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1 Department of Physiology and Functional Genomics, College of Medicine2 Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA
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Abstract |
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(Received 23 January 2004;
accepted after revision 4 March 2004; first published online 16 March 2004)
Corresponding author M. J. Katovich: College of Pharmacy, Box 100487 JHMHC, University of Florida, Gainesville, FL 32610, USA. Email: TUkatovich{at}cop.ufl.edu
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Introduction |
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It has been suggested that AT2 receptors mediate some cardiovascular effects that are opposite to those of the AT1 receptor. If true, then alteration of the ‘balance’ between AT1 and AT2 receptors could have significant ramifications in normal cardiovascular physiology and associated diseases. Most of our knowledge of the function of the AT2 receptor has been obtained from transgenic mouse models (Hein et al. 1995; Siragy et al. 1999; Ichiki et al. 1995; Hunley et al. 2000). These ‘knockout’ models are important to identify redundant systems that replace the AT2 receptor, but may not be the ideal model to understand the role of this receptor in normal cardiovascular control and in hypertension, since the AT2 receptor has been implicated in developmental processes. Animals born without these receptors may have some abnormal or compensatory physiology to maintain normal BP homeostasis. We and others have used a slightly different approach to elucidate functions of the RAS. That is, the use of gene therapy interventions to ‘knockdown’ components of the RAS after birth (Iyer et al. 1996; Lu et al. 1997; Martens et al. 1998; Wang et al. 2000; Pachori et al. 2000, 2002; Kagiyama et al. 2001; Kimura et al. 2001; Reaves et al. 2003). Using such an approach with antisense to the AT1 receptor (AT1R) we have shown that AT1R number is selectively decreased by 25–45% without affecting the AT2 receptor (Iyer et al. 1996; Lu et al. 1997). Therefore, the purpose of the present study was to use this gene therapy approach to reduce, not eliminate, AT2R number after the completion of embryonic development, and to determine the effects of this under expression on BP and dipsogenic responses.
Early studies using pharmacological antagonists of the AT2 receptors concluded that this receptor was not involved in BP regulation in normotensive or hypertensive animals (Tofovic et al. 1991; Toney & Porter, 1993). Results obtained from an AT2R ‘knockout’ mouse, however, suggested a cardiovascular role for the AT2 receptor. Adult knockout mice exhibit an increased BP response to Ang II, while basal BP was reported to increase or remain unchanged (Hein et al. 1995; Ichiki et al. 1995; Siragy et al. 1999; Hunley et al. 2000). These knockout animals also displayed an altered dipsogenic response (Hein et al. 1995), as well as other behavioural modifications (Hein et al. 1995; Ichiki et al. 1995; Siragy et al. 1999; Hunley et al. 2000). However, these observed changes in BP and dipsogenic responses in the AT2 receptor mutant mice may be caused by compensatory changes in the expression of other genes rather that the loss of the AT2 receptor. The goal of the present study was to determine whether use of the aforementioned antisense ‘knockdown’ approach could provide insight into the role of the AT2 receptor in the normal BP and dipsogenic responses in the rat. This approach should obviate some of the compensatory mechanisms associated with abolishment of the entire receptor population.
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Methods |
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Antisense (AT2R-AS) and missense (AT2R-MS) constructs of the AT2 receptor were constructed by the creation of a linker (Hind III and Cla I) on the 5' and 3' ends. The following primers were used to generate these AT2 receptor constructs.
Primers 1 and 2 were used for the AT2R-AS construct. Primers 3 and 4 were used to produce an AT2R-S (sense) construct, and an AT2R-MS construct was produced from this by a one base pair deletion at N-terminal position (+1). Cloning was carried out as described elsewhere for AT1R-AS constructs (Lu & Raizada, 1995; Lu et al. 1995). Characterization and orientation of clones was established by restriction enzyme analysis and by sequencing the AT2 receptor constructs (Lu & Raizada, 1995; Lu et al. 1995).
Recombinant retroviral vector construction
Figure 1 illustrates the cloning strategy used to insert the AT2R-AS gene into the murine leukemia virus (MLV)-based long terminal repeats, Neomycin selection, Simian Virus (LNSV) genome. cDNA was inserted into the vector using the Hind III and Cla I sites. The insert was under the control of the SV40 promoter. Neo is a G418-resistant gene and served as a drug selection marker. AT2R-S and AT2R-MS constructs were cloned in a similar manner.
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To create a stable virus -producing cell line, recombinant LNSV retroviral vector DNA was transfected into PA317 packaging cells. Forty-eight hours after transfection, cells were subcultured and subjected to G418 (800 µg ml–1) selection, the Neo-resistant cell clones were isolated and expanded. Vial medium titre was determined by infection of NIH3T3 cells. Culture medium containing 106–107 cfu ml–1 virus particles was collected and concentrated to produce 107–109 cfu ml–1 virus particles at 20 000 g for 2 h at 4°C, essentially as previously described by Wang et al. (2000).
Cell cultures.
PC12 rat pheochromocytoma cells were obtained frozen from American Type Culture Collection (Manassas, VA, USA) and were cultured at 37°C in Ham's F12K medium with 2 mm L-glutamine adjusted to contain 1.5 g l–1 sodium bicarbonate, 15% horse serum and 2.5% fetal bovine serum according to the directions supplied by the company. Cultures were used when confluent (
10 days). Neuronal cocultures of newborn normotensive rat hypothalamus and brainstem were prepared and maintained as detailed previously (Sumners et al. 1990, 1991). Cultures were grown for 10–14 days in media containing 10% plasma-derived horse serum (Sigma, St Louis, MO, USA) before use. Chinese Hamster ovary cells (CHO) were obtained from Dr Peter Sayeski (University of Florida) and infected with the AT2 receptor. Cells were grown in Ham's F-12 media supplemented with 10% fetal bovine serum (Cellgro, Herndon, VA, USA). Once grown to confluency, cells were split, plated and transduced with the LNSV containing either the AT2R or GFP (green fluorescent protein; 6 x 105 cfu ml–1) in the growth media for 48 h before the determination of AT2 receptor number. PBS in the growth media was used as a control.
Infection of PC12 cells and expression of AT2R-AS. PC12 cells were infected with viral particles (1 x 106 cfu ml–1) containing either AT2R-MS or AT2R-AS for 48 h (Lu & Raizada, 1995). Cells were dissociated with trypsin and subcultured. Five days following subculture, total RNA was isolated and subjected to RT-PCR (Lu & Raizada, 1995). In brief, 5 µg of total RNA was subjected to reverse transcriptase reaction at 42°C for 50 min in a total 20 µl. Two microlitres of this RT solution was used for polymerase chain reaction (PCR). The profile of PCR is initially 94°C for 2 min, and then 30 cycles of 94°C for 50 s, 58°C for 50 s and finally 74°C for 1 min. The expression of AT2R-MS and AT2R-AS was determined using primer pairs 5/6 and 5/7, respectively.
Primer 5 was located in the viral vector just in front of the insert. Primers 6 and 7 were located in the end of the antisense and missense insert. Thus, the primer combination will not pick up endogenous AT2R, but will pick the transcript from the vector. PA317 cells transfected with AS or MS constructs were used as positive controls while uninfected cells were used as negative controls.
Infection of neuronal cultures and expression of endogenous AT2R mRNA. Primary neuronal cultures from newborn normotensive rat brain were infected with viral particles (1 x 109 cfu ml–1) containing either AT2R-MS or AT2R-AS. After day 4 of cell culture, endogenous AT2 receptor mRNA was analysed by semiquantitative RT-PCR using primer pairs 8 and 9 (Lu & Raizada, 1995).
Angiotensin II receptor binding. Specific binding of 125I-SI-Ang II to the AT2R was performed as previously described (Raizada et al. 1993) in CHO-AT2 cells. Cells were treated with PBS, LNSV congugated to green fluorescent protein (GFB) or LNSV-AT2R-AS for 48 h. Briefly, transduced CHO cells were washed with Dulbecco's phosphate-buffered saline (Cellgro, Herndon, VA, USA). Cells were incubated, in triplicate, for 30 min in 0.4 ml PBS containing 0.1 nmol l–1125I-SI-Ang II (Washington University) and 0.5% bovine serum albumin (Sigma). All incubations were performed in the presence of 1 µM Losartan to block AT1 receptors, so the specific[125I]-Ang II receptor binding represents entirely AT2 receptors. Following incubations, cultures were rapidly rinsed 3 times in ice-cold PBS, removed from the dishes and placed into a plastic tube and radioactivity levels were determined using a Beckman 5500 gamma counter.
In vivo studies
Animals. The University of Florida Institutional Animal Care and Use Committee approved the use of animals for these experiments. Neonatal Sprague–Dawley (SD) rats were divided into three or four experimental groups depending on the specific experiment: virus alone (LNSV) containing no transgene; virus containing AT2R-AS; virus containing AT2R-MS; and saline controls (Saline). Previously we have demonstrated that neither of the control groups differed from saline treatment (Iyer et al. 1996; Lu et al. 1997; Pachori et al. 2000, 2002). All animals were littermates and the treatments were administered in a 25 µl volume into the ventricular chamber (cardiac administration) while animals were anaesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL, USA) as previously described (Iyer et al. 1996; Lu et al. 1997; Martens et al. 1998; Pachori et al. 2000; Reaves et al. 2003). Following injections the animals were tagged and returned to their original mothers. The survival rate 24–48 h after viral administration was near 95%. There were no mortalities in any group after the pups were accepted by the mothers.
Two separate in vivo studies were performed. In the first pilot study, male and female SD animals were administered either AT2R-AS or AT2R-MS 5 days after birth. The concentration of the viral titre was 5 x 108 cfu ml–1. When animals were approximately 90 days old, direct BP and pressor responses to I.V. Ang II (0.02, 0.04, 0.08 and 0.16 µg kg–1) and Ang I (0.1 and 1.0 µg kg–1) were determined in instrumented, awake, unrestrained animals as previously described (Iyer et al. 1996; Lu et al. 1997; Martens et al. 1998; Pachori et al. 2000). Briefly, animals were anaesthetized with a rodent cocktail containing ketamine (100 mg ml–1) and xylazine (20 mg ml–1), administered I.M. (0.7 ml kg–1). A SilasticTM catheter (Helix Medical) was implanted into the jugular vein for drug infusion, and the carotid artery was cannulated with a PE-50 catheter (Clay Adams) for direct BP determination. Both catheters were exteriorized between the shoulder blades, filled with a heparin solution (10 U ml–1; Elkins-Sinn, Inc.), and sealed with stylets. After a recovery period of 24–48 h, direct BP was recorded in free-moving, non-restrained animals with a pressure transducer coupled to a Digi-Medical BP analyser (Micro-Medical). After a 30 min equilibration period, the pressor response to Ang II or Ang I (administered I.V. in a volume of 250 µl (kg body weight)–1) was determined. After each dose of Ang, either saline or the next higher dose of Ang was administered when the animals returned to baseline. No significant pressor responses (0–6 mmHg) were observed with the administration of saline in any group, as we have previously demonstrated (Lu et al. 1997). Maximal pressor responses for Ang II over baseline BP were calculated to produce a response curve for the two groups. Expression of AT2R-MS and AT2R-AS transcript was determined by RT-PCR in selected tissue at the time of killing as described above.
In the second study, two separate groups of SD male rats were evaluated. Group 1 animals were administered a high viral titre (1.4 x 108 cfu ml–1) of AT2R-AS 5 days after birth, while group 2 animals were administered a lower viral titre of the AT2R-AS (8.2 x 107 cfu ml–1) on day 5. All animals were littermates and two different controls were used: saline or empty LNSV. When animals reached adulthood (75–90 days old) physiological studies were conducted. Initially, all animals were dehydrated for 20 h and water intake measured hourly for the subsequent 2 h. Dipsogenic responses were also evaluated to peripheral administration of angiotensin I (150 µg kg–1, S.C.) and angiotensin II (100 and 200 µg kg–1, S.C.). Water intake was determined as previously described (Iyer et al. 1996) and expressed as millilitres consumed per kilogram body weight for the first hour. When animals were approximately 90 days of age, direct BP measurements were made as previously described (Iyer et al. 1996; Lu et al. 1997; Martens et al. 1998; Pachori et al. 2000).
Statistics
All results are expressed as means ±S.E.M. Direct BP and the dipsogenic responses were analysed by one-way ANOVA. In vivo pressor responses to Ang II were also analysed by repeated-measures ANOVA. Values of P
0.05 were considered statistically significant. Number of animals in each group/treatment of each experiment consisted of at least 6, unless otherwise indicated.
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Results |
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As shown in Fig. 2A, infection with the LNSV viral vector/AT2R constructs causes a robust expression of AT2R-AS and AT2R-MS in PC12 cells. The transgene expression was considered long term since it was observed in five subsequent subcultures of these cells. The data shown in Fig. 2B indicate that AT2R-AS, but not AT2R-MS, causes a 70–80% decrease in the endogenous AT2 receptor mRNA levels in normotensive rat neuronal cultures. Figure 3 summarizes the result from AT2 binding studies in the CHO-AT2 cell line. LNSV-AT2R-AS, but not LNSV-GFP, produced a significant reduction in the levels of AT2 receptor binding (Fig. 3). In summary, these data indicate that cells in culture can be infected with the AT2R constructs, and that the AT2R-AS is effective in reducing AT2R expression in vitro.
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In the first in vivo study, AT2R-AS treatment (5 x 108 cfu ml–1) was without effect on body weight; however, direct BP measurements did reveal that the AT2R-AS-treated animals had significantly elevated BP compared to the missense controls. Significance was observed with systolic (Fig. 4A, P < 0.01) and mean BP (121.2 ± 5.3 mmHg in AT2R-AS versus 103.3 ± 4.9 mmHg in AT2R-MS; P < 0.03) but not heart rate (346 ± 11 versus 347 ± 10 beats min–1 in AT2R-AS and AT2R-MS- treated groups, respectively; P= 0.956) or pulse pressure (29.1 ± 2.2 versus 34.6 ± 5.2 mmHg in the two groups, respectively; P= 0.40). Diastolic BP was elevated to 105.9 ± 6.3 mmHg in the AT2R-AS-treated animals (n= 6), when compared to the missense controls (84.3 ± 7.6 mmHg, n= 8, P= 0.056). Figure 4B summarizes the systolic BP response to dose-related increases in Ang II. Repeated-measures ANOVA revealed significant dose (P= 0.0007) and treatment (P= 0.04) effects. The AT2R-AS-treated animals displayed an enhanced pressor response to Ang II compared to the AT2R-MS-treated rats. When challenged with a 0.1 µg kg–1 dose of Ang I, AT2R-AS-treated animals displayed a 16 mmHg elevation in BP over that of the missense controls, and a 10 mmHg increase at a 1.0 µg kg–1 dose; however, these differences did not reach statistical significance. This lack of significance may be related to the small numbers of animals and the use of both males and females in this particular study. Expression of the transgenes (AS and MS) was maintained in heart, kidney, lung and brain 90 days after viral administration (Fig. 5).
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Discussion |
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The in vitro studies demonstrated that using a retrovirus -mediated delivery approach we can introduce AT2R-AS into cell culture and have the transcript expressed. Additionally, this transgene can be expressed in infected cells in culture and significantly reduce both the mRNA and number of AT2R. These results parallel those that we have previously reported with retroviral antisense treatment with AT1R-AS (Lu et al. 1995, Lu & Raizada, 1995a. In vivo studies with the AT1R-AS demonstrated that expression of AT1R was selectively decreased by 40–60% when a titre of 5 x 108 cfu ml–1 was administered to 5-day-old rats (Iyer et al. 1996; Lu et al. 1997). The spontaneously hypertensive rats (SHR) harbouring the AT1R-AS failed to develop hypertension and cardiac hypertrophy (Iyer et al. 1996; Lu et al. 1997; Martens et al. 1998). Likewise, Pachori et al. (2002) demonstrated that in the renin-transgenic rat (TGR, mRen2), treated with a 1 x 108 cfu ml–1 titre of the AT1R-AS, systolic BP was significantly reduced and these animals did not develop cardiac hypertrophy. Recently, Reaves et al. (2003) also demonstrated a reduced dipsogenic response to Ang II and a reduced BP and cardiac hypertrophy in deoxycorticosterone acetate/salt (DOCA/NaCl) animals harbouring the AT1R-AS. The results reported in the present study are the first using the AT2R-AS in place of the AT1R-AS transgene.
Inagami's group (Ichiki et al. 1995) was the first to report that mice lacking the angiotensin type 2 receptor developed a significantly higher BP (about 24 mmHg) than control mice. These knockout mice also displayed an increased sensitivity to the pressor actions of Ang II. At the same time, Hein et al. (1995) also deleted the AT2 gene and also observed an increased sensitivity to Ang II; however, they did not see any increase in basal BP. These differences may be due to differences in mouse strains or in the measurement techniques used to measure BP. Our results suggests that a reduction in the AT2R in rats elevates systolic BP by about 10 mmHg with a viral titre of 1.4 x 108 cfu ml–1 and a 17 mmHg increase with a slightly higher titre of antisense. Further support for a BP-lowering effect of the AT2R comes from a recent study using antisense oligodeoxynucleotides (AS-ODN) to the AT2 receptor, which also resulted in a 21 mmHg increase in systolic BP in the conscious rat (Moore et al. 2001). These animals had a 40% decrease in the expression of AT2 receptors in the treated kidney. In the present study no BP effects were observed when the lowest titre of antisense was administered. This may be a result of either insufficient integration of the antisense or less of a reduction in AT2 receptor number. Further experiments would be required to determine this lack of response; although similar lack of effects have been observed in other systems when lower concentrations of pharmacological antagonists are used.
The increase in pressor responses to Ang II observed in our study is similar to that observed by several others using the AT2 null mouse (Hein et al. 1995; Ichiki et al. 1995; Siragy et al. 1999; Hunley et al. 2000). These results would suggest that the AT2 receptor could serve to limit the vascular response of the AT1 receptor to Ang II. Unfortunately, studies using the AT2-specific antagonist PD123319 have not been able to consistently observe an effect on BP (Tofovic et al. 1991; Toney & Porter, 1993). Lack of consistency may be related to the relatively short half-life of the antagonist and/or the dose of antagonist used or even the age of the animal. The fact that our results are similar to those of the AT2 null mice would suggest that the AT2 receptor does play an opposing role to AT1R in the vasculature. Further support for this role of AT2 receptors on vascular response is the inhibition of Ang II pressor actions in animals that overexpress the AT2R (Matsubara, 1998). The mechanism(s) have yet to be elucidated; however, several recent studies have demonstrated that AT2R-mediated activation of the kinin and nitric oxide systems occurs in several tissues of the rat (Carey et al. 2000a; Siragy et al. 1999; Moore et al. 2001; Matsubara, 1998). It also has been suggested that NO production is involved in the AT2R-mediated pressure diuresis and natriuresis (Carey et al. 2000a; Siragy et al. 1999). In addition, Hunley et al. (2000) suggested that inhibition of AT2 receptor function increases ACE activity. Thus, BP could be elevated in the AT2-deficient animal by increasing ACE levels. Tanaka et al. (1999) also suggested that the increased vascular response observed in AT2R knockout mice might be a result of an up-regulation of AT1 receptors. Recently Zisman et al. (2003) demonstrated a direct correlation with the AT2 receptor number and Ang(1–7) concentration. Thus, a reduction in AT2 receptor may decrease the vasodilatory actions of Ang(1–7) and thus elevate BP. We did not assess the concentration of AT2R in the present study. The basal level in adult animals is low and, since we are not sure of the precise mechanism of action of this ‘knockdown’ approach, we were not able to assess which tissue to evaluate. In future studies with this AT2‘knockdown’ model these potential mechanism(s) need to be evaluated.
Hein et al. (1995) initially reported that AT2R null mice had an impaired drinking response to 40 h water deprivation, whereas there was no difference in the daily water intake. We observed similar effects in our antisense-treated rats following 20 h water deprivation. This simple, noninvasive testing procedure may be a quick way to screen for reduction of AT2R. Similar depression in water intake after dehydration has been reported in rats pretreated with specific antagonists of the AT2R (Rowland & Fregly, 1993; Lee et al. 1996). The reduced dipsogenic response to Ang II observed in the present study also has been reported in animals treated with a selective AT2R antagonist (Alova et al. 1999). These results would suggest that both the traditional AT1 as well as the AT2 receptor might mediate the effects of angiotensin on water balance. Thus, thirst would be one variable where the two receptor subtypes do not appear to work in opposition to each other. This hypothesis was recently confirmed by Li et al. (2003). They used Ang II receptor-deficient mice and demonstrated that there was an antagonistic action between central AT1 and AT2 receptors in the regulation of BP, but that they act synergistically in the regulation of water intake induced by Ang II. Lastly, the altered dipsogenic effect observed in the present study suggests that the retroviral antisense treatment has central effects. This is supported by the fact that the AS transcript is found in the hypothalamus and may suggest that the BP effects are also mediated at least in part by some central mechanism. One aspect not addressed in the present study, nor in the previous dipsogenic studies using AT2R antagonists or the AT2R knockout mouse, is the BP response to Ang II and how it may effect the dipsogenic response. Intracerebroventricular administration of Ang II may reduce the possible confounding cardiovascular effects of peripheral administration of angiotensin on the dipsogenic responses. Future studies also need to evaluate the specific tissue localization of this transgene and to determine concentration of both the AT1 and AT2 receptor numbers in the particular tissue(s) in order to obain a better understanding of the physiological role of the AT2 receptor in vivo.
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References |
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  W. Vongpatanasin, G. D. Thomas, R. Schwartz, L. A. Cassis, S. Osborne-Lawrence, L. Hahner, L. L. Gibson, S. Black, D. Samols, and P. W. Shaul C-Reactive Protein Causes Downregulation of Vascular Angiotensin Subtype 2 Receptors and Systolic Hypertension in Mice Circulation, February 27, 2007; 115(8): 1020 - 1028. [Abstract] [Full Text] [PDF]
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 S. McMullen and S. C. Langley-Evans Sex-Specific Effects of Prenatal Low-Protein and Carbenoxolone Exposure on Renal Angiotensin Receptor Expression in Rats Hypertension, December 1, 2005; 46(6): 1374 - 1380. [Abstract] [Full Text] [PDF]
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T. Matsuura, H. Kumagai, H. Onimaru, A. Kawai, K. Iigaya, T. Onami, K. Sakata, N. Oshima, T. Sugaya, and T. Saruta Electrophysiological Properties of Rostral Ventrolateral Medulla Neurons in Angiotensin II 1a Receptor Knockout Mice Hypertension, August 1, 2005; 46(2): 349 - 354. [Abstract] [Full Text] [PDF]
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 M. J. Katovich, J. L. Grobe, M. Huentelman, and M. K. Raizada Angiotensin-converting enzyme 2 as a novel target for gene therapy for hypertension Exp Physiol, May 1, 2005; 90(3): 299 - 305. [Abstract] [Full Text] [PDF]
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