Recently discovered, angiotensin-converting enzyme-2 (ACE2) is an important therapeutic target in the control of cardiovascular diseases as a result of its proposed central role in the control of angiotensin peptides. Thus our objective in the present study was to determine whether ACE2 gene transfer could decrease high blood pressure (BP) and would improve cardiac dysfunctions induced by hypertension in the spontaneously hypertensive rat (SHR) model. Five-day-old SHR and normotensive WKY rats received a single intracardiac bolus injection of lentiviral vector containing either murine ACE2 (ACE2) or control enhanced green fluorescent protein (EGFP) genes. Systolic BP, cardiac functions, and perivascular fibrosis were evaluated 4 mo after ACE2 gene transduction. ACE2 gene transfer resulted in a significant attenuation of high BP in the SHR (149 +/- 2 mmHg in lenti-ACE2 vs. 180 +/- 9 mmHg in lenti-EGFP, P < 0.01). In contrast, no significant effect of lenti-ACE2 on BP of WKY rats was observed. Lenti-ACE2-treated SHR showed an 18% reduction in left ventricular wall thickness (1.52 +/- 0.04 vs. 1.86 +/- 0.04 mm in lenti-EGFP, P < 0.01). In addition, there was a 12% increase in left ventricular end diastolic and a 21% increase in end systolic diameters in lenti-ACE2-treated SHR. Finally, lenti-ACE2 treatment resulted in a significant attenuation of perivascular fibrosis in the SHR. In contrast, ACE2 gene transfer did not influence any of these parameters in WKY rats. These observations demonstrate that ACE2 overexpression exerts protective effects on high BP and cardiac pathophysiology induced by hypertension in the SHR.

Abstract-Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT 1a -, AT 1b -, and AT 2 -receptors. Because of a high degree of similarity among these G-protein-coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT 1a receptor (AT 1a R) subtype. A dsRNA, AT 1 47, was found to be highly selective and efficient in reducing the levels of AT 1a R subtype. Transfection of AT 1a R-expressing CHO cells with dsRNA AT 1 47 resulted in an 80% decrease in the AT 1a R expression. In contrast, dsRNA AT 1 47 showed no significant effects on both AT 1b R and AT 2 R subtypes. A ngiotensin II (Ang II) exerts profound physiological effects on the cardiovascular system by regulating such diverse functions as increases in blood pressure (BP), extracellular fluid volume, hormone secretion, vascular and cardiac remodeling, stimulation of sympathetic nerve activity, and damping of baroreflexes. 1,2 Most, if not all, of these effects are mediated by activation of a single angiotensin receptor subtype (AT 1 R). Since the discovery of 2 subtypes of AT 1 R, AT 1a R, and AT 1b R in rodents, it has been proposed that this diversity of Ang II actions resides in these 2 subtypes. 3 Because AT 1a R and AT 1b R R share 94% sequence similarity, 4 it has been difficult to develop subtype specific antagonists to link AT 1a R and AT 1b R to distinct cardiovascular effects of Ang II. Thus, investigators have relied on the use of genetic targeting of these receptors in mouse models to link them with various phenotypes. 5-8 For example, AT 1a R knockout studies have indicated the role of this receptor in BP regulation, sodium handling, and central dipsogenic responses. 9 However, knockout studies indicate that AT 1b R can partially replace the BP regulatory functions of AT 1a R. 10 These observations indicate that although knockout technology enables us to delineate an overall physiological perspective, it is limited because of the possible expression of compensatory mechanisms during development. Therefore, new and more selective alternatives need to be developed in which the expression of AT 1a R and AT 1b R could be attenuated in adult animals after development.We have taken advantage of recent advances in RNA interference (RNAi) technology to determine whether it can be used to selectively silence the expression of these receptor subtypes. 11 Thus, our objective in this study was to determine whether we could identify a double stranded RNA (dsRNA) sequence that is highly effective and selective for the AT 1a R subtype. Materials and MethodsComplete coding sequence of Rattus rattus AT 1a R (Gen Bank accession number X62295) was used for the selection of target sequences. The sequence was used in the Target Finder and Design Tool provided by Ambion Inc (Austin, Tex). For screening, 3 target sequences spaced throughout the gene were ch...

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