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(Hypertension. 2005;45:115.)
© 2005 American Heart Association, Inc.

Scientific Contributions

Selective Silencing of Angiotensin Receptor Subtype 1a (AT_1aR) by RNA Interference

Jorge Vázquez; María F. Correa de Adjounian; Colin Sumners; Aaron González; Carlos Diez-Freire; Mohan K. Raizada

From the Department of Physiology and Functional Genomics (J.V., C.S., A.G., C.D.-F., M.K.R.), University of Florida College of Medicine and McKnight Brain Institute, Gainesville, Fla; and the Instituto de Medicina Experimental (M.F.C.d.A.), Universidad Central de Venezuela, Caracas, Venezuela.

Correspondence to Mohan K. Raizada, PhD, Professor, Department of Physiology and Functional Genomics, College of Medicine, University of Florida, McKnight Brain Institute, Gainesville, FL 32610. E-mail mraizada{at}phys.med.ufl.edu

	Abstract

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Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT_1a-, AT_1b-, and AT₂-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_1aR) subtype. A dsRNA, AT₁ 47, was found to be highly selective and efficient in reducing the levels of AT_1aR subtype. Transfection of AT_1aR-expressing CHO cells with dsRNA AT₁ 47 resulted in an 80% decrease in the AT_1aR expression. In contrast, dsRNA AT₁ 47 showed no significant effects on both AT_1bR and AT₂R subtypes. Thus, AT₁ 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.

Key Words: receptors, angiotensin II • gene therapy • receptors, angiotensin

	Introduction

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Angiotensin 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 (AT₁R). Since the discovery of 2 subtypes of AT₁R, AT_1aR, and AT_1bR in rodents, it has been proposed that this diversity of Ang II actions resides in these 2 subtypes.³ Because AT_1aR and AT_1bR R share 94% sequence similarity,⁴ it has been difficult to develop subtype specific antagonists to link AT_1aR and AT_1bR 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_1aR 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_1bR can partially replace the BP regulatory functions of AT_1aR.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_1aR and AT_1bR 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_1aR subtype.

	Materials and Methods

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Complete coding sequence of Rattus rattus AT_1aR (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 chosen from the list of candidate siRNAs. Selected sequences did not show near-exact matches to any other known sequence on a BLAST search, confirming their sequence specificity to the AT_1aR. Synthetic duplexed–deprotected siRNAs were purchased from Dharmacon Research, Inc (Lafayette, Col). Three 21-bp dsRNA sequences were designated as AT₁ 9 (5'-AAACAGCTTGGTGGTGGTGATTGT'-3) directed to nucleotides 135 to 156, AT₁ 39 (5'-AACAACTGCCTGAACCCTCTG-3') directed to nucleotides 880 to 901, and AT₁ 47 (5'-AAAGGCCAAGTCCCACTCAAG-3') directed to nucleotides 966 to 987. In addition, a scrambled sequence with no near-exact match to any known sequence, Scr (5'-AATGTACTCACTACGAGTGCG-3') was designed as control for AT₁ 9, AT₁ 39, and AT₁ 47 dsRNAs.

Cell Culture and Transfection
Chinese hamster ovary cells (CHO) stably expressing either rat AT_1aR, AT_1bR, or AT₂R were maintained at 37°C in Ham F12 medium supplemented with 10% fetal bovine serum. Cells were seeded in 12-well plates at a cell density of 1x10⁵ cells/well, 24 hours before their use in transfection. Oligofectamine-mediated (Invitrogen Corporation) transfection of the dsRNA was performed after manufacturer’s instructions. Briefly, 2 µL of oligofectamine was mixed with 8 µL/well of OptiMEM (Invitrogen Corporation); similarly, the dsRNA was diluted a final volume of 90 µL/well in OptiMEM. Both solutions were incubated at room temperature for 10 minutes, mixed, and further incubated for 10 to 15 minutes. Cells were washed with phosphate-buffered saline (PBS) and 400 µL/well of serum-free Ham F12 media added. After transfection complexes were formed, the transfection mixture was added to the cells and incubation continued for 4 hours at 37°C. This was followed by addition of 250 µL/well of Ham F12 to 30%. Cells were maintained for 3 days and used for measurements of Ang II binding activity.

Measurement of [¹²⁵I]-Sar¹-Ile⁸ Ang II Binding to AT₁R and AT₂R
Cell cultures were rinsed with PBS (pH 7.2) and incubated with the Binding Buffer (PBS; pH 7.2; 0.16% heat-inactivated bovine serum albumin) containing 10 nmol/L [¹²⁵I]-Sar¹-Ile⁸ Ang II for 1 hour at room temperature in the absence and presence of 1 µmol/L of either losartan (AT₁R-specific antagonist) or PD-123319 (AT₂R-specific antagonist). Unbound radioligand was removed by rinsing the cells 3 times with ice-cold PBS pH 7.2. Cells were removed from the dish by incubation with NaOH–SDS (0.1 mol/L NaOH and 10% SDS solution) for 2 hours at room temperature. Bound radioactivity was measured in a DP5500 gamma counter (Beckman Coulter, Fullerton, Calif). Specific binding was calculated by subtracting the [¹²⁵I]-Sar¹-Ile⁸ Ang II bound in the presence of either losartan or PD123319 from that bound in its absence. Scatchard analysis was performed from saturation binding experiments with increasing Ang II concentration for the calculation of K_d and B_max values.

Semi-Quantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated using 4-PCR RNA isolation kit (Ambion, Austin, Tex) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) specific primers for AT₁R and AT₂R sequence were obtained from GenoMechanix LLC (Gainesville, Fla). Sequences for these primers were as follows: AT₁R: sense, 5'-TACGCTATGCAGATGGTGATGGG-3'; antisense, 5'-CTGGCTGATGGCTGGCTTGG-3'; AT₂R: sense, 5'-GCACCAATGAGTCCGCATTTA-3'; antisense, 5'-CAGAAAAGGGTAGATAACCGATTGG-3'. We used as internal control the amplification of 18 rRNA using the primer set provided by on the TaqMan Ribosomal RNA Control Reagents kit (Applied Biosystems, Foster City, Calif). Reverse transcription reactions were performed using 2 µg of total RNA in a volume of 100 µL. The PCR conditions were: 95°C for 5 minutes after of 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, with a final extension step of 72°C for 10 minutes. The resulting PCR products were subjected to a 2% agarose gel electrophoresis. Bands representing PCR products for the AT₁ and AT₂R were quantitated and data normalized with PCR bands for 18S rRNA.

Calcium Uptake
Cells were transfected with either dsRNA AT₁ 47 or a dsRNA Scr, as described. Two days after transfection, Ang II-stimulated [⁴⁵Ca²⁺] uptake was tested as described elsewhere.¹² Briefly, cells were washed 3 times with Tyrodes solution (137 mmol/L NaCl, 2.68 mmol/L KCl, 1.36 mmol/L CaCl₂, 0.46 mmol/L MgCl₂, 5.6 mmol/L glucose, 12 mmol/L NaHCO₃, 0.36 mmol/L NaH₂PO_4, pH 7.4) and equilibrated in this solution for 1 hour at 37°C. Cells were incubated for 20 seconds in Tyrode solution containing 490 mCi/mmol [⁴⁵Ca²⁺] in the presence or absence of 0.1 µmol/L Ang II. Immediately after incubation, the assay medium was removed and the cells were quickly rinsed twice with 1 mL of ice-cold Wash solution (100 mmol/L MgCl₂, 10 mmol/L HEPES, 10 mmol/L CdCl₂, 10 mmol/L MnCl₂) and then rinsed rapidly with 10 mL of ice-cold 100 mmol/L MgCl₂ containing 10 mmol/L HEPES. Cells were solubilized in 1 mL of 0.1 mol/L NaOH and [⁴⁵Ca²⁺] was quantified in a LS6500 liquid scintillation counter (Beckman Coulter, Fullerton, Calif).

Statistical Analysis
Data are shown as mean±SEM. Statistical differences between 2 means were determined by unpaired Student t test.

	Results

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In the first series of experiments, our objective was to identify target sequences that would trigger a highly efficient and specific silencing of the AT₁R. For this, we selected 3 dsRNA sequences targeting the 5' middle and 3' regions of AT_1aR coding sequence.

Of these dsRNAs, 2 (AT₁ 9 and AT₁ 47) significantly reduced specific Ang II binding in CHO cells that overexpress AT₁R (Figure 1A). These dsRNAs were without effect on Ang II binding in the CHO cells that overexpress AT₂R (Figure 1A). Whereas AT₁ 47 was able to decrease binding of Ang II to cells expressing AT₁R by 95%, AT₁ 9 caused a decrease of only 60% (Figure 1A). Scrambled dsRNA failed to influence the binding of Ang II. In contrast, the AT₁ 39 sequence was found to be nonselective, decreasing the binding of Ang II in both AT₁R- and AT₂R-expressing cells (Figure 1A). These data indicate that AT₁ 47 sequence is the most effective and selective for AT₁R silencing. Next, we determined the AT₁R subtype specificity of dsRNA AT₁ 47. For this, both AT₁a and AT₁b overexpressing cells were transfected with either AT₁ 47 or Scr dsRNA. Figure 1B shows that AT₁ 47 was able to completely silence AT_1aR subtype with only modest effect on the AT_1bR subtype. Transfection with Scr dsRNA did not influence binding of either receptor (data not shown). This indicated that AT₁ 47 was predominantly selective for the AT_1aR subtype.

View larger version (18K): [in this window] [in a new window]   Figure 1. Specificity of dsRNA targets for the AT1a receptor subtype. A, CHO cells overexpressing AT1a or AT2 receptors were transfected with 100 nmol/L AT1 9, AT1 39, AT1 47, or Scr dsRNA essentially as described in Methods. Two days after transfection, cultures were used to determine the specific binding of [125I]-Sar1-Ile8 Ang II to cell surface receptors. Nonspecific binding (in presence of 1 µmol/L of Losartan for the AT1R or 1 µmol/L PD 123319 for the AT2 R) was subtracted from the total binding. Data are presented as percent binding over Scr-transfected cells (control). Binding after Scr dsRNA transfection was comparable to the binding in mock-transfected cells. Data are mean±SE (n=6; *P<0.001 vs Scr-treated control). B, CHO cells overexpressing AT1a or AT1b receptor subtypes were transfected with 100 nmol/L AT1 47. Two days after transfection, cultures were used to measure [125I]-Sar1-Ile8 Ang II binding as described in Methods. Data are presented as percent binding over Scr control dsRNA-transfected cells (mean±SE; n=6; *P<0.001 vs Scr-treated control).

Next, we characterized the effect of AT₁ 47 on AT₁a R. dsRNA AT₁ 47 caused a dose-dependent decrease in the specific binding of [¹²⁵I]-Sar¹-Ile⁸ Ang II to AT_1aR-expressing CHO cells, with a maximal inhibition of 90% observed at 50 nmol/L dsRNA (Figure 2A). The IC50 was calculated to be 1.2 nmol/L. No effect was seen on AT₂ R-expressing CHO cells with these concentrations of dsRNA. Incubation with 100 nmol/L dsRNA AT₁ 47 caused a time-dependent silencing of AT_1aR expression (Figure 2B). An 80% decrease in binding was observed within 1 day, followed by a 97% decrease at 2 days after transfection. This level of decrease was maintained for an additional 24 hours. The receptor levels returned to control levels 7 days after transfection.

View larger version (19K): [in this window] [in a new window]   Figure 2. Dose-response (A) and time course (B) of AT1a 47 dsRNA-mediated inhibition of [125I]-Sar1-Ile8 Ang II binding in AT1aR-expressing CHO cells. A, AT1aR expressing CHO cells were transfected with 100 nmol/L AT1a 47 dsRNA. Binding assays were performed at the indicated time periods and data are presented as a percentage of the binding on Scr dsRNA-transfected cells (mean±SE, n=3). B, CHO cells were transfected with the indicated concentrations of AT1a 47 dsRNA with fixed concentration of oligofectamine (variable charge ratio). Specific binding of [125I]-Sar1-Ile8 Ang II to AT1aR was determined in the presence of 1 µmol/L losartan. Data are presented as percentage of binding on Scr dsRNA-transfected cells (mean±SE; n=3; *P<0.001 vs Scr-treated control).

Saturation analyses of [¹²⁵I]-Sar¹-Ile⁸ Ang II binding were performed in Scr and AT₁ 47-transfected AT_1aR CHO cells (Figure 3). Scatchard analyses revealed a 20-fold decrease in the Bmax for AT_1aR in the AT₁ 47-treated cells when compared with Scr controls (0.46±0.01 versus 8.80±0.46 pmol/mg protein). In contrast, the Kd values for both treatments remained comparable (0.9±0.09 versus 2.0±0.30 nmol/L). The observed decrease in the number of AT_1aR was also reflected in a significant decrease in AT_1aR mRNA levels (Figure 4A). An 80% decrease in the mRNA was observed 48 hours after transfection (Figure 4B).

View larger version (18K): [in this window] [in a new window]   Figure 3. Saturation analysis of [125I]-Sar1-Ile8 Ang II binding to AT1aR–CHO cells transfected with AT1 47 dsRNA. CHO–AT1a R cells were transfected with 100 nmol/L Scr or AT1 47 dsRNA. Seventy-two hours after transfection, cells were used to determine specific binding at the indicated concentrations of [125I]-Sar1-Ile8 Ang II. Saturation data were subjected to Scatchard analysis.

View larger version (32K): [in this window] [in a new window]   Figure 4. Semi-quantitative reverse-transcription PCR analysis for AT1 and AT2 mRNA. A, Representative agarose gels of AT1R and AT2R reverse-transcription PCR products of mRNA from CHO AT1aR or CHO AT2R cells transfected either with Scr or AT1 47 dsRNA. B, Densitometry analysis of semi-quantitative reverse-transcription PCR from (A). Data are means±SE; n=3; *P<0.001 vs Scr-treated control.

Finally, we determined the effect of AT₁ 47 on Ca²⁺ uptake. To find out if decreases in AT_1aR-specific binding was associated to a reduction in functional AT_1aR, we determined the effects of dsRNA transfection on Ang II-stimulated calcium uptake. Ang II (100 nmol/L) caused a 54% increase in [⁴⁵Ca²⁺] uptake in CHO cells expressing AT_1aR. Transfection of these cells with dsRNA Scr did not influence this stimulation and it was comparable to that of nontransfected cells. In contrast, the Ang II-induced increase in [⁴⁵Ca²⁺] uptake was completely abolished in by dsRNA AT₁ 47 transfected cells (Figure 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. Ang II-stimulated [45Ca2+] uptake in CHO AT1a cells. Two days after transfection, cells were incubated for 20 seconds with Tyrodes solution containing 490 mCi/mmol [45Ca2+] in the presence or absence of 0.1 µmol/L Ang II. After washing, cells were solubilized and [45Ca2+] was quantified. Results are expressed as [45Ca2+] uptake in cpm/µg protein (mean±SE; n=4. P<0.05 vs Scr-treated control).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most significant finding of our study is that it establishes, for the first time to our knowledge, that RNAi technology can be used to silence the AT_1aR subtype. This provides us with a powerful tool for elucidating the respective roles of AT_1aR and AT_1bR subtypes in the diverse and profound physiological effects of Ang II in the cardiovascular system in normal animals. The observed reduction in [¹²⁵I]-Sar¹-Ile⁸ Ang II binding, AT_1aR mRNA levels, and Ang II-stimulated [⁴⁵Ca²⁺] uptake demonstrates that at least 1 of the 3 dsRNA targeted for the AT_1aR subtype is selective and efficient in silencing the AT_1aR subtype gene. The dsRNA AT₁ 47 is more efficient in reducing AT_1aR numbers compared with its sister subtype, the AT_1bR, despite the fact that these subtypes share 96% similarity. The dsRNA AT₁ 47 is highly efficient and produces AT_1aR silencing that lasts for 3 days. Until now, the popular technology to reduce angiotensin receptors has been the use of antisense oligonucleotides.^13–18 RNAi technology not only is highly selective for the subtypes of AT_1aR and AT_1bR but also is more effective than antisense oligonucleotides. For example, a previous study¹⁹ demonstrated a reduction of AT₁R by 57% to 73% with 1 µmol/L concentration of AT₁R-specific antisense oligonucleotides. In contrast, in the present study, a concentration of 100 nmol/L of dsRNA was found to silence AT₁R by 95% in 48 hours.

The efficacy of the dsRNA 47 to silence AT₁R in a physiologically relevant cell was confirmed with the use of astroglia cells in primary cultures. Our observations indicated an 80% decrease in AT₁ binding elicited by this dsRNA in these cultures, confirming the validity of the silencing data obtained for the CHO cells.

In summary, our observations demonstrate that RNAi technology can be successfully used to silence 2 closely related genes for Ang II receptors. This provides us with a powerful tool to selectively silence 1 or more subtypes of AT₁R and to study their role in cardiovascular physiology and pathophysiology in a way that was not previously possible.

Perspectives
Delineating the roles of the AT₁R subtypes in various cardiovascular functions has been difficult in the past because of lack of selective antagonists or antisense targeting. This study uses RNAi technology and successfully demonstrates the effectiveness of a dsRNA that selectively targets AT_1aR subtype. Thus, our study provides a well-needed tool to dissect AT_1aR functions in vivo.

Received July 22, 2004; first decision August 19, 2004; accepted October 31, 2004.

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This Article Abstract Full Text (PDF) All Versions of this Article: 45/1/115    most recent 01.HYP.0000150161.78556.c6v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vázquez, J. Articles by Raizada, M. K. Search for Related Content PubMed PubMed Citation Articles by Vázquez, J. Articles by Raizada, M. K. Related Collections ACE/Angiotension receptors Other hypertension Functional genomics Hypertension - basic studies Gene therapy