Prolonged Reduction in High Blood Pressure With β1 Antisense Oligodeoxynucleotides
Abstract—β-Blockers are widely used for hypertension treatment but must be taken daily. We have developed a novel β-blocker by targeting β1-adrenergic receptor (β1-AR) mRNA with antisense oligodeoxynucleotides (β1-AS-ODN). A single intravenous injection of β1-AS-ODN significantly reduced cardiac contractility and blood pressure (38±5 mm Hg, P<0.05) in spontaneously hypertensive rats for 3 weeks. In the present study, we improved the antihypertensive effect of β1-AS-ODN by delivery with the cationic liposomes DOTAP/DOPE and studied its impact on the peripheral renin-angiotensin system. Five charge ratios (±) of liposome/ODN from 0 to 3.5 were tested to deliver 0.5 mg/kg β1-AS-ODN intravenously in spontaneously hypertensive rats (n=30). On the basis of the magnitude and duration of hypotension, 2.5 was determined to be the optimal charge ratio, which decreased blood pressure by up to 35 mm Hg for 20 to 33 days (P<0.05). The effects were specific for β1-AR, because radioligand binding assay and quantitative autoradiography showed a 35% reduction in β1-AR levels in kidney but no change in β2-AR. β1-AS-ODN diminished the preprorenin mRNA levels in renal cortex by 37% 4 days after administration. This transient effect was followed by a delayed yet marked diminution of plasma renin activity and plasma angiotensin II levels on days 10 and 17 (P<0.01). The results show that β1-AS-ODN has an effective long-term antihypertensive effect up to 33 days with a single intravenous injection. The mechanism appears to be through reduced β1-AR number specifically and reduced cardiac contractility. The inhibition of the renin-angiotensin system is probably a second mechanism to produce the sustained antihypertensive effect of β1-AS-ODN.
Since the introduction of propranolol in 1965, β-blockers have become major first-line drugs for hypertension. Through the inhibition of β-adrenergic receptors in heart and kidney, β-blockers lower high blood pressure via the reduced response to the sympathetic nervous system. However, all current β-blockers have to be taken daily. Also, most have central nervous system side effects that lead to poor patient compliance. Furthermore, the mechanism of β-blockade in hypertension is not well understood.1 Antisense oligonucleotides have been successfully constructed to components of the renin-angiotensin system (RAS) to decrease blood pressure.2 In view of this, we developed a novel antisense oligonucleotide targeted to β1-adrenergic receptors (β1-ARs), with the goal of producing a long-term effect with a single dose and avoiding central nervous system effects.3 It produced a profound and prolonged reduction in blood pressure of spontaneously hypertensive rats (SHR) without affecting heart rate, β2-adrenergic receptors (β2-ARs), or the brain. Therefore, it is likely to have fewer side effects and longer-lasting action.
We have previously shown that antisense oligodeoxynucleotide (β1-AS-ODN) significantly inhibits β1-AR expression in the cardiac ventricles, which results in suppressed inotropic response to adrenergic activation and thereby contributes to hypotension.3 In addition to inducing positive inotropy and chronotropy in the heart, β1-ARs are also responsible for mediating the sympathetic stimulation of renin expression and secretion from juxtaglomerular cells of the renal cortex. Infusion of isoproterenol has been shown to increase renin expression and secretion and plasma renin activity (PRA) in rats.4 β-Blockers reduce PRA in patients.5 However, despite the evidence that β-blockers are more effective in patients with high renin profiles,6 the importance of β-blocker–induced decreases in renin release has been debated.7 The present study had 2 goals. One was to investigate whether β1-AS-ODN reduces renin expression and secretion and whether the RAS is involved in the antihypertensive impact of β1-AS-ODN. The second was to test whether the antihypertensive effect of β1-AS-ODN could be improved by delivery with cationic liposomes and to determine the optimal charge ratio of liposome:ODN.
Antisense Sequence and Delivery
AS-ODN and inverted ODN control were 15-mer and targeted to the AUG start codon of rat β1-AR mRNA.8 The sequence of AS-ODN is 5′-CCGCGCCCATGCCGA-3′, and the inverted ODN is 5′-AGCCGTACCCGCGCC-3′.3 These ODNs were modified by backbone phosphorothioation. The cationic lipid 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) mixed with the helper lipid l-α-dioleoyl phosphatidylethanolamine (DOPE, Avanti Polar Lipids) at 1:1 molar ratio was used to deliver ODNs in a single intravenous injection into the tongue vein. ODN-liposome complex was prepared on the day of use by mixing desired amounts of ODNs with DOTAP/DOPE to a final DNA concentration of 300 μg/mL in 5% (wt/vol) dextrose in water and incubating at room temperature for 60 minutes.
Adult male SHR (4 to 6 months old, Harlan, Indianapolis, Ind) were kept in cages in a room with a 12-hour light-dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum. Tail blood was collected for determination of PRA and angiotensin II (Ang II) levels. All procedures were approved by the University of Florida Animal Care Committee.
Blood Pressure Measurement
Blood pressure was measured by the tail-cuff method as previously described.3 Systolic blood pressure (SBP) was determined as the first pulsatile oscillation on the descending side of the pressure curve. Data values of each rat were taken as an average of ≥4 stable readings. Baseline was determined by averaging 3 days of measurements before antisense administration.
Membrane Preparation and β-Adrenergic Receptor Binding Assay
Four days after intravenous injection of saline (n=6) or 0.5 mg/kg inverted ODN (n=6) or 4, 10, 18, and 40 days after injection of 0.5 mg/kg β1-AS-ODN (n=24), animals were euthanized, and membranes were prepared from the renal cortex of left kidneys as previously described.9 For saturation experiments, 100 μg membrane protein was incubated in triplicate with 6 concentrations of [125I](−)-iodocyanopindolol (ICYP, NEN Life Science, 6.25 to 100 pmol/L) in a total volume of 250 μL containing 50 mmol/L Tris-HCl (pH 7.4) and 5 mmol/L MgCl2 at 36°C for 60 minutes. The nonspecific and β2-adrenergic receptor binding levels were determined in the presence of 1 μmol/L (±)-alprenolol and 150 nmol/L CGP20712A (RBI), respectively. Then the reaction mixture was passed through a Whatman GF/B glass fiber filter with a Brandel harvester, and the bound radioactivity was counted for 1 minute.
Tissue Preparation and Quantitative Autoradiography
Four days after injection of 0.5 mg/kg β1-AS-ODN (n=6) or saline (n=6), rats were euthanized, and the right kidneys were removed and frozen in dry ice. Sagittal sections of kidney (20 μm) were cut on a cryostat (Microm) at −20°C and mounted on microscope slides. Every seventh slide was stained with hematoxylin and eosin for histology. Receptor autoradiography was performed as described10 with 100 pmol/L ICYP at 25°C for 150 minutes in the presence of 1 μmol/L (−)-propranolol, 100 nmol/L ICI118,551 (β2-selective antagonist), or 100 nmol/L CGP20712A (β1-selective antagonist) to distinguish nonspecific, β1-, and β2-bindings. The images were quantified with a computerized image analysis system (MCID, Imaging Research) and normalized with 125I standards. Nonspecific binding was <10% of total binding.
Reverse Transcription–Polymerase Chain Reaction and Southern Blotting
At different time points after the single injection of β1-AS-ODN or inverted ODN, rats were euthanized, and the renal cortex was dissected from the left kidneys, immediately dipped into RNAlater tissue storage buffer (Ambion), and stored at −20°C. Total RNA was extracted with RNAwiz reagent (Ambion) and quantified by spectrophotometer. RNA samples from 4 to 5 rats from each time point were pooled. RNA (5 μg) was digested by DNase I and reverse-transcribed by Superscript reverse transcriptase (GIBCO BRL) at 42°C for 50 minutes, and 1/20 of the reverse transcription (RT) product was used to run a polymerase chain reaction (PCR) for 20 cycles. PCR primers for β1-AR were 5′-CTCCGAAGCTCGGCATGG-3′ (forward) and 5′-GCACGTCTACCGAAGTCCAGA-3′ (reverse) and yielded products of 432 bp, which spanned the AUG start codon. Primers for preprorenin were 5′-AGGCAGTGACCCTCAACATTACCAG-3′ (forward) and 5′-CCAGTATGCACAGGTCATCGTTCCT-3′ (reverse) and yielded products of 362 bp. Primers for GAPDH were 5′-ATCAAATGGGGTGATGCTGGTGCTG-3′ (forward) and 5′-CAGGTTTCTCCAGGCGGCATGTCAG-3′ (reverse) and yielded products of 505 bp.11 RT-PCR products were subjected to Southern blotting, hybridized with psoralen-biotin–labeled cDNA probes, and detected with nonisotopic kits (Ambion). After the membranes had been exposed to x-ray films, the intensity of β1-AR and preprorenin mRNAs was quantified by densitometry and normalized with GAPDH mRNA levels. The experiments were repeated at least twice.
PRA and Plasma Ang II Levels
PRA was determined with an angiotensin I (125I) radioimmunoassay kit (DuPont). Plasma Ang II levels were measured by radioimmunoassay as previously described.12
Values were expressed as mean±SEM. Differences were considered statistically significant at a value of P<0.05. One-way repeated ANOVA and Tukey’s test were used to compare blood pressure before and after AS-ODN treatment. Unpaired t test was used to compare Bmax, PRA, and plasma Ang II levels in 2 groups.
Optimization of β1-AS-ODN Delivery by Cationic Liposomes
Systemic delivery of AS-ODN was optimized with the commercially available cationic lipid DOTAP mixed with neutral lipid DOPE. Previous studies reported that a charge ratio of DOTAP:DNA of ≈2.0 achieved the best gene delivery in vitro and in vivo.13 14 Therefore, we chose to test 5 charge ratios of DOTAP:ODN ranging from 0 to 3.5 to deliver 0.5 mg/kg β1-AS-ODN intravenously. It was noticed that different batches of liposome mixture varied slightly in structure and particle size, which may influence the delivery efficiency. Figure 1⇓ shows the effect of different liposome:ODN charge ratios on blood pressure of SHR (n=6 for each ratio) in a representative experiment. β1-AS-ODN alone, ie, at ratio 0, did not change SBP, whereas ratio 0.5 significantly reduced SBP by up to 38 mm Hg for 7 to 8 days. When the ratio was increased, the duration of the hypotensive impact was drastically prolonged to 20 days at ratio 1.5 and 33 days at ratio 2.5 and 3.5, varying with liposome preparations. But the maximum drop in SBP was greater at ratio 1.5 and 2.5 (≈35 mm Hg) than at ratio 3.5 (≈25 mm Hg) (Table⇓). Accordingly, the optimal charge ratio of DOTAP:ODN was determined to be 2.5. In the subsequent experiments, SHR (n=24) injected with 0.5 mg/kg β1-AS-ODN with liposomes at a charge ratio of 2.0 were analyzed for the time course of changes in SBP, receptor levels, and peripheral RAS.
Effects of β1-AS-ODN on β-Adrenergic Receptors in Renal Cortex
Scatchard analysis of β-AR binding in renal cortex (Figure 2⇓) indicated that β1-AR was the major subtype in the control rats, composing 70% of total β-ARs. After β1-AS-ODN injection, the Bmax of β1-ARs was diminished significantly, by 35% on day 4 (P<0.05), 29% on day 10 (P<0.05), and 23% on day 18, and completely restored on day 40. β1-AR reduction in kidney coincided with that in heart,3 and both were accompanied by a significant drop in SBP (P<0.01) (Figure 2A⇓). In contrast, the β2-AR level was not affected (Figure 2B⇓), nor was the affinity of either subtype (data not shown). Inverted ODN had no effect on either subtype (Figure 2C⇓).
Kidney slices from the same rats were subject to quantitative autoradiography to display the structural distribution of β-ARs (Figure 3⇓). β1-Subtype composed ≈60% of the β-AR levels, which was localized predominantly in the renal cortex and the outer band of the medulla. β2-Subtype was more diffusely distributed in the kidney at a lower level. This result was consistent with previous reports.15 Four days after β1-AS-ODN treatment, the overall density of β1-subtype in kidney was significantly reduced from 23.5±2.1 to 15.4±3.3 fmol/mg (P<0.05). The diminution in renal cortex was particularly conspicuous because of the higher basal level. As expected, the distribution and concentration of β2-subtype remained unchanged in accord with binding results. This further confirms the specificity of the inhibitory effect of β1-AS-ODN on β1-subtype.
In an effort to demonstrate whether β1-AS-ODN decreases the mRNA level of β1-AR by inducing RNase H digestion, a pair of primers flanking the AUG start codon where β1-AS-ODN was targeted was used to run a semiquantitative RT-PCR for 20 cycles, followed by Southern blotting. Figure 4⇓ shows that β1-AS-ODN did not reduce the level of steady-state β1-AR mRNA in renal cortex, indicating that the inhibition of β1-AR expression was not at the transcriptional level.
Effects of β1-AS-ODN on Peripheral RAS
RT-PCR (Figure 4⇑) revealed that the preprorenin mRNA level in renal cortex was transiently decreased to 62% of control 4 days after β1-AS-ODN injection. It was completely reversed by day 18 (Figure 5A⇓). Conversely, PRA and plasma Ang II levels showed different patterns of reduction, which were significantly decreased on day 10 and day 18 (P<0.01) but not on day 4. Thus, PRA and Ang II seemed to have a delayed action relative to the reduction in renin mRNA (Figure 5⇓).
We previously reported the development of a specific and effective β1-AS-ODN that was able to lower blood pressure in SHR by 38±5 mm Hg after a single intravenous administration. Its significant hypotensive effect was associated with the rapid decrease in the positive inotropic response of cardiac β1-adrenergic receptors to sympathetic stimulation.3 Because β1-adrenergic receptors are also involved in renin expression and secretion from the kidney, the present study was designed to evaluate the effect of β1-AS-ODN on peripheral RAS and its contribution to the reduction in blood pressure. In addition, a significant improvement of hypotensive action up to 33 days was achieved by optimizing the delivery of β1-AS-ODN with cationic liposomes.
β-Blockers have been used to treat hypertension for 3 decades. The reasons for their antihypertensive effects remain largely unclear, but the inhibition of renin release is regarded as a primary mechanism. Many β-blockers can reduce PRA in patients and experimental animals.5 16 They are found to be more effective in patients with higher renin profiles.6 In our study, we find that β1-AS-ODN effectively decreased PRA and Ang II in the long term. But the decrease in PRA and Ang II did not occur until ≈10 days after β1-AS-ODN injection, in contrast to the rapid drop in cardiac output 2 days after injection.3 Thus, it appears that the effects of β1-AS-ODN on the kidney renin and the circulating RAS are more delayed than cardiac action. We speculate that the suppression of cardiac output may account for the early phase of the antihypertensive effect of β1-AS-ODN, whereas the inhibition of renin-angiotensin activity acts as the secondary mechanism underlying the sustained reduction of blood pressure in SHR.
Receptor binding assay showed that β1-AS-ODN reduced the β1-AR levels in renal cortex by ≈30% for 18 days. This is consistent with the decrease of β1-AR in heart ventricles in magnitude and time course.3 This suggests that β1-AS-ODN delivered by cationic liposomes is rapidly transported to peripheral organs after intravenous injection and effectively taken up into heart and kidney cells to a comparable extent. Several mechanisms have been proposed for the AS-ODN inhibition of the expression of target proteins. One involves the decrease in mRNA levels resulting from RNase H digestion of the RNA strand of the RNA-DNA duplex.17 18 To test this hypothesis, we designed a pair of primers that flanked the AUG start codon of β1-AR mRNA where AS-ODN bound to perform semiquantitative RT-PCR. As shown in the Results, there was no reduction in the RT-PCR products, indicating the absence of RNase H action. Therefore, the inhibition of β1-AR expression probably occurs in posttranscriptional steps.
By decreasing β1-AR levels in kidney, β1-AS-ODN significantly reduced PRA and the subsequent plasma Ang II levels. This is unlikely to be through the inhibition of renin expression, however, because there is no long-term diminution of renin mRNA levels. Instead, β1-AS-ODN may exert its inhibitory impact on renin secretion or the conversion of inactive renin to active renin. This hypothesis is consistent with the observation that β-adrenergic stimulation of renin expression had a time course different from that of renin secretion.4 19 Furthermore, β-blockers have been shown to reduce prorenin processing to active renin without changing total renin (prorenin+PRA) levels in plasma.5
Efficient gene delivery is vital to the therapeutic application of AS-ODN in vivo. Among nonviral vectors, cationic liposomes are the most widely used. They are safe, nonimmunogenic, and easy to produce on a large scale. However, relatively low transfection efficiency has been obtained after intravenous administration, mainly because of the inactivation of cationic liposome by serum. It was recently shown that increasing the charge ratio (±) of liposome to DNA and inducing the maturation of liposome-DNA complex by prolonging incubation time can overcome this problem.13 20 The optimal charge ratio of DOTAP:DNA was demonstrated to be ≈2.13 14 Thus, we chose to test 5 charge ratios ranging from 0 to 3.5 to optimize the AS-ODN delivery. As shown in the results, increasing the charge ratio not only improved the delivery efficiency but also prolonged the duration of β1-AS-ODN action. The best antihypertensive result (−35 mm Hg for 33 days) was consistently achieved at ratio 2.5.
In summary, β1-AS-ODN delivered with cationic liposomes at a single intravenous injection achieves a marked and sustained hypotensive effect (30 to 35 mm Hg for 33 days) in SHR. The β1-AS-ODN is clearly longer-lasting than any current drug, does not inhibit β2-adrenergic receptors or cross the blood-brain barrier, and has negligible effect on heart rate.3 Therefore, the antisense is likely to have fewer side effects than currently used β-blockers. Inhibition of cardiac contractility initially followed by reduced renin release are important mechanisms contributing to its antihypertensive effects.
This study was supported by NIH Merit Award HL-27344 and American Heart Association Fellowship 9850002FL.
- Received September 14, 1999.
- Revision received October 29, 1999.
- Accepted November 16, 1999.
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