear. eNOS plays a key role in this pathway. Giaid et al. detected decreased eNOS protein expression in human lungs with PAH [9]. Additionally, exhaled NO has been found to be lower in patients with PAH than in controls [10]. Subsequent studies have reported increased eNOS protein expression in plexiform lesions in PAH [11] and increased eNOS activity in idiopathic PAH (IPAH) lungs, despite no change in NOS expression [12]. The membrane protein caveolin-1 (CAV1) is a crucial negative regulator of eNOS activity. The CAV1 expression is decreased in IPAH lungs, which might lead to persistent eNOS activation, the accumulation of dysfunctional (i.e., uncoupled) eNOS, the formation of peroxynitrite, and the impairment of PKG

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**Figure 4.**

**Figure 3.**

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension*

*NO solution (0.16–0.2 mM NO) caused the relaxation responses in isolated normal and pulmonary hypertensive arterial rings. Pulmonary artery rings were obtained from normal control air rats (A), rats exposed to 10 days of hypoxia with chronic hypoxic pulmonary hypertension (PH) (B), and rats after 28 days of recovery in room air from chronic hypoxia (C). NO solution was made by bubbling 10% NO through deoxygenated distilled water, which results in 0.16–0.2 mM concentration. Aliquots (0.5 ml) of this solution were applied to the organ bath. Papaverine (Pap) was introduced to obtain maximal relaxation. Relaxation responses to NO solution to prostaglandin F2a-precontracted rings were recorded. (A) NO-induced relaxation in pulmonary artery rings from normal rats. (B) Response to NO was depressed in pulmonary artery rings from chronic hypoxic rats. (C) The relaxation response returned to normal after 28 days of recovery from chronic hypoxic pulmonary hypertension. The result of (B) showed that NO could dilate hypertensive pulmonary vascular smooth muscles, although depressed compared to normal.*

*Inhaled NO as selective pulmonary vasodilator in pulmonary hypertensive rats. A pulmonary artery catheter was introduced via the right external jugular vein into the pulmonary artery by a closed chest technique. Pulmonary arterial pressure(PAP) was recorded with rat fully awake in a pulmonary hypertensive rat (19 days after the single injection of monocrotaline). About 20 ppm NO inhalation decreased PAP with no change* 

*of arterial pressure. When NO inhalation was discontinued, the PAP returned to baseline.*

*DOI: http://dx.doi.org/10.5772/intechopen.89381*

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.89381*

#### **Figure 3.**

*NO solution (0.16–0.2 mM NO) caused the relaxation responses in isolated normal and pulmonary hypertensive arterial rings. Pulmonary artery rings were obtained from normal control air rats (A), rats exposed to 10 days of hypoxia with chronic hypoxic pulmonary hypertension (PH) (B), and rats after 28 days of recovery in room air from chronic hypoxia (C). NO solution was made by bubbling 10% NO through deoxygenated distilled water, which results in 0.16–0.2 mM concentration. Aliquots (0.5 ml) of this solution were applied to the organ bath. Papaverine (Pap) was introduced to obtain maximal relaxation. Relaxation responses to NO solution to prostaglandin F2a-precontracted rings were recorded. (A) NO-induced relaxation in pulmonary artery rings from normal rats. (B) Response to NO was depressed in pulmonary artery rings from chronic hypoxic rats. (C) The relaxation response returned to normal after 28 days of recovery from chronic hypoxic pulmonary hypertension. The result of (B) showed that NO could dilate hypertensive pulmonary vascular smooth muscles, although depressed compared to normal.*

#### **Figure 4.**

*Inhaled NO as selective pulmonary vasodilator in pulmonary hypertensive rats. A pulmonary artery catheter was introduced via the right external jugular vein into the pulmonary artery by a closed chest technique. Pulmonary arterial pressure(PAP) was recorded with rat fully awake in a pulmonary hypertensive rat (19 days after the single injection of monocrotaline). About 20 ppm NO inhalation decreased PAP with no change of arterial pressure. When NO inhalation was discontinued, the PAP returned to baseline.*

kinase activity via tyrosine nitration [12]. Increased eNOS activity and/or expression might not be associated with NO production in PAH.

## *2.2.2 eNOS-NO pathway in animal models of pulmonary arterial hypertension*

Animal studies using a monocrotaline (MCT)-induced PAH rat model, which is characterized by pulmonary endothelial damage and perivascular inflammation in the early pathological stage, have shown decreased eNOS expression [13] and/or phosphorylated eNOS activity [14] as well as decreases in sGC and PKG. Vasodilation induced by ACh, an endothelium-dependent NO-related vasodilator, was also impaired. Another adult rat model of severe PAH with precapillary obliterative lesion (SU/Hx model) shows similarities in the pulmonary vascular pathology to that of PAH in adults. This SU/Hx model, induced by combined SUGEN5416 (a vascular endothelial growth factor receptor II antagonist) and exposure to chronic hypoxia, showed a reduction of ACh-induced NO production and/or release in pulmonary arteries [15]. Another recent study has reported decreased CAV1 expression in the same model [16]. eNOS also translocates from cell surface caveolae to cytoplasmic and perinuclear regions in pathological state [17]. Consequently, the amount of NO production is decreased. Accordingly, the pathogenesis and progression of PAH may be partially induced by endothelial dysfunction associated with suppression of the eNOS-NO-related pathway.

#### *2.2.3 eNOS-NO-cGMP pathway and BMPRII*

Genetic variants in the eNOS-NO-cGMP pathway might cause PAH. CAV1 plays an important role in NO signaling in PAH. Mutations in CAV1 have been identified in PAH [18]. The gene encoding bone morphogenetic protein receptor 2 (BMPRII) is frequently mutated in heritable PAH [19, 20] and adult IPAH [19, 21]. BMPRII is a member of the transforming growth factor (TGF)-β receptor superfamily, localized to caveolae, and interacts with CAV1 in vascular smooth muscle cells [22, 23]. Recent studies have demonstrated that BMPRII deficiency promotes SRC-dependent caveolar trafficking defects [24]. In addition, CAV1-deficient mice have shown reduced BMPRII expression after exposure to chronic hypoxia [16]. In MCT-treated pulmonary arterial endothelial cells, BMPRII was increasingly trapped intracellularly together with increased trapping CAV1 and eNOS. These results suggest that NO-cGMP-related dysfunction and BMPRII deficiency are closely related to and play a significant role in the pathogenesis of PAH.

### **2.3 Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis**

Pulmonary veno-occlusive disease (PVOD), classified as a PAH subgroup, is inextricably associated with pulmonary capillary hemangiomatosis (PCH) [25]. Pulmonary vascular lesions in this condition are mainly detected in postcapillary venules and veins but are also found in pulmonary capillaries and arteries [25]. The pathogenesis is heterogeneous and poorly understood [25]. These are rare diseases, and few studies have focused on the pathogenesis and pathophysiology. Kradin et al. reported that eNOS expression in abnormal capillary lesions is significantly decreased in patients with PCH with pulmonary vascular remodeling and concomitant pulmonary hypertension and is minimally decreased or not decreased in patients without pulmonary vascular remodeling [26]. These results suggest

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*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension*

that the alteration of eNOS expression is associated with the pathogenesis of these complicated conditions. Further experiments are necessary to determine the precise

Biallelic mutations in eukaryotic translation initiation factor 2α kinase 4 (*EIF2AK4*) have been identified in familial and idiopathic PVOD/PCH. *EIF2AK4* encodes general control nonderepressible 2 (GCN2) [27]. The most common experimental models of these conditions are mitomycin C (MMC)-treated rats and mice [28]. Interestingly, MMC dose dependently induces pulmonary GCN2 depletion [28]. *EIF2AK4* mutations are also found in sporadic PVOD/PCH [27]. Mutation carriers have distinct histological features, including strong muscular hyperplasia of the interlobular septal vein as well as arterial severe intimal fibrosis [29]. *EIF2AK4* is activated by amino acid depletion. Because l-arginine, a substrate of NOS, is depleted during NO production, *EIF2AK4* activation can be induced by eNOS activ-

The pathophysiologic features of lung diseases include chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) and mixed pathologic diseases, including combined pulmonary fibrosis and emphysema. All involve alveolar hypoxia and subsequent hypoxic pulmonary vasoconstriction. eNOS expression is upregulated in acute hypoxia in rat lungs [30]. eNOS expression increases in a time-dependent manner in rats during the development of hypoxiainduced PH [31–33], while eNOS activity is impaired [34]. The production of tetrahydrobiopterin (BH4), an obligatory cofactor for generating the active dimer form of eNOS, was altered in hypoxic conditions [34]. An imbalance between BH4 and dihydrobiopterin (BH2) may cause eNOS uncoupling and inactive monomer formation [34]. Several studies have reported decreased eNOS expression and/or activity in patients with COPD [35, 36], with severity of endothelial dysfunction correlated with degree of airflow obstruction [36]. eNOS is also absent in pulmonary arteries of patients with IPF [37]. As the histological features of this disease differ from those of COPD, the pathogenesis of IPF-induced PH may include a multifactorial and complex process involving proinflammatory cytokines and growth factors.

In 1988, at the international conference of the American Thoracic Society, Higenbottam presented his team's paper titled "Inhaled endothelium-derived relaxing factor (EDRF) in primary pulmonary hypertension (PPH)," including the first description of NO inhalation in humans with pulmonary arterial hypertension for laboratory use [38]. In 1991, Lancet published the study [39], showing that 40 ppm NO inhalation selectively reduces PAP, with no changes in systemic pressure. Frostell et al. also showed that inhaled NO (5–80 ppm) causes selective pulmonary arterial dilatation, without changes in systemic arterial pressure in sheep [40], where PAP elevation was induced by hypoxic pulmonary vasoconstriction. Both research groups referenced the studies by Yoshida, Kasama, and Kitabatake about the metabolic fate of iNO [41, 42] because toxicity and retention in the human body should be minimal. NO has been a focus in air pollution research and thus provides

*DOI: http://dx.doi.org/10.5772/intechopen.89381*

ity associated with l-arginine depletion.

**3. Inhaled nitric oxide**

**3.1 Inhaled NO as a selective pulmonary vasodilator**

role of the eNOS-NO-cGMP pathway in PVOD/PCH.

**2.4 Lung disease-related and/or alveolar hypoxia-induced pH**

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.89381*

that the alteration of eNOS expression is associated with the pathogenesis of these complicated conditions. Further experiments are necessary to determine the precise role of the eNOS-NO-cGMP pathway in PVOD/PCH.

Biallelic mutations in eukaryotic translation initiation factor 2α kinase 4 (*EIF2AK4*) have been identified in familial and idiopathic PVOD/PCH. *EIF2AK4* encodes general control nonderepressible 2 (GCN2) [27]. The most common experimental models of these conditions are mitomycin C (MMC)-treated rats and mice [28]. Interestingly, MMC dose dependently induces pulmonary GCN2 depletion [28]. *EIF2AK4* mutations are also found in sporadic PVOD/PCH [27]. Mutation carriers have distinct histological features, including strong muscular hyperplasia of the interlobular septal vein as well as arterial severe intimal fibrosis [29]. *EIF2AK4* is activated by amino acid depletion. Because l-arginine, a substrate of NOS, is depleted during NO production, *EIF2AK4* activation can be induced by eNOS activity associated with l-arginine depletion.

### **2.4 Lung disease-related and/or alveolar hypoxia-induced pH**

The pathophysiologic features of lung diseases include chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) and mixed pathologic diseases, including combined pulmonary fibrosis and emphysema. All involve alveolar hypoxia and subsequent hypoxic pulmonary vasoconstriction. eNOS expression is upregulated in acute hypoxia in rat lungs [30]. eNOS expression increases in a time-dependent manner in rats during the development of hypoxiainduced PH [31–33], while eNOS activity is impaired [34]. The production of tetrahydrobiopterin (BH4), an obligatory cofactor for generating the active dimer form of eNOS, was altered in hypoxic conditions [34]. An imbalance between BH4 and dihydrobiopterin (BH2) may cause eNOS uncoupling and inactive monomer formation [34]. Several studies have reported decreased eNOS expression and/or activity in patients with COPD [35, 36], with severity of endothelial dysfunction correlated with degree of airflow obstruction [36]. eNOS is also absent in pulmonary arteries of patients with IPF [37]. As the histological features of this disease differ from those of COPD, the pathogenesis of IPF-induced PH may include a multifactorial and complex process involving proinflammatory cytokines and growth factors.
