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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 447-450
Nitric oxide synthase 3 and endothelin 1 immunoreactivity in pulmonary hypertension

Department of Pathology, Seth G.S. Medical College & KEM Hospital, Mumbai, India

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Date of Web Publication22-Oct-2010


Context: Pulmonary hypertension (PH) is a serious and sometimes life-threatening event that occurs as a complication of various cardiopulmonary disorders, of which rheumatic heart disease (RHD) is an important example in our country. The pathogenesis of PH is a complex, multistep process in which "pulmonary endothelial dysfunction" (PED) is widely regarded as the central pathogenetic event. PED is, in turn, influenced by several local and systemic factors, of which nitric oxide synthase 3 (NOS3) and endothelin 1 (ET1) are 2 prime candidates, and are the subject of our study. Aims: Our aim was to study the immunoreactivity of NOS3 and ET1 in the pulmonary vasculature of PH patients of various etiologies, with emphasis on RHD cases. Settings and Design: A retrospective, autopsy-based study. Subjects and Methods: A total of 49 autopsy cases (39 patients and 10 controls) were chosen for our study. Of the 39 patients, 20 had PH secondary to RHD, whereas the remaining 19 patients had non-RHD etiologies as the basis of their PH. Lung sections taken from all the 49 cases were subjected to routine H and E, elastic van Gieson, and immunohistochemical staining (with NOS3 and ET1 separately). The intensity of immunostaining in all the cases and controls were then graded as focal/diffuse and weak/strong. Results: Controls showed positivity for both NOS3 (bronchiolar epithelium) and ET1 (endothelium of pulmonary arteries). Characteristic changes of PH on H and E were seen in 14 out of 19 non-RHD cases, which matched with the number of ET1 positivity cases. Similarly, for the RHD cases, 14 out of 20 cases showed changes of PH on H and E, but only 2 cases showed mild, focal positivity for ET1. Surprisingly, NOS3 positivity was largely absent in both the non-RHD and RHD cases. Conclusions: Our study showed NOS3 negativity and ET1 positivity in the lung vasculature of patients with PH, a conclusion more or less in line with the predominant view of the other investigators in this field. But at the same time, our study could not conclude an unequivocal role of NOS3 in PH, whereas it could, in the case of ET1.

Keywords: Endothelin 1, nitric oxide synthase 3, pulmonary hypertension, rheumatic heart disease

How to cite this article:
Gupta RK, Vaideeswar P. Nitric oxide synthase 3 and endothelin 1 immunoreactivity in pulmonary hypertension. Indian J Pathol Microbiol 2010;53:447-50

How to cite this URL:
Gupta RK, Vaideeswar P. Nitric oxide synthase 3 and endothelin 1 immunoreactivity in pulmonary hypertension. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Sep 26];53:447-50. Available from: https://www.ijpmonline.org/text.asp?2010/53/3/447/68270

   Introduction Top

Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure of more than 25 and 30 mmHg at rest and during exercise, respectively. [1] It is a serious and at times, a life-threatening complication that often develops secondary to various cardiopulmonary disorders. The pathogenesis of PH is a complex multistep process. It begins with "pulmonary endothelial dysfunction" (PED), which, depending on the severity of PH, ultimately culminates into various morphologically identifiable forms of vascular remodeling. [2],[3] PED is influenced by several local and systemic factors acting singly or in concert. Among these, endothelial nitric oxide synthase (eNOS or NOS3), and endothelin 1 (ET1) are widely believed to be the prime candidates responsible for the vascular changes of PH. [2],[3] Most of the studies have been directed toward primary PH or PH secondary to congenital heart disease. One of the major causes of PH in our country is rheumatic heart disease (RHD, particularly mitral stenosis) that produces varying degrees of venous PH. In this report, we have undertaken an autopsy-based study to document the intensity of eNOS and ET1 staining on immunohistochemistry in the lungs and have compared it with the staining pattern in other subsets of PH.

   Subjects and Methods Top

This is an autopsy-based retrospective study of the immunoreactivity of eNOS and ET1 in different subsets of PH. A total of 49 cases (39 patients and 10 controls) were chosen for the study. Of the 39 patients, 20 had PH (documented clinically and/or pathologically) secondary to RHD, while the remaining 19 patients had PH due to other causes (congenital left to right shunts in 10 patients, transposition of great arteries [TGA] in 4 patients, primary plexogenic arteriopathy [PPH] in 4 patients, and persistence of fetal circulation [PFC] in 1 patient). Ten age-matched controls without clinical or histopathologic evidence of PH were selected for comparison.

In all the 49 cases, 3-7 lung sections were taken from lungs that had been perfused with 10% buffered formalin. The sections were then processed and stained with hematoxylin and eosin (H and E) and elastic van Gieson. The presence and severity (graded subjectively as mild, moderate, and severe) of PH were assessed in all the sections. In each case, 1 section showing the maximum changes of PH on routine staining was selected for immunohistochemical (IHC) demonstration of eNOS (US Biologicals, Swamscott, MA, USA) and ET1 (US Biologicals, Swamscott, MA, USA) [Figure 1]. For the controls, a random block was selected for IHC staining. IHC was carried out using the labeled streptavidin biotin method, and the complex was visualized with the chromogen diaminobenzidine. The intensity of immunohistostaining was graded semiquantitatively as focal or diffuse and weak or strong. Positivity was also correlated with the internal controls as eNOS immunostaining in the bronchial epithelium or pneumocytes and ET1 immunostaining in smooth muscle cells of arterial walls, lung neuroendocrine cells, or some inflammatory cells. [4],[5],[6]
Figure 1 :(a) Small muscular artery showing endothelial nitric oxide synthase positi vity in the endothelial cells (immunohistochemistry [IHC], ×200); (b) endothelin-positi ve endothelial cells of an elasti c pulmonary artery (IHC, ×400) all the best

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   Results Top

In the controls, eNOS was largely positive in the bronchiolar epithelium, whereas endothelin 1 showed positivity in the endothelium of the pulmonary arteries. Among the 19 non-RHD cases [Table 1], the changes of PH were seen on H and E in 14 out of 19 cases. They were mild in 6 cases and moderate in 4. Two cases each of ASD and TGA did not show any histologic evidence of PH, and 1 was a case of PFC. Severe changes in the form of plexiform lesions were observed in 4 patients with PPH. The results of ET1 immunoreactivity showed mild, focal staining in about half of the cases (9 out of 19); moderate, focal staining in 1 case; strong, focal staining in 2 cases, and strong, diffuse staining in 3 cases. Four cases did not show any positivity for ET1 at all. The results of eNOS immunoreactivity for the same non-RHD cases were largely negative (18 out of 19 cases), with only 1 case of ASD showing mild, focal positivity.

In the RHD cases,14 out of 20 cases showed different grades of PH on H and E, with 8 cases showing mild changes, 3 cases showing moderate changes, and 2 cases showing severe changes (with plexiform changes in one of them). On IHC, all the 20 cases showed uniform negativity for eNOS immunoreactivity, and mild, focal positivity for ET1 in 2 cases [Table 2].
Table 1 :Comparison of H and E and IHC fi ndings in non-RHD cases

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Table 2 :Comparison of H and E and IHC fi ndings in RHD cases

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   Discussion Top

PH is a cardiopulmonary disorder with potential serious complications, affecting patients of all ages, right from neonates to the elderly. RHD, although rare in the Western world, is still quite a common cause of PH in the Southeast Asian countries, including India, acting as an important cause of morbidity and mortality in both pediatric and adult age groups. [7] The exact pathogenesis of PH is complex and open to debate with several theories abounding. However, there is little doubt that endothelial dysfunction (ED) plays a central unifying role in initiating and maintaining the early or advanced changes of pulmonary vascular remodeling, the histologic sine qua non of PH. Vasoactive factors, especially eNOS and ET1, are now being looked upon with great interest as 2 of the most potential candidates, [2] and were the subjects of this study.

The roles of eNOS and ET1 in PH are opposing; eNOS is a vasodilator and inhibitor of vascular smooth muscle cell (SMC) proliferation, whereas ET1 is a potent vasoconstrictor and mitogenic for vascular smooth muscle. [2],[3] Although few studies have been carried out in the West, [4],[6],[8],[9] to investigate the above line of pathogenesis in the lungs of PH patients, no such study, to our knowledge, has been done in India till date. We made an autopsy-based retrospective study of the intensity of immunoreactivity of eNOS and ET1 in the lung tissues of a cohort of 49 cases (39 PH patients and 10 controls) belonging to pediatric and adult age groups. We then tried to compare and contrast the grade of PH (based on H and E) with the intensity of staining of eNOS and ET1 in the lungs of these patient subsets.

In our results, we failed to show any eNOS reactivity in the lung vasculature of patients with PH, a finding more consistent with the study of Giaid et al[4] who have suggested a protective role of eNOS and have found normal or increased eNOS reactivity in control lungs and reduced or absent staining in the lungs of PH patients. Our study did not fall in line with the findings of those few investigators who proposed a prohypertensive role of eNOS in PH, [8],[9] that is, reduced or absent staining of eNOS in control lungs, and increased staining in PH lungs. Mason et al[9] in addition, have noted a distinct regional distribution of eNOS with reduced immunoreactivity in hypertensive arterioles but a high eNOS level in plexiform lesions.

However, our study could not clearly validate either a vasoprotective [4] (the predominant view) or a prohypertensive role[ 8],[9 ] (alternative, albeit minority view) of eNOS in cases of PH (in spite of showing eNOS positivity in the bronchiolar epithelium of controls). Whether the low levels of nitric oxide in PH lungs documented by many of the workers could be due to overexpression of arginase and not due to direct reduction of eNOS expression per se in the pulmonary vasculature (as pointed out by Xu et al[10] ) thus remains a novel possibility.

The reactivity of ET1, on the other hand, has been more consistent with the findings of other investigators. [6],[11],[12] We have got ET1 positivity of differing grades (mild, moderate, or strong) in 15 of a total of 19 cases in the various subsets of non-RHD-related PH. One significant observation was that in the 4 cases of PPH, although all the cases showed evidence of marked PH on H and E with 2 showing plexiform lesions, none of them showed any strong, diffuse or strong, focal reactivity for ET1, with the predominant reactivity being mild, focal. Thus, although overall our results for ET1 reactivity matched those of Western studies, we could not show any consistent positive correlation between the severity of vascular changes in PH and the intensity of ET1 staining, as documented by Western studies. [4],[6]

Another surprising observation was that although we saw differing grades of histologic changes of PH in the 3 subsets of RHD cases, with 2 cases showing marked changes of PH on H and E, we could not match it with concomitant positive staining for ET1 in any of the cases of RHD-related PH. Only 2 cases of mild PH (on H and E) among the 20 RHD cases showed mild, focal positivity for ET1, and the rest were all negative. The reason for this predominant absence of ET1 staining in the RHD-related PH cases, is unknown to us, and open to speculation. Till date, no similar study on RHD cases for eNOS and ET1 has been documented in Western literature or by Indian investigators. So, further research is needed in this direction, and whether a different pathway or a new molecular mediator (or both) is responsible for the development of PH in RHD is a question whose definite answer is yet to be known.

   References Top

1.Rich S, McLaughlin VV. Pulmonary hypertension. In: Zipes DP, Libby P, Bonow RO, Braunwald E, editors. Braunwald's Heart Disease. 7th ed. Elsevier Saunders; 2005. p. 1807-42.  Back to cited text no. 1      
2.Budhiraja R, Tuder RM, Hassoun PM. Endothelial dysfunction in pulmonary hypertension. Circulation 2004;109:159-65.   Back to cited text no. 2      
3.Said SI. Mediators and modulators of pulmonary arterial hypertension Am J Physiol Lung Cell Mol Physiol 2006;291:L547-58.  Back to cited text no. 3      
4.Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214-21.  Back to cited text no. 4      
5.Barberΰ JA, Peinado VI, Santos S, Ramirez J, Roca J, Rodriguez-Roisin R. Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med 2001;164:709-13.  Back to cited text no. 5      
6.Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, et al. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Eng J Med 1993;328:1732-39.  Back to cited text no. 6      
7.Deshpande J, Vaideeswar P, Amonkar G, Vasandani S. Rheumatic heart disease in the past decade: an autopsy analysis. Indian Heart J 2002;54:676-80.  Back to cited text no. 7      
8.Xue C, Johns RA. Endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:1642-44.  Back to cited text no. 8      
9.Mason NA, Springall DR, Burke M, Pollock J, Mikhail G, Yacoub MH, et al. High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension. J Pathol 1998;185:313-8.  Back to cited text no. 9      
10.Xu W, Kaneko FT, Zheng S, Comhair SA, Janocha AJ, Goggans T, et al. Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J 2004;18:1746-8.  Back to cited text no. 10      
11.Stelzner TJ, O'Brien RF, Yanagisawa M, Sakurai T, Sato K, Webb S, et al. Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 1992;262:L614-20.  Back to cited text no. 11      
12.Li H, Chen SJ, Chen YF, Meng QC, Durand J, Oparil S, et al. Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia J Appl Physiol 1994;77:1451-9.  Back to cited text no. 12      

Correspondence Address:
Pradeep Vaideeswar
Department of Pathology, Cardiovascular & Thoracic Division, Seth G. S. Medical College and KEM Hospital, Parel, Mumbai - 400 012
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Source of Support: The work has been supported by the Diamond Jubilee Society Trust and the Dr. P. K. Sen Research Society, Seth GS Medical College & KEM Hospital., Conflict of Interest: None

DOI: 10.4103/0377-4929.68270

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