J Korean Med Sci.  2012 Dec;27(12):1507-1516. 10.3346/jkms.2012.27.12.1507.

Effect of Small Hairpin RNA Targeting Endothelin-Converting Enzyme-1 in Monocrotaline-Induced Pulmonary Hypertensive Rats

• Son JS
• Kim KC
• Kim BK
• Cho MS
• Hong YM

• KMID: 2157974
• DOI: http://doi.org/10.3346/jkms.2012.27.12.1507

Abstract

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.

Keyword

Hypertension, Pulmonary; Monocrotaline; Small Hairpin RNA; Lentivirus; Endothelin-Converting Enzyme; RNA, Small Interfering

MeSH Terms

Animals
Aspartic Acid Endopeptidases/*antagonists & inhibitors/blood/genetics
Body Weight
Heart Ventricles/physiopathology
Hypertension, Pulmonary/chemically induced/*enzymology/mortality
Lentivirus/genetics
Lung/anatomy & histology/metabolism/pathology
Male
Metalloendopeptidases/*antagonists & inhibitors/blood/genetics
Monocrotaline/toxicity
Pulmonary Artery/drug effects/physiopathology
RNA, Small Interfering/*metabolism
Rats
Rats, Sprague-Dawley
Receptor, Endothelin A/genetics/metabolism
Survival Rate
Tumor Necrosis Factor-alpha/metabolism
Vascular Endothelial Growth Factor A/metabolism
RNA, Small Interfering
Receptor, Endothelin A
Tumor Necrosis Factor-alpha
Vascular Endothelial Growth Factor A
Monocrotaline
Aspartic Acid Endopeptidases
Metalloendopeptidases

Figure

• 

  Fig. 1 Medial wall thickening of pulmonary arterioles in three groups (H&E staining, × 200). Medial layer of pulmonary muscular arteries is progressively thickened after MCT injection. The shRNA group shows an improved medial thickening on days 14 and 28 compared to the MCT group. MCT, monocrotaline; shRNA, small hairpin RNA.

• 

  Fig. 2 Green fluorescent protein expression in the lung endothelium tranduced with shRNA-encoding lentivirus. Two groups transduced with lentivirus shows uniform endothelial expression of GFP, demonstrating a lentiviral transduction is efficient and well preserved throughout whole experiment (magnification × 400). MCT, monocrotaline; shRNA, small hairpin RNA.

• 

  Fig. 3 Relative expression levels of ECE-1 (A), ET-1 (B) and ETA (C) mRNA in the lung tissues in weeks 2 and 4. The expression levels on ECE-1, ET-1, and ETA mRNA in the lung tissues are significantly increased in the MCT group compared with the control group in weeks 2 and 4. The expression levels of ET-1 and ETA mRNA are significantly decreased in the shRNA group compared with the MCT group in week 4. *P < 0.05 compared with the control group; †P < 0.05 compared with the MCT group. ECE, endothelin-converting enzyme; ET, endothelin; ETA, endothelin receptor type A; C, control group; M, MCT group; shRNA, small hairpin RNA.

• 

  Fig. 4 Protein contents of ECE-1 (A), ET-1 (B), and ETA (C) in the lung tissues in weeks 2 or 4. The protein levels on ECE-1, ET-1 and ETA in the lung tisssues are significantly increased in the MCT group compared with the control group in weeks 2 and 4. The protein levels of ETA are significantly decreased in the shRNA group compared with the MCT group in week 2. *P < 0.05 compared with the control group; †P < 0.05 compared with the MCT group. ECE, endothelin-converting enzyme; ET, endothelin; ETA, endothelin receptor A; C, control group; M, MCT group; shRNA, small hairpin RNA.

• 

  Fig. 5 Protein contents of TNF-α (A) and VEGF (B) in the lung tissues in weeks 2 and 4. The protein levels of TNF-α and VEGF in the lung tissues are significantly increased in the MCT group compared with the control group in weeks 2 and 4. The protein levels of TNF-α and VEGF are significantly decreased in the shRNA group compared with the MCT group in week 4. *P < 0.05 compared with the control group; †P < 0.05 compared with the MCT group. TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; C, control group; M, MCT group; shRNA, small hairpin RNA.

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