Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase

Abstract

Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2).

Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats

“…PHT (24,35,40) by resulting in reduced NO availability (impaired vasodilatation) and increased peroxynitrite formation leading to vasoconstriction. BH 4 and HSP90 may also be oxidized and/or nitrated by peroxynitrite, which may cause, or further contribute to, eNOS uncoupling (36,37).…”

“…Pulmonary vascular ROS were detected in situ utilizing DHE, a dye that fluoresces upon reaction with ROS [predominantly O2 •Ϫ but also H2O2 and hydroxyl radicals (50)], using a method recently described by others (22,24). Briefly, the lungs of two animals per group and time point were snap frozen in Tissue-Tek OCT, as previously described (28), and stored at Ϫ80°C.…”

“…A major pathological role for increased generation of reactive oxygen species (ROS) in the pathogenesis of experimental PHT is evidenced by antioxidant intervention studies performed in fetal lambs (24,39), in hyperoxia-exposed neonatal rats (30), and in hypoxia- (25,38,41) or monocrotalineexposed (9) adult rodents. A putative role for oxidative stress is also supported by observational studies in adult humans with idiopathic PHT (7,10).…”

“…Major enzymatic sources described in adult rats and mice chronically exposed to hypoxia have included xanthine oxidase (XO) (25) and reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase (42), respectively. Furthermore, both NAD(P)H oxidase and uncoupled endothelial nitric oxide synthase (eNOS) have been described as major vascular sources of ROS in fetal lambs with ductal ligation-induced PHT (24,36). Under normal conditions, the enzyme xanthine oxidoreductase (XOR) exists primarily in its dehydrogenase form, serving as the rate-limiting step in purine degradation to uric acid.…”

Role of Oxidized Lipids in Pulmonary Arterial Hypertension

“…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.…”