Our previous studies demonstrated that peripheral overexpression of angiotensin II (ANG II) type 2 receptors (AT2R) prevents hypertension-induced cardiac hypertrophy and remodeling without altering high blood pressure. This, coupled with the observations that AT2R play a role in the antihypertensive actions of ANG II type 1 receptor (AT1R) blockers (ARBs), led us to propose that peripheral overexpression of AT2R would improve the antihypertensive action of losartan (Los) in Sprague-Dawley (SD) rats made hypertensive via chronic infusion of ANG II. Here we utilized adenoviral vector-mediated AT2R gene transfer to test this hypothesis. A single intracardiac injection of adenoviral vector containing genomic AT2R (G-AT2R) DNA and enhanced green fluorescent protein (EGFP) gene controlled by cytomegalovirus (CMV) promoters (Ad-G-AT2R-EGFP; 5 × 109 infectious units) into adult SD rats produced robust AT2R overexpression in cardiovascular tissues (kidney, lung, heart, aorta, mesenteric artery, and renal artery) that persisted for 3–5 days postinjection. By 7 days post viral injection, the overexpressed AT2R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels). In two separate protocols, we demonstrated that the hypotensive effect of Los (0.125, 0.5, and 1.0 mg/kg iv) was significantly greater in the AT2R-overexpressing animals (−40.7 ± 4.3, −41.8 ± 4.8, and −48.1 ± 2.6 mmHg, respectively) compared with control vector (Ad-CMV-EGFP)-treated rats (−12.4 ± 2.2, −20.2 ± 3.4, and −27.3 ± 3.4 mmHg, respectively). These results provide support for a depressor role of AT2R and the proposal that combined AT2R agonist and ARB treatment may be an improved therapeutic strategy for controlling hypertension.
- angiotensin II
- angiotensin receptor blocker
- gene transfer
it is well established that angiotensin II (ANG II) has a major role in the control of blood pressure and cardiovascular function and that its hyperactivity leads to hypertension and cardiovascular disease (19). The physiological actions of ANG II include vasoconstriction and increases in aldosterone secretion, sympathetic nerve activity, and water and sodium intake (19). In pathological situations such as hypertension, the vasoconstrictor actions of ANG II are exacerbated, and this peptide also displays a powerful trophic action and contributes to cardiac hypertrophy and fibrosis (37, 40). These physiological and pathological actions of ANG II are mediated via its type 1 receptors (AT1R) (19), and AT1R blockers (ARBs) such as losartan (Los; Cozaar) are in common use as antihypertensive agents (11, 51).1
In comparison with the AT1R, a role for ANG II type 2 receptors (AT2R) in the control of blood pressure and cardiovascular function is not established. A number of studies have failed to demonstrate any role of endogenous AT2R in blood pressure regulation (15, 35, 42, 48), but they may have been hampered by the fact that AT2R exist in adult cardiovascular tissues in low concentrations (19). An increasing amount of evidence now indicates that ANG II can influence cardiovascular function via its AT2R, producing a vasodilator action that is opposite to the vasoconstrictor effect of ANG II via AT1R (12). This AT2R-mediated vasodilator action was more apparent in situations where the renin-angiotensin system was upregulated. For example, Carey and colleagues (12, 14) demonstrated that, in the kidney, ANG II acts at the AT2R to stimulate a vasodilator cascade of bradykinin, nitric oxide, and cyclic guanosine 5′-monophosphate, which is tonically activated only during conditions of increased ANG II, such as sodium depletion and ANG II infusion-induced hypertension. It is now apparent that these conditions produce increased sensitivity and/or expression of vascular and renal AT2R (9, 25, 46). The vasodilator action of ANG II via AT2R was also revealed in some studies in the presence of ARB to prevent the vasoconstrictor actions of the peptide (6, 13, 17, 23, 54). However, other studies have demonstrated that the hypotensive action of the ARB Los is not dependent on AT2R activation (16, 30). The controversy that surrounds the vasodilatory effect of ANG II via AT2R also extends to the site of action. In addition to the above renal vasodilator actions of ANG II, other studies (8, 28, 32, 36) have demonstrated AT2R-mediated vasodilatory actions of ANG II in resistance microvessels and in large capacitance vessels. By contrast, another study (41) indicated that coronary but not renal AT2R stimulation results in vasodilation.
The above studies that suggest a role of native AT2R in blood pressure regulation are supported by experiments using transgenic animal models. For example, transgenic mice that overexpress AT2R within aortic vascular smooth muscle cells exhibit vasodilation in response to chronic infusion of ANG II (49). It is also clear that AT2R knockout mice display increased basal blood pressures and increased vasoconstrictor responses to ANG II via AT1R (3, 26, 27, 31, 45). These studies are supported by our previous gene transfer studies, which demonstrated that overexpression of AT2R-antisense in cardiovascular tissues of neonatal normotensive rats, and subsequent knockdown of AT2R, resulted in elevated blood pressure in these animals during adulthood (52). Collectively, these data imply that AT2R normally antagonize the AT1R-mediated rise in blood pressure. However, the increase in pressor sensitivity may also be explained by the increased vascular AT1R expression in AT2R knockout mice (47).
On the basis of the available data, it is difficult at this point to assign a role for AT2R in hypertension. However, a number of studies have reported that the expression of AT2R is increased following renal ablation and in pathological conditions such as vascular injury, cardiac remodeling, congestive heart failure, and myocardial infarction (2, 29, 39, 50). Furthermore, it appears that the expressed AT2R have a protective role within cardiovascular and renal tissues (29, 39, 50). In concert with these findings, a virally mediated gene transfer approach allowed us to demonstrate that overexpression of AT2R in cardiac tissues attenuates cardiac hypertrophy and fibrosis in spontaneously hypertensive rats (SHR) and in normotensive rats made hypertensive by ANG II infusion (21, 38). Nonetheless, there are also conflicting data that argue that the AT2R elicits cardiac growth and does not antagonize AT1R-mediated cardiac hypertrophy (5, 18). From a more clinical perspective, a recent study has demonstrated that, in patients with systemic hypertension, there is an association between left ventricular hypertrophy and a common intronic polymorphism of the AT2R gene, which results in less effective transcription (4). This supports the idea of an antigrowth/cardioprotective role of the AT2R.
Therefore, the actions of AT2R described to this point are largely counteractive to those of ANG II via AT1R. A number of studies have also suggested that the decrease in blood pressure produced by ARBs is partially mediated by high circulating levels of ANG II acting via AT2R (20, 44). In fact, all of the available clinical data suggest that the beneficial effects of ARBs on hypertension and cardiovascular disease are due in part to their antagonism of the AT1R and in part by activation of the AT2R as a result of their ability to increase circulating ANG II (22, 43, 53). Considering the conflicting reports in this area and that cardiovascular tissues of adult animals contain only low levels of AT2R, we set out to test this idea in a novel way by overexpressing the AT2R in cardiovascular tissues. The rationale was that we would be able to enhance their participation to a level that can be physiologically studied. Our data indicate that increased peripheral expression of AT2R produced by virally mediated gene delivery is associated with enhancement of the hypotensive action of the ARB Los.
MATERIALS AND METHODS
For the cell culture experiments described here we utilized neonatal Sprague-Dawley (SD) rat pups, derived from our breeding colony. For the physiological experiments, we used adult male SD rats. Breeders and adult male rats were purchased from Charles River Farms (Wilmington, MA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee (protocols 0746 and 0758).
Collagenase and trypsin were purchased from Worthington Biochemical (Lakewood, NJ). RNeasy kits, DNeasy tissue kits, and OneStep RT-PCR kits were obtained from Qiagen (Valencia, CA). Primer Express and TaqMan PCR Master Mix were from Applied Biosystems (Foster City, CA). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin mix, and fetal bovine serum were from Invitrogen (Grand Island, NY). 125I-labeled (Sar1, Ile8)-ANG II was obtained from the University of Mississippi (Oxford, MS). Los was a gift from Merck (Rahway, NJ). ANG II, PD-123,319, heparinized 0.9% saline, and all other chemicals were obtained from Sigma-Aldrich Chemical (St. Louis, MO).
Recombinant Adenoviral Constructs
Preparation of the adenoviral constructs [adenoviral vector containing enhanced green fluorescent protein gene controlled by a cytomegalovirus promoter (Ad-CMV-EGFP) and adenoviral vector containing genomic AT2R (G-AT2R) DNA with introns 1 and 2 and the encoding region and enhanced green fluorescent protein gene controlled by cytomegalovirus promoters (Ad-G-AT2R-EGFP)] was performed exactly as detailed previously (34).
Adenovirally Mediated Gene Transduction
To assess adenovirally mediated transduction, Ad-G-AT2R-EGFP and Ad-CMV-EGFP [both 5 × 109 infectious units (ifu)/rat] were injected intracardially (left ventricle) into SD rats (n = 6/group) using the same procedures as detailed previously by our group (21, 38, 40, 52). On days 3, 5, and 7 after the injections, two rats from each group were euthanized and the liver, heart, kidney, lung, aorta, and mesenteric and renal arteries were removed. All samples were immediately frozen in dry ice and stored at −80°C. The biodistribution of adenoviral vector and the expression of AT2R in each tissue were determined as described in the following two sections.
RT-PCR Analysis of AT2R Transgene Expression in Rat Tissues
The forward primer and the reverse primer were designed from AT2R and simian virus 40 poly(A), respectively, to amplify a specific fragment of the transduced exogenous AT2R cDNA. The primers were as follows: AT2-F (forward primer): 5′-ACAGAATTACCCGTGACCAA-3′; poly(A)-R (reverse primer): 5′-GGCTGATTATGATCAGTTATC-3′.
Total RNA was prepared from the transduced tissues using an RNeasy kit. RT-PCR reactions were performed using a OneStep RT-PCR kit under the following conditions: 1 cycle of 50°C for 30 min, 95°C for 15 min; 40 cycles of 94°C for 40 s, 50°C for 1 min, 72°C for 2 min, and 1 cycle of 72°C for 8 min. PCR products were separated by electrophoresis on a 2.0% agarose gel and identified by restriction endonuclease analysis.
Real-Time PCR for the Detection of Adenoviral Vectors in Rat Tissues
Oligonucleotide primers and probe specific for EGFP were designed to detect the Ad vectors in rat tissues using Primer Express. The primers and probe used were as follows: EGFP-F (forward): 5′-AGAACGGCATCAAGGTGAAC-3′; EGFP-R (reverse): 5′-TGCTCAGGTAGTGGTTGTC-3′; EGFP probe: 5′-FAMCCGGCAGATAAGCAT-TAMRA-3′.
DNA was extracted from the frozen tissue samples using DNeasy tissue kits. The procedures used were according to the manufacturer's instructions of the kits. About 10 mg tissue of each sample was used for extraction of total DNA.
Real-time PCR was performed according to the protocols of the manufacturer with an ABI Prism 7000 HT Detection System (Applied Biosystems) using TaqMan PCR Master Mix. Relative quantification was performed using the comparative method as described in Applied Biosystems User Bulletin 2. No template controls were used to monitor for any contaminating amplification.
Preparation of Cardiac Fibroblast Cultures
Neonatal rat cardiac fibroblasts were isolated using a method adapted from Zhang et al. (56). Briefly, 5-day-old SD rat pups were anesthetized by halothane inhalation. Heart ventricles were removed by blunt dissection, cleaned of atria and adherent tissue, and washed in ice-cold PBS. Ventricles were then minced with scissors and resuspended in 20 ml of 1% collagenase and incubated at 37°C for 2 h. The suspension was centrifuged at 2,000 rpm for 5 min, and the supernatant was aspirated. The pellet was resuspended in 20 ml of 0.25% trypsin and incubated at 37°C for 1 h. This suspension was centrifuged as before, and the pellet was resuspended in 10 ml of growth media consisting of DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 5 μg/ml ascorbic acid. This suspension was plated in a 75-cm2 flask. Cells were maintained with a 95% air-5% CO2 gas mixture in a 37°C humidified incubator. Media was changed every 48 h until confluence. Cells were split using a standard trypsin method and seeded into 12-well plates at 3,000 cells/cm2. Only cells in passages 2 and 3 were used for experiments.
ANG II Receptor Binding
Cardiac fibroblasts grown in 12-well plates were transduced with 4.0 × 105 ifu per well of either Ad-CMV-EGFP or Ad-G-AT2R-EGFP for 24 h or were incubated with PBS for the same time period. This was followed by analysis of ANG II receptor binding as described by us previously (34). In brief, transduced cultures were washed with PBS (pH 7.4) and incubated for 1 h at room temperature with 0.1 nM 125I-labeled (Sar1, Ile8)-ANG II (2,176 Ci/mmol) in the absence or presence of unlabeled ANG II to assess nonspecific binding. Parallel incubations were performed in the presence of the AT2R-selective blocker PD-123,319 (1 μM), to assess AT2R-specific binding, or the AT1R receptor antagonist Los (1 μM), to assess AT1R-specific binding. Following these incubations, cells were washed four times with PBS, lysed in 0.5 M NaOH, and the bound 125I-labeled (Sar1, Ile8)-ANG II in the lysate under each condition was quantified in a gamma counter (CliniGamma 1272; LKB-Wallac, Turku, Finland). Data are expressed as counts per minute per well.
In Vivo Physiological Procedures and Measurements
−1·min−1 for 4 wk. After three and a half weeks of treatment, rats were anesthetized by inhalation of metaphane, and Ad-CMV-EGFP (n = 6 rats) or Ad-G-AT2R-EGFP (n = 8 rats) was delivered by a single intracardiac injection (1.1 ml of 5 × 1010
Eighteen male SD rats weighing 175–200 g were implanted with telemetry transducers (Data Sciences, St. Paul, MN) into the abdominal aorta under isoflurane anesthesia [O2-isoflurane (2%) mixture] as described previously (21−1·min−1 for ∼4 wk. Mean arterial pressure (MAP) and heart rate were recorded via the telemetry devices, with raw data analyzed using Dataquest IV software (Data Sciences). Sixteen days following implantation of the minipumps, rats were randomly assigned to three groups. Each rat was anesthetized with the above O2-isoflurane (2%) mixture and received a single intracardiac injection (1.0 ml of 5 × 109 ifu) of either Ad-CMV-EGFP (GFP; n = 7 rats), Ad-G-AT2R-EGFP (AT2R; n = 7 rats), or 0.9% saline (n = 4 rats). This was followed 1 day later by implantation of a silicone elastomer cannula (Helix Medical) into the jugular vein, as in protocol 1. At 3 days after the viral or 0.9% saline injections, rats received an intravenous injection of 1.0 mg/kg body wt of Los in a volume of 1 ml. MAP was recorded via telemetry before and during the 2 h following Los administration. This procedure was repeated using respective intravenous doses of 0.125 and 0.5 mg/kg body wt of Los on day 4 after the viral or 0.9% saline injections. Note that the second injection of Los (0.5 mg/kg) was made at least 3 h after MAP had returned to baseline following the first Los injection (0.125 mg/kg).
On day 5 after viral injections, rats from the GFP and AT2R groups were anesthetized with a mixture of ketamine, xylazine, and acepromazine as above in protocol 1. Anesthetized rats received an intravenous injection of 1.0 mg/kg body wt of Los in a volume of 1 ml, followed 20 min later by intravenous infusion of 1.0 mg/kg body wt for 30 min of the AT2R-selective blocker PD-123,319. MAP was recorded via telemetry before and for 30 min following PD-123,319 administration.
At 7 days after the viral injections (at which time AT2R expression has declined toward control values), conscious rats received an intravenous injection of 0.5 mg/kg body wt of Los in a volume of 1 ml, and MAP was recorded via telemetry as above.
Data are expressed as means ± SE. Statistical significance was evaluated with the use of a one- or two-way ANOVA as appropriate, followed by a Bonferroni post hoc test to compare individual means. Differences were considered significant at P < 0.05; individual P values are noted in the figure legends.
Adenovirally Mediated Overexpression of AT2R
Our previous studies demonstrated that Ad-G-AT2R-EGFP elicits significant expression of AT2R within various cell lines (e.g., C2C12 mouse muscle myoblasts, NIH/3T3 fibroblasts, CATH.a locus ceruleus tumor cells) (34). Here, in the first set of experiments we tested the ability of Ad-G-AT2R-EGFP (4.0 × 105 ifu per well) to elicit AT2R expression in primary cultures of neonatal rat cardiac fibroblasts. The fluorescence micrograph presented in Fig. 1A demonstrates the localization of GFP within these cells at 24 h following transduction with 4.0 × 105 ifu per well of Ad-G-AT2R-EGFP. Similar localization of GFP was obtained following transduction with the control vector Ad-CMV-EGFP at 4.0 × 105 ifu per well (data not shown). At this titer, Ad-G-AT2R-EGFP produced a large increase in the level of AT2R-specific binding when compared with the levels of AT2R present in control (PBS-treated) or Ad-CMV-EGFP-transduced cells (Fig. 1B). Neither Ad-CMV-EGFP nor Ad-G-AT2R-EGFP produced any change in the levels of AT1R-specific binding (Fig. 1B). Similar results were obtained from cultured cardiac myocytes, i.e., Ad-G-AT2R-EGFP provided robust transduction and resulted in a large increase in the level of AT2R-specific binding, with no changes in AT1R-specific binding (data not shown). These data are consistent with our previous studies that indicate that lentivirally mediated overexpression of AT2R in rat heart produces no changes in AT1R levels in cardiomyocytes (38).
The above findings indicate that Ad-G-AT2R-EGFP is highly efficient at increasing the expression of AT2R in cardiac cells, without having any effect on the level of AT1R binding. Next we tested whether this vector produces expression of AT2R within peripheral tissues following intracardiac administration. SD rats received an intracardiac injection of either Ad-G-AT2R-EGFP (5 × 109 ifu; n = 6 rats) or Ad-CMV-EGFP (5 × 109 ifu; n = 6 rats). At days 3, 5, and 7 after the injections, two rats from each group were euthanized, and the liver, heart, kidney, lung, aorta, mesenteric artery, and renal artery were removed for detection of adenoviral vectors and analysis of AT2R transgene expression. The data presented in Fig. 2 demonstrate that viral (Ad-G-AT2R-EGFP) DNA was detected in all of the tissues examined at 3 and 5 days after intracardiac injections. At day 7, each tissue demonstrated reduced level of Ad-G-AT2R-EGFP DNA. This ranged from reductions of (for example) ∼80-fold from day 3 to day 7 in the liver to ∼16-fold in the mesenteric artery over the same time period (Fig. 2). Thus viral DNA persists within all of the infected tissues at 7 days post intracardiac injections, albeit at lower levels. However, the expression pattern obtained for the AT2R transgene (i.e., the overexpressed AT2R) is different. The data in Fig. 3 show that AT2R transgene was expressed in liver, heart, lung, aorta, kidney, and mesenteric arteries at 3 and 5 days post viral injections. Thus Ad-G-AT2R-EGFP transduces and elicits AT2R overexpression in tissue sites such as heart, kidney, and blood vessels that express native AT2R (19). AT2R transgene persists in the liver and lung at 7 days but is sharply reduced according to real-time RT-PCR analyses (not shown). In addition, it is clear from Fig. 3 that the AT2R transgene is either reduced (heart) or undetectable (kidney, aorta, and mesenteric artery) at day 7 after viral injections. The reduction in AT2R transgene expression may reflect silencing of the CMV promoter, as observed in other tissues following adenoviral administration (7, 10). On the basis of this expression pattern, analyses of the effects of the AT1R antagonist Los on MAP in AT2R-overexpressing animals were assessed at 3 to 5 days following viral administration.
Enhanced Hypotensive Action of Los in SD Rats Overexpressing AT2R
The effects of overexpression of AT2R on Los-induced decreases in blood pressure were assessed via two separate experimental protocols as set out in materials and methods.
In this protocol, SD rats that were implanted subcutaneously with osmotic minipumps containing ANG II (152.0 ± 1.6 ng·kg−1·min−1) developed a significant increase in blood pressure within 2–3 wk of treatment. During the 3rd wk of treatment, animals received a single intracardiac injection of either Ad-CMV-EGFP or Ad-G-AT2R-EGFP (1.1 ml of 5 × 1010 ifu/rat). Basal mean blood pressures (analyzed via direct monitoring from the carotid artery) were not different between the animals treated with Ad-CMV-EGFP versus the Ad-G-AT2R-EGFP vector-treated rats (124.9 ± 8.5 and 125.7 ± 6.6 mmHg, respectively). Heart rate values were also not different between the Ad-CMV-EGFP and Ad-G-AT2R-EGFP treatments (349.1 ± 49.4 and 384.8 ± 49.1 beats/min, respectively). Blood pressure responses to acute injection of Los (1 mg/kg iv) were significantly enhanced in animals treated with the Ad-G-AT2R-EGFP virus. MAP decreased by 9.0 ± 4.0 mmHg in the Ad-CMV-EGFP-treated rats and by 29.5 ± 5.9 mmHg in the Ad-G-AT2R-EGFP-treated rats in response to Los, as shown in Fig. 4.
Having established in protocol 1 that rats that overexpress AT2R display an enhanced hypotensive response to intravenously administered Los, we had three objectives in protocol 2: 1)to determine the hypotensive effects of different doses of Los in the AT2R-overexpressing rats; 2) to determine the hypotensive effect of Los following the return of AT2R toward basal values; and 3) to investigate whether the enhanced hypotensive action of Los involved activation of AT2R. Subcutaneous infusion of ANG II (200 ng·kg−1·min−1) via osmotic minipump produced a time-dependent increase in MAP in all rats within 2 wk. Intracardiac administration of either Ad-CMV-EGFP, Ad-G-AT2R-EGFP (both 1.0 ml of 5 × 109 ifu/rat), or 0.9% saline produced no significant change in MAP (156.2 ± 3.1, 151.2 ± 3.8, and 157.3 ± 3.3 mmHg, respectively) or heart rate (335.8 ± 3.7, 341.7 ± 3.7, and 323.9 ± 3.1 beats/min, respectively) 3 days later, at which time AT2R expression is maximal (Fig. 3). Intravenous injection of Los (0.125, 0.5, or 1.0 mg/kg) into the Ad-CMV-EGFP-treated rats produced significant dose-related decrease in MAP (Fig. 5A). A similar significant decrease in MAP was produced by Los (1.0 mg/kg iv) in the rats that had been injected intracardially with 0.9% saline (Fig. 5A). This hypotensive action of Los was significantly enhanced, at all three doses, in the Ad-G-AT2R-EGFP-treated rats (Fig. 5A). However, in the Ad-G-AT2R-EGFP-treated rats, the effect of Los was not particularly dose related. It is possible that the increased levels of AT2R in this group have shifted the response curve such that it would take a broader range of doses to see a dose-dependent action of Los. Closer inspection of the data also revealed that the decrease in MAP produced by Los persisted for a longer period in the Ad-G-AT2R-EGFP-treated rats when compared with the control (Ad-CMV-EGFP) group of animals. This is illustrated by the example given in Fig. 5B, which shows the hypotensive action of Los (0.5 mg/kg iv) as a function of time. Seven days after injection of the viral vectors, at which time the overexpressed AT2R are no longer detectable in kidney and blood vessels (Fig. 3), there was no significant difference in the effects of Los (0.5 mg/kg iv) between the Ad-CMV-EGFP- and Ad-G-AT2R-EGFP-treated rats (Fig. 5C).
To determine whether the hypotensive action of Los included an AT2R influence, we tested the effects of AT2R antagonism during the reduction in MAP produced by Los. In rats that had received either Ad-CMV-EGFP or 0.9% saline intracardially 5 days earlier, Los (1.0 mg/kg iv) produced a fall in MAP that was unaltered by infusion of the AT2R-selective blocker PD-123,319 (1.0 mg/kg iv for 30 min) (Fig. 6). However, in the Ad-G-AT2R-EGFP-treated rats, similar administration of PD-123,319 prevented the enhanced hypotensive action of Los (1.0 mg/kg iv) (Fig. 6). Upon cessation of PD-123,319 infusion, the MAP of the Ad-G-AT2R-EGFP-treated rats began to fall again (Fig. 6).
The major findings of this study are the following: 1) the Ad-G-AT2R-EGFP viral construct utilized here produces high levels of AT2R expression in cardiac cells and transduces peripheral tissues following intracardiac injection; 2) the transduction of AT2R in peripheral tissues is transient, reaching a peak at 3 to 5 days; at 7 days post viral injection, the overexpressed AT2R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels); 3) the increased expression of AT2R produces no alterations in AT1R expression; 4) in two separate protocols, peripheral overexpression of AT2R via Ad-G-AT2R-EGFP does not alter basal MAP in the rats made hypertensive by infusion of ANG II but enhances the hypotensive action of Los in rats made hypertensive via subcutaneous infusion of ANG II; 5) the enhanced hypotensive action of Los is no longer apparent when systemic AT2R have returned almost to control (basal) values; and 6) the enhanced hypotensive action of Los produced by Ad-G-AT2R-EGFP is reversed by the AT2R blocker PD-123,319.
Thus the Ad-G-AT2R-EGFP system that has been used here to transiently overexpress AT2R in peripheral tissues has been valuable in that it has helped to provide further evidence for a depressor role of the AT2R. This is consistent with previous pharmacological, gene knockout, and overexpression approaches that have argued for a blood pressure-lowering role of AT2R (3, 6, 13, 17, 23, 26, 27, 31, 45, 54). More importantly, the ability to transiently increase the expression of AT2R in cardiovascular tissues has revealed that the hypotensive action of Los can be increased during elevated expression of AT2R. These findings provide solid evidence to support the idea that concurrent activation of AT2R along with AT1R blockade may be beneficial for hypertensive patients (43, 53). The results presented here also complement previous work from our lab that demonstrated that reduced expression of AT2R resulted in an increase in blood pressure (52).
Despite the above findings, a number of important questions remain. First and foremost concerns the site(s) of action of AT2R in enhancing the hypotensive action of Los. The gene systemic delivery approach that is used here, which involves intracardiac injections of viral vectors, produces overexpression of AT2R at a variety of peripheral tissue sites, including blood vessels and the heart and kidney (Figs. 2 and 3). Thus it is difficult to assess which of these sites may be important for the AT2R action in enhancing the effects of Los. On the basis of the current literature, we might expect that vasodilator actions of the expressed AT2R involve vascular actions at the kidney or at specific vessel beds (8, 28, 32, 36), and targeted gene delivery to blood vessels or other cardiovascular organs would help to reveal which site or sites are more important for this AT2R effect. However, even though the particular site of action of AT2R in increasing the antihypertensive action of Los is not known, the fact remains that AT2R are effective in this regard. A second issue relates to the length of time that the AT2R are overexpressed within cardiovascular tissues. The Ad5 vector system employed here allows for transient overexpression that is useful in experimental situations in that it allows determination of physiological parameters during elevated levels of AT2R and then again when AT2R have returned toward control levels. Such a system may prove useful clinically where transient expression of a protein is required but only if the immune response produced by Ad vectors can be tempered. Clearly, it will be interesting to determine whether longer-term overexpression of AT2R can result in a greater and more sustained enhancement of the hypotensive action of Los. We plan to achieve this through the use of an adeno-associated virus-CMV-AT2R construct that has been developed in our laboratory. Combined use of this construct along with targeted delivery to specific organs/sites may allow us to identify the areas that are most important in this AT2R action. A third issue concerns the mechanism of action of AT2R in lowering MAP. The data presented in the current study do not allow for us to determine the relative contributions of AT2R-mediated alterations in cardiac output and total peripheral resistance to the fall in MAP. However, it appears from the literature that either (or both) mechanism(s) may mediate the observed blood pressure effects of AT2R overexpression (3, 23, 33, 55).
Whereas the data demonstrate that overexpression of AT2R can enhance the decrease in MAP produced by AT1R antagonism, the mechanisms involved are not established. One possibility is that, with the AT1R blocked, the increased levels of plasma ANG II derived from the subcutaneous administration are free to act at the AT2R and produce a depressor action. In addition, it is well known that AT1R blockade via Los results in high circulating levels of ANG II because of a lack of feedback inhibition at kidney juxtaglomerular cells and increased renin secretion (20, 44). Thus we may speculate that the increased vasodilator action of Los in the AT2R-overexpressing (Ad-G-AT2R-EGFP-treated) rats is due to the raised endogenous levels of ANG II (as well as the exogenous subcutaneously delivered ANG II) acting at the overexpressed AT2R. Either case is certainly supported by our data (Fig. 6), which demonstrate that the enhanced hypotensive action of Los in these rats is seen acutely and is rapidly reversed by the AT2R-selective blocker PD-123,319. The observation that PD-123,319 has no effect on the Los-induced decreases in MAP in the control ANG II-infused groups of rats [0.9% saline and Ad-CMV-EGFP] suggests that, in the experimental model of hypertension used here, activation of native AT2R is not at a sufficient level to contribute to the hypotensive action of Los. This is consistent with a report that demonstrates that AT2R do not contribute to the blood pressure-lowering effects of Los in SHR and two-kidney, one-clip hypertensive rats (20).
Another contributing factor that may be considered is that the increased levels of AT2R bind to or dimerize with the AT1R (1) in cardiovascular tissues, further preventing the pressor action of ANG II via AT1R but allowing free AT2R to respond to ANG II with a vasodilator action. However, we think that this possibility is unlikely since the Ad-G-AT2R-EGFP induced-increased expression of AT2R does not alter AT1R binding, at least in vitro (Fig. 1). Furthermore, if AT2R/AT1R dimers had a role to play in this response, we might expect Ad-G-AT2R-EGFP to decrease basal MAP. However, this is clearly not the case since basal MAP and heart rate are unaltered in the AT2R-overexpressing rats when compared with both control groups.
To summarize, the present data support previous findings that describe a depressor role of AT2R. However, the observation that increased expression of AT2R in cardiovascular tissues does not alter basal MAP may indicate that even the higher levels of this receptor are not adequate to offset or influence the actions of ANG II via AT1R or that the increased expression produced here is too transient. Rather, it appears that AT2R actions are unmasked in situations where AT1R activity is depressed, in this case by the AT1R antagonist Los.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-068085 (to C. Sumners, M. K. Raizada, and M. J. Katovich) and the American Heart Association (to M. J. Katovich and J. L. Grobe).
We gratefully acknowledge the technical assistance of Adam Mecca and Jillian Stewart.
↵1 This paper was presented at the 9th Cardiovascular-Kidney Interactions in Health and Disease Meeting at Amelia Island Plantation, Florida, on May 26–29, 2006.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society