Korean Circ J.  2011 Feb;41(2):83-90. 10.4070/kcj.2011.41.2.83.

Gene Expressions of Nitric Oxide Synthase and Matrix Metalloproteinase-2 in Monocrotaline-Induced Pulmonary Hypertension in Rats After Bosentan Treatment

• Koo HS
• Kim KC
• Hong YM

• KMID: 2225140
• DOI: http://doi.org/10.4070/kcj.2011.41.2.83

Abstract

BACKGROUND AND OBJECTIVES
Nitric oxide (NO) is a major endothelium dependent vasomediator and growth inhibitor. NO synthesis is catalyzed by endothelial nitric oxide synthase (eNOS), and NO can also produce peroxynitrite, which activates matrix metalloproteinases (MMPs). The purpose of this study was to determine the gene expression of eNOS and MMP-2 in the lungs of a rat model of pulmonary hypertension after bosentan treatment.
MATERIALS AND METHODS
Six-week-old male Sprague-Dawley rats were treated as follows: control group, subcutaneous (sc) injection of saline; monocrotaline (MCT) group, sc injection of MCT (60 mg/kg); and bosentan group, sc injection of MCT (60 mg/kg) plus 20 mg/day bosentan orally. The rats were sacrificed after 1, 5, 7, 14 and 28 days.
RESULTS
The right ventricle/(left ventricle+septum) ratio significantly increased in the MCT group compared to the control group on day 14 and 28. The expression of eNOS messenger ribonucleic acid was significantly increased in the MCT group compared to the control group on day 28. MMP-2 gene expression was significantly increased in the MCT-treated rats compared to the control group on day 5 and 28. Following bosentan treatment to reduce pulmonary hypertension, the expression levels of MMP-2 gene were significantly decreased on day 7 and 28. eNOS and tissue inhibitor of MMPs genes were also significantly decreased on day 28 after bosentan treatment.
CONCLUSION
These results suggest that elevated eNOS expression may be responsible for MMP-2 activation. The causal relationship between eNOS and MMP-2 and their role in pulmonary hypertension require further investigations.

Keyword

Pulmonary hypertension; Monocrotaline; Gene expression; Nitric oxide synthase; Matrix metalloproteinases

MeSH Terms

Animals
Endothelium
Gene Expression
Humans
Hypertension, Pulmonary
Lung
Male
Matrix Metalloproteinase 2
Matrix Metalloproteinases
Monocrotaline
Nitric Oxide
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Peroxynitrous Acid
Rats
Rats, Sprague-Dawley
RNA
Sulfonamides
Matrix Metalloproteinase 2
Matrix Metalloproteinases
Monocrotaline
Nitric Oxide
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Peroxynitrous Acid
RNA
Sulfonamides

Figure

• 

  Fig. 1 Typical example of RT-PCR products are shown for the level of inducible nitric oxide synthase endothelial nitric oxide synthase, matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinases messenger ribonucleic acid in lung tissue. The RT-PCR products from the transcripts of NOS2, NOS3, MMP2, TIMP and GAPDH were 107 bp, 140 bp, 137 bp, 133 bp and 89 bp. NOS 2: inducible nitric oxide synthase, NOS3: endothelial nitric oxide synthase, MMP2: matrix metalloproteinase 2,TIMP1: tissue inhibitor of matrix metalloproteinases, RT-PCR: reverse transcription-polymerase chain reaction.

• 

  Fig. 2 Gene expression of inducible nitric oxide synthase after bosentan treatment.

• 

  Fig. 3 Gene expression of endothelial nitric oxide synthase after bosentan treatment. *p<0.05 significantly different from control.

• 

  Fig. 4 Gene expression of matrix metalloproteinase 2 after bosentan treatment. *p<0.05 significantly different from control.

• 

  Fig. 5 Gene expression of tissue inhibitor of matrix metalloproteinases -1 after bosentan treatment. *p<0.05 significantly different from control.

Reference

1. Murata T, Sato K, Hori M, Ozaki H, Karaki H. Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension. J Biol Chem. 2002; 277:44085–44092. PMID: 12185080.

2. Everett AD, Le Cras TD, Xue C, Johns RA. eNOS expression is not altered in pulmonary vascular remodeling due to increased pulmonary blood flow. Am J Physiol. 1998; 274:L1058–L1065. PMID: 9609746.

3. 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. 2001; 358:1119–1123. PMID: 11597664.

4. Clozel M, Hess P, Rey M, Iglarz M, Binkert C, Qiu C. Bosentan, sildenafil, and their combination in the monocrotaline model of pulmonary hypertension in rats. Exp Biol Med (Maywood). 2006; 231:967–973. PMID: 16741032.

5. Frisdal E, Gest V, Vieillard-Baron A, et al. Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension. Eur Respir J. 2001; 18:838–845. PMID: 11757635.

6. Lepetit H, Eddahibi S, Fadel E, et al. Smooth muscle cell matrix metalloproteinases in idiopathic pulmonary arterial hypertension. Eur Respir J. 2005; 25:834–842. PMID: 15863640.

7. Morrell NW, Adnot S, Archer SL, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol. 2009; 54(1 Suppl):S20–S31. PMID: 19555855.

8. Lim KA, Shim JY, Cho SH, Kim KC, Han JJ, Hong YM. Effect of endothelin receptor blockade on monocrotaline-induced pulmonary hypertension in rats. Korean J Pediatr. 2009; 52:689–695.

9. Miyauchi T, Yorikane R, Sakai S, et al. Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension. Circ Res. 1993; 73:887–897. PMID: 8403258.

10. Itoh T, Nagaya N, Fujii T, et al. A combination of oral sildenafil and beraprost ameliorates pulmonary hypertension in rats. Am J Respir Crit Care Med. 2004; 169:34–38. PMID: 14525801.

11. Lim KA, Kim KC, Cho MS, Lee BR, Kim HS, Hong YM. Gene expression of endothelin-1 and endothelin receptor A on monocrotaline-induced pulmonary hypertension in rats after bosentan treatment. Korean Circ J. 2010; 40:459–464. PMID: 20967148.

12. Hampl V, Herget J. Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension. Physiol Rev. 2000; 80:1337–1372. PMID: 11015616.

13. 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. PMID: 7540722.

14. Komai H, Naito Y, Aimi Y, Kimura H. Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease. Cardiovasc Pathol. 2001; 10:29–32. PMID: 11343992.

15. Kim MY, Kim YK, Jung YW, Kim WT, Kwon TH, Lee DS. The protective effect of simvastatin on monocrotaline-induced pulmonary hypertension in rats. Korean Circ J. 2008; 38:313–319.

16. Hessel MH, Steendijk P, den Adel B, Schutte CI, van der Laarse A. Characterization of right ventricular function after monocrotaline-induced pulmonary hypertension in the intact rat. Am J Physiol Heart Circ Physiol. 2006; 291:H2424–H2430. PMID: 16731643.

17. Umar S, Hessel M, Steendijk P, et al. Activation of signaling molecules and matrix metalloproteinases in right ventricular myocardium of rats with pulmonary hypertension. Pathol Res Pract. 2007; 203:863–872. PMID: 17913382.

18. Tyler RC, Muramatsu M, Abman SH, et al. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol. 1999; 276:L297–L303. PMID: 9950892.

19. Resta TC, Gonzales RJ, Dail WG, Sanders TC, Walker BR. Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension. Am J Physiol. 1997; 272:H806–H813. PMID: 9124442.

20. Mathew R, Yuan N, Rosenfeld L, Gewitz MH, Kumar A. Effects of monocrotaline on endothelial nitric oxide synthase expression and sulfhydryl levels in rat lungs. Heart Dis. 2002; 4:152–158. PMID: 12028599.

21. Chou TF, Wu MS, Chien CT, Yu CC, Chen CF. Enhanced expression of nitric oxide synthase in the early stage after increased pulmonary blood flow in rats. Eur J Cardiothorac Surg. 2002; 21:331–336. PMID: 11825745.

22. Dai ZK, Tan MS, Chai CY, Chen IJ, Jeng AY, Wu JR. Effects of increased pulmonary flow on the expression of endothelial nitric oxide synthase and endothelin-1 in the rat. Clin Sci. 2002; 103(Suppl 48):289S–293S. PMID: 12193106.

23. 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:H1643–H1651. PMID: 9815072.

24. Xue C, Johns RA. Upregulation of nitric oxide synthase correlates temporally with onset of pulmonary vascular remodeling in the hypoxic rat. Hypertension. 1996; 28:743–753. PMID: 8901818.

25. Le Cras TD, Xue C, Rengasamy A, Johns RA. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol. 1996; 270:L164–L170. PMID: 8772540.

26. Resta TC, Chicoine LG, Omdahl JL, Walker BR. Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia. Am J Physiol. 1999; 276:H699–H708. PMID: 9950873.

27. Le Cras TD, McMurtry IF. Nitric oxide production in the hypoxic lung. Am J Physiol Lung Cell Mol Physiol. 2001; 280:L575–L582. PMID: 11237994.

28. Fagan KA, Morrissey B, Fouty BW, et al. Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension. Respir Res. 2001; 2:306–313. PMID: 11686901.

29. Mukhopadhyay S, Xu F, Sehgal PB. Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging. Am J Physiol Heart Circ Physiol. 2007; 292:H1373–H1389. PMID: 17071725.

30. Wang QD, Li XS, Pernow J. The nonpeptide endothelin receptor antagonist bosentan enhances myocardial recovery and endothelial function during reperfusion of the ischemic rat heart. J Cardiovasc Pharmacol. 1995; 26(Suppl 3):S445–S447. PMID: 8587441.

31. Winkler MK, Foldes JK, Bunn RC, Fowlkes JL. Implications for matrix metalloproteinases as modulators of pediatric lung disease. Am J Physiol Lung Cell Mol Physiol. 2003; 284:L557–L565. PMID: 12456387.