Received December 3, 2007
Revised January 3, 2008
Accepted after revision February 25, 2008
Cardiovascular Control [210]
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RNA interference shows interactions between brainstem angiotensin AT1 receptors and ACE2
Zhanyi Lin 1,
Yanfang Chen 2,
Wenfeng Zhang 2,
Alex F. Chen 3,
Shuguang Lin 1,
Mariana Morris 2*
1 Guangdong Cardiovascular Institute
2 Wright State University, Boonshoft School of Medicine
3 Michigan State University School of Medicie
* To whom correspondence should be addressed. E-mail: mariana.morris{at}wright.edu.
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Abstract |
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Angiotensin (Ang) AT1 receptors and Ang converting enzymes (ACE and ACE2) are expressed in the dorsal vagal complex (DVC) of the brainstem. The goal was to examine in vivo interactions between brainstem Ang AT1 receptors, ACE and ACE2 using shRNA gene silencing methods. The study takes advantage of the bilateral brainstem expression of renin angiotensin system (RAS) markers. Adenovirus vectors (Ad, 2.0x109 cfu/ml, 200nl) carrying interference small, hairpin RNA (shRNA) for either AngAT1a (Ad-AT1a-shRNA) or AngAT1b (Ad-AT1b-shRNA) were microinjected into the right side of the brainstem DVC. The Ad-LacZ control was injected into the left side. Brainstems were processed with in situ hybridization (ISH) and immunochemistry. Results showed: 1) Ad-AT1a-shRNA down-regulated Ang AT1a mRNA by 61.2 ± 6.8% (p<0.01). Ad-AT1b-shRNA down-regulated Ang AT1b mRNA by 51.6 ± 5.2% (p<0.01); 2) Down regulation of Ang AT1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 ± 14.5 %, p<0.01) while reduction in Ang Ad-AT1b had no effect; 3) ACE mRNA expression was not altered by either RNAi treatment; 4) Immunochemical staining for Ang AT1 receptors, ACE and ACE2 were in agreement with the mRNA changes observed. These results demonstrate the utility of in vivo gene silencing to examine functional specificity. Ad-AT1a-shRNA and Ad-AT1b-shRNA induced site and subtype specific down-regulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT1a receptors and the RAS regulatory enzyme, ACE2.
Key Words:
Cardiovascular, Medulla oblongata, Peptide
