Pulmonary hypertension (PH) is a progressive disease characterized by increased pulmonary vascular resistance and pulmonary artery pressures ^[1]. The hemodynamic definition of PH has recently been updated and is currently defined as an increase in mean pulmonary arterial pressure (mPAP) >20 mmHg at rest ^[2]. This chronic pressure overload due to PH, leads to the development of right ventricular hypertrophy (RVH), heart failure and, ultimately, death [1,3]^[1][3]. Structural remodeling of the vasculature, resulting in reduced vessel lumen, is related to increased pulmonary vascular resistance and increased pulmonary pressure ^[1].

Clinical classification of PH categories clinical conditions associated with PH into five groups according to similarities in clinical presentation, pathological findings, hemodynamic characteristics, and therapeutic management ^[4]. Group I is a rare condition known as pulmonary arterial hypertension (PAH), with a prevalence of 48–55 cases per million adults ^[4]. PAH can be idiopathic (iPAH), heritable (hPAH), drug- and toxin-induced, associated with several conditions or diseases, with features of venous/capillary involvement or persistent PH of the newborn, with iPAH being the most common subtype (50–60% of all cases) ^[4]. hPAH includes patients with mutations in BMPR2 (bone morphogenetic protein receptor type 2), a member of the transforming growth factor (TGF-β) superfamily, seen in 70–80% of hPAH ^[5]. Group II PH or left heart disease (PH-LHD) is the main cause of PH, accounting for 75% of all cases of PH ^[6]. It is caused by increased left atrial pressure, normally occurring as a consequence of an underlying cardiac disorder ^[7]. In group III, PH is related to lung disease or hypoxemia. Chronic obstructive pulmonary disease (COPD) is the most common lung disease associated with PH, accounting for about 80% of cases ^[8]. Group IV, chronic thromboembolic PH (CTEPH), is a progressive disease caused by the obstruction of major pulmonary arteries as a consequence of flow-limiting organized thrombi ^[9]. Finally, group V encompasses a complex group of disorders associated with PH. The cause can be related to multifactorial mechanisms and can be secondary to increased pre- and post-capillary pressure or direct effects on pulmonary vasculature ^[4].

2. Oxidative Stress

Aerobic metabolism involves the production of reactive oxygen species (ROS), even under basal conditions, where it plays an essential role in some physiologic signaling pathways, such as inflammation. Therefore, there is a continuous requirement for the inactivation of these reactive oxygen species ^[10]. The term ROS describes a variety of small molecules characterized by high reactivity and biological activity. It mostly includes superoxide anion (O2^•−), hydroxyl radical (OH^•), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), peroxynitrite (ONOO⁻), or hypochlorous acid (HOCl) ^[11]. ROS can be generated from various sources. In human cells, there are several H₂O₂ and O₂⁻ generating enzymes, the NADPH oxidases (NOXs) the major endogenous enzymatic source of H₂O₂ and O₂⁻ together with the mitochondrial electron transport chain (ETC) ^[12]. Mitochondrial ROS (mtROS) is generated as a consequence of electron transfer during ATP production. Electrons that leak out of the ETC at complex I and III, can react with oxygen producing O₂^{− [13]}. Apart from NOXs and the mitochondrial ETC, H₂O₂ can be generated by oxidase enzymes found in other subcellular locations, mainly in the endoplasmic reticulum (ER) and peroxisomes ^[12]. Several superoxide dismutases (SOD1–SOD3) can also produce H₂O₂ from O₂⁻ [12,13]^[12][13]. In addition to intracellular sources, ROS can be also generated by cumulative environmental exposure, such as molecular factors (drugs, pollution and nutrients), physical (UV, X-ray and other ionizing radiation), and psychological stressors (lifestyle) ^[12]. The excessive production of ROS associated with mitochondrial, enzymatic, or exogenous ROS sources can result in an imbalance between ROS production and the cells’ defense systems, inducing oxidative stress, resulting in subsequent alteration of biological molecules, including DNA, lipids, proteins, and carbohydrates [10,13]^[10][13]. Consequently, oxidative stress could be involved in processes such as mutagenesis, carcinogenesis, membrane damage, lipid peroxidation, protein oxidation and fragmentation, carbohydrate damage, as well as in the pathogenesis of several diseases [10,13]^[10][13]. The following sections will describe in detail the published evidence of oxidative stress in the different subtypes of PH.

3. Oxidative Stress in the Different Subtypes of PH

3.1. Oxidative Stress in PAH (Group I PH)

Vasoconstriction promoted by oxidative stress is probably one of the most critical factors in the early stages of PAH ^[14]. Oxidative stress plays a key role in impairing endothelial cell function, producing an increase in the synthesis and release of endothelium-derived constrictor factors such as endothelin-1 (ET-1) and a decrease in relaxing factors such as NO, contributing to the alteration of vascular tone and vascular permeability [15,16]^[15][16]. Reduction in endogenous NO levels, an important signaling molecule involved in the modulation of vascular tone, blood pressure, and the regulation of smooth muscle proliferation and migration, may contribute to the development of PAH [14,17]^[14][17]. This reduction in NO bioavailability is achieved when ROS, principally O₂⁻ that reacts readily with NO, forms the intermediate peroxynitrite (ONOO⁻), which reacts with available tyrosine residues of proteins producing 3-nitrotyrosine, causing lung epithelial damage [18,19]^[18][19]. Therefore, most animal models of PH aim to reproduce the two principal pathological characteristics in the pulmonary vasculature, common to most PH groups, which are excessive vasoconstriction and pulmonary vascular remodeling (PVR) ^[20]. In animal studies, Guo et al. ^[21] found that in monocrotaline (MCT)-treated rats, which develop severe PAH, the observed increased oxidative stress caused decreased pyruvate kinase isoenzyme type M2 (PKM2) activity, resulting in increased proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, to confirm that PKM2 was triggered by ROS, they treated MCT-PAH rats with the antioxidant N-acetylcysteine (NAC), showing an attenuation of PKM2 activity, thus demonstrating the role of ROS in cell signaling for the pathogenesis of PAH. In human studies, Sun et al. ^[22] showed that monoamine oxidase (MAO), an important ROS source implicated in different vascular diseases, specifically MAO-A expression, was increased in the medial and intimal layers of patients with PAH. They also determined that this increase was involved in the progression of PAH and that MAO-A inhibitors could reverse PVR. Similarly, Cracowski et al. ^[23] established that urinary levels of isoprostaglandin F_2α type III (iPF_2α-III), a stable and specific product of lipid peroxidation, were 2.3 times higher in patients with PAH and other types of PH than in healthy controls, showing that oxidative stress is increased in patients with PH. Other studies have shown increased oxidative stress levels in persistent PH of the newborn (PPHN). Brennan et al. ^[24] demonstrated increased superoxide formation, without a simultaneous increase in cellular antioxidant capacity, in PPHN lungs compared with controls. In addition, elevated levels of H₂O₂ in PPHN pulmonary arteries, associated with decreases in cGMP signaling, have been shown to contribute to development of the pathology ^[25].

3.2. Pulmonary Hypertension Caused by Left Heart Disease (Group II PH)

PH-LHD is the most common form of PH, accounting for 65–80% of all cases [26,27]^[26][27]. PH-LHD develops mainly due to the sustained elevation of left-sided filling pressure because of left-ventricular (LV) dysfunction, producing an increase in pulmonary arterial pressure and subsequently pulmonary vascular remodeling ^[28]. This pulmonary venous congestion may promote additional pathophysiological changes, such as pulmonary vasoconstriction, decreased NO availability, increased expression of ET-1 and desensitization to natriuretic peptide-induced vasodilation [26,27]^[26][27]. Several studies have demonstrated increased oxidative stress in patients with PH, but there is little evidence of oxidative stress in PH group II. Ravi et al. ^[28] found high levels of peroxynitrite and superoxide in left-heart failure induced rats, and showed that this mediated the downregulation of PTEN expression, a phosphatase-and-tensin homolog on chromosome 10 and a modulator of the phosphoinositide 3-kinase activity related to vascular remodeling. Decreased PTEN expression resulted in smooth muscle cell (SMC) proliferation and subsequent vascular remodeling, demonstrating the association between oxidative stress and the pathogenesis of PH-LDH. Using a synthetic analogue of curcumin, HO-3867, a molecule with antioxidant and antiproliferative properties, they showed significant attenuation of oxidative stress, resulting in upregulation of PTEN expression and inhibition of vascular remodeling. Furthermore, Sunamura et al. ^[29] also demonstrated that ROS was involved in the pathogenesis of PH-LDH. They found that mice deficient in ROCK1 (cROCK1^−/−), a rho-kinase member of the serine/threonine protein kinase family, showed pressure-overload-induced cardiac dysfunction and postcapillary PH. Additionally, they showed upregulation of ROS levels by cyclophilin A (CyPA) and basigin (Bsg), two common molecules that augment heart failure (HF) and PH. However, downregulation of ROS and CyPA and Bsg proteins has also been reported in cROCK2^−/− mice with attenuated cardiac dysfunction and postcapillary PH. Moreover, ROCK1 deficiency destroyed the balance between mitochondrial fission and fusion, resulting in impaired mitochondrial homeostasis and the generation of ROS. Interestingly, upregulation of ROCK2 in cROCK1^−/− mice produced an increase in ROS levels after pressure overload, showing that ROCK2 specifically increases ROS production mediated by the downstream effectors CyPA and Bsg. Additionally, celastrol, a compound with antioxidant and anti-inflammatory effects, reduces CyPA and Bsg expression, reducing ROS production and improving pressure-overload-induced cardiac dysfunction and postcapillary PH, showing the role of oxidative stress in the development of PH-LHD.

3.3. Pulmonary Hypertension Caused by Lung Diseases and/or Hypoxia (Group III PH)

PH associated with hypoxia and lung disease is the second most common type of PH. The most common lung diseases causing PH are COPD and interstitial lung disease, but it can also be associated with other pathologies such as cystic fibrosis and high altitude exposure ^[30]. The origin of PH in hypoxic lung disease is multifactorial. Chronic lung diseases produce periods of continuous or intermittent hypoxia, increasing the release of vasoconstrictors causing pulmonary artery vasoconstriction (HPV), leading to vascular remodeling, which increases vascular resistance and pulmonary artery pressure [30,31]^[30][31]. Moreover, under hypoxic conditions, the expression of oxidative stress biomarkers increases, causing cellular damage ^[31]. Liu et al. ^[32] demonstrated that increased generation of O₂⁻ or ROS derived from this anion acting on PASMCs was required for HPV. Treatment of distal porcine pulmonary artery PASMCs with SOD or SOD with catalase (CAT), inhibited constriction induced by hypoxia, suggesting that ROS play an essential role in HPV during hypoxia. Weissmann et al. ^[33] provided evidence that superoxide formation, perhaps derived from a NAD(P)H oxidase, and the subsequent generation of H₂O₂ is the underlying mechanism of acute HPV, having a role in the signaling cascade linking hypoxia sensing and vasoconstrictor phenomena and suggesting that hypoxia produces an increase rather than a decrease in ROS levels. Many studies have shown that hypoxic exposure might cause oxidative stress in lung tissue. Hoshikawa et al. ^[34] found that lung tissue levels of PCOOH, a primary peroxidation product of phosphatidylcholine, increased after hypoxic exposure, and that administration of the antioxidant NAC reduced hypoxia-induced cardiopulmonary alterations and inhibited the increase in PCOOH levels. Inhibition of xanthine oxidase (XO)-hypoxanthine, an important pathway in generating oxidative stress in vivo, decreased PCOOH levels in hypoxia exposed rats, which showed attenuation of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular media thickening. In a hypoxia-induced PH model You et al. ^[35] observed increased NOX4 and VPO1 expression, as well as HOCl production. VPO1, a member of the peroxidase family, uses chloride and NOX-derived H₂O₂ to produce HOCl, a more potent oxidizer that accelerates the increase of oxidative stress in the vasculature. They also observed enhanced proliferation, apoptosis resistance, and migration of PASMCs, demonstrating that NOX4/VPO1 pathway-mediated oxidative stress promotes vascular remodeling. Furthermore, Pu et al. ^[36] reported that chronic high-altitude exposure produced pulmonary hypertension, increasing the generation of both malondialdehyde (MDA) and ROS, and decreasing glutathione peroxidase and SOD activities, which was accompanied by pulmonary vessel remodeling.

3.4. Oxidative Stress in CTEPH (Group IV PH)

CTEPH is produced by unresolved blood clots associated with fibrosis that obstruct pulmonary arteries ^[37]. However, the mechanisms leading to the lack of pulmonary thrombo-emboli resolution remain unclear ^[38]. It has been suggested that vascular dysfunction, such as endothelial dysfunction, caused by a decrease in NO availability due to the overproduction of ROS, may contribute to the progression of the pathology [39,40]^[39][40]. CTEPH-derived endothelial cells (CTEPH-EC) presented a significant increase in oxidative stress levels, specifically mtROS production, and reduced expression of SOD-2 compared to healthy human pulmonary artery ECs used as control cells ^[41]. Nukala et al. also showed that CTEPH-EC exhibited an increase in intracellular ROS, advanced oxidation protein products and total protein carbonyl content (PCO), and a downregulation of GPX4 and GPX1 proteins, demonstrating dysregulation of the oxidative stress response and highlighting the involvement of oxidative stress in CTEPH ^[42]. Brandt et al. ^[38] found that expression of NADPH oxidase and superoxide formation increased in mice with induced PE, resulting in endothelial dysfunction in pulmonary arteries. Furthermore, Stam et al. ^[43] using a swine-CTEPH model, in addition to other changes, observed increased expression of genes associated with oxidative stress (ROCK2, NOX-1, and NOX-4) in the right ventricle (RV) which contributed to RV hypertrophy and dysfunction. In another study, Smukowska-Gorynia et al. ^[37] evaluated antioxidant status in patients with deteriorating or stable CTEPH, analyzing serum oxidative stress biomarkers including: MDA, a lipid peroxidation indicator; total antioxidant capacity (TAC), for the evaluation of antioxidant status; and CAT and SOD activities, two of the main cellular antioxidant systems. MDA was higher in the deteriorating group compared with stable patients, while TAC and CAT were lower, with no significant difference in SOD, suggesting that MDA concentration and TAC and CAT activities correlated with adverse clinical outcomes, showing an imbalance between the generation of ROS and the biological detoxification system. Zhang et al. ^[44] measured oxidative-antioxidant biomarker levels, and asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor implicated in some cardiovascular diseases, to assess the association of these biomarkers with the development and prognosis of CTEPH. Comparing healthy controls and CTEPH patients, they found a significant increase in MDA and ADMA levels in the CTEPH group, as well as an important decrease in the biological antioxidant potential and SOD levels, suggesting that ROS contributes to the pathogenesis of CTEPH.

3.5. Pulmonary Hypertension with Unclear and/or Multifactorial Mechanisms (Group V)

Group 5 PH includes several diseases in which the mechanisms leading to the development of PH are unclear ^[45]. PH can be generated as a major complication of hematological disorders (sickle cell disease (SCD), beta-thalassemia, chronic hemolytic anemia, myeloproliferative disorders or splenectomy), systemic disorders (sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (glycogen storage disease, Gaucher’s disease, or thyroid disorders), and other disorders such as chronic renal failure or tumoral obstruction [45,46]^[45][46]. Several mechanisms are thought to contribute to the pathophysiology of PH in hematological disorders. Sickle erythrocytes have shown high levels of ROS, which disrupt NO homeostasis ^[47]. Moreover, the impaired glutathione pathway and iron overload contribute to the increased ROS in SCD ^[47]. The overproduction of ROS promotes vasoconstriction and pulmonary vascular remodeling, resulting in the development of PH ^[47]. Novelli et al. ^[48] demonstrated that oxidative stress contributes to SCD-associated PH. They reported increased thrombospondin-1 levels, which contributed to ROS generation via binding to the CD47 receptor using a Berkeley model of SCD and patients with SCD-associated PH. Oxidative stress also plays an important role in PH associated with thalassemia, which is exacerbated by hemolysis and the iron overload present in patients receiving transfusion therapy ^[49]. Additionally, alterations in the glutathione system in thalassemia make it difficult to remove reactive oxygen species in erythrocytes, contributing to their hemolysis. This increase in oxidative stress level, in addition to other pathogenic mechanisms, results in endothelial dysfunction and vascular damage ^[49]. It has also been reported that ROS levels of vascular ECs are increased in diabetes. The hyperglycaemic environment enhances EC permeability due to ROS-mediated upregulation of cell adhesion molecules exacerbating leukocyte adhesion and migration into the vascular wall, resulting in vascular damage ^[50]. In light of the observed involvement of ROS in the pathophysiology of PH, targeting these reactive species through the use of antioxidant therapy may present a viable and promising avenue for the treatment of PH.