J Korean Soc Hypertens.  2012 Sep;18(3):105-116.

Effect of Small Hairpin RNA Molecules Targeting Angiotensin-converting Enzyme Gene in Spontaneously Hypertensive Rats

• Hong YM
• Lee HR
• Kim KC

Abstract

BACKGROUND
Interfering RNA (iRNA) represents a recent breakthrough in effective blocking of the target genes in mammalian cells. Angiotensin-converting enzyme (ACE) has been shown to play an important role in the pathogenesis of hypertension. The purposes of this study were to investigate the effects on blood pressure, myocardial hypertrophy and gene expressions of iRNA targeting ACE.
METHODS
Twelve week old male Wistar-Kyoto rats were grouped as follows: control group (C group), spontaneously hypertensive rat (SHR) group (H group), and ACE-iRNA group (A group) in which SHR was treated with recombinant lentiviral vectors carrying small hairpin RNA targeting ACE. Reverse transcription-polymerase chain reaction and western blot analysis of ACE, endothelin (ET)-1, angiotensin (AT) II receptor type 1A, neutrophil cytosolic factor, caspase 3, Bax, and Bcl-2 were performed in the heart tissues. Serum AT, ACE, and high sensitive-C reactive protein were estimated.
RESULTS
Systolic blood pressure was significantly decreased in the A group compared with the H group in weeks 3 and 5. Serum AT level was significantly lower on day 1, weeks 3 and 5 after ACE-iRNA treatment. ACE protein contents were significantly lower after ACE-iRNA treatment in week 5. ET-1 and Bcl-2 protein contents were significantly lower after ACE-iRNA treatment in weeks 3 and 5. Bax protein contents were significantly lower after ACE-iRNA treatment in week 3.
CONCLUSIONS
Recombinant lentiviral vectors carrying shRNA targeting ACE prevented hypertension. Serum AT and gene expressions such as ACE, ET-1, Bax, and Bcl-2 were significantly decreased after ACE-iRNA treatment.

Keyword

Hypertension; Small hairpin RNA; Lentivirus; RNA interference

MeSH Terms

Angiotensins
Animals
bcl-2-Associated X Protein
Blood Pressure
Blotting, Western
Caspase 3
Cytosol
Endothelins
Gene Expression
Heart
Humans
Hypertension
Hypertrophy
Lentivirus
Lifting
Male
Neutrophils
Rats
Rats, Inbred SHR
RNA
RNA Interference
RNA, Small Interfering
Angiotensins
Caspase 3
Endothelins
RNA
RNA, Small Interfering
bcl-2-Associated X Protein

Figure

• 

  Fig. 1 Construction of shRNA-expression lentiviral vector for targeting rat angiotensin-converting enzyme (ACE). The shRNA-expression lentiviral vector for targeting rat ACE was constructed by inserting synthetic double strand oligonucleotides. CMV, cytomegalovirus; cPPT, central polypurine tract; hCMV, human cytomegalovirus; GFP, green fluorescent protein; IRES, internal ribosome entry site.

• 

  Fig. 2 Systolic blood pressure after angiotensin-converting enzyme (ACE)-interfering RNA (iRNA) treatment. Systolic blood pressure was significantly decreased in the A group compared with the H group in weeks 3 and 5. C, control; H, hypertension; A, ACE-iRNA group. *p < 0.05 significantly different C group vs. H group. †p < 0.05 significantly different H group vs. A group.

• 

  Fig. 3 Change of collagen by Masson's Trichrome staining after angiotensin-converting enzyme (ACE)-interfering RNA (iRNA) treatment (×200). (A, B) Collagen penetration in the H group was significantly higher than that in the C group. (C) There was no statistically significant difference between the H and the A group. C, control; H, hypertension; A, ACE-iRNA group.

• 

  Fig. 4 Angiotensin-converting enzyme (ACE) and endothelin (ET)-1 protein content were significantly higher in the H group compared with that in the C group in weeks 3 and 5. (A) ACE protein contents were significantly lower in the A group compared with the H group in week 5. (B) ET-1 protein contents were significantly lower in the A group compared with the H group in weeks 3 and 5. C, control; H, hypertension; A, ACE-interfering RNA group. *p < 0.05 significantly different C group vs. H group. †p < 0.05 significantly different H group vs. A group.

• 

  Fig. 5 (A) Angiotensin II receptor (AT II R) type 1A and (B) neutrophil cytosolic factor (NCF) 1 protein levels were significantly higher in the H group compared with the C group in weeks 3 and 5 (p < 0.05) and not significantly different in the A group compared with the H group in weeks 3 and 5. C, control: H, hypertension: A, angiotensin-converting enzyme- interfering RNA group. *p < 0.05 significantly different C group vs. H group.

• 

  Fig. 6 Caspase-3, Bax, and Bcl-2 protein contents by westernblot analysis after angiotensin-converting enzyme (ACE)-interfering RNA (iRNA) treatment. (A) Caspase-3 protein contents were significantly higher in the H group compared with that in the C group in weeks 3 and 5 and not significantly different in the A group compared with the H group in weeks 3 and 5. (B) Bax protein contents were significantly lower in the A group compared with the C group in week 3. (C) Bcl-2 protein contents were significantly higher in the H group compared with that in the C group in weeks 3 and 5 and significantly lower in the A group compared with the A group in weeks 3 and 5. C, control: H, hypertension: A, ACE-iRNA group. *p < 0.05 significantly different C group vs. H group. †p < 0.05 significantly different H group vs. A group.

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