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Integrative Physiology |
From the Departments of Pediatrics (L.A.B., R.H.S., S.W., E.M.-G., E.A.R., S.M.B.) and Molecular Pharmacology (S.M.B.), Northwestern University, Chicago, Ill, and the Department of Physiology (J.A.R.), State University of Buffalo, New York.
Correspondence to Stephen M. Black, PhD, Department of Pediatrics, Northwestern University, Ward 12-191, 303 E Chicago Ave, Chicago, IL 60611-3008. E-mail steveblack{at}northwestern.edu
| Abstract |
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Key Words: reactive oxygen species vascular remodeling smooth muscle
| Introduction |
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Recently, production of reactive oxygen species (ROS) has been demonstrated in fibroblasts,10 endothelial cells,11,12 and smooth muscle cells (SMCs).13 This oxidant production plays an important role both in the normal functioning of vascular systems as well as in the pathogenesis of vascular disease. Every cell type in the vascular wall has been shown to produce and be regulated by ROS. We, and others, have demonstrated that a vascular equivalent of the phagocytic NADPH oxidase plays an important role in generating ROS in cells of the vessel wall.1316 This vascular NAD(P)H oxidase enzyme complex appears to be membrane associated, catalyzing the one electron reduction of oxygen using NADPH or NADH as the electron donor: NAD(P)H+2O2
NAD(P)++H+ +2O2·-. One of the important attributes of the vascular oxidase is that it responds to external signals to generate superoxide. However, the role of superoxide and NADPH oxidase in the development of PPHN has not been evaluated. Thus, the purpose of our study was to determine whether the development of PPHN is associated with increased superoxide generation, determine a possible mechanism for this effect, and establish whether superoxide scavenging could restore NO-mediated vessel relaxation and reduce SMC proliferation.
| Materials and Methods |
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Prenatal Ligation of the Ductus Arteriosus
The technique for creating pulmonary hypertension by prenatal ligation of the ductus arteriosus has been described previously.9,1719 Briefly, time-dated pregnant ewes (mixed breed; Swartz family sheep farm, Attica, NY) were operated on at 127 days of gestation (term=146 days). After either 2 or 9 days, the fetus was delivered by cesarean section and the umbilical cord was clamped (n=8). Twin fetuses served as controls (n=8). Before the first breath, lambs were killed by rapid exsanguination through a direct cardiac puncture.
Tissue Preparation and Western Blotting
Peripheral lung parenchyma and fifth-generation pulmonary arteries and veins were rapidly isolated and frozen in liquid nitrogen. Protein extracts, prepared as previously described,9 were separated on 12% (superoxide dismutase [SOD] enzymes) or 4% to 20% (copper chaperone for superoxide dismutase [CCS-1] protein and p67phox) polyacrylamide gels, then transferred onto enhanced chemiluminescence nitrocellulose (Amersham Pharmacia). After blocking, the membranes were incubated at room temperature for 1 hour with the appropriate dilution of antibody: CuZnSOD and MnSOD (1:1000), CCS-1 protein (1:250), and p67phox (1:500). After washing blots to remove excess primary antibody binding, blots were incubated for 1 hour with the appropriate horseradish peroxidaselinked secondary antibody: rabbit (1:1000) for SOD and CCS-1 and mouse (1:1000) for p67phox. Antibody binding was detected using enhanced chemiluminescence (Amersham Pharmacia).
Immunohistochemistry
Immunohistochemistry was performed as previously described.20 Studies were done on serial sections of control and ductal ligation lung, using polyclonal antiserum raised against the p67phox subunit of NADPH oxidase. The tissue sections were incubated in antiserum (1:500 dilution) for 12 to 18 hours at 4°C. Appropriate biotin-conjugated secondary antibodies were used and detected with an anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC). Sections were then viewed under a fluorescent microscope (Olympus BX40) and imaged using a digital camera (Pixera DiRactor).
In Situ Analysis of Superoxide Generation
Lung segments and vessels were obtained from control and hypertensive lambs, embedded in tissue freezing medium (TBS, Fisher), frozen, cut into 30-µm-thick sections, placed on glass slides, and cover-slipped. The sections were exposed to 2 µmol/L dihydroethidium (DHE, Molecular Probes) in Krebs/HEPES buffer. Slides were incubated in a light-protected humidified chamber at 37°C for 30 minutes. Ethidium-stained cells were observed with an Olympus BX40 fluorescent microscope after excitation at 518 nm and emission at 605 nm. Captured fluorescent images were quantified using Metamorph imaging software (Fryer). Control and hypertensive tissue sections were processed and imaged in parallel. Specificity of DHE for superoxide was demonstrated by preincubation with PEG-SOD (50 U) quenching the ethidium signal.
SOD Activity
Total SOD activity was measured using a SOD activity kit from Oxis Research.
Isolated Vessel Protocol
We have previously described the techniques for study of isolated vessels in detail.18,19 In the present study, we used fifth-generation pulmonary arteries with inside diameters of
500 µm. Vessel rings were mounted on stainless steel hooks and placed in water-jacketed chambers containing Krebs-Ringer solution. The buffer was maintained at 37°C and aerated with a gas mixture of 94% O2 and 6% CO2 to maintain a pH of 7.40, a PCO2 of 38 torr, and a PO2 of >500 torr. Vessels were pretreated for 20 minutes with 10-5 mol/L indomethacin to prevent the formation of vasoactive prostaglandins and with 10-6 mol/L propranolol to block activation of ß-adrenergic receptors. Vessels were preconstricted with a submaximal concentration of norepinephrine. Cumulative concentration-response curves for S-nitrosyl-acetyl-penicillamine (SNAP, 10-9 to 3x10-6 mol/L) were developed. Some vessels were pretreated for 20 minutes with polyethylene glycolbound SOD (37.5 U/mL), Tiron (10-5 mol/L), or the NADPH oxidase inhibitor diphenyliodonium (2x10-6 mol/L) before relaxation with SNAP.
Statistical Analysis
All data are expressed as mean±SE. Band intensities from Western blot analysis were analyzed densitometrically using the Kodak 1SD software. Comparisons of Western blot band densities, ethidium fluorescence intensities, and SOD activity were compared by ANOVA. Comparisons of vascular responses were made by repeated-measures ANOVA. A value of P<0.05 was considered statistically significant.
| Results |
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Using whole lung and isolated vessel homogenates, we determined whether there were changes in lung antioxidant enzymes. Western blot analysis showed that CuZnSOD (Figures 2A and 2B) and MnSOD (Figures 2C and 2D) protein levels were not different in lung and isolated vessels from control lambs compared with PPHN lambs after either 2 days or 9 days of ductal ligation. Total SOD activity was unchanged in lung parenchyma or isolated pulmonary vessels after 2 days of ductal ligation (Figure 3). However, pulmonary arteries, but not pulmonary veins, isolated from the 9-day ductal ligation lambs exhibited a 44% decrease in SOD activity compared with control lambs (Figure 3; P<0.05).
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CuZnSOD activity is dependent on copper transfer from the CCS-1 protein. Thus, a potential mechanism for the apparent decrease in SOD activity in the pulmonary arteries from ligated lambs could be a decrease in CCS-1 expression. However, results demonstrated that CCS-1 was abundantly expressed in lung and isolated vessels and that the only significant change in CCS-1expression was a decrease in the pulmonary veins of the PPHN lambs after 2 days of ductal ligation (Figure 4).
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To investigate potential sources of superoxide, we carried out Western blot analysis of lung and isolated vessels using an antibody to the p67phox subunit of NADPH oxidase (Figure 5). We found significant increases in p67phox expression in lung and pulmonary arteries from PPHN relative to control lambs as early as 2 days of ductal ligation, and these increases in expression were maintained at 9 days (P<0.05). There was no difference in p67phox expression in pulmonary veins at either time point examined. Immunostaining of lung sections confirmed this Western blot data with increased p67phox expression observed in vessels in the lung sections prepared from PPHN lambs relative to controls after both 2 days and 9 days of ductal ligation (Figure 6).
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We then performed functional assays using fifth-generation pulmonary arteries from control and PPHN lambs. Pulmonary arteries from PPHN lambs showed significantly blunted relaxations to the NO donor SNAP (Figure 7A), but only after 9 days of ductal ligation. Pretreatment with the cell-permeable superoxide scavengers Tiron (Figure 7B) and PEG-SOD (Figure 7C) enhanced the relaxations to SNAP in pulmonary arteries from PPHN lambs after 9 days of ductal ligation but had no effect on SNAP-induced relaxations in pulmonary arteries isolated from PPHN lambs after 2 days of ductal ligation. Similarly, pretreatment with diphenyliodonium (DPI) to inhibit NADPH oxidase also enhanced relaxations to SNAP in pulmonary arteries isolated from PPHN lambs but only after 9 days of ductal ligation (Figure 7D). Pretreatment with Tiron, SOD, and DPI had no significant effect on SNAP relaxations in pulmonary arteries isolated from control lambs at either time point (Figures 7B through 7D). In addition, the relaxation curves obtained from pulmonary arteries isolated from lambs after 2 days of ductal ligation were equivalent to those obtained from control lambs (Figures 7A through 7D).
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| Discussion |
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Little is known about the levels and activity of lung antioxidant enzymes in PPHN. We investigated whether the enzymes responsible for clearance of superoxide were altered in PPHN. These enzymes, CuZnSOD and MnSOD, are metalloenzymes that catalyze the dismutation of the superoxide anion to hydrogen peroxide and oxygen. Using Western blotting, we demonstrated that, while both CuZnSOD (Figures 2A and 2B) and MnSOD (Figures 2C and 2D) were present in fetal lung, there was no difference in PPHN lambs compared with controls at either time point examined. Therefore, we conclude that PPHN is associated with an overproduction of the oxidant superoxide without a simultaneous increase in cellular antioxidant capacity. This hypothesis is strengthened when we compared the SOD activities in fetal and adult lung. Our preliminary results indicate that SOD activity is 4-fold less in fetal compared with adult lung. Although these data will need to be confirmed at the protein expression levels, they suggest that the fetal lung may be especially sensitive to oxidative stress. Further studies will be required to investigate the mechanism by which increased oxidative stress reduces SOD activity during the progression of pulmonary hypertension in the fetal lung.
Activity of CuZnSOD requires copper. To limit spontaneous fenton-type reactions, copper availability is limited to very low levels in vivo, and delivery of copper to this enzyme is the task of the CCS-1 protein.22 Because SOD protein levels were not altered in the PPHN lung, we determined whether alterations in CCS-1 expression explained the increased superoxide formation seen in the PPHN lungs. Recent evidence has shown that there is an oxygen requirement for copper transfer to CuZnSOD, and it has been suggested that excess superoxide may inhibit copper transfer by CCS-1 and inactivate the CuZnSOD enzyme.23 These changes would enhance superoxide formation in the cytosol. However, our data indicate that CCS-1 expression was not altered in lung or isolated pulmonary arteries in PPHN lambs, although a reduction in CCS-1 was observed after 2 days of ductal ligation in pulmonary veins, but this was not maintained after 9 days (Figures 4A and 4B). Thus, although SOD activity is significantly less than that in adult sheep lung, this is not likely to be due to a lack of CCS-1.
We did find that SOD activity was decreased in pulmonary arteries, but not pulmonary veins, isolated from PPHN lambs relative to controls after 9 days, but not 2 days, of ductal ligation (Figure 3). The reason for this decrease in activity after 9 days, which occurred without reduction in the level of SOD protein, is unclear. However, Cys55 in bovine CuZnSOD (or Cys57 in human) is involved in the formation of an internal disulfide bond that is essential for activity. Without the Cys55 to Cys144 disulfide bond, a key hydrogen bond (essential for SOD activity) cannot form between the carbonyl group of Cys55 and the amine group of Arg141. It has been suggested that this cysteine residue is sensitive to oxidative modification, which can result in enzyme inhibition.24 Because the sheep CuZnSOD protein has not yet been analyzed, it is unclear whether the Cys55 equivalent residue in the ovine CuZnSOD enzyme would be sensitive to this type of oxidative inhibition.
We next investigated the potential sources of the increased superoxide generated in the PPHN lungs identifying an increase in the p67phox subunit of the NADPH oxidase enzyme complex by Western blot analysis (Figure 5) or immunohistochemistry (Figure 6). The presence of a superoxide-generating enzyme similar to neutrophil NADPH oxidase has been identified in cultured calf pulmonary artery SMCs.25 Vascular smooth muscle cell accumulation and hypertrophy are characteristic of hypertensive vascular diseases, and it is also thought that many other cells that comprise the vessel wall generate ROS. In phagocytic cells, the NADPH oxidase system consists of the cytosolic components, p47phox and p67phox,26,27 a low molecular weight G protein, Rac1 or Rac2,28,29 and a membrane-associated cytochrome b558.30 In phagocytes, cytochrome b558 functions as the final electron transporter from NADPH to molecular oxygen, and this protein consists of a 22-kDa subunit (p22phox) and a glycosylated 91-kDa subunit (gp91phox).30 Messenger RNAs for p22phox, p47phox and p67phox, gp91phox, and Rac1 have been identified in endothelial cells while smooth muscles cells appear to express p22phox, p47phox, p67phox, and Rac1 with the expression of gp91phox less certain.31 It has been suggested that the recently identified mox-1 protein (for mitogenic oxidase) may represent the gp91phox homologue in SMCs.31
Cultured endothelial cells have been shown to produce ROS in response to LDL exposure.32,33 In these cell lysates, increased expression of gp91phox and p67phox was demonstrated by Western blotting.34 Upregulation of p67phox and gp91phox has also been shown in the aorta of mice with hypertension after infusion of angiotensin II.35 NADPH oxidase is a highly regulated enzyme complex, and its activation requires the assembly of both cytosolic- and membrane-bound factors.36 Using Western blot analysis, we demonstrated that the p67phox subunit was increased in whole-lung homogenates and pulmonary arteries of PPHN lambs as early as 2 days after ductal ligation, and this increase was maintained at 9 days (Figures 5A and 5B). Further, this increase appeared to be localized to the adventitia and smooth muscle layers (Figure 6), the same layers where we localized the increases in superoxide and the areas that have been previously reported to exhibit vascular remodeling.68 Although our studies are highly suggestive that the increased superoxide generated in the fetal lung during the development of pulmonary hypertension is due to an increase in the NADPH oxidase expression further studies will be required to confirm that there is an increase in NADPH oxidase activity. In addition, although NADPH oxidase expression was found to be elevated after only 2 days of ductal ligation, this did not correlate with an increase in the levels of superoxide (Figure 1). This suggests that at this early time period the antioxidant capacity of the lung is sufficient to scavenge the excess superoxide generated by the increase in NADPH oxidase expression. In agreement, our vessel studies (Figure 7) determined that pulmonary arteries isolated after only 2 days of ductal ligation relaxed normally to SNAP.
One of the important attributes of the vascular oxidase is that it appears to respond to external signals to generate superoxide. We have previously demonstrated that ET-1 stimulates the activity of the vascular NADPH oxidase complex in pulmonary arterial SMCs from fetal sheep, which leads to an increase in proliferation.16 PPHN is associated with increased levels of ET-1 in both infants37 and in the ductal ligation model.9 Thus, we speculate that the elevated ET-1 concentrations activate NADPH oxidase, which in turn increases superoxide generation. Superoxide could lead to an increase in smooth muscle proliferation, producing the anatomic findings of increased thickness in the smooth muscle layer of the pulmonary arteries, as well as muscularization of the nonmuscularized arteries. Further studies will be required to determine whether ET-1 also increases the expression of the subunits of NADPH oxidase.
Our next set of studies investigated whether the increased levels of superoxide in the PPHN lung had functional consequences on the relaxation responses of pulmonary arteries (Figure 7). We have previously shown that pulmonary veins isolated from PPHN lambs relax normally to endogenous and exogenous NO.18 Therefore, we focused our present studies on pulmonary arterial responses. We demonstrated that fifth-generation pulmonary arteries isolated from PPHN lambs had significantly decreased relaxations to the NO donor SNAP compared with controls, but only after 9 days of ductal ligation (Figure 7A). The addition of the superoxide scavengers Tiron (Figure 7B) or PEG-SOD (Figure 7C) to pulmonary arteries isolated from PPHN lambs significantly enhanced their relaxations to SNAP, but again only after 9 days of ductal ligation. Little effect of these scavengers was seen in control pulmonary arteries, indicating this enhancement was unique to PPHN. Further, the inhibition of NADPH oxidase with DPI enhanced relaxations to SNAP in pulmonary arteries from PPHN but not control lambs (Figure 7D). However, this was again limited to the lambs subjected to 9 days of ductal ligation. These data indicate that excess superoxide inhibits relaxations to exogenous NO in PPHN, and that scavengers of superoxide restore these relaxations. In addition, our studies indicate that pulmonary vessels isolated from lambs exposed to only 2 days of ductal ligation relax normally to SNAP, suggesting that the vascular remodeling normally associated with PPHN may not have occurred at this early time point.
ROS appear to be important for SMC proliferation and survival, and studies have demonstrated that suppression of endogenous ROS levels will inhibit SMC proliferation and promote apoptosis.38,39 Thus, the NADPH oxidase enzyme complex may play an important role in fetal pulmonary artery SMC proliferation by producing a required level of superoxide within the cell. Therefore, we speculate that antioxidant therapy could potentially be used to inhibit vascular SMC proliferation, although the concomitant effects of antioxidants on fetal pulmonary artery endothelial cell proliferation remain to be established.
In conclusion, although PPHN is a condition resulting from a diverse set of circumstances, and caution must be exercised when applying the results of experimental models to humans, this study provides a strong rationale to further explore therapeutic intervention strategies for PPHN based on antioxidants.
| Acknowledgments |
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Received January 24, 2002; revision received June 5, 2002; accepted February 12, 2003.
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K. N. Farrow, B. S. Groh, P. T. Schumacker, S. Lakshminrusimha, L. Czech, S. F. Gugino, J. A. Russell, and R. H. Steinhorn Hyperoxia Increases Phosphodiesterase 5 Expression and Activity in Ovine Fetal Pulmonary Artery Smooth Muscle Cells Circ. Res., February 1, 2008; 102(2): 226 - 233. [Abstract] [Full Text] [PDF] |
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Y.-X. Shi, Y. Chen, Y.-Z. Zhu, G.-Y. Huang, P. K. Moore, S.-H. Huang, T. Yao, and Y.-C. Zhu Chronic sodium hydrosulfide treatment decreases medial thickening of intramyocardial coronary arterioles, interstitial fibrosis, and ROS production in spontaneously hypertensive rats Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2093 - H2100. [Abstract] [Full Text] [PDF] |
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K. A. Sanders and J. R. Hoidal The NOX on Pulmonary Hypertension Circ. Res., August 3, 2007; 101(3): 224 - 226. [Full Text] [PDF] |
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G. G. Konduri, I. Bakhutashvili, A. Eis, and K. Pritchard Jr. Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1812 - H1820. [Abstract] [Full Text] [PDF] |
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K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF] |
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R. H Steinhorn and K. N Farrow Pulmonary Hypertension in the Neonate NeoReviews, January 1, 2007; 8(1): e14 - e21. [Abstract] [Full Text] [PDF] |
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S. Lakshminrusimha, J. A. Russell, S. Wedgwood, S. F. Gugino, J. A. Kazzaz, J. M. Davis, and R. H. Steinhorn Superoxide Dismutase Improves Oxygenation and Reduces Oxidation in Neonatal Pulmonary Hypertension Am. J. Respir. Crit. Care Med., December 15, 2006; 174(12): 1370 - 1377. [Abstract] [Full Text] [PDF] |
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A. C. Grobe, S. M. Wells, E. Benavidez, P. Oishi, A. Azakie, J. R. Fineman, and S. M. Black Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1069 - L1077. [Abstract] [Full Text] [PDF] |
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A. Sturrock, B. Cahill, K. Norman, T. P. Huecksteadt, K. Hill, K. Sanders, S. V. Karwande, J. C. Stringham, D. A. Bull, M. Gleich, et al. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L661 - L673. [Abstract] [Full Text] [PDF] |
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E. Mata-Greenwood, C. Jenkins, K. N. Farrow, G. G. Konduri, J. A. Russell, S. Lakshminrusimha, S. M. Black, and R. H. Steinhorn eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L232 - L241. [Abstract] [Full Text] [PDF] |
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H. Cai A new mechanism for flow-mediated vasoprotection? Focus on "Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species" Am J Physiol Cell Physiol, January 1, 2006; 290(1): C35 - C36. [Full Text] [PDF] |
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J. Q. Liu, I. N. Zelko, E. M. Erbynn, J. S. K. Sham, and R. J. Folz Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox) Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L2 - L10. [Abstract] [Full Text] [PDF] |
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C.-F. Lam, T. E. Peterson, A. J. Croatt, K. A. Nath, and Z. S. Katusic Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2334 - H2341. [Abstract] [Full Text] [PDF] |
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S. Wedgwood, R. H. Steinhorn, M. Bunderson, J. Wilham, S. Lakshminrusimha, L. A. Brennan, and S. M. Black 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, October 1, 2005; 289(4): L660 - L666. [Abstract] [Full Text] [PDF] |
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C. D. Fike, Y. Zhang, and M. R. Kaplowitz Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets J Appl Physiol, August 1, 2005; 99(2): 670 - 676. [Abstract] [Full Text] [PDF] |
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E. Mata-Greenwood, A. Grobe, S. Kumar, Y. Noskina, and S. M. Black Cyclic stretch increases VEGF expression in pulmonary arterial smooth muscle cells via TGF-{beta}1 and reactive oxygen species: a requirement for NAD(P)H oxidase Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L288 - L289. [Abstract] [Full Text] [PDF] |
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J. P. Khoo, L. Zhao, N. J. Alp, J. K. Bendall, T. Nicoli, K. Rockett, M. R. Wilkins, and K. M. Channon Pivotal Role for Endothelial Tetrahydrobiopterin in Pulmonary Hypertension Circulation, April 26, 2005; 111(16): 2126 - 2133. [Abstract] [Full Text] [PDF] |
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T. Djordjevic, R. S. BelAiba, S. Bonello, J. Pfeilschifter, J. Hess, and A. Gorlach Human Urotensin II Is a Novel Activator of NADPH Oxidase in Human Pulmonary Artery Smooth Muscle Cells Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 519 - 525. [Abstract] [Full Text] [PDF] |
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S. Wedgwood and S. M. Black Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide Am J Physiol Lung Cell Mol Physiol, March 1, 2005; 288(3): L480 - L487. [Abstract] [Full Text] [PDF] |
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S. Patil, M. Bunderson, J. Wilham, and S. M. Black Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth Am J Physiol Lung Cell Mol Physiol, December 1, 2004; 287(6): L1314 - L1322. [Abstract] [Full Text] [PDF] |
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N. L. Jernigan, B. R. Walker, and T. C. Resta Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L801 - L808. [Abstract] [Full Text] [PDF] |
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N. L. Jernigan, T. C. Resta, and B. R. Walker Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia Am J Physiol Lung Cell Mol Physiol, May 1, 2004; 286(5): L947 - L955. [Abstract] [Full Text] [PDF] |
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P. Minuz, P. Patrignani, S. Gaino, F. Seta, M. L. Capone, S. Tacconelli, M. Degan, G. Faccini, A. Fornasiero, G. Talamini, et al. Determinants of Platelet Activation in Human Essential Hypertension Hypertension, January 1, 2004; 43(1): 64 - 70. [Abstract] [Full Text] [PDF] |
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S. Wedgwood and S. M. Black Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L305 - L312. [Abstract] [Full Text] [PDF] |
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R. D. Bland, C. Y. Ling, K. H. Albertine, D. P. Carlton, A. J. MacRitchie, R. W. Day, and M. J. Dahl Pulmonary vascular dysfunction in preterm lambs with chronic lung disease Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L76 - L85. [Abstract] [Full Text] [PDF] |
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