It is generally accepted that an imbalance of the vascular effectors causes vasoconstriction, vascular smooth muscle cell proliferation, and thrombosis in pulmonary arterial hypertension (PAH).1,2 Recent studies demonstrate that patients with severe PAH show diminished expression of lung endothelial nitric oxide (NO)3 and prostacyclin4 as well as overexpression of endothelin-1.5 Although prostacyclin and endothelin receptor antagonists are suggested as first line of therapeutic strategy of PAH,6-9 there are still several potential limitations in these modalities, including side effects of drugs, complexity of administration methods, and high medical costs, and the development of additional novel therapeutic approaches focusing on the components of multifactorial pulmonary vascular pathobiology is necessary.2,10-16
Nicorandil is an adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener with a nitrate-like action. This agent dilates peripheral and coronary resistance arterioles as well as systemic veins and epicardial coronary arteries through an increase in intracellular cyclic guanosine monophosphate (cGMP) in vascular smooth muscle cells, hyperpolarization of the smooth muscle membrane due to increased potassium conductance, and calcium antagonistic action.17,18 Nicorandil also acts to exert cardioprotective effects on ischemic myocardium probably due to its ability to mimic the phenomenon of ischemic preconditioning by opening of the KATP channel.19,20 Recently some investigators have revealed that nicorandil increases endothelial NO synthase (eNOS) in normal rat heart.21 Although accumulating evidence suggests that nicorandil shows a number of beneficial cardiovascular actions in both clinical and experimental settings, its effect on PAH has not yet been clarified.
The present study was designed to test our hypothesis that nicorandil might enhance eNOS expression in the lung tissue and prevent development of monocrotaline (MCT)-induced PAH in rats. We also investigated whether or not nicorandil could exert regressive effects on pathobiology of established PAH once it had been induced by MCT.
Male Sprague-Dawley rats (aged 4 weeks, weighing 100-120 g) were injected with 50 mg/kg of MCT subcutaneously and randomized to either 1 of the 3 doses of nicorandil (5 mg/kg/d, 7.5 mg/kg/d, or 10 mg/kg/d in drinking water) (nicorandil group) or placebo (placebo group) for 3 weeks. The normal male Sprague-Dawley rats (aged 4 weeks, weighing 100-120 g) served as controls (normal control group). One hundred five rats subjected to MCT (26 in the 5 mg/kg/d of nicorandil group, 26 in the 7.5 mg/kg/d of nicorandil group, 25 in the 10 mg/kg/d of nicorandil group, and 28 in the placebo group) and 25 normal control animals were observed for 3 weeks to examine survival rate. Another group of male Sprague-Dawley rats (aged 4 weeks, weighing 100-120 g) was treated with 7.5 mg/kg/d of nicorandil in drinking water in combination with 5 mg/kg/d of glibenclamide by gastric gavage for 3 weeks (nicorandil + glibenclamide group, n = 24). Thirteen rats in the placebo group, 11 in the 5 mg/kg/d of nicorandil group, 13 in the 7.5 mg/kg/d of nicorandil group, 12 in the 10 mg/kg/d of nicorandil group, 13 in the nicorandil + glibenclamide group, and 12 normal control animals underwent measurement of the pulmonary arterial pressure, blood pressure, and heart rate and histologic, biochemical, immunohistochemical, and Western blot analyses of the lung tissue. The doses of nicorandil and glibenclamide in the present study were determined according to those in the previous study.22
Ninety male Sprague-Dawley rats (aged 4 weeks, weighing 100-120 g) injected with 50 mg/kg of MCT subcutaneously and survived for 3 weeks were randomly assigned to either nicorandil (7.5 mg/kg/d in drinking water) (n = 45) or placebo (n = 45) for the next 3 weeks. Animals survived for 6 weeks also had measurement of the pulmonary arterial pressure, blood pressure, and heart rate and histologic, biochemical, immunohistochemical, and Western blot analyses of the lung tissue.
The experimental protocol was approved by the Animal Subjects Committee of Shinshu University School of Medicine and the investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO.85-23, revised 1996). Nicorandil was kindly provided by Chugai Pharmaceutical, Co Ltd (Tokyo, Japan) and glibenclamide was from Sigma (St. Louis, MO).
Physiologic, Morphologic, and Biochemical Analyses
After treatment, blood pressure, and heart rate were measured in the conscious state by using the tail-cuff method with a sphygmomanometer (Riken Development, Co Ltd, Tokyo, Japan). Each value was the average of at least 3 consecutive data. Rats were then anesthetized with 60 mg/kg sodium pentobarbital given intraperitoneally, and pulmonary arterial pressure was measured with a fluid-filled catheter inserted into the pulmonary artery via the right jugular vein. Rats were euthanized and wet weights of the individual cardiac chambers and lungs were measured. Plasma level of rat brain natriuretic peptide (BNP) was measured by using sensitive and specific radioimmunoassay kits (Peninsula Laboratories, San Carlos, CA) as previously described.23 Serum level of NOx (NO2 + NO3) was then determined by gas chromatography.23
Histologic and Immunohistochemical Analyses
Lung tissue sections were prepared as previously described elsewhere23,24 and stained with elastic hematoxylin-eosin. Morphometric analysis was performed on muscular arteries with external diameter in the ranges of 50 to 99 μm and 100 to 200 μm, using a color digital camera mounted on a computer-interfaced light microscope. Medial wall thickness was measured as the distance between the external and internal elastic laminae of each artery, and external diameter was measured as the diameter of the external lamina, using a calibrated eyepiece micrometer. The percent medial wall thickness of each artery was calculated by using the following formula as previously described23,25:
For each rat, 10 to 15 vessels were counted and the average was calculated. The remaining tissue sections of the lung were subjected to immunostaining with antibodies against P-selectin (Santa Cruz Biotechnology, Santa Cruz, CA) and proliferating cell nuclear antigen (PCNA) (Dako, Inc., Osaka, Japan). A Vectastain ABC Kit (Vector Laboratories, Inc) was then used with the following primary MAbs: MAb S789G and MAb OX-1 (mouse IgG1, PharMingen), which reacts with leukocyte common antigen CD45 on all hematopoietic cells except erythrocytes. The average numbers of CD45-positive cells and those of PCNA-positive cells were calculated to identify leukocyte recruitment and proliferative activity in the lung tissue in 100 arbitrary fields under ×160 magnification.
Western Blot Analysis
Proteins were prepared from the lung tissue. Immunoblotting was performed by means of monoclonal antibodies to eNOS (Transduction Laboratories, Lexington, KY). The eNOS protein was detected using the enhanced chemiluminescence immunoblotting detection kit (Amersham Life Science, Piscataway, NJ). Densitometric analysis was performed and the protein levels in each specimen were expressed relative to those of the normal control animals.23
Data are presented as mean ± SEM. Analysis of variance with Bonferroni's multiple comparison test was used to analyze the differences among the groups. Survival data were analyzed by Kaplan-Meier method with log-rank test and χ2 analysis. A probability value of <0.05 was accepted as statistically significant.
Effects on Survival
During the follow-up of 3 weeks, 42 of the 105 animals subjected to MCT died, including 9 in the 5 mg/kg/d of nicorandil group, 7 in the 7.5 mg/kg/d of nicorandil group, 9 in the 10 mg/kg/d of nicorandil group, and the remaining 17 in the placebo group. Thus, the survival rate at 3 weeks after treatment was significantly greater in the 7.5 mg/kg/d of nicorandil-treated group than in the placebo group (73% versus 39%, P < 0.05). The survival rates in the 5 mg/kg/d of nicorandil and in the 10 mg/kg/d of nicorandil groups were 65% and 64%, respectively. In the nicorandil + glibenclamide group, 15 of the 24 rats died, resulting in 38% of the survival rate (Fig. 1). All 57 animals died of severe right heart failure with massive pericardial and pleural effusions and ascites.
Physiologic and Morphologic Data
Mean blood pressure and heart rate in the awake condition were not different among the groups. Mean pulmonary arterial pressure was significantly elevated in the placebo and nicorandil + glibenclamide groups compared with the nicorandil-treated and normal control groups. In the nicorandil-treated animals, 7.5 mg/kg/d of the nicorandil-treated group showed maximal effects. There were no significant differences in the mean pulmonary arterial pressure among the normal controls and nicorandil-treated groups. The ratio of the right ventricular weight to the weight of left ventricle plus septum was significantly increased in the placebo and nicorandil + glibenclamide groups compared with the normal control animals. The nicorandil-treated groups showed intermediate values for the ratio among the other 3 groups (Table 1).
Histologic, Immunohistochemical, Western Blot, and Biochemical Analyses
Compared with the normal control animals, marked medial hypertrophic and hyperplastic responses in the medium- to small-sized pulmonary arteries were evident in the placebo group 3 weeks after MCT injection. These features were markedly suppressed in the nicorandil-treated groups. In contrast, glibenclamide in combination with nicorandil completely suppressed improvement of pulmonary vascular remodeling (Table 1).
eNOS protein level in the lung tissue was significantly decreased in the placebo group compared with the normal control animals, and it was prevented after nicorandil (P < 0.001) (Fig. 2). P-selectin was intensely expressed on the endothelium of the pulmonary arteries in the placebo group. This was markedly attenuated in the nicorandil-treated groups (Fig. 3). The numbers of the CD45-positive cells and those of the PCNA-positive cells in the lung tissue were significantly increased in the placebo group compared with the normal control group, and the increases in these cells were significantly prevented after 3 weeks of nicorandil (Figs. 3 and 4). However, there were still significant differences in the number of these cells between the normal control and nicorandil-treated groups (Fig. 4). In the placebo group, most of the CD45-positive cells were found to be mononuclear leukocytes and infiltrated throughout the lung tissue. Nuclear staining with PCNA antibody was observed in mononuclear cells and spindle-shaped nuclear cells around the alveolar walls and small pulmonary vessels (Fig. 3). These immunohistochemical and Western blot findings induced by nicorandil were completely suppressed by glibenclamide (Figs. 2-4).
Plasma level of BNP was markedly increased in the placebo group compared with the normal control group, and the increase was significantly suppressed after nicorandil. Glibenclamide prevented a decrease in plasma level of BNP by nicorandil (Table 1). Serum NOx concentration was significantly reduced in the placebo group compared with the normal control animals, but there were no significant changes after nicorandil and after glibenclamide in combination with nicorandil (Table 1).
Twelve (6 with nicorandil-treated animals and 6 with placebo-treated animals) of the 90 rats survived for 6 weeks. Mean pulmonary arterial pressure, cardiac weights, and the extent of pulmonary vascular remodeling were not different between the 2 groups (Table 2). There were also no significant differences in the results of biochemical, immunohistochemical, and Western blot analyses of the lung tissue between the groups (Table 2 and Fig. 5).
To our knowledge, this is the first study to assess treatment effects of nicorandil on experimental PAH induced by MCT. The novel findings of the present study were:
- Nicorandil, a KATP channel opener, just after MCT injection, suppressed development of severe PAH with pulmonary vascular structural remodeling and prolonged survival in rats.
- These effects were associated with up-regulation of extremely diminished expression of the lung eNOS protein along with marked improvement of pulmonary vascular endothelial activation and anti-inflammatory and anti-proliferative effects in the lung tissue.
- A KATP channel blocker, glibenclamide, reversed these beneficial effects induced by nicorandil.
- Late treatment with nicorandil did not palliate PAH nor improved survival in this model.
Effects of Nicorandil on Development of Monocrotaline-Induced Pulmonary Arterial Hypertension
Nicorandil is the first clinically available KATP channel opener and has been shown to possess an anti-anginal effect and safety profile comparable to those of nitrates, β-blockers, and calcium antagonists.26,27 In the clinical setting, nicorandil is widely used for the management of ischemic heart disease without causing nitrate tolerance.28 In a recent, large clinical trial, named the Impact Of Nicorandil in Angina (IONA) study, nicorandil caused a significant improvement in outcome owing to decreased major coronary events for patients with stable angina.29 Although the precise mechanisms for the beneficial effects of nicorandil remains uncertain in the IONA study, cardioprotective effects mimicking the phenomenon of the powerful ischemic preconditioning are thought to be the most plausible explanation.19,20 In a rat model of acute myocardial infarction, nicorandil has been shown to protect against fatal ischemic ventricular arrhythmias with enhancement of myocardial eNOS expression via activation of the KATP channel, which is the most prominent in the endothelium of the coronary arteries of the non-ischemic area.30
In hypertensive pulmonary vascular disease, the most characteristic pathologic changes involve medial hypertrophy, intimal proliferation, and thrombosis,31,32 accounting for occlusion of small pulmonary arteries. When rats are injected with MCT, a hepatic metabolite of this agent, monocrotaline pyrrole, causes damage of pulmonary arterial endothelial cells,33,34 which is followed by an inflammatory response and medial hypertrophy, leading to subsequent vascular occlusion by smooth muscle cells. Thus, the pathogenesis of PAH induced by MCT is not the same as that of atherosclerosis in coronary and brain arteries.
In the present study, 3 doses of nicorandil (5, 7.5, and 10 mg/kg/d in drinking water) for 3 weeks immediately after MCT markedly suppressed development of PAH and prolonged survival. This was accompanied by improvement of severe structural changes of the pulmonary arteries, right ventricular hypertrophy, right-sided heart failure, and elevated plasma BNP levels. The maximal effects were achieved with 7.5 mg/kg/d of nicorandil. Extremely diminished expression of eNOS protein in the lung tissue of the placebo group was dramatically up-regulated after nicorandil without significant changes in systemic hemodynamics. In addition, strong expression of P-selectin on the endothelium of the pulmonary arteries seen in the placebo group was largely attenuated after nicorandil, suggesting that nicorandil suppressed platelet activation on the endothelial cells in experimental PAH induced by MCT. We observed that in the placebo group, the recruitment of CD45-positive leukocytes, which were characterized by inflammatory leukocytes, and of PCNA-positive leukocytes was markedly enhanced and that this was inhibited by nicorandil. These findings suggest anti-inflammatory and anti-proliferative properties of nicorandil in the lung tissue of MCT-treated rats. No significant changes in systemic hemodynamics after nicorandil may imply relatively selective pulmonary vasodilation by this agent in the model. The beneficial effects of nicorandil were completely suppressed by a KATP channel blocker, glibenclamide, indicating that the effects were mediated by the KATP channel.
It is generally recognized that NO mediates vasodilation mainly through activation of guanylate cyclase and an increase in cGMP, inhibits platelet activation and vascular smooth muscle cell proliferation, and suppresses leukocyte adhesion to the vascular endothelial cells.15,35 On the basis of the results of the present study, marked enhancement of NO production in the lung tissue by opening of KATP channel may contribute to the beneficial effects of nicorandil in the setting of MCT-induced PAH. This may account for marked improvement of pulmonary vascular endothelial activation and inflammatory and proliferative lesions in the lung tissue of the model.
Effects of Nicorandil on Established Pulmonary Arterial Hypertension
In the clinical setting, most human subjects have already developed a relatively full-blown pathologic process by the time PAH is discovered, and clinically apparent PAH is an end-stage phenotype. It has been reported that serine elastase inhibitors can reverse advanced pulmonary vascular disease in MCT-treated rats.25 In a recent study, Nishimura et al36 found dramatic reversal of the established PAH with a 100% survival after simvastatin in a rat model PAH produced by MCT and pneumonectomy. They observed that simvastatin reduced proliferation and increased apoptosis of pathologic smooth muscle cells in the neointima and medial walls of pulmonary arteries along with down-regulation of the inflammatory genes.37 More recently, Abe et al37 demonstrated that long-term inhibition of Rho-kinase with fasudil improved survival and regressed pulmonary vascular lesions even after development of MCT-induced PAH in rats. Thus, it would be of great interest to evaluate whether nicorandil could reverse the pathobiology of PAH once it had been induced by MCT. Unfortunately, our results indicate that oral nicorandil has no regressive effects on established PAH and pulmonary vascular remodeling. Failure to maintain eNOS may be the reason for the lack of regression of PAH in the model.
Limitations and Clinical Implications
Recent advances in our understanding of pathobiology of PAH have facilitated the evolution of newer therapeutic options. Although nicorandil given just after MCT injection showed beneficial pulmonary vascular protective effects in a rat model of PAH, there were several limitations in the present study. First, this agent could not rescue the advanced disease nor completely inhibit development of pathobiology of MCT-induced PAH. Second, it should be noted that NO-modulating property is not specific for nicorandil because other drugs, such as statins, angiotensin-converting enzyme inhibitors, and phosphodiesterase type 5 inhibitors, also exert increased NO-producing effects.9,38 Finally, to our knowledge, whether nicorandil could increase blood flow in the lungs remains uncertain. So, we could offer no definite conclusion on the relation between the up-regulation of eNOS and pulmonary blood flow in the present study.
Nicorandil would be promising for treatment of patients with less advanced disease, such as idiopathic PAH, PAH associated with connective tissue diseases, and chronic pulmonary thromboembolism, in which inflammatory mechanisms with endothelial dysfunction may contribute to the development and progression of PAH. The combined use of drugs with different mechanisms of action is also an emerging option for the clinical benefit. Thus, large clinical trials are necessary to provide convincing evidence of efficacy and safety of nicorandil either solely or adjunctively in the management of PAH.
1. Farber HW, Loscalzo JL. Pulmonary arterial hypertension
. N Engl J Med
2. Galie N, Torbicki A, Barst R, et al. Guidelines on diagnosis and treatment of pulmonary arterial hypertension
: The Task Force on Diagnosis and Treatment of Pulmonary Arterial Hypertension
of the European Society of Cardiology. Eur Heart J
3. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide
synthase in the lungs of patients with pulmonary hypertension. N Engl J Med
4. Tuder RM, Cool CD, Gearci MW, et al. Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension. Am J Respir Crit Care Med
5. Giaid A, Yanagisawa M, Langleben D, et al. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med
6. McLaughlin VV, Genthner DE, Panella MM, et al. Reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension. N Engl J Med
7. Channick RN, Simonneau G, Sitbon O, et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo- controlled study. Lancet
8. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan in patients with pulmonary artery hypertension: a randomized, placebo controlled, multicenter study. N Engl J Med
9. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension
. N Engl J Med
10. Jeffery TK, Morrell NW. Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Prog Cardiovasc Dis
11. Hoeper MM, Galie N, Simonneau G, et al. New treatments for pulmonary arterial hypertension
. Am J Respir Crit Care Med
12. Tuder RM, Zaiman AL. Prostacyclin analogs as the brakes for pulmonary artery smooth muscle cell proliferation: Is it sufficient to treat severe pulmonary hypertension? Am J Respir Cell Mol Biol
13. Galie N, Manes A, Branzi A. Emerging medical therapies for pulmonary arterial hypertension
. Prog Cardiovasc Dis
14. Sharma S. Treatment of pulmonary arterial hypertension
: a step forward. Chest
15. Budhiraja R, Tuder RM, Hassoun PM. Endothelial dysfunction in pulmonary hypertension. Circulation
16. Humbert M, Morreli NW, Archer SL, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension
. J Am Coll Cardiol
17. Hongo M, Takenaka H, Uchikawa S, et al. Coronary microvascular response to intracoronary administration of nicorandil
. Am J Cardiol
18. Akai K, Wang Y, Sato K, et al. Vasodilatory effect of nicorandil
on coronary arterial microvessels: its dependency on vessel size and the involvement of the ATP-sensitive potassium channels. J Cardiovasc Pharmacol
19. Patel DJ, Purcell HJ, Fox KM, on behalf of the CESAR 2 investigation. Cardioprotection by opening of the KATP
channel in unstable angina: Is this a clinical manifestation of myocardial preconditioning? Eur Heart J
20. Sato T, Sasaki N, O'Rourke B, et al. Nicorandil
, a potent cardioprotective agent, acts by opening mitochondrial ATP-dependent potassium channels. J Am Coll Cardiol
21. Horinaka S, Kobayashi N, Higashi T, et al. Nicorandil
enhances cardiac endothelial nitric oxide
synthase expression via activation of adenosine triphosphate-sensitive K channel in rat. J Cardiovasc Pharmacol
22. Sanada H, Node K, Asanuma H, et al. Opening of the adenosine triphosphate-sensitive potassium channel attenuates cardiac remodeling induced by long-term inhibition of nitric oxide
synthesis. J Am Coll Cardiol
23. Hironaka E, Hongo M, Sakai A, et al. Serotonin receptor antagonist inhibits monocrotaline-induced pulmonary hypertension and prolongs survival in rats. Cardiovasc Res
24. Hongo M, Ryoke T, Schoenfeld J, et al. Effects of growth hormone on cardiac dysfunction and gene expression in genetic murine dilated cardiomyopathy. Basic Res Cardiol
25. Cowan KN, Heilbut A, Humpl T, et al. Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med
26. Doring G. Antianginal and anti-ischemic efficacy of nicorandil
in comparison with isosorbide-5-mononitrate and isosorbide dinitrate: results from two multicenter, double-blind, randomized studies with stable coronary heart disease patients. J Cardiovasc Pharmacol
. 1992;20(Suppl 3):S74-S81.
27. Di Somma S, Liguori V, Verdecchia P, et al. A double-blind comparison of nicorandil
and metoprolol in patients with effort stable angina. Cardiovasc Drugs Ther
28. Taira N. Nicorandil
as a hybrid between nitrates and potassium channel activators. Am J Cardiol
29. The IONA Study Group. Effect of nicorandil
on coronary events in patients with stable angina: the Impact Of Nicorandil
in Angina (IONA) randomized trial. Lancet
30. Horinaka S, Kobayashi N, Yabe A, et al. Nicorandil
protects against lethal ischemic ventricular arrhythmias and up-regulates endothelial nitric oxide
synthase expression and sulfonylurea receptor 2 mRNA in conscious rats with acute myocardial infarction. Cardiovasc Drugs Ther
31. Fishman AP, Fishman MC, Freeman BA, et al. Mechanisms of proliferative and obliterative vascular diseases: insights from the pulmonary and systemic circulations. NHLBI Workshop Summary. Am J Respir Crit Care Med
32. Yi ES, Kim H, Ahn H, et al. Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension: a morphometric and immunohistochemical study. Am J Respir Crit Care Med
33. Wilson DW, Segall HJ, Pan LC, et al. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit Rev Toxicol
34. Lame MW, Jones AD, Wilson DW, et al. Protein targets of monocrotaline pyrrole in pulmonary artery endothelial cells. J Biol Chem
35. Ghofrani HA, Pepke-Zaba J, Barbera JA, et al. Nitric oxide
pathway and phosphodiesterase inhibitors in pulmonary arterial hypertension
. J Am Coll Cardiol
36. Nishimura T, Vaszar LT, Faul JL, et al. Simvastatin rescues rats from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle cells. Circulation
37. Abe K, Shimokawa H, Morikawa K, et al. Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats. Circ Res
38. Rakhit RD, Marber MS. Nitric oxide
: an emerging role in cardioprotection? Heart
Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
nicorandil; nitric oxide; pulmonary arterial hypertension