New β-Blocker

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.

Animals

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

Statistical Analysis

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 B_max, 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.

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Figure 1.

Improving the antihypertensive effect of β₁-AS-ODN by optimization of liposome:ODN charge ratios. SHR received a single intravenous injection of β₁-AS-ODN or inverted ODN. β₁-AS-ODN 0.5 mg/kg was delivered by DOTAP/DOPE at charge ratios from 0 to 3.5. Inverted ODN 0.5 mg/kg delivered by DOTAP/DOPE at charge ratio 2.0 served as control. Data represent mean values of each group (n=6). Standard errors were omitted for clarity.

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 B_max 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⇓).

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Figure 2.

Effects of β₁-AS-ODN on blood pressure and β-AR levels in renal cortex. SHR were injected with 0.5 mg/kg β₁-AS-ODN or inverted ODN with liposomes at charge ratio 2. A, Effect on blood pressure. B, Time course of the changes in B_max of β₁-AR (•) and β₂-AR (○). C, B_max of β-AR 4 days after intravenous injection of saline (solid bar), inverted ODN (open bar), or β₁-AS-ODN (shaded bar). Data represent mean±SEM of each point (n=6). *P<0.05, **P<0.01 vs saline control.

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.

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Figure 3.

Representative autoradiography of β-AR levels in kidney of control (n=6) or β₁-AS-ODN–treated (n=6) SHR 4 days after injection.

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.

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Figure 4.

RT-PCR of β₁-AR, preprorenin, and GAPDH mRNAs in renal cortex of SHR. Lane 1, saline control; 2, inverted ODN–treated; 3, β₁-AS-ODN–treated day 4; 4, β₁-AS-ODN day 10; 5, β₁-AS-ODN day 18; 6, β₁-AS-ODN day 40. Every point represents a pool of total RNA extracted from 4 or 5 rats.

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⇓).

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Figure 5.

β₁-AS-ODN at a single injection exerts a delayed suppression on RAS. A, Effect on preprorenin mRNA levels in renal cortex. B, Effect on PRA. C, Effect on plasma Ang II levels. Data represent mean±SEM of each point (n=6). *P<0.01, **P<0.001 vs control.