Correspondence
Endothelial Nitric Oxide Synthase in the Lungs of Patients with Pulmonary Hypertension
N Engl J Med 1995; 333:1642-1644December 14, 1995
- Article
To the Editor:
In their paper in the July 27 issue,1 Giaid and Saleh demonstrate reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. Although these data are extremely provocative, we have several reservations about this study. First, the paper does not present a balanced view of the current literature on the role and expression of nitric oxide in normal lung and in pulmonary hypertension. For example, the authors state that “nitric oxide plays an important part in . . . maintaining low pressure in the normal pulmonary circuit.” This remains controversial. Nishiwaki et al.2 have demonstrated that nitric oxide is not important in controlling the low pulmonary vascular tone of conscious dogs, and we have demonstrated that in normal rats nitric oxide synthase is not expressed in small resistance vessels of the lung. There is also an evolving literature demonstrating that, in animal models of pulmonary hypertension, nitric oxide synthase is up-regulated rather than down-regulated.3,4
Furthermore, there are several technical problems with the way the study was performed. There is no demonstration of the specificity of the antibody used. Of more concern is the fact that there is no indication of which photomicrographs represent paraffin sections and which ones cryostat sections (both are said to have been used). This is critical information because the intensity of immunostaining is different with the two methods.
Although we have shown that inhaled nitric oxide prevents pulmonary hypertension and vascular remodeling in a model of chronic hypoxia,5 we have also shown that endothelial nitric oxide synthase is up-regulated in this model of pulmonary hypertension and is absent in small resistance vessels from normal lung.4 Indeed, in human lungs, we observed strong immunostaining of endothelial nitric oxide synthase in the endothelium of abnormally thickened large (data not shown), medium (Figure 1CFigure 1
Representative Tissue Samples Removed from Human Lungs during Surgical Procedures., Figure 1D, and Figure 1F), and small (Figure 1E) pulmonary arteries that showed typical characteristics of pulmonary hypertension. In contrast, normal human pulmonary vessels showed a lower degree of immunostaining of endothelial nitric oxide synthase (Figure 1A and Figure 1B).5 ReferencesChun Xue, M.D., Ph.D.
Roger A. Johns, M.D.
University of Virginia Health Sciences Center, Charlottesville, VA 229081
Giaid A ,Saleh D . Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214-221
Full Text | Web of Science | Medline2
Nishiwaki K ,Nyhan DP ,Rock P , et al. Nω-nitro-l-arginine and pulmonary vascular pressure-flow relationship in conscious dogs. Am J Physiol 1992;262:H1331-H1337
Web of Science | Medline3
Hampl V ,Archer SL ,Nelson DP ,Weir EK . Chronic EDRF inhibition and hypoxia: effects on pulmonary circulation and systemic blood pressure. J Appl Physiol 1993;75:1748-1757
Web of Science | Medline4
Le Cras TD ,Xue C ,Daily GC ,Johns RA . Endothelial NO synthase mRNA and protein, and inducible NO synthase mRNA are upregulated in the rat lung following chronic hypoxia. Am J Respir Crit Care Med 1995;151:Suppl:A729-A729 abstract.5
Roos CM, Frank DU, Xue C, Johns RA, Rich GF. Chronic inhaled nitric oxide: effects on pulmonary vascular endothelial function and pathology in hypoxic and normoxic rats. J Appl Physiol (in press).
Author/Editor Response
The authors reply:
To the Editor: In their letter, Xue and Johns state that our article did not present a balanced review of the literature on the role of nitric oxide in normal and diseased lungs. We are aware of their findings and those of others suggesting that, at least in animals, nitric oxide synthase is not important in maintaining low pulmonary vascular tone. There are other reports, however, as we stated in our paper, suggesting an important role for nitric oxide synthase in maintaining low vascular tone in humans. Our finding of an inverse correlation between pulmonary vascular resistance and endothelial nitric oxide synthase expression in patients with plexogenic pulmonary arteriopathy suggests a role for nitric oxide synthase in maintaining normal pulmonary vascular tone.
The finding that nitric oxide synthase is up-regulated in animal models of pulmonary hypertension may not be directly related to the pathologic role of nitric oxide synthase in pulmonary hypertension in humans. First, this finding is based on one form of pulmonary hypertension (i.e., hypoxia). Second, it is apparent that the cellular expression of nitric oxide synthase in the lungs of normal rats is different from that in normal humans. Xue and Johns have previously shown that nitric oxide synthase was absent in small resistance vessels from normal rat lungs. In contrast, we observed strong endothelial nitric oxide synthase expression in these vessels in normal human lungs. Indeed, Xue and Johns claim that they observed stronger immunostaining of nitric oxide synthase in the endothelium of abnormally thickened pulmonary arteries than in normal human pulmonary arteries. Their finding is based on studies of two patients with pulmonary hypertension secondary to chronic bronchitis and cystic fibrosis, with no mention of the severity of pulmonary hypertension in these patients.
In addition, we have several reservations about the photomicrographs they have included. The photomicrographs are shown at different magnifications and therefore do not allow an impartial comparison of the intensity of immunostaining between the normal and diseased arteries. Furthermore, the vessel indicated by arrows in Panels A and B resembles a venule rather than a small arteriole, as they claim. Panels C and D show an artery with intimal fibrosis and no evidence of muscular hypertrophy, which classifies it as grade 1 on the Heath and Edwards scale on which we based our histologic analysis. It is noteworthy that Figure 4 of our article shows the presence of a rather strong staining for nitric oxide synthase in pulmonary arteries with histologic abnormalities of grade 1. In Panel F of Xue and Johns's figure, it is very difficult to understand why the authors used eosin as a counterstain for the cytoplasm, where the brown color of immunostaining resides. Nuclear staining would have been a more appropriate technique to demonstrate the intensity of immunostaining of the mature protein in the cytoplasm of endothelial cells.
The specificity of the antiserum used in our study was demonstrated in Panel D of our Figure 1. Panels D and E of Figure 2 show paraffin sections. The remaining panels show cryostat sections. Finally, adjustment of the concentration of the antiserum samples results in similar intensities of immunostaining regardless of the embedding technique.
Adel Giaid, Ph.D.
Dina Saleh, B.Sc.
Montreal General Hospital, Montreal, QC H3G 1A4, Canada- Citing Articles (23)
Citing Articles
1
Jan F. Gielis, Judy Y. Lin, Kirstin Wingler, Paul E.Y. Van Schil, Harald H. Schmidt, An L. Moens. (2011) Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders. Free Radical Biology and Medicine 50:7, 765-776
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Guy Hagan, Joanna Pepke-Zaba. (2011) Pulmonary hypertension, nitric oxide and nitric oxide-releasing compounds. Expert Review of Respiratory Medicine 5:2, 163-171
CrossRef3
B. S. Zuckerbraun, P. George, M. T. Gladwin. (2011) Nitrite in pulmonary arterial hypertension: therapeutic avenues in the setting of dysregulated arginine/nitric oxide synthase signalling. Cardiovascular Research 89:3, 542-552
CrossRef4
Beena G. Sood, Susan Wykes, Marina Landa, Lilia De Jesus, Raja Rabah. (2011) Expression of eNOS in the lungs of neonates with pulmonary hypertension. Experimental and Molecular Pathology 90:1, 9-12
CrossRef5
Ivan M. Robbins, Anna R. Hemnes, J. Simon Gibbs, Brian W. Christman, Luke Howard, Sharon Meehan, Ines Cabrita, Rochelle Gonzalez, Tracy Oyler, Lan Zhao, Rui-Hong Du, Lisa A. Mendes, Martin R. Wilkins. (2011) Safety of sapropterin dihydrochloride (6r–bh4) in patients with pulmonary hypertension. Experimental Lung Research 37:1, 26-34
CrossRef6
Gerhard-Paul Diller, Thomas Thum, Martin R. Wilkins, John Wharton. (2010) Endothelial Progenitor Cells in Pulmonary Arterial Hypertension. Trends in Cardiovascular Medicine 20:1, 22-29
CrossRef7
N. J. Russell, P. J. Irwin, B. J. Hopper, T. Olivry, P. K. Nicholls. (2008) Acute necrotising pulmonary vasculitis and pulmonary hypertension in a juvenile dog. Journal of Small Animal Practice 49:7, 349-355
CrossRef8
Theresa A John, Basil O Ibe, J Usha Raj. (2007) REGULATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE: INVOLVEMENT OF PROTEIN KINASE G 1?, SERINE 116 PHOSPHORYLATION AND LIPID STRUCTURES. Clinical and Experimental Pharmacology and Physiology 0:0, 070924173348005-???
CrossRef9
Jian Yang, Jack Campitelli, Gang Hu, Yanhe Lin, Jin Luo, Chun Xue. (2007) Increase in DPP-IV in the intestine, liver and kidney of the rat treated with high fat diet and streptozotocin. Life Sciences 81:4, 272-279
CrossRef10
James R. Klinger. (2007) The Nitric Oxide/cGMP Signaling Pathway in Pulmonary Hypertension. Clinics in Chest Medicine 28:1, 143-167
CrossRef11
Rachel E. Nisbet, Roy L. Sutliff, C. Michael Hart. (2007) The Role of Peroxisome Proliferator-Activated Receptors in Pulmonary Vascular Disease. PPAR Research 2007, 1-10
CrossRef12
Kan Puikuan, Zhang Chunyu, Fan Jin, Tang Chaoshu, Du Junbao. (2006) Inhalation of nebulized nitroglycerin, a nitric oxide donor, for the treatment of pulmonary hypertension induced by high pulmonary blood flow. Heart and Vessels 21:3, 169-179
CrossRef13
Richard Kradin, Osamu Matsubara, Eugene J. Mark. (2005) Endothelial nitric oxide synthase expression in pulmonary capillary hemangiomatosis. Experimental and Molecular Pathology 79:3, 194-197
CrossRef14
Zen-Kong Dai, Mian-Shin Tan, Chee-Yin Chai, Jwu-Lai Yeh, Shah-Hwa Chou, Chaw-Chi Chiu, Arco Y. Jeng, Ing-Jun Chen, Jiunn-Ren Wu. (2004) Upregulation of endothelial nitric oxide synthase and endothelin-1 in pulmonary hypertension secondary to heart failure in aorta-banded rats. Pediatric Pulmonology 37:3, 249-256
CrossRef15
Pinar Yildiz, Huseyin Oflaz, Naci Cine, Nihan Erginel-Ünaltuna, Faruk Erzengin, Veysel Yilmaz. (2003) Gene polymorphisms of endothelial nitric oxide synthase enzyme associated with pulmonary hypertension in patients with COPD. Respiratory Medicine 97:12, 1282-1288
CrossRef16
Gilbert Blaise, David Langleben, Bernard Hubert. (2003) Pulmonary Arterial Hypertension. Anesthesiology 99:6, 1415-1432
CrossRef17
Rajamma Mathew, Ning Yuan, Louis Rosenfeld, Michael H Gewitz, Ashok Kumar. (2002) Effects of Monocrotaline on Endothelial Nitric Oxide Synthase Expression and Sulfhydryl Levels in Rat Lungs. Heart Disease 4:3, 152-158
CrossRef18
Graeme A Deuchar, Andrew Docherty, Margaret R MacLean, Martin N Hicks. (2002) Pulmonary hypertension secondary to left ventricular dysfunction: the role of nitric oxide and endothelin-1 in the control of pulmonary vascular tone. British Journal of Pharmacology 135:4, 1060-1068
CrossRef19
Hiroshi Ozaki, Hideaki Karaki. (2002) Organ Culture as a Useful Method for Studying the Biology of Blood Vessels and Other Smooth Muscle Tissues. The Japanese Journal of Pharmacology 89:2, 93-100
CrossRef20
Takahisa Murata, Hideyuki Yamawaki, Masatoshi Hori, Koichi Sato, Hiroshi Ozaki, Hideaki Karaki. (2001) Hypoxia impairs endothelium-dependent relaxation in organ cultured pulmonary artery. European Journal of Pharmacology 421:1, 45-53
CrossRef21
Karen A. Fagan, Brian W. Fouty, Robert C. Tyler, Kenneth G. Morris, Lisa K. Hepler, Koichi Sato, Timothy D. LeCras, Steven H. Abman, Howard D. Weinberger, Paul L. Huang, Ivan F. McMurtry, David M. Rodman. (1999) The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia. Journal of Clinical Investigation 103:2, 291-299
CrossRef22
MARIA ESTERLITA T. VILLANUEVA, FADI M. ZAHER, DAVID M. SVINARICH, G. GANESH KONDURI. (1998) Decreased Gene Expression of Endothelial Nitric Oxide Synthase in Newborns with Persistent Pulmonary Hypertension. Pediatric Research 44:3, 338-343
CrossRef23
Nicola A. Mason, David R. Springall, Margaret Burke, Jennifer Pollock, Ghada Mikhail, Magdi H. Yacoub, Julia M. Polak. (1998) High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension. The Journal of Pathology 185:3, 313-318
CrossRef
