Published online before print February 27, 2003, doi: 10.1161/01.RES.0000063424.28903.BB
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Increased Superoxide Generation Is Associated With Pulmonary Hypertension in Fetal Lambs
A Role for NADPH Oxidase
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
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Abstract |
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Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (P<0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (P<0.05). Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67phox, a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (P<0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (P<0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth.
Key Words: reactive oxygen species • vascular remodeling • smooth muscle
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Introduction |
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With initiation of ventilation and oxygenation at birth, pulmonary vascular resistance decreases and pulmonary blood flow increases.1 In some neonates, this normal transition does not occur, resulting in persistent pulmonary hypertension of the newborn (PPHN). PPHN can occur idiopathically or as a complication of a variety of cardiorespiratory disorders, including hyaline membrane disease, asphyxia, meconium aspiration, and congenital diaphragmatic hernia.2,3 In newborns that die with PPHN, there is an increase in both the thickness of the smooth muscle layer within small pulmonary arteries and an extension of this muscle to nonmuscular arteries.4 There is also proliferation of adventitial tissue.5 These structural changes indicate that in utero events have altered the pulmonary circulation. However, the mechanism underlying these changes is unclear. Surgical ligation or constriction of the ductus arteriosus in the fetal lamb several days before delivery at term results in PPHN after delivery.6–9 This lamb model closely resembles the human condition, with suprasystemic pulmonary arterial pressure and anatomic changes including muscularization of the normally nonmuscular intra-acinar arteries.
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.13–16 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.
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Materials and Methods |
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The Laboratory Animal Care Committee at the State University of New York at Buffalo and Northwestern University approved all protocols.
Prenatal Ligation of the Ductus Arteriosus
The technique for creating pulmonary hypertension by prenatal ligation of the ductus arteriosus has been described previously.9,17–19 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 peroxidase–linked 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 glycol–bound 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.
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Results |
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We measured superoxide levels in lung sections from control and PPHN lambs using the superoxide-sensitive dye dihyroethidium (Figure 1A). We found that there was a 2-fold increase in ethidium fluorescent intensity in PPHN sections compared with lung sections from control lambs after 9 days, but not after 2 days, of ductal ligation (Figure 1B; P<0.05).
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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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In the present study, we used a lamb model of PPHN to investigate the potential role of superoxide in the pathophysiology. We found a 2-fold increase in superoxide formation in lungs from PPHN lambs compared with controls after 9 days of ductal ligation (Figure 1B). Superoxide formation in the hypertensive lambs was evident in the SMCs and the adventitia of pulmonary arteries and arterioles, sites where the anatomic changes of vascular remodeling are most evident.6 PPHN is also associated with endothelial dysfunction, and endothelial cells have been shown to increase their generation of superoxide both from the "uncoupling" of endothelial NOS and from the activation of NADPH oxidase.21 Thus, it is possible that some of the increased superoxide we have observed in the ligated lung may be generated by the endothelium. Further studies will be required to examine this possibility.
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.6–8 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.
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Acknowledgments |
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This research was supported in part by grants HL60190 (to S.M.B.), HD398110 (to S.M.B.), HL67841 (to S.M.B.), HL70061 (to S.M.B.), HL072123 (to S.M.B.), and HL54705 (to R.H.S.) from the NIH, FY00-98 from the March of Dimes (to S.M.B.), and 0051409Z (to S.M.B.) from the American Heart Association, Midwest Affiliate. R.H.S. is an Established Investigator of the American Heart Association. The authors also gratefully acknowledge Sylvia Gugino for expert technical assistance in performing the isolated vessel experiments.
Received January 24, 2002; revision received June 5, 2002; accepted February 12, 2003.
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References |
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|---|
1. Fineman JR, Soifer SJ, Heymann MA. Regulation of pulmonary vascular tone in the perinatal period. Annu Rev Physiol. 1995; 57: 115–134.[CrossRef][Medline] [Order article via Infotrieve]
2. Graves EDIII, Redmond CR, Arensman RM. Persistent pulmonary hypertension in the neonate. Chest. 1988; 93: 638–641.
3. Steinhorn R, Millard S, Morrin FIII. Persistent pulmonary hypertension of the newborn: role of nitric oxide and endothelin in pathophysiology and treatment. Clin Perinatol. 1995; 22: 405–428.[Medline] [Order article via Infotrieve]
4. Haworth S, Reid L. Persistent fetal circulation: newly recognized structural features. J Pediatr. 1976; 88: 614–620.[CrossRef][Medline] [Order article via Infotrieve]
5. Mecham R, Whitehouse L, Wren D, Parks W, Griffin G, Senior R, Crouch E, V
lkel N, Stenmark K. Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension. Science. 1987; 237: 423–426.
6. Wild LM, Nickerson PA, Morin FCIII. Ligating the ductus arteriosus before birth remodels the pulmonary vasculature of the lamb. Pediatr Res. 1989; 25: 251–257.[Medline] [Order article via Infotrieve]
7. Morin FCIII. Ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb. Pediatr Res. 1989; 25: 245–250.[Medline] [Order article via Infotrieve]
8. Abman SH, Shanley PF, Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J Clin Invest. 1989; 83: 1849–1858.[Medline] [Order article via Infotrieve]
9. Black SM, Johengen MJ, Soifer SJ. Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs. Pediatr Res. 1998; 44: 821–830.[Medline] [Order article via Infotrieve]
10. Meier B, Radeke HH, Selle S, Younes M, Sies H, Resch K, Habermehl GG. Human fibroblasts release ROS in response to interleukin-1 or tumour necrosis factor-
. Biochem J. 1989; 263: 539–545.[Medline]
[Order article via Infotrieve]
11. Matsubara T, Ziff M. Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore. J Cell Physiol. 1986; 127: 207–210.[CrossRef][Medline] [Order article via Infotrieve]
12. Matsubara T, Ziff M. Increased superoxide anion release from human endothelial cells in response to cytokines. J Immunol. 1986; 137: 3295–3298.[Abstract]
13. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.
14. Marumo T, Schini-Kerth VB, Fisslthaler B, Busse R. Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-
B and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells. Circulation. 1997; 96: 2361–2367.
15. Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism underlying tolerance and cross-tolerance. J Clin Invest. 1995; 95: 187–194.[Medline] [Order article via Infotrieve]
16. Wedgwood S, Dettman R, Black S. ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species. Am J Physiol Lung Cell Mol Physiol. 2001; 281: L1058–L1067.
17. Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RH, Morin FCIII. Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol. 1997; 272: L1005–L1012.[Medline] [Order article via Infotrieve]
18. Steinhorn RH, Russell JA, Morin FCIII. Disruption of cGMP production in pulmonary arteries isolated from fetal lambs with pulmonary hypertension. Am J Physiol. 1995; 268: H1483–H1489.[Medline] [Order article via Infotrieve]
19. Steinhorn RH, Dukarm RC, Morin FC, Gugino SF, Russell JA. Dipyridamole enhances relaxations and cGMP generation to nitric oxide in lambs with persistent pulmonary hypertension. Pediatr Res. 1995; 39: 351a.Abstract.
20. Black SM, Fineman JR, Steinhorn RH, Bristow J, Soifer SJ. Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol. 1998; 275(5 pt 2): H1643–H1651.[Medline] [Order article via Infotrieve]
21. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.
22. Casareno RL, Waggoner D, Gitlin JD. The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase. J Biol Chem. 1998; 273: 23625–23628.
23. Rae TD, Torres AS, Pufahl RA, O’Halloran TV. Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS. J Biol Chem. 2001; 276: 5166–5176.
24. Rypniewski WR, Mangani S, Bruni B, Orioli PL, Casati M, Wilson KS. Crystal structure of reduced bovine erythrocyte superoxide dismutase at 1.9 A resolution. J Mol Biol. 1995; 251: 282–296.[CrossRef][Medline] [Order article via Infotrieve]
25. Marshall C, Mamary AJ, Verhoeven AJ, Marshall BE. Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol. 1996; 15: 633–644.[Abstract]
26. Nunoi H, Rotrosen D, Gallin JI, Malech HL. Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science. 1988; 242: 1298–1301.
27. Volpp BD, Nauseef WM, Clark RA. Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science. 1988; 242: 1295–1297.
28. Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991; 353: 668–670.[CrossRef][Medline] [Order article via Infotrieve]
29. Knaus UG, Heyworth PG, Kinsella BT, Curnutte JT, Bokoch GM. Purification and characterization of Rac 2: a cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem. 1992; 267: 23575–23582.
30. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ. Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest. 1987; 80: 732–742.[Medline] [Order article via Infotrieve]
31. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
32. Galle J, Heinloth A, Wanner C, Heermeier K. Dual effect of oxidized LDL on cell cycle in human endothelial cells through oxidative stress. Kidney Int. 2001; 78: S120–S123.[CrossRef]
33. Galle J, Schneider R, Heinloth A, Wanner C, Galle PR, Conzelmann E, Dimmeler S, Heermeier K. Lp(a) and LDL induce apoptosis in human endothelial cells and in rabbit aorta: role of oxidative stress. Kidney Int. 1999; 55: 1450–1461.[CrossRef][Medline] [Order article via Infotrieve]
34. Meyer JW, Holland JA, Ziegler LM, Chang MM, Beebe G, Schmitt ME. Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells: a potential atherogenic source of reactive oxygen species. Endothelium. 1999; 7: 11–22.[Medline] [Order article via Infotrieve]
35. Cifuentes ME, Rey FE, Carretero OA, Pagano PJ. Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice. Am J Physiol Heart Circ Physiol. 2000; 279: H2234–H2240.
36. Jones RD, Hancock JT, Morice AH. NADPH oxidase: a universal oxygen sensor? Free Radic Biol Med. 2000; 29: 416–424.[CrossRef][Medline] [Order article via Infotrieve]
37. Rosenberg AA, Kennaugh J, Koppenhafer SL, Loomis M, Chatfield BA, Abman SH. Elevated immunoreactive endothelin-1 levels in newborn infants with persistent pulmonary hypertension. J Pediatr. 1993; 123: 109–114.[CrossRef][Medline] [Order article via Infotrieve]
38. Brown M, Miller F, Li W, Ellingson A, Mozena J, Chatterjee P, Engelhardt J, Zwacka R, Oberley L, Fang X, Spector A, Weintraub N. Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells. Circ Res. 1999; 85: 524–533.
39. Tsai J, Jain M, Hsieh C, Lee W, Yoshizumi M, Patterson C, Perralla M, Cook C, Wang H, Haber E, Schlegal R, Lee M. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. J Biol Chem. 1996; 271: 3667–3670.
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  K. E. Dennis, J. L. Aschner, D. Milatovic, J. W. Schmidt, M. Aschner, M. R. Kaplowitz, Y. Zhang, and C. D. Fike NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets Am J Physiol Lung Cell Mol Physiol, October 1, 2009; 297(4): L596 - L607. [Abstract] [Full Text] [PDF]
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S. de Frutos, C. H. Nitta, E. Caldwell, J. Friedman, and L. V. Gonzalez Bosc Regulation of soluble guanylyl cyclase-{alpha}1 expression in chronic hypoxia-induced pulmonary hypertension: role of NFATc3 and HuR Am J Physiol Lung Cell Mol Physiol, September 1, 2009; 297(3): L475 - L486. [Abstract] [Full Text] [PDF]
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 V. G. DeMarco, J. Habibi, A. T. Whaley-Connell, R. I. Schneider, J. R. Sowers, B. T. Andresen, A. A. Gutweiler, L. Ma, M. S. Johnson, C. M. Ferrario, et al. Rosuvastatin ameliorates the development of pulmonary arterial hypertension in the transgenic (mRen2)27 rat Am J Physiol Heart Circ Physiol, September 1, 2009; 297(3): H1128 - H1139. [Abstract] [Full Text] [PDF]
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M. Chester, P. Tourneux, G. Seedorf, T. R. Grover, J. Gien, and S. H. Abman Cinaciguat, a soluble guanylate cyclase activator, causes potent and sustained pulmonary vasodilation in the ovine fetus Am J Physiol Lung Cell Mol Physiol, August 1, 2009; 297(2): L318 - L325. [Abstract] [Full Text] [PDF]
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R.-J. Teng, A. Eis, I. Bakhutashvili, N. Arul, and G. G. Konduri Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, July 1, 2009; 297(1): L184 - L195. [Abstract] [Full Text] [PDF]
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S. Ismail, A. Sturrock, P. Wu, B. Cahill, K. Norman, T. Huecksteadt, K. Sanders, T. Kennedy, and J. Hoidal NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-{beta}1 and insulin-like growth factor binding protein-3 Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L489 - L499. [Abstract] [Full Text] [PDF]
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 X. Wang, H. Chai, P. H. Lin, Q. Yao, and C. Chen Roles and Mechanisms of Human Immunodeficiency Virus Protease Inhibitor Ritonavir and Other Anti-Human Immunodeficiency Virus Drugs in Endothelial Dysfunction of Porcine Pulmonary Arteries and Human Pulmonary Artery Endothelial Cells Am. J. Pathol., March 1, 2009; 174(3): 771 - 781. [Abstract] [Full Text] [PDF]
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K. N. Farrow, S. Lakshminrusimha, W. J. Reda, S. Wedgwood, L. Czech, S. F. Gugino, J. M. Davis, J. A. Russell, and R. H. Steinhorn Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, December 1, 2008; 295(6): L979 - L987. [Abstract] [Full Text] [PDF]
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J. G. Lopez-Lopez, J. Moral-Sanz, G. Frazziano, M. J. Gomez-Villalobos, J. Flores-Hernandez, E. Monjaraz, A. Cogolludo, and F. Perez-Vizcaino Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L727 - L732. [Abstract] [Full Text] [PDF]
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C. D. Fike, J. C. Slaughter, M. R. Kaplowitz, Y. Zhang, and J. L. Aschner Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L881 - L888. [Abstract] [Full Text] [PDF]
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S. Kumar, X. Sun, S. Wedgwood, and S. M. Black Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity Am J Physiol Lung Cell Mol Physiol, August 1, 2008; 295(2): L370 - L377. [Abstract] [Full Text] [PDF]
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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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