Abstract Chronic infusion of atrial natriuretic peptide (ANP) has been shown to cause natriuresis, diuresis, and hypotension in rats and humans. We explored the effect of a continuous supply of ANP by somatic ANP delivery on genetically hypertensive rats. A DNA construct containing the human ANP gene fused to the Rous sarcoma virus 3′-long terminal repeat (RSV-LTR) was injected intravenously into spontaneously hypertensive rats (SHR) through the tail vein. Expression of human ANP in SHR was identified in the heart, lung, and kidney by radioimmunoassay and reverse transcription–polymerase chain reaction followed by Southern blot analysis. A single injection of naked ANP plasmid DNA (12.3 kb) caused a significant reduction of systemic blood pressure in young SHR (4 weeks old), and the effect continued for 7 weeks. The differences were significant at 1 to 2 weeks (n=6, P<.05) and 3 to 6 weeks after injection (n=6, P<.01) A maximal blood pressure reduction of 21 mm Hg in young SHR was observed 5 weeks after injection with ANP DNA (159.4±3.02 mm Hg, mean±SEM, n=6) compared with SHR injected with vector DNA alone (180.2±3.02 mm Hg, mean±SEM; n=6; P<.01). Somatic gene delivery of human ANP DNA had no effect on the blood pressure of adult SHR (12 weeks old). After ANP gene delivery, there were significant increases in urinary volume and urinary potassium output (n=6, P<.05) but not in body weight, heart rate, water intake, urinary sodium output, urinary creatinine, and urinary protein. Antibodies to human ANP or plasmid ANP DNA were not detected in rat sera. These results indicate that somatic delivery of the human ANP gene induces a sustained reduction of systemic blood pressure in young hypertensive rats and raise the feasibility of using ANP gene therapy for the treatment of human hypertension.
ANP is a 28–amino acid peptide hormone secreted predominantly by atrial cardiomyocytes.1 2 The administration of exogenous ANP resulted in numerous physiological responses, including a rapid natriuresis and diuresis as well as a reduction in arterial blood pressure.3 4 5 6 7 8 9 10 The use of ANP as a therapeutic agent has been explored extensively. ANP has been shown to cause blood pressure reduction in animals and hypertensive human subjects when applied as a bolus injection or in short-term infusions.11 12 13 14 15 16 17 Long-term infusion of ANP often raised ANP concentrations to the upper range of physiological levels and caused a sustained reduction in blood pressure.11 12 13 14 15 16 17 18 When ANP was infused at a low rate of 0.5 to 1.0 pmol/kg per minute, plasma renin activity, aldosterone, and catecholamines were suppressed, with a concomitant reduction of blood pressure.11 18 Infusion of ANP at the rate of 5 to 10 pmol/kg per minute was found to be effective in primary aldosteronism, glomerulonephritis, and renovascular hypertension by reducing blood pressure, lowering plasma aldosterone, and increasing natriuresis.19 Recent studies suggest that transgenic mice overexpressing mouse ANP develop sustained hypotension without causing diuretic and natriuretic effects.20 Monoclonal antibody against ANP was used chronically to block endogenous ANP and to accelerate the development of hypertension in SHR and deoxycorticosterone acetate salt–hypertensive rats.21 A genetic defect in ANP production could lead to salt-sensitive hypertension in mice with a disruption of the mouse ANP gene.22
To investigate the role of ANP in blood pressure regulation in vivo, we applied the human ANP gene linked with Rous sarcoma virus 3′-long terminal repeat (RSV-LTR) to overexpress human ANP in young SHR. In this study, the results revealed that direct delivery of the human gene into SHR via a single intravenous injection led to a sustained lowering of blood pressure for 7 weeks. In addition, the apparent diuretic and slight natriuretic effects occurred 5 weeks after delivery of the human ANP gene. These findings suggest that it is feasible to use gene therapy for introducing ANP DNA to alleviate human hypertensive diseases.
The eukaryotic expression vector pREP8 was purchased from Invitrogen. Twelve young SHR (male; 4 weeks old; body weight, 50 to 100 g) and 12 adult SHR (male; 12 weeks old; body weight, 270 to 300 g) were used (Harlan Sprague Dawley, Harlan, Indianapolis, Ind). The rats were housed at a constant room temperature with a 12-hour light/dark cycle and had free access to tap water and rat chow.
RSV-cANP Plasmid DNA Preparation
Human ANP cDNA (cANP) encoding the entire coding sequence23 (nucleotides 1 through 456) was amplified from total RNA of human atrium by RT-PCR. The sense primer used was 5′-ATGAGCTCCTTCTCCACCAC-3′ from 1 to 20 of human ANP cDNA and the antisense primer was 5′-TCAGTACCGGAAGCTGTTAC-3′ from 456 to 437 of the termination codon and exon 2. The amplified product was cloned into pCR II vector (Invitrogen) according to the manufacturer’s instructions. To construct plasmid RSV-cANP, the human ANP cDNA insert was released from pCR II vector by Xho I and HindIII digestion and cloned into the plasmid pREP8 at Xho I and HindIII sites (Fig 1⇓). The mammalian episomal expression vector pREP8 includes ori P and EBNA-1 gene (the Epstein-Barr virus replication origin and nuclear antigen gene), which permit extrachromosomal replication in a wide variety of primate and canine cell lines.24 25 26 The expression of human ANP cDNA encoding the entire coding sequence was under the transcriptional control of the RSV-LTR promoter in plasmid RSV-cANP.
Transient Transfection of Human Hepatoma G2 Cells
Human hepatoma G2 cells (Hep G2 cells) at 80% confluence were transfected according to the manufacturer’s instructions with 10 μg of plasmid RSV-cANP DNA and pREP8 by Lipofectamine reagent (BRL). Seventy-two hours after transfection, the transfected Hep G2 cells were washed with 1× PBS and harvested. Hep G2 cells were lysed by three cycles of freeze-thawing in PBS. The immunoreactive human ANP in cell lysates and medium was assayed by a direct RIA specific for human ANP.
Seven weeks after injection of ANP or vector DNA, rats were anesthetized intraperitoneally with pentobarbital at a dose of 50 mg/kg body wt and perfused with normal saline (0.9% NaCl) by cardiac puncture. Tissues were homogenized in normal saline with a Polytron (Brinkmann Instruments). The homogenate was centrifuged at 600g for 10 minutes. The supernatant was incubated in 0.5% sodium deoxycholate and then centrifuged at 10 000g for 30 minutes. Total protein in the supernatant was determined by Lowry’s method.27
RIA for Human ANP
The level of human ANP in each tissue extract was determined by an RIA specific for human ANP. Ten micrograms of human synthetic ANP-(Ser99-Tyr126) (Sigma Chemical Co) was labeled with 1 mCi of 125I, which was iodinated with iodogen for 10 minutes at room temperature. The iodinated ANP in 250 mmol/L sodium phosphate buffer, pH 7.0, was separated on a reversed-phase C18 high-performance liquid chromatography column in an acetonitrile gradient. 125I-ANP labeled tracer, which was eluted from the column at 19 to 20 minutes after injection, was identified by antibody titration. Serial dilutions of standard ANP (10 to 1280 pg) or tissue extracts (100 μL) were incubated with goat anti-human ANP antiserum (1:1500 dilution, Sigma) in a solution containing 0.01 mol/L PBS (pH 7.4), 0.3% bovine serum albumin, 0.1% Triton X-100, 0.1 mmol/L EDTA and 0.1% sodium azide, and 125I-ANP tracer (10 000 cpm in 100 μL) in a total volume of 400 μL for 18 to 24 hours at 4°C. The reaction was stopped by adding 800 μL of 25% polyethylene glycol in PBS containing 0.1% sodium azide and 400 μL of 1% bovine γ-globulin in PBS containing 0.1% sodium azide. The radioactivity of the precipitate was determined in a gamma counter.
RT-PCR Southern Blot Analysis of Human ANP mRNA in Transfected Hep G2 Cells and in Tissues of SHR
Total RNA was extracted from fresh rat tissues by guanidine isothiocyanate–cesium chloride gradient ultracentrifugation.28 The reaction mixture for RT contained 1 μg of total RNA from SHR, 10 pmol of 3′ primer (5′-CACTGAGCACTTGTGGG-3′ located at the second exon of the human ANP gene), 20 nmol of dNTP, 0.2 μmol of dithiothreitol, 4 μL of 5× RT buffer (250 mmol/L Tris-HCl, pH 8.3; 375 mmol/L KCl; and 15 mmol/L MgCl2), and 200 U of Moloney’s murine leukemia virus reverse transcriptase (BRL) in a total volume of 20 μL. The RT reaction mixture was incubated at 37°C for 1 hour to synthesize the first strand of cDNA. Ten picomoles of 5′ primer (5′-CACCGTGAGCTTCCTCCTTT-3′ from the first exon of the human ANP gene), 5 μL of 10× PCR buffer, and 0.5 U of Taq DNA polymerase were added to the RT mixture to a total volume of 50 μL followed by 35 cycles of hot-start PCR (94°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute) with wax. Twenty microliters of the RT-PCR products was subjected to a Southern blot analysis. A specific oligonucleotide (5′-TAGGTCAGACCAGAGCT-3′ from the first exon of the human ANP gene) was used as an internal probe for hybridization at 45°C. The filter was washed twice in 6× SSC at room temperature for 15 minutes each time and then washed in 3× SSC at 45°C for 30 minutes each time. The blot was washed to a final stringency of 1× SSC with 0.1% sodium dodecyl sulfate at 50°C for 30 minutes once and exposed to Kodak X-Omat film at −80°C.
Direct DNA Delivery
The plasmid DNA of pREP8 vector and RSV-cANP was purified with a plasmid purification kit (Qiagen) according to the manufacturer’s instructions. Twelve 4-week-old SHR and twelve 12-week-old SHR were each divided into two groups. One group of young and adult rats was injected with a plasmid pREP8 construct through the tail vein, and the other was injected with RSV-cANP. The constructs were diluted to 1 mg/mL in PBS before injection and administered at a dose of 500 μg per young rat and 1 mg per adult rat.
Blood Pressure Measurement
Systolic pressure of SHR was measured with a programmed electrosphygmomanometer PE-300 (Narco Bio-Systems, Division of International Biomedical, Inc) with the use of the indirect tail-cuff method.29 Unanesthetized rats were placed in a plastic holder that was mounted on a thermostatically controlled warm plate maintained at 37°C during measurement. An average of 10 readings was taken for each animal.
Assays for Serum Antibodies in Rats
Immunologic responses were monitored by measuring the production of antibodies against human ANP or ANP plasmid DNA (RSV-cANP) in the sera of experimental rats. Serum antibodies binding to human ANP protein or ANP DNA were measured by ELISA. Microtiter ELISA plates were coated with either synthetic human ANP at 5 μg/mL in PBS at 4°C or with the purified ANP DNA construct at 5 μg/mL in 1× SSC (150 mmol/L sodium chloride, 15 mmol/L sodium citrate, pH 7.0) at 37°C overnight.30 Serially diluted rat serum in PBS containing 0.05% Tween-20 was added to the plates. After incubation for 45 minutes at room temperature, plates were washed and peroxidase-conjugated goat anti-rat IgG was added. Substrate solution was added, and the plates were read at 414 nm on a Titertek plate reader.
Urine Collection and Analysis of Physiological Parameters
Twenty-four–hour urine of rats was collected using metabolic cages 5 weeks after injection. Controls and ANP-injected animals (n=6 each) were fed regular food for 3 hours before they were placed in metabolic cages supplied with drinking bottles. To eliminate contamination of urine samples, animals received only water during the 24-hour collection period. Urine was collected 24 hours later and centrifuged in a microfuge at 1000g to remove particles. The volume of the supernatant was measured and stored for analysis. One milliliter of each sample was used to measure urinary sodium and potassium output, urinary creatinine, and total protein.
Data were analyzed with the use of standard statistical methods. Repeated blood pressure measurements were taken after gene delivery for comparison between control and RSV-cANP groups with the use of ANOVA and Fisher’s protected least significant difference. Group data are expressed as mean±SEM. Values were considered significantly different at a value of P<.05.
Expression of Human ANP in Hep G2 Cells Transfected with RSV-cANP DNA
Human ANP cDNA construct that was linked to RSV-LTR promoter (RSV-cANP) is shown in Fig 1⇑. The human ANP cDNA construct was transiently transfected in Hep G2 cells, and expression of human ANP was measured by an RIA specific for human ANP. Immunoreactive human ANP secreted from Hep G2 cells into culture medium reached a level of 337±7 pg/mL (n=4). A serial dilution of the culture medium showed parallelism with the human ANP standard, indicating their immunologic identity (Fig 2⇓). No immunoreactive ANP could be detected in the culture medium of Hep G2 cells transfected with control vector DNA.
Expression of Human ANP and its mRNA in SHR after Gene Delivery
Blood pressure was monitored weekly, and rats were killed 7 weeks after injection. Expression of human ANP in SHR was analyzed by an RIA for human ANP. The results showed that immunoreactive human ANP is detected in the heart, lung, and kidney after gene delivery. Linear displacement curves for immunoreactive human ANP in heart, lung, and kidney extracts paralleled the human ANP standard curve, indicating their immunologic identity (Fig 2⇑). A serial dilution of kidney, lung, and heart extracts from control rats injected with the vector DNA did not show parallelism with the human ANP standard. These results indicate that goat anti-human ANP antibody has some cross-reactivity with rat ANP; however, human and rat ANP are not immunologically identical and are distinguished in the RIA.
Total RNAs prepared from the heart, lung, liver, and kidney of SHR after gene delivery were analyzed for the human ANP transcript by using specific oligonucleotide probes for human ANP. RT-PCR followed by Southern blot analysis showed that the human ANP gene is expressed in the heart, lung, and kidney of rats receiving human ANP DNA injection (Fig 3⇓). The RT-PCR products from control rats showed weak, nonspecific binding but did not hybridize to the specific human ANP probe. These results indicate that expression of the human ANP gene was detected in SHR after somatic ANP gene delivery.
Lack of Immunoresponse With Intravenous Injection of Human ANP DNA
Direct delivery of the human ANP gene into SHR induces expression of exogenous human ANP. To examine the possibility of antibody production against human ANP or its DNA in rats, we used ELISA to measure antibody levels in rat sera injected with ANP DNA and vector DNA. Using serial dilutions of rat sera from 1:10 to 1:1000, we did not detect any antibodies against either human ANP protein or ANP DNA at 7 weeks after injection. From our previous ELISA assays using the same procedures, the lower limit of detecting the concentration of antibody for human kallistatin antibody is 5 ng IgG/mL.
Hypotensive Effect of ANP Gene Delivery in Young but not Adult SHR
The effect of human ANP DNA (500 μg) on the blood pressure of young SHR (4 weeks old) was monitored weekly from 1 to 7 weeks after intravenous injection. The plasmid DNA of pREP8 vector was injected intravenously as a control. The results show that the ANP construct produced a significant reduction in the systolic pressure 1 to 7 weeks after injection compared with control rats receiving vector DNA injection (Fig 4⇓). The differences were significant at 1 to 2 weeks (n=6, P<.05) and 3 to 6 weeks (n=6, P<.01) after injection. A maximal blood pressure reduction of 21 mm Hg in young SHR was observed 5 weeks after injection with ANP DNA (159.4±3.02 mm Hg, mean±SEM, n=6) compared with SHR injected with vector DNA alone (180.2±3.02 mm Hg, mean±SEM, n=6, P<.01). However, there is no significant difference of blood pressure in adult SHR (12 weeks old) injected with 1 mg human ANP DNA versus pREP8 vector DNA (Fig 5⇓). The hypotensive effects of ANP gene delivery in young SHR were observed in three separate experiments.
Effects of Human ANP Gene Delivery on the Physiological Parameters in SHR
The Table⇓ shows results of physiological analysis of urine samples of young SHR injected with RSV-cANP compared with control rats receiving vector DNA at 5 weeks after injection. At the time of urine collection, a reduction of blood pressure was observed from 180.2±3.02 mm Hg in the control group to 159.4±3.02 mm Hg (n=6, P<.01) in the experimental group. Significant increases of urine volume (5.62±0.96 versus 3.68±0.53 mL/100 g body wt per day, mean±SEM; n=6, P<.05) and urinary potassium output (0.90±0.06 versus 0.76±0.05 mmol per rat per day, mean±SEM; n=6, P<.05) were observed in SHR after delivery of the ANP DNA. No apparent changes in heart rate, body weight, water intake, urinary sodium output, urinary creatinine, and urinary protein levels were observed in SHR after delivery of the human ANP gene.
The present study shows that somatic gene delivery of naked human ANP DNA resulted in a sustained decrease in the systolic pressure of young SHR (4 weeks old) without having an effect on the blood pressure of adult SHR (12 weeks old). The maximal reduction of blood pressure was 21 mm Hg after ANP gene delivery compared with control rats receiving vehicle DNA injection. Hypotension was sustained for 7 weeks after a single injection of ANP DNA, and the expression of human ANP was detected in the heart, lung, and kidney of SHR by an RIA and by RT-PCR Southern blot analysis. Human ANP gene delivery resulted in a significant increase in diuresis. These findings suggest that it is feasible to apply ANP gene therapy for treating human hypertensive diseases.
Previous studies suggest that ANP administration causes several biological responses: reduction in arterial blood pressure and rapid natriuresis and diuresis.3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Garcia et al31 reported that chronic infusion of active ANP (ANP-[Arg101-Tyr126]) reduced blood pressure and increased diuresis and natriuresis in conscious one-kidney, one-clip hypertensive rats. Hamet and colleagues evaluated the effects of prolonged ANP infusion on blood pressure and humoral parameters in normotensive and hypertensive African green monkeys (Cercopithecus aethiops). They also found that prolonged ANP infusion caused a significant decrease in blood pressure, plasma renin activity, aldosterone, and hematocrit in normotensive and hypertensive monkeys without increasing plasma cGMP levels.32 Furthermore, recent studies have shown that transgenic mice overexpressing mouse ANP exhibit sustained hypotension,20 whereas ANP knockout mice are hypertensive after salt loading.22 The present study shows that a continuous supply of ANP by somatic gene delivery has a prolonged effect on blood pressure reduction. Collectively, these findings are consistent with our recent studies of other vasodilators of the tissue kallikrein-kinin system. Previously, we showed that transgenic mice overexpressing human tissue kallikrein are hypotensive29 and that somatic gene delivery of human tissue kallikrein into SHR by intravenous and intramuscular injection induces reduction of blood pressure in these rats.30 33 The present studies showed that ANP and kallikrein gene therapies could potentially be used to treat complex hypertensive diseases.
It is interesting to note that direct gene delivery of human ANP causes a reduction in the blood pressure of young SHR (4 weeks old) without causing any effect on adult SHR (12 to 14 weeks old). Our results are comparable with those reported by Mulvany,34 who showed that a synthetic ANP induces dilatation of isolated renal resistance vessels in young but not in adult SHR. We injected human ANP DNA construct into young SHR and observed a blood pressure–lowering effect 1 week after gene delivery that lasted for 7 weeks. This result is consistent with a previous study by Harrap et al,35 who showed that an angiotensin-converting enzyme inhibitor prevents full expression of hypertension in 6- to 10-week-old SHR as a result of permanent reduction in total peripheral resistance.36 It has been suggested that 6- to 10-week-old SHR are in the most critical phase in the development of hypertension.36 At the present time, it is not known whether ANP receptors gradually alter their sensitivity to ANP on renal resistance vessels during the development of hypertension in SHR. It is interesting to observe that tissue kallikrein gene delivery into SHR induces a sustained reduction of blood pressure in adult SHR.30 33 These results suggest that therapeutic usage of both ANP and tissue kallikrein gene delivery are effective in reducing blood pressure, but each gene construct may be unique in exerting the greatest impact during a specific developmental stage.
Rats receiving a single intravenous injection of human ANP DNA have a 65% increase in daily urine production and a significant increase of urinary potassium output. Although there is a slight increase of urinary sodium output and water consumption in RSV-cANP–injected SHR, no significant differences were detected in water consumption, urinary sodium output, urinary creatinine, and urinary protein. These results are similar to those of Steinhelper et al,20 who showed that several physiological parameters including water intake and daily sodium output of ANP transgenic mice are not significantly different from those of control littermates. Since SHR injected with ANP were fed a normal salt diet during the experimental period, it is possible that sodium excretion is not in excess as a result of bodily fluid and systemic sodium homeostasis. Therefore, salt-sensitive hypertensive rats might serve as a good model for studying the relation between natriuretic effects and human ANP expression.
Somatic gene transfer techniques using various constructs and vectors have been developed extensively in recent years.37 38 39 40 41 In this study, we show that a single intravenous injection of unprotected human ANP DNA construct produced a sustained reduction in blood pressure. The RSV-cANP construct harbors the EBNA-1 gene, which makes it possible to replicate as an episome in primate and canine cells but not in rodent cells.24 25 26 Therefore, somatic delivery of RSV-cANP construct in human gene therapy could prolong the expression period even more. One possible way to achieve long-term expression is to use retrovirus-mediated gene transfer, which promotes chromosomal integration. However, the site of integration is random and carries the risk of gene inactivation.42 In SHR injected with the human ANP gene construct, there were no obvious changes in body weight, heart rate, or rat activity, indicating that DNA injection was nontoxic to the recipient rats. In comparison with long-term daily drug intake or human ANP infusion for controlling hypertensive symptoms, a major therapeutic advantage of ANP gene therapy is that a single injection provides a long-lasting effect without causing any apparent side effects or immunologic responses.
These findings suggest that ANP gene delivery could be an effective and feasible alternative for treating human hypertensive diseases. Efficiency and uptake of naked ANP plasmid DNA by direct intravenous injection might be quite low. Other methods such as liposome-mediated delivery or virus vector-mediated transfer for improving efficiency of gene delivery and expression will be the subject of future studies.
Selected Abbreviations and Acronyms
|ANP||=||atrial natriuretic peptide|
|EBNA||=||Epstein Barr virus nuclear antigen|
|ELISA||=||enzyme-linked immunosorbent assay|
|LTR||=||long terminal repeat|
|PCR||=||polymerase chain reaction|
|RSV||=||Rous sarcoma virus|
|SHR||=||spontaneously hypertensive rat|
This work was supported by National Institutes of Health grants HL-29397 and DE-09731. We thank Dr Carmelann Zintz for critical review of the manuscript.
- Received May 22, 1995.
- Revision received June 14, 1995.
- Accepted July 25, 1995.
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