Hyperoxia Increases Phosphodiesterase 5 Expression and Activity in Ovine Fetal Pulmonary Artery Smooth Muscle Cells
In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase (PDE), PDE5. Infants with persistent pulmonary hypertension of the newborn (PPHN) are often mechanically ventilated with high oxygen concentrations. The effects of hyperoxia on the developing pulmonary vasculature and PDE5 are largely unknown. Here, we demonstrate that exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to high levels of oxygen for 24 hours leads to decreased responsiveness to exogenous NO, as determined by a decreased intracellular cGMP response, increased PDE5 mRNA and protein expression, as well as increased PDE5 cGMP hydrolytic activity. We demonstrate that inhibition of PDE5 activity with sildenafil partially rescues cGMP responsiveness to exogenous NO. In FPASMCs, hyperoxia leads to increased oxidative stress without increasing cell death. Treatment of normoxic FPASMCs with H2O2 is sufficient to induce PDE5 expression and activity, suggesting that reactive oxygen species mediate the effects of hyperoxia in FPASMCs. In support of this mechanism, a chemical antioxidant, N-acetyl-cysteine, is sufficient to block the hyperoxia-mediated increase in PDE5 expression and activity and rescue cGMP responsiveness to exogenous NO. Finally, ventilation of healthy neonatal sheep with 100% O2 for 24 hours leads to increased PDE5 protein expression in the resistance pulmonary arteries and increased PDE5 activity in whole lung extracts. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone in response to common clinical treatments for PPHN, such as oxygen and inhaled NO.
Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that results from a failure of the pulmonary vasculature to transition to extrauterine life. Infants typically present shortly after birth with respiratory distress and cyanosis but a structurally normal heart. The estimated incidence of PPHN is 0.2% of liveborn term infants.1 PPHN is a manifestation of a heterogeneous group of disorders, and the response to therapy frequently depends on the underlying disease. Despite advances in clinical therapy, including high frequency ventilation, inhaled nitric oxide (iNO), and extracorporeal membrane oxygenation, there is still significant morbidity and mortality associated with this disease. Furthermore, iNO does not improve survival, and many infants do not respond or sustain their response to iNO for reasons that are unclear.2,3
One factor that confounds the treatment of these infants is the use of supplemental oxygen. Oxygen has long been considered a vasodilator in the pulmonary circulation, and thus, 100% O2 is considered a first-line therapy in infants with PPHN.1,4 However, data from the adult acute respiratory distress syndrome literature,5 from the neonatal bronchopulmonary dysplasia literature,6 and from the neonatal resuscitation literature7,8 suggest that exposure to high levels of oxygen may cause lasting lung injury, oxidative stress, and pulmonary vascular remodeling. Thus, the rationale for high inspired oxygen concentrations has come under question, although the mechanisms by which oxygen affects PPHN therapy are not known.
Phosphodiesterases (PDEs) constitute a superfamily of enzymes comprised of 11 different PDE families. Each family of PDEs has unique enzymatic properties, as well as specific tissue and cellular distributions. The prevalent PDE within the lung is PDE5, although PDE3 and PDE4 are also expressed in pulmonary tissue.9,10 In the fetal lung, PDE5 activity and expression is highest in pulmonary arteries (PAs). In lambs, it decreases shortly after birth and then rises again 4 to 7 days later, suggesting developmental regulation.11,12 Moreover, as the primary enzyme responsible for regulating cGMP, PDE5 represents an important regulator of NO-mediated vascular relaxation.
Endogenous NO and cGMP are important in the normal pulmonary vascular transition after birth,13 and PDE5 activity may be increased in a lamb model of PPHN.14–17 All of the studies that have examined PDE5 expression or activity in the pulmonary vasculature have been conducted in fetal animals exposed to low oxygen tension in utero16,17 or in fetal animals exposed to high levels of inspired oxygen for less than 2 hours.14,15 There are few data regarding the effects of longer hyperoxia exposures on PDE5 expression and activity in the pulmonary vasculature. Recently published data demonstrate that ventilation of newborn sheep with 100% oxygen increases contractile responses to norepinephrine.7 Additionally, in a hyperoxic rat model of bronchopulmonary dysplasia, treatment with sildenafil, a PDE5 inhibitor, decreased pulmonary vascular resistance and improved lung angiogenesis, suggesting that PDE5 activity plays a critical role in mediating pulmonary vascular tone in the context of hyperoxia.18 We propose that increased PDE5 expression and activity in pulmonary vascular smooth muscle in response to hyperoxia may explain abnormal baseline vasoreactivity, as well as iNO resistance and rebound pulmonary hypertension after iNO withdrawal. A greater understanding of the mechanisms of PDE5 regulation in the pulmonary vasculature will be of critical importance to identifying improved therapies for infants with PPHN.
Materials and Methods
Primary cultures of FPASMCs were prepared from intrapulmonary arteries as described in the expanded Materials and Methods section in the online data supplement, available http://circres.ahajournals. org. FPASMCs were treated in incubators with 21% O2–5% CO2, 50% O2–5% CO2, or 95% O2–5% CO2 with or without DETANONOate (100 μmol/L; Cayman Chemical, Ann Arbor, Mich), sildenafil (100 nmol/L, provided by Dr Sharron Francis and Dr Jackie Corbin), or N-acetylcysteine (NAC) (500 μmol/L, Sigma, St Louis, Mo); or with 21% O2–5% CO2 with or without a single dose of H2O2 (50 μmol/L; Sigma). FPASMCs were harvested for analysis after 24 hours.
Cyclic GMP Enzyme-Linked Immunoassay
Treated cells were lysed, and cGMP content was measured by enzyme-linked immunoassay in triplicate according to the protocol of the manufacturer and as described in the expanded Materials and Methods section (Cayman Chemical). Results are shown as picomoles of cGMP per milligram of total protein.
FPASMCs were harvested for RNA using the Aurum Total RNA Mini Kit (Bio-Rad, Hercules, Calif), and RNA was quantified using the Quant-it RiboGreen assay (Molecular Probes/Invitrogen, Carlsbad, Calif). cDNA was prepared from total RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed using the iQ SYBR Green Supermix (Bio-Rad) with the iCycler iQ real-time PCR detection system (Bio-Rad), with 50 cycles of real-time data collection using 95°C for 10 seconds and 46.1°C for 45 seconds, followed by melt curve analysis to verify the presence of a single product. Primers were designed using commercially available software and are described in the expanded Materials and Methods section. Relative PDE5 amounts were normalized to β-actin expression using the ΔΔCt method.19
After treatment, FPASMCs were harvested for total protein (40 μg), which was subjected to Western blot analysis as described in the expanded Materials and Methods section using commercially available antibodies: PDE5 (BD Transduction, San Jose, Calif) and β-actin (Sigma). Bands were visualized via chemiluminescence (Pierce, Rockford, Ill), using a Digital Science Image Station (Kodak, Rochester, NY). Expression of PDE5 was normalized to β-actin expression.
FPASMCs were plated onto collagen-treated glass coverslips and treated as above. After treatment, cells were fixed, permeabilized, and stained for immunocytochemistry with anti-PDE5 used in conjunction with a fluorescent secondary antibody (Molecular Probes/Invitrogen) as described in the expanded Materials and Methods section.
PDE5 Activity Assay
After treatment, FPASMCs were harvested for total protein (5 μg), which was assayed for cGMP hydrolytic activity using a commercially available colorimetric cyclic nucleotide phosphodiesterase assay kit (Biomol, Plymouth Meeting, Pa) as described in the expanded Materials and Methods section. Assays were performed with and without sildenafil (100 nmol/L) to determine PDE5-specific cGMP hydrolytic activity. Results are shown as PDE5-specific picomoles of cGMP hydrolyzed per milligram of total protein per minute.
Detection of Reactive Oxygen Species
FPASMCs were infected with 100 plaque-forming units per cell of a Ro-GFP adenoviral construct as described in the expanded Materials and Methods section. Ro-GFP is a previously characterized ratiometric fluorescent probe sensitive to cellular oxidative stress.20 To create this probe, surface-exposed residues in green fluorescent protein (GFP) were replaced with cysteine residues capable of forming disulfide bonds. Assessment of fluorescence ratios therefore provide a real-time measure of cysteine thiol redox status in live cells.20 Ro-GFP-infected FPASMCs were exposed to 21% O2–5% CO2 or 95% O2–5% CO2 for 24 hours, and subsequently, their oxidative status was analyzed using multilaser flow cytometry as described in the expanded Materials and Methods section.
Ventilation Protocols for Neonatal Sheep
The Laboratory Animal Care Committees at the State University of New York at Buffalo and at Northwestern University approved this study. Near-term gestation pregnant ewes (134 days gestation) were anesthetized with pentothal and halothane, and lambs were delivered by cesarean section (n=5). Lambs were intubated and ventilated with 100% O2 for 24 hours as described in the expanded Materials and Methods section. Control groups included newborn lambs euthanized ≈24 hours after spontaneous delivery (1 day nonventilated, n=4) and fetal lambs delivered and euthanized at 136 days of gestation (fetal control, n=6). For tissue harvest, all lambs were anesthetized with pentothal sodium and killed by rapid exsanguinations through a direct cardiac puncture. The heart and lungs were removed en bloc and fifth-generation PAs (inner diameters of 500 μm) were dissected and isolated.7 Total protein was isolated from lung and PA tissue using the commercially available PARIS kit (Ambion, Austin, Tex). Total protein was then subjected to Western blot analysis for PDE5 protein expression or to measurement of PDE5-specific cGMP hydrolytic activity as described above.
Tissue blocs were snap frozen, cut into thin sections (8 μm), and collected on glass slides. Sections were fixed with acetone and stained with anti-PDE5, in conjunction with a fluorescent secondary antibody (Molecular Probes/Invitrogen), as described in the expanded Materials and Methods section.
Data are presented as means±SEM. Data were analyzed by paired t test or 1-way repeated measures ANOVA with Bonferroni multiple comparison test where appropriate, using Prism (GraphPad Software Inc, San Diego, Calif). P<0.05 was considered statistically significant.
Hyperoxia Decreases cGMP Induced by Exogenous NO
As recent reports show that ventilation of sheep with 100% oxygen diminishes PA relaxations to an NO donor,7 we sought to determine the effect of hyperoxia on cGMP levels in FPASMCs from resistance PA. FPASMCs were exposed to 21% O2–5% CO2, 50% O2–5% CO2, or 95% O2–5% CO2 with or without DETANONOate (100 μmol/L), a long-acting NO donor, for 24 hours (Figure 1). Treatment with exogenous NO in normoxia led to a significant increase in cGMP levels (446±64% of control; P<0.001). However, treatment with exogenous NO in the context of hyperoxia blunted the increase in cGMP levels (P<0.001 for 21% O2+NO versus 50% O2+NO and 21% O2+NO versus 95% O2+NO). Thus, exposure to hyperoxia for 24 hours makes the FPASMCs less responsive to exogenous NO, the primary clinical therapy for PPHN.
Hyperoxia Increases PDE5 Expression in FPASMCs
We hypothesized that the blunting of NO-mediated cGMP responsiveness in hyperoxia represents a PDE5-mediated desensitization. FPASMCs were exposed to 21% O2–5% CO2, 50% O2–5% CO2, or 95% O2–5% CO2 for 24 hours. FPASMCs exposed to hyperoxia showed increased PDE5 mRNA expression (2.7±0.5-fold; P<0.005; Figure 2A) and increased PDE5 protein expression, as determined by immunocytochemistry (Figure 2B) and Western blot (139±9% of control in 50% O2; 185±33% of control in 95% O2; P<0.001; Figure 2C and 2D). Thus, FPASMC exposure to hyperoxia for 24 hours leads to increased PDE5 mRNA and protein expression.
Hyperoxia Increases PDE5 Activity in FPASMCs in a Dose-Dependent Fashion
The hypothesis that increased PDE5 expression leads to decreased cGMP responsiveness in FPASMCs exposed to hyperoxia is valid only if increased PDE5 expression correlates with increased activity. One of the key events required for PDE5 activity is cGMP binding directly to PDE5, followed by phosphorylation by protein kinase G (PKG).21–23 Hyperoxia for 24 hours led to significantly increased PDE5 phosphorylation in FPASMCs (Figure I in the online data supplement). Similarly, hyperoxia for 24 hours led to significantly increased PDE5 activity in FPASMCs (161±26% of control in 50% O2; 309±94% of control in 95% O2; P<0.05; Figure 3). Furthermore, the increase in PDE5 activity in 95% O2–5% CO2 is significantly greater than that seen in 50% O2–5% CO2 (P<0.05; Figure 3). Therefore, the hyperoxia-induced increase in PDE5 protein expression correlates with increased PDE5 phosphorylation and activity, suggesting that increased PDE5 expression and activity contribute to decreased intracellular cGMP responsiveness to exogenous NO in hyperoxia.
Sildenafil Partially Rescues cGMP Responsiveness in FPASMCs in Hyperoxia
If increased PDE5 activity leads to decreased cGMP responsiveness in hyperoxia-exposed FPASMCs, these effects should be blocked by a PDE5 inhibitor, sildenafil. FPASMCs were exposed to 21% O2–5% CO2 or 95% O2–5% CO2 for 24 hours with or without sildenafil (100 nmol/L). In baseline conditions, addition of sildenafil led to a significant increase in PDE5 protein expression (246±30% of control; P<0.05 versus 21% O2; Figure 4A and 4B), likely as a result of increased cGMP levels activating the PDE5 promoter, which has been described previously.24 Hyperoxia for 24 hours led to significantly increased PDE5 protein expression in FPASMCs (192±40% of control; P<0.05 versus 21% O2; Figure 4A and 4B), and in contrast to the effects of sildenafil in 21% O2, this effect is blocked with sildenafil (116±21% of control; P<0.05 versus 95% O2; Figure 4A and 4B).
As expected, sildenafil significantly blocks PDE5 activity in both 21% O2 and 95% O2 (P<0.05 versus untreated cells; Figure 4C). As in Figure 1, treatment with exogenous NO in normoxia led to a significant increase in cGMP that was blunted in the context of hyperoxia (P<0.05 for 21% O2+NO versus 95% O2+NO; Figure 4D). As expected, treatment with sildenafil led to a significant increase in cGMP levels at baseline in both 21% O2 and 95% O2 (P<0.05 versus untreated cells; Figure 4D). Treatment of cells with exogenous NO and sildenafil led to a marked increase in cGMP in 21% O2 (46.2±18.2-fold relative to 21% O2; P<0.001 versus 21% O2; Figure 4D) and in 95% O2 (26.2±8.8-fold relative to 95% O2; P<0.001 versus 95% O2; Figure 4D), but the 95% O2 cGMP levels were still significantly less than those seen in 21% O2 (P<0.01; Figure 4D). Therefore, sildenafil is sufficient to partially rescue the effects of exogenous NO on FPASMC cGMP levels in hyperoxia.
Reactive Oxygen Species Increase PDE5 Expression and Activity in FPASMCs
The effects of hyperoxia are likely mediated via reactive oxygen species (ROS), and others have shown that ROS may play an important role in the underlying pathophysiology of PPHN.25–27 We used a ratiometric fluorescent ROS-sensitive probe, Ro-GFP, to measure the oxidative state of the cell, using dual-laser flow cytometry.20 Exposure of FPASMCs to hyperoxia for 24 hours significantly increased the basal oxidation state of cytosolic proteins within the cell, as measured by the percentage maximal oxidation of the Ro-GFP probe (18.8±4.6% in 21% O2–5% CO2 versus 33.8±7.9% in 95% O2–5% CO2; P<0.05; Figure 5A), without increasing cell death (supplemental Figure II). Thus, ROS likely represents a critical mediator for the hyperoxia effects on PDE5 expression and activity in FPASMCs.
We next sought to determine whether treatment of FPASMCs with exogenous oxidants in the context of normoxia was sufficient to replicate the hyperoxia effects on PDE5 expression, phosphorylation, and activity. Treatment of FPASMCs with a single dose of H2O2 (50 μmol/L) led to a significant increase in PDE5 protein expression (311±87% versus untreated; P<0.05; Figure 5B and 5C), PDE5 phosphorylation (supplemental Figure III), and PDE5 activity (163±32% versus untreated; P<0.05; Figure 5D). Similarly, pretreatment with sildenafil significantly blocked PDE5 activity in both untreated and H2O2-treated FPASMCs (supplemental Figure IV). These data strongly suggest that ROS, in general, and H2O2, in particular, are sufficient to induce effects similar to hyperoxia on PDE5 expression and activity.
NAC Rescues cGMP Responsiveness in FPASMCs in Hyperoxia
The hypothesis that increased ROS leads to decreased cGMP responsiveness in FPASMCs exposed to hyperoxia would suggest that these effects can be blocked by an antioxidant, NAC. FPASMCs were exposed to 21% O2–5% CO2 or 95% O2–5% CO2 for 24 hours with or without NAC (500 μmol/L). Hyperoxia for 24 hours led to significantly increased PDE5 protein expression in FPASMCs (304±100% of control; P<0.01 versus 21% O2; Figure 6A and 6B), and this effect is completely blocked with NAC (106±46% of control; P<0.01 versus 95% O2; Figure 6A and 6B).
Furthermore, NAC significantly blocks PDE5 activity in 95% O2 (P<0.05 versus untreated cells; Figure 6C). Treatment with exogenous NO in normoxia led to a significant increase in cGMP levels that was blunted in the context of hyperoxia (P<0.01 for 21% O2+NO versus 95% O2+NO; Figure 6D). Treatment of cells with exogenous NO and NAC in 95% O2 restored normal cGMP responsiveness relative to 95% O2 with exogenous NO alone (P<0.05 versus 95% O2+NO; Figure 6D). Therefore, NAC, an antioxidant, is sufficient to rescue the effects of exogenous NO on cGMP levels in FPASMCs in hyperoxia.
Ventilation of Healthy Neonatal Sheep With 100% O2 for Twenty-Four Hours Increases PDE5 Expression and Activity
To verify that the effects of hyperoxia on PDE5 were not confined to isolated FPASMCs, we mechanically ventilated healthy near-term sheep with 100% O2 for 24 hours and then euthanized the lambs to harvest lung and resistance PA tissue. Ventilation with 100% O2 for 24 hours led to increased PDE5 protein expression in resistance PAs as compared with both control nonventilated fetal lambs and 1-day nonventilated lambs (1.8±0.4-fold versus fetal lambs; 3.2±0.7-fold versus 1-day lambs; P<0.05; Figure 7A). There was a trend toward decreased PDE5 protein expression in 1-day lambs versus fetal lambs, but this did not reach statistical significance. Similarly, ventilation with 100% O2 for 24 hours significantly increased lung PDE5 activity relative to fetal and 1-day lambs (3.9±0.9-fold versus fetal lambs; 2.6±0.6-fold versus 1-day lambs; P<0.05; Figure 7B). Immunohistochemistry confirmed that ventilation with 100% O2 for 24 hours leads to increased PDE5 expression, which is primarily seen in the smooth muscle of the resistance PAs (Figure 7C). Thus, the increase in PDE5 expression and activity in neonatal sheep ventilated with 100% O2 confirms that our findings are physiologically valid and may explain the impaired vasoreactivity previously reported in sheep after exposure to 100% O2.7
Our results demonstrate that in primary cultures of FPASMCs exposed to hyperoxia, there is decreased cGMP responsiveness to exogenous NO, which is an important therapeutic vasodilator for infants with pulmonary hypertension. We propose that this is attributable, in part, to a PDE5-mediated increase in cGMP degradation. In support of that theory, we demonstrate that exposure to hyperoxia for 24 hours increases PDE5 mRNA and protein expression, as well as phosphorylation and activity. Inhibition of the hyperoxia-induced PDE5 activity with sildenafil, a PDE5 inhibitor, was sufficient to partially rescue the cGMP response to exogenous NO, indicating that PDE5 is a critical regulator of cGMP in the context of hyperoxia.
As might be expected, exposure to hyperoxia for 24 hours leads to increased oxidative stress with the FPASMCs, as detected using a novel ratiometric fluorescent probe sensitive to changes in oxidation. This probe represents a valuable new tool to investigate the oxidation state of cells without relying on fluorescent dyes with their accompanying technical limitations. Using this tool, it is not surprising that 24 hours of hyperoxia increased the oxidation of cellular proteins. However, this oxidative stress was not sufficiently severe to cause an increase in cell death (supplemental Figure II). The FPASMCs show significant changes in cellular signaling pathways during hyperoxia, as evidenced by the increase in PDE5 expression, phosphorylation, and activity. As such, we hypothesized that ROS may serve as critical mediators in the crosstalk between oxygen and PDE5. In support of that, a single dose of exogenous H2O2 was sufficient to induce long-lasting changes in PDE5 expression, phosphorylation, and activity, which mirror those seen after exposure to hyperoxia. Similarly, the changes in PDE5 expression and activity, as well as the decreased cGMP responsiveness in hyperoxia, are all reversed with a chemical antioxidant, NAC. This confirms that ROS, in general, and H2O2, in particular, are sufficient to induce significant changes in PDE5 expression and activity, which negatively impact the pulmonary vasculature. Finally, we demonstrate that these results are recapitulated in an in vivo model, where ventilation of healthy neonatal sheep with 100% O2 for 24 hours produced an increase in PDE5 expression and activity.
PDE5 is the key enzyme for cGMP hydrolysis in the pulmonary vasculature. The observation that the response to exogenous NO in FPASMCs is diminished in hyperoxia indicates that the increase in PDE5 expression and activity is biologically significant (Figure 1). However, because sildenafil only partially rescues the cGMP response to exogenous NO (Figure 4D), other key signaling mediators may also be impacted by hyperoxia in fetal PASMC. Soluble guanylyl cyclase (sGC) is the primary enzyme responsible for cGMP production. sGC protein expression is decreased in nonventilated fetal lambs with PPHN, suggesting that it may also be an important regulator of neonatal vascular tone.28 Recent studies suggest that sGC protein expression in FPASMCs is decreased by ROS, specifically H2O2.29 Little is known regarding the impact of hyperoxia on sGC, although it is possible that changes in sGC further contribute to the decreased cGMP responsiveness to exogenous NO after exposure to hyperoxia. Similarly, atrial natriuretic polypeptide and C-type natriuretic peptide signal through their respective natriuretic peptide receptors, natriuretic peptide receptor-A and -B, to produce vasorelaxation. These receptors are coupled to particulate guanylate cyclase activity, which can thus impact cGMP concentrations and thereby affect relaxation of the PAs. Although these molecules play a role in the intact vessels and animals,30 they are not likely to influence the changes seen in the isolated fetal PASMCs because there are no adjacent endothelial cells to produce the natriuretic peptides. Thus, although other signaling mediators such as sGC may contribute, the present study demonstrates that PDE5 represents a critical factor for the regulation of intracellular cGMP levels in the context of hyperoxia.
Finally, the mechanism for the interplay among oxygen, ROS, and PDE5 is not fully understood. In corpus cavernosum, the regulation of PDE5 expression is largely cGMP dependent.31 However, this would seem an unlikely mediator between oxygen and PDE5, given our finding of impaired cGMP signaling in FPASMCs under hyperoxic conditions. Promoter analysis shows both an Sp-1 and an activator protein-1 site in the human PDE5 promoter region.31 Both of these transcription factors have previously been shown to be redox sensitive and may represent potential downstream targets of the ROS-mediated signaling that impacts PDE5.32 In contrast, studies of PDE5 activity in gastric and aortic smooth muscle demonstrate that increased cGMP causes activation of PKG, which in turn phosphorylates and activates PDE5.21,22 Consistent with these data, we demonstrate that oxygen leads to increased PDE5 phosphorylation, using an antibody raised to a previously characterized PKG site (supplemental Figure I) and increased PDE5 activity (Figure 3), suggesting that a likely mechanism for PDE5 activation by oxygen is via PKG-mediated phosphorylation (Figure 8). Unlike PDE5, there is a growing body of literature that PKG is regulated by cellular redox status within aortic smooth muscle as well as the pulmonary vasculature, confirming that it is a likely candidate for the intermediary between ROS and PDE5.33,34
In conclusion, we have demonstrated that the ability of FPASMCs to respond to exogenous NO, a critical therapeutic vasodilator, is impaired when FPASMCs are exposed to hyperoxia for 24 hours. We have further demonstrated that hyperoxia is sufficient to induce PDE5 expression, phosphorylation, and activity in FPASMCs, which is likely a critical mediator of impaired cGMP responsiveness under these conditions. These findings are novel, because they represent the first description of redox regulation of PDE5. PDE5 appears to be exquisitely sensitive to redox regulation, because even a short burst of oxidative stress such as a single dose of H2O2 is sufficient to produce changes in PDE5. These studies provide key insights into the basic regulation of PDE5 in the pulmonary vasculature and its crosstalk with oxygen, as well as provide a potential mechanism for the impaired vasoreactivity seen in the oxygen-exposed pulmonary vasculature.
We thank Robert D. Guzy for Ro-GFP protocol assistance, Gabriel Loor for cell death assay assistance, and Sharron H. Francis and Jackie D. Corbin for providing the sildenafil used in this study.
Sources of Funding
This work was supported by NIH grants K12 HD052902 and K08 HL086715 (to K.N.F.), R01 HL079650 and R01 HL35440 (to P.T.S.), and R01 HL54705 (to R.H.S.).
Original received August 7, 2006; resubmission received August 6, 2007; revised resubmission received September 9, 2007; accepted October 30, 2007.
Roberts JD, Fineman JR, Morin FC 3rd, Shaul PW, Rimar S, Schreiber MD, Polin RA, Zwass MS, Zayek MM, Gross I, Heymann MA, Zapol WM, Thusu KG, Zellers TM, Wylam ME, Zaslavsky A. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. N Engl J Med. 1997; 336: 605–610.
Vento M, Asensi M, Sastre J, Garcia-Sala F, Pallardo FV, Vina J. Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates. Pediatrics. 2001; 107: 642–647.
Maclean MR, Johnston ED, McCulloch KM, Pooley L, Houslay MD, Sweeney G. Phosphodiesterase isoforms in the pulmonary arterial circulation of the rat: changes in pulmonary hypertension. J Pharmacol Exp Ther. 1997; 283: 619–624.
Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem. 2004; 279: 13044–13053.
Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, Beavo JA. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem. 2002; 277: 3310–3317.
Turko IV, Francis SH, Corbin JD. Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation. Biochem J. 1998; 329: 505–510.
Wedgwood S, McMullan M, Bekker JM, Fineman JR, Black SM. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res. 2001; 89: 357–364.
Brennan LA, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, Black SM. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. Circ Res. 2003; 92: 683–691.
Wedgwood S, Steinhorn RH, Bunderson M, Wilham J, Lakshminrusimha S, Brennan LA, Black SM. Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn. Am J Physiol Lung Cell Mol Physiol. 2005; 289: L660–L666.
Lakshminrusimha S, D’Angelis CA, Russell JA, Nielsen LC, Gugino SF, Nickerson PA, Steinhorn RH. C-type natriuretic peptide system in fetal ovine pulmonary vasculature. Am J Physiol Lung Cell Mol Physiol. 2001; 281: L361–L368.
Liu MY, Eyries M, Zhang C, Santiago FS, Khachigian LM. Inducible platelet-derived growth factor D-chain expression by angiotensin II and hydrogen peroxide involves transcriptional regulation by Ets-1 and Sp1. Blood. 2006; 107: 2322–2329.
Sander F, Gao Y, Raj JU. Hypoxia down-regulates cyclic guanidine monophosphate-dependent protein kinase in fetal pulmonary vascular smooth muscle cell through generation of reactive oxygen species and promotes development of pulmonary hypertension. Chest. 2005; 128: 577S–578S.
Schulz E, Tsilimingas N, Rinze R, Reiter B, Wendt M, Oelze M, Woelken-Weckmuller S, Walter U, Reichenspurner H, Meinertz T, Munzel T. Functional and biochemical analysis of endothelial (dys) function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment. Circulation. 2002; 105: 1170–1175.