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Published ahead of print on December 30, 2003, doi:10.1164/rccm.200301-147OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 764-769, (2004)
© 2004 American Thoracic Society

Original Article

Oxidative Stress in Severe Pulmonary Hypertension

Rebecca Bowers, Carlyne Cool, Robert C. Murphy, Rubin M. Tuder, Matthew W. Hopken, Sonia C. Flores and Norbert F. Voelkel

Pulmonary Hypertension Center, University of Colorado Health Sciences Center, National Jewish Medical and Research Center, Denver, Colorado; and Cardiopulmonary Section, Department of Pathology, Johns Hopkins University, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Severe pulmonary hypertension (PH) occurs in a primary or "unexplained" form and in a group of secondary forms associated with a number of diseases. Because the lung tissue from patients with severe PH demonstrates complex vascular lesions, which contain inflammatory cells, we wondered whether the lung tissue from patients with severe PH was "under oxidative stress." We used immunohistochemistry to localize nitrotyrosine and 8-hydroxy guanosine in the lung tissue sections from patients with primary and secondary PH. In some lung tissue extracts, the eicosanoid metabolites 5-oxo-eicosatetraenoic acid, leukotriene B4 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 15-HETE were measured using mass spectroscopy, and superoxide dismutase amount and activity were measured. Nitrotyrosine expression was ubiquitous in all PH lungs, and 5-oxo-eicosatetraenoic acid and HETE levels were elevated in the lungs of patients with severe PH but not in those lungs that were from the patients with severe PH treated chronically with prostacyclin. We conclude that indeed the lungs from patients with severe PH are under oxidative stress and that chronic prostacyclin infusion has an antiinflammatory effect on the lung tissue.

Key Words: nitrotyrosine • superoxide dismutase • 5-oxo-eicosatetraenoic acid • pulmonary hypertension

Microarray gene expression profiling of lung tissue samples from patients with primary pulmonary hypertension (PPH) (1) and analysis of their lung lavage samples (2) suggest that the lung tissue of these patients undergoes oxidative stress. For example, our gene expression survey showed increased expression of the antiapoptotic thioredoxin, which plays a role in the redox regulation of transcription factors such as nuclear factor-{kappa}B (3, 4) and increased expression of metallothionein in the tissues from patients with sporadic PPH (1), suggesting that these tissues are responding to oxidative stress by upregulating their antioxidant defenses.

Oxidative stress is characterized by increased production of oxidants (e.g., superoxide, hydrogen peroxide, nitric oxide [NO]) and/or decreased concentrations of antioxidants and antioxidant enzymes. Oxidants may damage tissues either by direct oxidation of key biological molecules (i.e., lipid peroxidation, DNA damage) or by alteration of transcription factors such as nuclear factor-{kappa}B, Sp1, or AP-1 (5, 6). Reactive oxygen species could be produced in the lung tissue of patients with severe pulmonary hypertension (PH) as a consequence of tissue hypoxia (6, 7) or ischemia (8) or through the activation of inflammatory cascades and increased production of inflammatory cytokines (9, 10). Increased oxidant stress—if present in the lungs from patients with severe PH—could activate/inactivate transcriptional factors, alter the normal cell gene expression profile (11), and affect phosphorylation of critical enzymes (12). Foremost among the lines of biological defense against the superoxide radical are the superoxide dismutases (SODs). The manganese-containing enzyme (Mn-SOD), localized to the mitochondrial matrix, is tightly regulated by cytokines and cellular stress and rapidly inactivated by nitration.

Further support for increased oxidative stress in the lungs from PH patients could also be the presence of arachidonic acid–derived metabolites (13). However, whether simpler free radical products of the reactions of arachidonate with one molecule of oxygen, such as the hydroxyeicosatetraenoic acids (HETEs) and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) (14, 15), are also present in severe PH is unknown. The latter compound is of interest because it can be a product of both enzymatic and free radical events and is sufficiently lipophilic that it tenaciously remains associated with lipid membranes.

To determine whether lungs from patients with severe PH (16, 17), either primary or secondary, are under increased oxidant stress, we assessed biochemical markers such as 8-hydroxy guanosine (8-HG), nitrotyrosine, and 5-oxo-ETE (18–20). Furthermore, we examined lung tissue protein levels and activity of the enzyme Mn-SOD. Our results suggest that the oxidant/antioxidant balance is altered in the lungs from patients with severe PH (21).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lung Tissue Source
Details are discussed in the online supplement.

8-HG Immune Histochemistry
Immunohistochemical detection of 8-HG.
Paraffin-embedded sections were processed; the primary antibody mouse-anti-8-HG (QED Bioscience, San Diego, CA) was applied at 1:250 or 1:1,000 dilution, and negative control consisted of isotype-matched control serum (19). Biotinylated universal secondary antibody and Elite ABC reagent were applied at room temperature for 30 minutes each. After washing with Tris-buffered saline with 0.05% tween 20 (Sigma, St. Louis, MO), Vector RED substrate was used as chromogen.

Nitrotyrosine Immune Histochemistry
All tissues were in 10% formaldehyde and were processed and embedded in paraffin. Five micron sections were cut, stained with hematoxylin and eosin, and immunostained for nitrotyrosine using a monoclonal antibody (Upstate Biotechnology, Lake Placid, NY) in a dilution of 1:500. The sections were deparaffinized, dried overnight, and decloaked in citrate buffer for 10 minutes. After washing in distilled water, the Ventana ES automatic immunostaining device was used for the immunohistochemical staining. Incubations with unrelated antibodies were used as control for the previously mentioned methods.

5-oxo-ETE Measurement
Details are discussed in the online supplement.

SOD Western Blot
Details are discussed in the online supplement.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
8-HG Staining
Lung tissue from three patients with PPH and three patients with histologically normal lung structure were stained to immunolocalize 8-HG. Intense red staining was observed in the plexiform lesions and of the lumenal endothelial cells of concentric intima fibrosis lesions of all three patients, whereas in normal lung tissue, only occasional macrophages stained red (Figure 1) .



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  		Figure 1. Representative 8-hydroxy guanosine (8-HG) expression (red staining) detected by immunohistochemistry. (A) Plexiform lesion (PL) of a primary pulmonary hypertension (PPH) lung demonstrating strong expression of 8-HG in endothelial cells (arrow) forming the core of the lesion and the poorly organized, incipient vascular channels (inset). Perivascular macrophages (arrowhead) show intense 8-HG staining. (B) Concentric lesion (CO) of a PPH lung showing abundant 8-HG expression in lumenal endothelial cells (arrow), whereas endothelial and smooth muscle cells embedded in the subintimal area do not express detectable 8-HG. (C) Endothelial cells of a normal pulmonary artery do not show 8-HG (arrowheads). Alveolar macrophages show intense 8-HG expression (arrows). V = vessel. (A–C) Immunohistochemistry (x200).

 
Nitrotyrosine Staining
Lung tissue samples from 10 patients with sporadic primary PH were examined immunohistochemically, and areas staining positive for nitrotyrosine were found in all of the hypertensive lungs. Figures 2A–2F show representative examples of lung tissue sections. Tissue from all patients with PPH showed ubiquitous nitrotyrosine staining of the parenchyma and of vascular walls, as did the tissue from three patients with secondary PH. This intense tissue staining was not observed in normal lung tissue (n = 3) or in tissue from patients with emphysema (n = 3) (Figures 2C and 2E). In addition, nitrotyrosine expression was also observed in 18 of 25 plexiform lesions (Figures 2B and 2D). Two blinded observers (R.M.T. and C.C.) could not distinguish between lungs from patients treated with continuous intravenous prostacyclin infusion and those from patients not treated with prostacyclin.



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  		Figure 2. Nitrotyrosine expression (brown staining) detected by immunohistochemistry. (A) Low magnification of vascular structures in a lung from a patient with PPH (x100). There is abundant expression in the smooth muscle of the vessel walls (arrows). The adjacent bronchiolar smooth muscle (arrowheads) also shows abundant expression, as does much of the alveolated lung parenchyma. (B) PL from a patient with PPH (x200). There is prominent positive staining (arrows) in the cells that surround the core of the lesion. By comparison, many of the core cells of the PL are negative for nitrotyrosine expression. (C) Compared with A, this low-power magnification (x100) of lung from a patient with emphysema shows relative absence of nitrotyrosine expression. The small pulmonary artery (arrow) is negative, as is the surrounding lung parenchyma. (D) High-power magnification (x400) of a PL from a patient with pulmonary hypertension (PH) secondary to an atrial–septal defect demonstrates marked nitrotyrosine expression in the endothelial cells that line the multiple lumens of this PL (arrows). There is background staining of the red blood cells within the lumens (arrowheads). Similar to B, the more centrally located cells (*) of the PL are relatively negative for nitrotyrosine expression. (E) This low-power magnification (x100) of a normal lung shows lack of nitrotyrosine staining, similar to the emphysematous lung in C. (F) High-power magnification of a muscularized pulmonary artery in a patient with PH secondary to an atrial–septal defect (x400). Note the intense positive staining of the endothelial cell layer (arrow) and many of the smooth muscle cells of the media (arrowheads).

 
Mass Spectroscopy
Basal levels of 5-oxo-ETE found in the lung tissue from nonhypertensive patients (Figure 3A ; patients 1–4) averaged 10.4 ± 2.9 (SEM) pg/mg lung tissue. In tissue samples isolated from patients with emphysema, the level of 5-oxo-ETE was not significantly altered (8.7 ± 2.1 pg/mg tissue). However, 5-oxo-ETE production was substantially higher in patients that had severe PH (28.7 ± 9.9 pg/mg tissue in PPH and 21.9 ± 4.0 pg/mg tissue in secondary PH). Of interest, the levels of 5-oxo-ETE were prominently decreased in patients who had been treated with continuous intravenous prostacyclin infusion (n = 5) (3.8 ± 1.0 pg/mg lung tissue).



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  		Figure 3. Amounts of 5-oxo-eicosatetraenoic acid (5-oxo-ETE) (A), 5-hydroxyeicosatetraenoic acid (5-HETE) (B), and leukotriene B4 (C) in human lung samples were determined by quantitative mass spectrometry. Lung tissue from a group of 28 subjects was analyzed. Tissues from patients treated with chronic intravenous prostacyclin infusion had statistically different (p < 0.05) amounts of 5-HETE when compared with the tissues from PPH and secondary PH not treated with prostacyclin. Tissues from patients with secondary severe PH had statistically greater amounts of 5-oxo-ETE when compared with those PPH patients treated with prostacyclin (p = 0.003). PGI2 = prostacyclin.

 
There was an increase in the average quantity of 5-HETE measured in the tissue samples from patients with primary and secondary PH. However, these average values were greatly affected by a few individuals found to have very high levels. As shown in Figure 3B, 5-HETE levels in nonhypertensive patients averaged 28.5 ± 13.4 pg/mg tissue as compared with primary pulmonary hypertensive patient levels that averaged 92.6 ± 32.4 pg/mg lung tissue (n = 8) and secondary PH levels that averaged 121.6 ± 42.0 pg/mg tissue (n = 7). The amount of leukotriene B4 found (Figure 3C) averaged 5.3 ± 1.5 in nonhypertensive patients and was not significantly higher in primary hypertensive patients or secondary hypertensive patient levels of leukotriene B4, which averaged 7.2 ± 1.3 and 9.3 ± 3.5 pg/mg tissue, respectively. Prostacyclin treatment of patients with primary PH resulted in a significant decrease of both 5-HETE (7.8 ± 3.1 pg/mg tissue) and leukotriene B4 (1.2 ± 0.1 pg/mg tissue).

Various other HETEs were analyzed in five additional lung tissues as shown (see Figure E2 in the online supplement). The levels of all of the HETEs were increased in the tissue from the two PPH patients not treated chronically with prostacyclin, but not in the tissues from the two PPH patients treated with prostacyclin. Notably, 12-HETE and 15-HETE, which are 12-lipoxygenase and 15-lipoxygenase products, followed similar patterns as the 5-lipoxygenase products.

Mn-SOD in Human Lungs
To examine whether levels of Mn-SOD protein or activity were altered in these lungs, a combination of immunoblot analyses or enzymatic activity assays was used. Figure 4A shows that PPH lungs have decreased total SOD activity and decreased amounts of immunoreactive Mn-SOD protein (see Figures E4 and E3 of the online supplement) but not of Cu,Zn-SOD (Figure E4) when compared with normal lung tissue. Native SOD activity gels confirmed that the decrease in total SOD could be attributed to decreased Mn-SOD with little change in Cu,Zn-SOD (Figure E4 of the online supplement).



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  		Figure 4. Superoxide dismutase (SOD) levels in human lung homogenates. (Top) Total SOD activity: lung tissues from either normal (n = 5) or patients with PPH (n = 3) were homogenized, cleared by centrifugation, and the total SOD activity in the soluble protein fraction determined spectrophotometrically by the ferricytochrome c reduction method of Fridovich and McCord. The total units of activity in the crude homogenate were normalized to total soluble protein. Note that this assay does not distinguish between the cytosolic (Cu,Zn-SOD) or mitochondrial (Mn-SOD) isoenzymes. (Results are expressed as mean ± SEM, *p < 0.05, Student's t test). (Bottom) Densitometric quantification of the SOD immunoreactivity in human lung samples. Images from the samples shown in Figure E2 of the online supplement were acquired with a Kodak DC290 digital camera and intensities of each sample quantified by densitometry with Kodak 1D image analysis software. The relative intensity for each band was expressed as a ratio of the relative intensity of the actin signal, used as an internal control. The numbers represent the average ± SEM of the ratio of Mn-SOD/actin (see Figure E3) or Cu,ZnSOD/actin (see Figure E4). *p < 0.03, Student's t test.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we combined immunohistochemistry, protein measurements, and mass spectroscopy to examine lung tissue samples from patients with severe PH. The data obtained with these different techniques support our hypothesis that the lung tissue from the patients with severe PH is undergoing oxidant stress, as we found increased expression of nitrotyrosine and 8-HG, decreased amounts of Mn-SOD protein, decreased Mn-SOD activity, and increased amounts of a number of arachidonic acid metabolites in the lung tissues of patients with severe PH.

Using antibodies directed against 8-HG, we demonstrated that cells stained positively in lungs from patients with severe PH, indicating that reactive oxygen species had been generated in these lungs. Conversion of guanine to 8-HG is a frequent result of reactive oxygen species and has been shown to affect the methylation of cytosines, providing a link between oxidative stress and DNA damage. Superoxide anion attacks guanine, and 8-HG has often been measured as an index of oxidative stress-induced DNA damage (22, 23).

Nitrotyrosine was ubiquitously and highly expressed in severe PH also at sites distant from inflammatory cell clusters and in pulmonary arteries regardless of whether the patients had had chronic prostacyclin infusion treatment. The cause of this expression could be endothelial cell and vascular smooth muscle cell peroxynitrite formation after NO and superoxide anion generation by these cells (24, 25); alternatively, nitrotyrosine expression may result from myeloperoxidase activation of neutrophils attached to the endothelium (26, 27). Clearly, in normal human lung tissue, nitrotyrosine staining was not observed, and less pronounced staining was observed in lung tissue from patients with emphysema (Figure 2).

In this context, it is of interest that recent studies reported diminished NO levels in exhaled air samples collected from patients with PPH (28, 29). Whereas decreased NO production is one possible explanation, either because of impaired NO synthase activity or substrate deficiency, another possibility is that the NO is continually consumed. Such consumption could occur by the interaction between NO and the superoxide anion resulting in peroxynitrite formation (25, 30, 31). We propose that the impressive nitrotyrosine expression in lung tissue from patients with PPH and with secondary forms of severe PH may be the result of NO consumption via a peroxynitrite generating chemical reaction. NO consumption in PPH was considered by Kaneko and colleagues (2) but was felt to be less likely than underproduction, perhaps because their study showed that the total SOD activity in the bronchoalveolar lavage fluid from patients with PPH was not reduced.

We did find that the amount and the activity of Mn-SOD were reduced in the lung tissue samples from the patients with PPH when compared with normal lung tissue extracts. Thus, both overproduction of superoxide anion and possibly impaired enzymatic removal of the oxidant could occur in the PH lungs. In this context, it is of interest that administration of recombinant SOD in an animal model of PH acutely reduced the pulmonary vascular resistance (32).

Using a direct analytical approach, we attempted to obtain evidence for oxidative stress by mass-spectroscopic analysis of frozen PH lung tissue extracts. Recently, isoprostane species have been identified as markers of oxidative stress (33, 34), and Cracowski and colleagues (35) reported elevated urinary isoprostane levels in patients with PH. Isoprostanes could not be detected by us in the analyzed extracts of stored frozen tissues; we speculate that isoprostanes, if present in the tissues, are very volatile and likely did not survive tissue handling, freezing, and rethawing. Rather unexpectedly, an increase in 5-oxo-ETE (Figure 3) and an increase in several HETEs (Figure 3) (see online supplement) were observed.

HETEs could be formed either by free radical reactions occurring within the lipid bilayer, by enzymatic reactions, or by a combination of both. There is also a connection between an increased hydroperoxide tone (one product of free radical oxidation of polyunsaturated lipids) and the activity of all of the known mammalian lipoxygenases (36). An increase in hydroperoxide tone as a result of free radical events could accelerate the production of eicosanoids generated by enzymatic pathways, and thus, increased amounts of HETEs could provide indirect evidence of oxidative stress. Platelet or macrophage 12-HETE, derived from action of 12-lipoxygenase on arachidonic acid, was one likely source of 12-HETE (37). Similarly, 15-HETE possibly resulted from airway epithelial cells that express 15-lipoxygenase (38).

Although 5-oxo-ETE is an enzymatic (5-lipoxygenase) product, 5-oxo-ETE can also be formed through free radical oxidation of arachidonic acid at carbon-5 (14). 5-oxo-ETE has profound biological activity as a chemotactic factor for eosinophils and neutrophils (39) and increases cell surface expression of the ß2-integrin CD11B, actin polymerization, as well as cell adherence (40). Possibly 5-oxo-ETE may promote granulocyte migration through basement membranes and therefore play an important role in lung inflammation. Irrespective of which mechanism is involved in the 5-oxo-ETE production, it is remarkable that the amount of this eicosanoid was decreased in those patients treated chronically with prostacyclin infusion. The decreased levels of eicosanoids, including 5-oxo-ETE levels and several of the HETEs in the lungs from prostacyclin-treated hypertensive patients, as compared with no treatment, suggest that prostacyclin decreases inflammation or oxidative stress or both in the lung tissues of treated PH patients.

We conclude that the lung tissue from patients with severe PH is under oxidant stress and suggest that peroxynitrite as well as several HETEs and 5-oxo-ETE are being formed in the lung tissue. Formation of peroxynitrite in the lung tissue, rather than diminished NO production, may explain the reduced exhaled NO concentration (28, 29) in patients with PPH. Whether ischemia/reperfusion or inflammation, or both, contribute to the lung tissue oxidative stress in severe PH presently remains unclear.


   Acknowledgments
 
The authors thank Professor Dr. Mark Gillespie, University of South Alabama, Mobile, Alabama, for stimulating discussions, and Kathy Wood for great help with the nitrotyrosine staining and manuscript preparation.


   FOOTNOTES
 
Supported in part by grants from the National Institutes of Health (HL36577).

Correspondence and requests for reprints should be addressed to Norbert F. Voelkel, M.D., Division of Pulmonary Sciences and Critical Care Medicine, Pulmonary Hypertension Center, 4200 East Ninth Avenue, C272, Denver, CO 80262. E-mail: norbert.voelkel{at}uchsc.edu

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: R.B. has no declared conflict of interest; C.C. has no declared conflict of interest; R.C.M. received $6,000 per annum for consulting with Cayman Chemical; R.M.T. has no declared conflict of interest; M.W.H. has no declared conflict of interest; S.C.F. has no declared conflict of interest; N.F.V. has no declared conflict of interest.

Received in original form January 31, 2003; accepted in final form December 22, 2003


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