Role of Oxidized Lipids in Pulmonary Arterial Hypertension

Role of oxidized fatty acids produced from arachidonic acid via the lipoxygenase pathway in PAH

In this section, we focus on oxidized lipids in PAH generated from linoleic acid (LA) and arachidonic acid (AA). LA can be oxidized by 5-lipoxygenase (5-LOX) and 15-LOX to form 9-hydroxyoctadecadienoic acid (9-HODE) and 13-HODE, respectively (Fig. 1A). Involvement of these oxidized products as contributing factors in oxidative stress in PAH has recently been demonstrated.^{16,28,29,56,57} LA is also the precursor of AA, which by enzymatic oxidation gives rise to different oxidized lipids, including 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE (Fig. 1A). These three oxidized metabolites are found to be elevated in the lung tissue samples obtained from patients with primary pulmonary hypertension (PH). ⁵⁸ The 12-HETE levels are increased in the lung SMCs cultured from hypoxia-treated rats, and exogenous 12-HETE treatment stimulated proliferation of SMCs. ¹⁷ Similarly, 15-HETE plays an important role in hypoxic PAH. ⁵⁹ Increased activity of 15-LOX in pulmonary arteries upon exposure to hypoxia catalyzes and enhances 15-HETE production. ⁵⁷ Vascular remodeling in hypoxic PAH is in part mediated by a positive feedback regulatory loop between 15-HETE and hypoxia-inducible factor 1α (HIF-1α), a critical oxygen-sensing transcription factor in PAH. ⁶⁰ Increased levels of 15-HETE stimulate proliferation of pulmonary artery SMCs (PASMCs); ¹⁸ pulmonary arterial vasoconstriction via K⁺ channels and the protein kinase C (PKC) signal transduction pathway;^23–26 the Rho/Rho-associated protein kinase (Rho/ROCK) signaling pathway; ²⁷ inhibition of apoptosis in PASMCs mediated via several signaling pathways, such as ERK1/2 (extracellular signal–regulated kinases), ¹⁹ PI3K (phosphatidylinositol 3-kinase)/Akt, ²⁰ the ROCK pathway, ²¹ and the nitric oxide synthase (iNOS) pathway; ²² and pulmonary vascular remodeling mediated via platelet-derived growth factor. ¹⁵ In addition, 15-LOX/15-HETE induces the p38 MAPK (mitogen-activated protein kinase)–dependent transforming growth factor (TGF) β1/Smad2/3 intracellular signaling pathways to mediate vascular fibrosis in the adventitia of the pulmonary arterial wall, resulting in pulmonary artery remodeling. ²⁸ Also, 15-HETE has been shown to mediate vascular medial hypertrophy and EC migration and angiogenesis contributing to hypoxic PH. ¹⁶ A mutual positive regulatory mechanism exists between telomerase reverse transcriptase and the 15-LOX/15-HETE pathway that could mediate migration, proliferation, and cell cycle distribution of PASMCs in hypoxia-induced pulmonary vascular remodeling. ⁶¹ Inhibition of 5-LOX by diethlycarbamazine, an enzyme responsible for 5-HETE and leukotriene synthesis (Fig 1B), has been demonstrated to improve PAH in the Sugen/hypoxia rat model. ⁶² Indeed, inhibition of 5-LOX improves RV function by decreasing RV systolic pressure (RVSP) and hypertrophy. Al-Husseini et al. ⁶² demonstrated that this improvement is mediated by a decrease in inflammation and pulmonary vascular wall thickness. Furthermore, we recently demonstrated that PH is associated with elevated plasma levels of 5-HETE, 12-HETE, 15-HETE, 9-HODE, and 13-HODE in the monocrotaline (MCT)-induced PH model in rodents. ⁵⁶ We also reported significantly higher levels of plasma HETEs and HODEs in PAH patients (idiopathic and associated PAH secondary to connective-tissue disease). ²⁹ Conversely, we showed that increasing oxidized lipids in vivo by feeding mice with 15-HETE leads to PH, establishing a strong connection between the levels of oxidized lipids and the pathophysiology of PH. ⁵⁶ Collectively, these studies reinforce a major role of oxidized lipids in the development of PH (Fig. 2).

OPEN IN VIEWER

Figure 1.

Pathways involved in linoleic and arachidonic acid metabolism. A, Linoleic acid is metabolized by 5- and 15-lipoxygenase (LOX) to form 9-hydroxyoctadecadienoic acid (9-HODE) and 13-HODE, respectively. Linoleic acid can also be metabolized to arachidonic acid. In turn, arachidonic acid is used to form prostaglandins (by cyclooxygenases) and epoxides (by the cytochrome p450 epoxygenase pathway). B, Arachidonic acid can also be metabolized to 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE by LOXs and to leukotrienes (LTs) such as LTA₄ (by LTA₄-synthase), LTB₄ (by LTA₄-hydrolase), LTC₄ (by LTC₄-synthase), LTD₄ (by γ-glutamyl-transferase), and LTE₄ (by peptidase).

OPEN IN VIEWER

Figure 2.

Role of oxidized lipids, oxidative stress, and lipid metabolism in promoting pulmonary arterial hypertension (PAH). A, Many altered pathways and abnormalities are involved in the progression and development of PAH. PASMC: pulmonary artery smooth muscle cell. B, Oxidized lipids (hydroxyeicosatetraenoic acids [HETE]s and hydroxyoctadecadienoic acids [HODEs]), leukotrienes (LTs), epoxyeicosatrienoic acids (EETs), and lipid metabolism contribute only to a selective subset of pathways (highlighted); pathways that are not involved in the pathogenesis of PAH are shaded (See Table 1 for references).

Role of other metabolites of AA in PAH

Many by-products of AA play an important role as mediators in PAH. For instance, epoxyeicosatrienoic acid (EET), which is derived from AA by cytochrome p450 epoxygenase (Fig. 1A), plays an essential role in vasoconstriction and the modulation of proliferative and angiogenic properties in pulmonary artery ECs (PAECs) in PH and other diseases.^63,64 It has been shown that the EET and JNK/c-Jun pathways are involved in pulmonary vascular remodeling caused by proliferation of ECs, inhibition of apoptosis, and stimulation of angiogenesis, culminating in pulmonary artery endothelial plexiform lesions (Fig. 2B). ³³ Kandhi et al. ⁶⁵ demonstrated that jugular administration of EET led to an increase in RVSP in a dose-dependent manner. Thus, the effect of EET on RVSP seems synergic with hypoxia. Wang et al. ⁶⁶ have clearly demonstrated that hypoxic pulmonary vasoconstriction response, which is impaired in PH, is modulated by endothelium calcium signaling via activation of EETs, further supporting the essential role of EETs in PH.

Leukotrienes (LTs) are another class of lipid mediators derived from the 5-LOX pathway of AA metabolism (Fig. 1B).^67,68 They trigger immune response by recruitment and activation of leukocytes and play an essential role as mediators in pulmonary inflammation.^31,32 Several studies have shown that leukotriene B₄ (LTB₄) is involved in PAH pathogenesis.^69–71 Rodent models of PH, including MCT- and SU5416 (a VEGFR2 [vascular endothelial growth factor receptor 2] inhibitor)-treated rat models, have shown elevated levels of LTB₄.^30,72 LTB₄ induces apoptosis of PAECs, proliferation of PASMCs, and fibroblast activation, three major pathologic events leading to PAH.^30,73 We have recently demonstrated that plasma levels of LTB₄, but not those of LTC₄ and LTE₄, are increased in the rat model of MCT-induced PH (Fig. 2B). ⁵⁶ Taken together, these data strengthen a critical role of oxidized lipids in the pathophysiological mechanism of PH (Table 1).

Plasma HDL and PAH

HDL cholesterol (HDL-C) is protective in coronary artery disease because of its antioxidant and anti-inflammatory properties.^74,75 Apolipoprotein A-I (ApoA-I), the major protein component of HDL-C, is present at lower levels in PAH, which correlates with increased endothelial dysfunction. ⁷⁶ Metabolic syndrome and insulin resistance are associated with low circulating HDL-C levels and may predispose to the development of pulmonary vascular disease.^77,78 Indeed, insulin resistance and RV hypertrophy with pulmonary vascular remodeling are observed in an apolipoprotein E–deficient mouse model. ⁷⁹ In addition, depressed circulating levels of HDL-C are associated with worse clinical outcomes in PAH patients and are independent of other cardiovascular risk factors. ⁸⁰ This observation is further supported by another independent study showing that circulating HDL cholesterol levels are depressed in a cohort of patients with idiopathic PAH (IPAH) and are associated with worse clinical outcomes. ⁸¹ On the whole, these data suggest a potential role of circulating HDL in metabolic syndrome and PH and are supported by the prevalence of subclinical PH in patients with metabolic syndrome. ⁸²

Role of lysophosphatidic acid

Studies have investigated the effects of lysophosphatidic acid (LPA) signaling and metabolism on vascular SMCs and ECs, indicating that LPA may also have implications for the remodeling of pulmonary vasculature.^83,84 LPA is a bioactive lipid molecule produced by the plasma lysophospholipase D enzyme autotoxin.^85,86 LPA has been shown to stimulate migration and proliferation of SMCs and to alter EC function; consequently, it plays a critical role in vascular development.^87–89 Mouse models with loss-of-function mutations in genes required for LPA production and signaling have been used to investigate the pathophysiological role of LPA metabolism and signaling in diverse settings, including pulmonary inflammation and hypoxia-induced vascular remodeling.^90,91

PAH is associated with oxidative stress and lipid peroxidation

PAH is associated with increased oxidative stress. This leads to tissue damage by oxidation of many important biological molecules, including DNA damage and lipid peroxidation. ⁵⁸ Increased reactive oxygen species (ROS) production is involved in the pathogenesis of PH in various animal models. Chronic hypoxia–induced PH in mice is associated with increased intrapulmonary superoxide levels and other pathophysiological changes, which are abolished by the antioxidant xanthine oxidase inhibitor allopurinol.^92,93 Elevated RV superoxide levels are also observed in the MCT rat model of PH. Antioxidant therapies with intratracheal administration of antioxidant superoxide dismutase or resveratrol suppressed the progression of PH.^94–97 Tissue hypoxia and an increase of inflammatory cytokines in the lungs of animal models of PH lead to elevated levels of ROSs.^58,97 PAH is associated with oxidative stress arising from the accumulation of ROSs, including superoxide and peroxide. Reaction of these highly reactive molecules with functional groups in membrane lipids and proteins can produce harmful oxidative-breakdown products. ⁹⁸ One such class of metabolites is the chemically stable bioactive lipid peroxidation product of AA known as “isoprostanes.” Levels of isoprostanes are elevated in many pulmonary vascular diseases, including PAH.^99–104 Isoprostanes not only serve as biomarkers of the disease but also act as signal transduction molecules exerting multiple biological effects through prostanoid receptors and other signaling pathways.^47,105–107 They can exert their effects on pulmonary vasculature in many ways, including pulmonary vasoconstriction,^48,108,109 induction of pulmonary endothelium to release endothelin 1, vasoconstriction in general,^49,110 and nonspecific effects on smooth muscle, such as hyperresponsiveness and hypertrophy,^50,51 thereby serving as important mediators in many lung pathologies including PAH.

Lipid peroxidation products of AA, including isoprostanes as well as other end products, such as malondialdehyde, are increased in patients with PAH.^103,111,112 Lungs of transgenic mice with bone morphogenetic protein receptor II (BMPR2) mutation showed an increase in isoprostanes, indicative of a rise in oxidative stress that was of mitochondrial origin and specific to vasculature. ¹¹³ Chronic hypoxic exposure of rats results in the production of ROSs, including phosphatidylcholine hydroperoxide, a primary peroxidation product of phosphatidylcholine, and serves as a contributing factor for pulmonary vascular thickening and development of PH. ⁹³ A reduction in the levels of antioxidants, including β-carotene and α-tocopherol, is observed in patients with IPAH compared to control subjects. ¹¹⁴ Studies have evaluated the role of NADPH oxidase in the development of PH. In a lamb model of PH of the newborn, it has been shown that the NADPH oxidase enzyme complex may contribute to proliferation of SMCs by producing increasing superoxide levels.^115,116 Moreover, the proliferative effect of endothelin 1 on fetal PASMCs is a consequence of increased generation of ROSs. ¹¹⁷ Regulation of the growth of PASMCs by the transcription factor GATA4 is inhibited by antioxidant serotonin via suppression of NADPH oxidase or monoamine oxidase A, further reinforcing the critical role of ROSs in altering signaling pathways involved in PH.^118,119 Several other studies have suggested that antioxidant therapy can suppress the progression of PH. Many compounds with antioxidant properties, including the superoxide dismutase mimetic tempol and resveratrol, are effective in preventing the development of PH in various animal models.^97,120 In addition, cell culture studies, transgenic mouse models, and human samples confirm that the heritable form of PAH caused by mutations in the BMPR2 pathway is also associated with increased oxidative stress that is very vascular specific and most likely of mitochondrial origin. Lectin-like oxidized low-density lipoprotein (oxLDLs) is involved in endothelial dysfunction and injury upon stimulation by many factors, including inflammation and shear stress; ⁵² oxLDL plays an important role in cardiovascular diseases such as myocardial infarction and atherosclerosis.^121,122 Overexpression of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), an endothelial receptor of oxLDL in the lungs of transgenic mice, promotes oxidative stress by ROS generation and induces PH in chronic hypoxia. ¹²³ Thus, oxidative stress and lipid peroxidation could make a major contribution to the pathogenesis of PAH (Fig. 2; Table 1).

Role of HDL (ApoA-I) mimetic peptides

HDL is a major lipid carrier in the bloodstream and plays a critical role in vascular disease. It is known that HDL protects against atherosclerosis through several mechanisms, including the ability to extract cholesterol and phospholipids from peripheral cells and transfer them to the liver for excretion. Moreover, HDL also protects against lipid oxidation and inflammation. However, under certain pathological conditions, as in PAH, these antioxidant and antiinflammatory properties of HDL decrease, accompanied by a drastic increase in the levels of oxidized lipids. Therefore, HDL can act as both an anti- and a proinflammatory molecule, depending on the context and environment. Indeed, we have determined the “inflammatory indices” of HDL and LDL in IPAH and associated PAH (APAH) patients. ²⁹ We found that LDL inflammatory indices were significantly higher in IPAH and APAH patients than in controls. Furthermore, HDL was proinflammatory in both IPAH and APAH. ²⁹

The major component of HDL in plasma is ApoA-I, which possesses anti-atherosclerotic, anti-inflammatory, and antioxidant properties. ¹⁴⁶ The mechanistic relationship between ApoA- and pulmonary function was highlighted by genetic deletion of ApoA-I in mice. ApoA-I-null (ApoA-I^−/−) mice show an increase in proinflammatory HDL, indicative of high oxidative stress and increased airway hyperresponsiveness as well as impaired pulmonary vascular function. ¹⁴⁷ PH in patients with sickle cell disease is associated with altered expression of ApoA-I, contributing to sickle cell disease–associated vasculopathy. ¹⁴⁸ ApoA-I concentrations were decreased in the lungs of idiopathic pulmonary fibrosis patients and in an experimental bleomycin-induced fibrosis model. ¹⁴⁹ The local treatment with ApoA-I has been shown to be very effective against the development of experimental lung injury and fibrosis. ¹⁴⁹ ApoA-I appears to be a promising therapeutic molecule, considering its therapeutic potential in reducing inflammation and fibrosis in the animal model of bleomycin-induced pulmonary fibrosis. ¹⁵⁰

Several HDL mimetic peptides, which mimic the lipid-binding properties of ApoA-I, have been engineered to mimic the antiinflammatory and antioxidant properties of HDL. Among these, the 4F peptide (18 amino acids with 4 phenylalanines, at positions 3, 6, 14, and 18) has received the most attention over the past decade. The 4F peptide is highly effective in improving vascular dysfunction implicated in the pathogenesis of many diseases and disorders, including atherosclerosis, diabetes, hypercholesterolemia, and sickle cell disease.^151–153 In the context of lung disease, 4F decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma. ¹⁵⁴ We have recently demonstrated that the levels of oxidized lipids are elevated in the plasma of PH rats, ⁵⁶ as well as in PAH patients, ²⁹ and may contribute to the inflammatory response and vascular changes involved in the progression of PH. Therapy with 4F has been shown to be very effective in restoring the levels of oxidized lipids and rescue of preexisting PH in animal models. ⁵⁶ We have examined the effect of 4F on HDL and LDL inflammatory indices in an arterial wall model and a monocyte migration assay in IPAH and APAH patients. ²⁹ HDL, as well as LDL, inflammatory indices were decreased significantly after ex vivo treatment with 4F to levels comparable to healthy controls. ²⁹

Role of the microRNA 193–oxidized lipids axis in PH

MicroRNAs (miRNAs) are small, regulatory, noncoding, single-stranded RNA molecules involved in the regulation of several physiological pathways, including apoptosis, cell migration, vascular development, and cell proliferation, via modulation of target genes.^155,156 Altered expression of miRNAs could result in a dysregulated expression of their target genes, consequently causing or exacerbating several pathological conditions, including cardiovascular diseases and PH.^157,158 Several miRNAs, including miR-21, miR-204, and miR-328, have been reported to regulate pathogenic signaling in the development and progression of PH.^159–161 We have recently demonstrated that PH is associated with increased plasma levels of oxidized lipids in rodents as well as in IPAH patients. ²⁹ Therapy with 4F was very effective in restoring their levels and led to rescue of preexisting PH in models of both hypoxia and MCT. Mechanistically, we identified the miRNA miR-193-3p (miR193) as a downstream effector molecule whose expression was significantly downregulated in the lungs in two experimental animal models of PH. The 4F therapy fully restored expression of miR193 to its level in the control group. ⁵⁶ Overexpression of miR193 in the lungs rescued preexisting PH induced by either MCT or hypoxia in animal models. ⁵⁶ We also found that overexpression of miR193 in SMCs isolated from small pulmonary arteries of PAH patients (confirmed by right catheterization) reduces proliferation, whereas knockdown of miR193 in SMCs isolated from small pulmonary arteries of control subjects with no PAH (discarded nondonor lungs) increases proliferation. ⁵⁶ Our data highlight the therapeutic role of miR193 in reversing pulmonary vascular remodeling.

We further showed that oxidized lipids regulate expression of miR193 through the transcriptional factor retinoid X receptor alpha (RXR-α). ⁵⁶ Oxidized lipids induce the expression of RXR-α in PASMCs. This induction results in an increased binding of RXR-α on the promoter of miR193, thus causing its subsequent downregulation and a net increase in the expression of lipoxygenases, the enzymes responsible for the production of oxidized lipids (Fig. 3). However, 4F can decrease the overall content and binding of RXR-α to an miR193 promoter by sequestering oxidized lipids, ultimately leading to miR193 induction. ⁵⁶ This study explored an essential aspect of oxidized lipid–induced pathology of PH, wherein miRNA (i.e., miR193) modulation is involved in the molecular and functional outcome. Future studies on the role of miRNAs in the oxidized lipid–mediated induction of PH with insight into novel targets are warranted to develop a multiple-miRNA therapeutic approach to tackle the disease effectively.