While it may not be the cause, evidence from several studies that supports the fact that increased oxidative stress and reactive oxygen species (ROS) together with decreased antioxidant activity can contribute to enhanced pulmonary vasoconstriction, vascular remodeling, and right heart dysfunction in pulmonary hypertension. Despite this evidence however, it is still unknown whether or not an oxidant/antioxidant imbalance contributes directly to the development of severe PAH. Answering this question is the aim of the recent paper reviewed in today’s post by Jernigan et al., “Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension”.
ROS in PH
Many studies have shown that there is an imbalance of oxidant production and antioxidant capacity in the pulmonary vasculature of PH patients. The authors of the present study, as well as several others, have previously shown that superoxide levels are elevated (NADPH oxidases, xanthine oxidases, and the mitochondria) and are a main source of ROS in the pulmonary vasculature and circulation under chronic hypoxia conditions. Other ROS species like hydrogen peroxide (H2O2) are also implicated in PH, although some studies show elevation in H2O2 and some studies show a decrease.
Antioxidants in PH
Antioxidant activity is decreased in both animal and human PH, as measured by the absence or decreased expression of the antioxidant enzyme superoxide dismutase (SOD), which acts to neutralize the harmful superoxide species: “SOD1 and SOD3 expression and activity are decreased in chronic hypoxia-induced pulmonary hypertensive mice, rats, calves, and piglets and in a lamb model of persistent pulmonary hypertension of the newborn. Furthermore, fawn-hooded rats, which have an epigenetic silencing of SOD2 expression/activity, and SOD1 knockout mice develop spontaneous pulmonary hypertension. These animal studies further correlate with evidence of significantly lower SOD mRNA and SOD activity in patients with idiopathic PAH compared to healthy individuals. Interestingly, SOD-1 immunoreactivity is markedly absent in neointimal lesions of hypoxia/SU5416 rats, suggesting a potential role for oxidative stress in the development of angioproliferative PAH.”
If internal antioxidant activity is decreased, and there are imbalances in ROS within the body during PH, than it makes sense to conclude that antioxidants (e.g. antioxidant supplements or drugs) may have a therapeutic impact on PH. Indeed, the therapeutic impact of some antioxidants on PH has actually already been studied. Most results indicate that antioxidants are effective for treating right heart failure associated with severe PAH, but whether they help alleviate the pathological pulmonary arterial remodeling associated with PAH is unknown, either because it has yet to be studied, or because no distinctive conclusions can be drawn on the studies that have already been done. A few examples of the antioxidants already studied in PH:
- N-acetylcysteine (NAC) – NAC attenuates PH in both chronic hypoxia and monocrotaline-induced pulmonary hypertension animal models.
- SOD – In addition to experiments mentioned previously whereby SOD is decreased or removed, studies have been done where SOD is overexpressed. When overexpressed, SOD reduces pulmonary hypertension in chronic hypoxia, monocrotaline, and lamb models of PH.
- Protandim – Protandim is a dietary supplement and a Nrf2 “activator”. It increases the expression of the antioxidant enzymes SOD and heme-oxygenase-1. In an animal model of severe PH, SUGEN/Hypoxia, Protandim prevented right ventricular hypertrophy and preserved right ventricular function.
Study & Results
In the study by Jernigan et al., the investigators tested the theory of the role of ROS and an oxidant/antioxidant imbalance in contributing to PAH by studying the effects of the antioxidant TEMPOL on two different animal model forms of PH: 1) the SUGEN/Hypoxia animal model (which represents Group I PAH), and 2) the chronic hypoxia rat animal model (which represents Group III PH). The SUGEN/Hypoxia model represents Group I PAH because it is the animal model that most closely resembles the human form of PH, since it is the only animal model that produces plexiform-like lesions in the lung vasculature. Plexiform lesions are the hallmark of human PAH, and other animal models, like the hypoxia only animal model, do not recapitulate the plexiform lesions observed in human PH.
Why use TEMPOL in this study? TEMPOL acts as a SOD mimetic, and neutralizes superoxide radicals, facilitates hydrogen peroxide metabolism, and limits formation of toxic hydroxyl radicals from Fenton reactions.
In brief, here is what the study found…
- TEMPOL effects on Right Ventricular Systolic Pressure (RVSP): TEMPOL prevented increases in RVSP in hypoxic rats. It also attenuated RVSP in the SUGEN/Hypoxia rats, but to a lesser degree.
- TEMPOL effects on Right Ventricular Hypertrophy: RVH in animal models is typically measured by assessing heart weight as well as the Fulton index, which is defined as [right/(left + septum) ventricular weight] or [RV/(LV+S)]. It essentially measures the ratio of the right ventricle to the left ventricle. Increases in this ratio indicate RVH. In the study, TEMPOL had no effect on RVH in either test group, the hypoxia treated rats, or the SUGEN/Hypoxia treated rats.
- TEMPOL effects on Pulmonary Arterial Remodeling – Medial Hypertrophy: A common hallmark of pulmonary arterial remodeling in PAH is hypertrophy of the medial layer of the blood vessel. In the hypoxia only animal model group, hypertrophy of the medial layer of the pulmonary artery was observed. In the SUGEN/Hypoxia group, this was also observed, but the extent of the hypertrophy was not as great in this group compared to hypoxia only rats. TEMPOL treatment had no effect on this hypertrophy in the hypoxia only group. Surprisingly, however, treatment with TEMPOL in the SUGEN/Hypoxia group resulted in an increase in hypertrophy in the medial layer.
- TEMPOL effects on Pulmonary Arterial Remodeling – Plexiform Lesions: Another hallmark of pulmonary arterial remodeling in PAH is the presence of plexiform lesions in the vasculature. In SUGEN/Hypoxia rats, two types of plexiform lesions were observed: 1) “plexiform-like neointimal lesions demonstrating Von Willebrand factor immunoreactivity combined with medial collagen deposition” and 2) “hypercellular lesions projecting outward from the medial and adventitial layers… extending into the adjacent lung parenchyma.” This second type of lesion was found to consist largely of myofibroblasts. As expected, no lesions were observed in hypoxia only treated rats. Again, surprisingly, TEMPOL treatment was found to increase the size and number of the type 2 lesions in the SUGEN/Hypoxia rat group.
- TEMPOL effects on Vasoconstriction: TEMPOL decreased the ET-1 mediated vasoconstriction in both hypoxia only rats and SUGEN/Hypoxia rats.
- TEMPOL effects on ROS: The amount of superoxide production in both hypoxic rats and SUGEN/Hypoxia rats were similar. However, TEMPOL treatment only decreased superoxide levels in hypoxic rats. TEMPOL also was shown to increase H2O2 induced oxidative stress, but it did so across all test groups, including the control normoxic rats.
Since the authors observed that TEMPOL attenuated RVSP in SUGEN/Hypoxia rats, and decreased vasoconstriction, it is tempting to conclude that ROS does indeed contribute to both pulmonary artery remodeling and vasoconstriction, respectively, in PH. Since TEMPOL is an antioxidant and scavenges ROS, and since RVSP is a function of pulmonary vascular resistance, which is a function of BOTH hypertrophy, blood vessel overgrowth, and vasoconstriction, and increases in RVSP and vasoconstriction could be indicative of pathological pulmonary arterial remodeling.
However, at odds with this is the fact that TEMPOL failed to reduce hypoxia-induced pulmonary arterial muscularization and right heart hypertrophy. Increases in these parameters are also indicative of pathological pulmonary arterial remodeling. Rather, “scavenging of ROS in hypoxia/SU5416-treated rats [by TEMPOL] caused an unexpected increase in arterial muscularization, vimentin, and HSP-47 expression, and severity of adventitial fibrotic lesions.” The fact that TEMPOL failed to reduce the parameters of medial hypertrophy and RVH, and actually increased muscularization and fibrotic lesions indicates that the antioxidants may actually promote pulmonary arterial remodeling.
How could TEMPOL possibly do this? As the authors explain, “ROS are essential signaling molecules that are tightly regulated to maintain physiological homeostasis… low levels (submicromolar) induce growth but higher concentrations induce apoptosis. It is possible that TEMPOL decreases superoxide levels thereby disrupting oxidative regulation of proliferation and host defense resulting in excessive proliferation and fibroblast activation.” If this is true, then the mechanism by which TEMPOL lowers RVSP in SUGEN/Hypoxia rats is via a reduction in vasoconstrictor reactivity, not because it decreases pulmonary arterial remodeling.
H2O2 is another ROS that is essential for physiological homeostasis, and the verdict is by no means out on H2O2… Small amounts are necessary to induce vasodilation (H2O2 binds to the potassium ion channel in the pulmonary artery smooth muscle cells, causing them to stay open, which causes relaxation/vasodilation in the cells), but large amounts, especially in the presence of iron, can be toxic due to formation of dangerous hydroxyl radicals. Furthermore, both contraction and relaxation have been observed in the presence of H2O2.
Since the antioxidant SOD neutralizes toxic superoxide into H2O2, by its very nature, reduced SOD activity, as observed in animal and human PH, leads to reduced H2O2. Under reduced SOD activity or hypoxic conditions, decreased H2O2 could contribute to proproliferative and antiapoptotic effects that are mediated by hypoxia-inducible factor 1α (HIF-1α).
However, other studies show that “H2O2 stimulates cell migration, proliferation, and differentiation in the pulmonary circulation.” And since TEMPOL is an SOD mimetic, it technically can lead to increased H2O2 levels and leading to excessive proliferation. The authors are not keen to accept this version of the theory however, since “TEMPOL induction of H2O2-specific oxidative stress was independent of chronic hypoxia or SU5416.”
Overall, the authors conclude that “despite a dramatic effect of the antioxidant, TEMPOL, to limit vasoconstrictor responsiveness and increases in RVSP in each rat model, we observed a paradoxical effect of TEMPOL to exacerbate both medial and adventitial remodeling in animals with severe PAH. Furthermore, TEMPOL had no effect to reduce RV hypertrophy. Together, these studies support a major role for ROS in mediating the vasoconstrictor component of PAH, however there may be therapeutic limitations of using TEMPOL in severe PAH due to exacerbation of medial remodeling and adventitial lesion formation.” The authors also stress that there may be therapeutic limitations to using SOD mimetics in general.
The limitations of this study are twofold:
- It relies on animal models. Even though the SUGEN/Hypoxia model closely resembles the human form of PH, it does not fully recapitulate the same exact types of features and lesions observed in human PAH. Nevertheless, useful information can still be gained.
- It only studied the effect of one antioxidant. Even though superoxide dismutase (SOD) is implicated in PH, and a key antioxidant enzyme, studying this effects of this one compound on ROS does not allow us to make comprehensive conclusive statements about the role of ROS in PAH. Furthermore, while TEMPOL may have promoted pulmonary arterial remodeling in an animal model, another antioxidant may not have the same effects.