Regulatable atrial natriuretic peptide gene therapy for hypertension

  1. Kurt J. Schillinger*,
  2. Sophia Y. Tsai*,
  3. George E. Taffet†,
  4. Anilkumar K. Reddy†,
  5. Ali J. Marian†,
  6. Mark L. Entman†,
  7. Kazuhiro Oka*,
  8. Lawrence Chan*, and
  9. Bert W. O'Malley*,‡
  1. Departments of *Molecular and Cellular Biology and †Medicine, Sections of Cardiovascular Sciences and Cardiology and DeBakey Heart Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030

  1. Contributed by Bert W. O'Malley, August 10, 2005

 
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Abstract

Hypertension (HTN) is a disease that begins with dysfunctional renal-sodium excretion and progresses to a syndrome of highly elevated systolic, diastolic, and mean arterial pressures. Inadequacies in the therapy of HTN have led to the investigation of the gene therapy of this disease by using systemic overproduction of vasodilatory peptides, such as atrial natriuretic peptide (ANP). However, gene-therapy approaches to HTN using ANP are limited by the need for long-term ANP gene expression and, most important, control of ANP gene expression. Here, we introduce a helper-dependent adenoviral vector carrying the mifepristone (Mfp)-inducible gene-regulatory system to control in vivo ANP expression. In the BPH/2 mouse model of HTN, Mfp-inducible ANP expression was seen for a period of >120 days after administration of vector. Physiological effects of ANP, including decreased systolic blood pressure, increased urinary cGMP output, and decreases in heart weight as a percentage of body weight were also under the control of Mfp. Given these capabilities, this vector represents a paradigm for the gene therapy of HTN.

Hypertension (HTN) is a disease that is characterized by chronically elevated arterial blood pressure that affects ≈25–35% of the population in developed countries, and it is one of the leading causes of morbidity and mortality in adults (1). Development of HTN is likely to involve defects in renal-sodium handling that are exacerbated by maladaptive activation of neurohormonal compensatory mechanisms. The morbidity and mortality caused by the hypertensive state are a consequence of detrimental effects that manifest predominantly in the heart, brain, and kidney and are referred to collectively as hypertensive end-organ disease. Typically, endorgan disease involves pathological left-ventricular hypertrophy, a propensity toward cerebrovascular accidents, and accelerated renal atherosclerosis (1, 2).

The current therapy of HTN has been rationally designed to address the recognized causes of hypertensive pathophysiology, including impaired urinary-sodium excretion and neurohormonal compensatory-mechanism activation. Also, in recognition of the fact that hypertensive end-organ disease represents the true danger of chronically elevated arterial blood pressure, the main goals of antihypertensive therapy have evolved to include a reduction in blood pressure as well as prevention of cardiovascular, cerebrovascular, and renal pathology associated with elevated blood pressure (2, 3). However, a multitude of studies have revealed that no available drug therapy for HTN has the capability to cure the hypertensive state or reverse or prevent the pathological changes associated with hypertensive end-organ disease fully (2). As a result, therapeutic approaches to HTN involving gene therapy have been of recent interest.

Atrial natriuretic peptide (ANP), a 27-aa peptide hormone with potent vasodilatory, natriuretic, diuretic, and antihypertrophic effects, is an attractive candidate gene product for antihypertensive therapy (4, 5). Studies involving the infusion of synthetic ANP to humans with HTN have demonstrated significant decreases in blood pressure and brisk natriuresis/diuresis associated with peptide administration (6, 7). Likewise, gene-therapy experiments conducted with adenoviral vectors in rodent models of HTN have shown equally impressive results (8, 9). However, these studies also have revealed the serious limitations that are associated with the use of ANP in a gene-therapy context. Chief among these limitations are the need for long-term ANP expression and, importantly, control of ANP gene expression. Here, we report the development and testing of a helper-dependent adenoviral (HD-Ad) vector, called iANP.HD, which was designed to address these current limitations for ANP gene therapy of HTN. Our strategy combines the long-term transgene-expression capability of an HD-Ad vector with the use of the mifepristone (Mfp)-inducible gene-regulatory system (MIGRS) for ANP-expression control. By application of iANP.HD to the BPH/2 mouse model of HTN, we demonstrate the ability of this vector to allow Mfp-inducible ANP expression in vivo at time points >120 days after a single administration of vector. Also, we demonstrate that certain physiological consequences of ANP overexpression from this vector are also regulatable with administration of Mfp.

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Methods

Preparation of iANP.HD. A three-piece ligation was performed with (i) a NotI–AscI fragment from a pBluescript II(+) backbone (Stratagene) containing a modified multicloning site; (ii) a PCR fragment generated with NotI and NcoI linkers encoding the promoter of the pEP1422 plasmid (Valentis, Burlingame, CA), which contains three tandem copies of the Gal4 upstream activation sequence, a TATA box, and an artificial intron; and (iii) a PCR fragment generated with NcoI and AscI linkers from the pUC9-preproANP plasmid (gift from David Vesely, University of South Florida, Health Sciences Center, Tampa), which contains a cDNA copy of the rat preproANP gene. The resulting plasmid, PNA-6xANP, contained the rat preproANP cDNA in the context of the pEP1422 Mfp-inducible promoter. A PCR fragment with ClaI and AscI linkers was then generated from the pCR3.1 plasmid (Stratagene), which contained the bovine-growth hormone poly(A) signal and was subcloned into the unique ClaI and AscI sites of PNA-6xANP to generate PNA-6xANPp(A). A NotI–AscI fragment from PNA-6xANPp(A) was then ligated to a NotI–AscI fragment from PAP-GH-GLp65 (10) encoding GLp65 driven by a transthyretin promoter-enhancer sequence (TTRB) (11) to generate plasmid PAP-6xANP-GLp65. A NotI fragment from this plasmid, ≈8.5 kbp, was then subcloned into the unique NotI site of the C4HSU HD-Ad backbone (12) to generate plasmid 6xANP.C4HSU. We then linearized 10 μg of 6xANP.C4HSU by digestion with PmeI, and recombinant HD-Ad particles, named iANP.HD, were generated and amplified as described (13).

In Vitro Studies. HepG2 cells were infected with 1 × 109 particles iANP.HD and treated with either 100% ethanol or 1 × 10–7 M Mfp in ethanol as described (10). At 24 h later, medium was removed from each well and frozen at –80°C until the time of assay. For assay, 50 μl of medium was used directly in a commercially available enzyme immunoassay for rat ANP (Kit S-1132; Bachem).

Mouse Studies. Three BPH/2 brother–sister mating pairs were acquired from The Jackson Laboratory (strain, BPH/2J; generation, F66). Animals were maintained at a constant temperature with 14:10-h light/dark cycle and ad libitum access to standard rodent food and tap water. Male BPH/2 mice were housed one animal per cage to avoid fighting. All animal protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

BPH/2 mice were infected with 5 × 1010 particles iANP.HD by tail-vein injection as described (10). For plasma collection, retroorbital blood was collected and transferred to Microtainer EDTA tubes (BD Biosciences). We combined 15 μl of plasma with 250 μl of 1× RIA buffer (RIKBUFF; Bachem) and stored it at –80°C until the time of the assay. For the assay, 100 μl of plasma–RIA buffer mixture was used directly in a commercially available RIA for rat ANP (Kit S-2039; Bachem).

Urine Studies. Model 650-0100 metabolic cages with mouse diuresis adaptors (MTB-0322, Nalge) were used for collection of urine. A Plexiglas divider was inserted into the domicile portion of the cage to prevent fighting. All collections were started at 0700–0730 hours. At the time of collection, two male mice were used in each cage (separated by the Plexiglas divider), and their combined urine was collected. Each mouse had ad libitum access to water but not to food. We split 9 h of total urine-collection time into three contiguous 3-h collection periods. At the end of each collection period, the urine was centrifuged for 5 min at 17,900 × g and room temperature. Volume was recorded, and urine was stored at –80°C until assay. The feces-separation screen was rinsed under tap water, dried, and replaced at the end of each 3-h collection period. For assay, urinary cGMP concentration was determined by using a commercially available ELISA (Kit 900-013, Assay Designs, Ann Arbor, MI). Simultaneously, urine samples were sent to the Pathology Department of Baylor College of Medicine Center for Comparative Medicine for determination of urine sodium and potassium by flame photometry.

Blood Pressure Studies. Mice were anesthetized by i.p. injection of 75 mg/kg pentobarbital (Nembutal, Abbott), and the left carotid artery was exposed and cannulated with a heparin–saline-filled catheter, as described in ref. 14. The catheter was composed of a 2-cm piece of polytetrafluoroethylene (PTFE) tubing (o.d./i.d., 0.41:0.25 mm) (SUBL-160, Braintree Scientific), which was joined with Superfast epoxy (Elmer's Products, Columbus, OH) to a 10-cm-long piece of PE-50 polyethylene tubing (o.d./i.d., 0.038:0.023 inches) (PE50, Braintree Scientific, Braintree, MA). Before insertion of the PTFE portion of the catheter into the carotid artery, the entire catheter was filled with a sterile 250 units/ml heparin–saline solution and connected to a pressure transducer (Meritrans MER100, Merit Medical Systems, South Jordan, UT) by 23G blunt tubing adaptor (Becton Dickinson). The catheter was flushed briefly with <50 μl of heparin–saline, the system was allowed to stabilize for ≈2 min, and then 10-s blood pressure recordings were made through a calibrated bridge amplifier (band width, 0–2 kHz) on a Dell workstation with signal-processing software (dspw, Indus Instruments, Houston) every 1 min for the next 5 min. At the end of recording, the catheter was removed, the left carotid artery was permanently ligated, and the neck incision was closed to allow the animal to recover. Blood pressure data acquired for 10 s during blood pressure measurement were exported to a worksheet in excel (Microsoft) at an interval of 0.5 ms (20,000 data points per 10 s) for accurate determination of systolic blood pressure and heart rate.

Tissue Preparation. At 14–16 h after blood pressure determination, animals were killed and weighed. Hearts were removed, cleaned under a dissecting microsope, rinsed, dried, and weighed. A cross-sectional ring through both ventricles was cut, placed in 10% zinc–formalin (Protocol, Fisher Scientific), and fixed overnight. Heart rings were then cleared through xylene and embedded in paraffin. Tissues were sectioned at 5 μm and stained with hematoxylin–eosin or Masson's trichrome (Kit 87019, Richard-Allan Scientific, Kalamazoo, MI).

mMode Echo and Doppler Analysis. Doppler and 2D mModeguided echocardiography studies were performed on isoflurane-anesthetized mice as described (15, 16).

Statistical Analysis. An independent t test was performed on sample data by using the spss 11.0 statistical-analysis software (Prentice Hall, Upper Saddle River, NJ). Levine test for equal variance (<0.05) was applied to all samples before evaluation with t test. In the absence of equal variance, statistical significance was not reported.

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Results

Design of iANP.HD. Prior attempts at using ANP for the gene therapy of HTN suffered from limitations, such as short duration of ANP expression and lack of control of ANP expression subsequent to vector delivery (9). iANP.HD, the HD-Ad vector used throughout the studies described here, was designed to address the limitations of earlier ANP gene-therapy approaches. To this end, the MIGRS, which is an expression system that couples control of a specific target gene to administration of the compound Mfp, was incorporated into iANP.HD.

Two expression cassettes have been incorporated into iANP.HD (Fig. 1). In the first cassette, termed the MIGRS cassette, expression of the Mfp-responsive chimeric transactivator GLp65 is driven by a liver-specific transthyretin promoter (TTRB) (11). GLp65 is composed of a truncated form of the human progesterone receptor ligand-binding domain, the DNA-binding domain of the yeast Gal4 transcription factor, and a transactivation domain derived from the p65 subunit of human NF-κB. In the absence of Mfp, GLp65 produced from this cassette exists in a largely monomeric, inactive state. The second expression cassette, called the iANP cassette, contains a cDNA of rat preproANP downstream of a Mfp-sensitive promoter (MSP). Upon binding Mfp, GLp65 undergoes a conformational change that allows the protein to dimerize, bind to the MSP in the iANP cassette, and transactivate expression of rat preproANP (Fig. 1). Because both the MIGRS and iANP cassettes have been subcloned simultaneously into a unique site in the C4HSU HD-Ad backbone of Sandig et al. (12) to create iANP.HD, this single vector has the capacity to enable liver-specific, Mfp-inducible ANP expression.

Fig. 1.
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Fig. 1.

The iANP.HD vector contains two expression cassettes in the C4HSU HD-Ad backbone, which contains the inverted terminal repeats (ITR) and packaging signal (ψ) of the Ad5 genome but is devoid of all adenoviral coding regions (12). The first expression cassette encodes the MIGRS and enables liver-specific expression of the GLp65 transactivator from a transthyretin promoter (TTRB) (11). The second expression cassette (iANP) encodes rat preproANP in the context of a Mfp-sensitive promoter (MSP). In the absence of Mfp, GLp65 exists in a predominantly monomeric, inactive state. Mfp binds to and activates GLp65, promoting dimerization, DNA binding, and transactivation of rat preproANP expression.


Testing iANP.HD in Vitro. To test Mfp-inducible ANP expression from iANP.HD, HepG2 cells were infected with 1 × 109 particles per well of vector. Treatment of infected cells with 1 × 10–7 M Mfp resulted in a 200-fold increase in ANP expression (Fig. 2A).

Fig. 2.
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Fig. 2.

iANP.HD enables Mfp-inducible ANP expression in vivo and in vitro. (A) Infection of HepG2 cells with iANP.HD and subsequent administration of Mfp resulted in a 213-fold increase in ANP expression. No increase in ANP expression occurred in cells treated with PBS (control) and Mfp. (B) At 4 weeks after introduction of 5 × 1010 particles of iANP.HD to mice (iANP.HD), a single oral dose of Mfp induced an average 134-fold increase in plasma ANP levels. Control mice demonstrated no increase in plasma ANP levels after Mfp. n = 6, for all groups.


Testing iANP.HD in Vivo. Preliminary investigation of Mfp-inducible ANP expression in vivo from iANP.HD was done by using two groups of male mice from the BPH/2 genetically hypertensive mouse strain (17). All mice in the first group received 5 × 1010 particles of iANP.HD by tail-vein injection, whereas all mice in the second group received only PBS by tail-vein injection. At 4 weeks after injection, administration of a single oral dose of Mfp at 330 μg/kg resulted in an average 134-fold increase in plasma ANP levels in mice that had received iANP.HD (Fig. 2B).

iANP.HD Enables Long-Term, Dose-Dependent ANP Expression in Vivo. Recognizing that any future potential application of iANP.HD to the gene therapy of HTN would require demonstration of constant, controllable expression of ANP, we next investigated the feasibility of inducing ANP expression over a 60-day period with the implantation of a single, biodegradable pellet containing Mfp. Also, to demonstrate true Mfp-regulable ANP expression, we investigated the possibility of inducing different plasma levels of ANP by using pellets containing different quantities of Mfp.

At 8 weeks of age, 30 male BPH/2 mice were divided into five groups of six mice. All mice in the first two groups received PBS by tail-vein injection, whereas mice in the remaining three groups received iANP.HD. To demonstrate that iANP.HD would enable inducible ANP expression >100 days after it was delivered, these long-term expression levels were not started until 11 weeks after vector delivery. At 11 weeks after tail-vein injection, all animals had blood drawn to measure baseline plasma ANP levels. At this time point, no difference in baseline plasma ANP levels was observed between mice that had received iANP.HD and mice that had received PBS (Fig. 3A, day 0).

Fig. 3.
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Fig. 3.

iANP.HD enables long-term, Mfp-inducible, bioactive ANP expression. (A) At 11 weeks after injection (day 0), and before implantation of pellets, no difference in plasma ANP levels was apparent between groups receiving iANP.HD (vector) and groups receiving PBS. Groups receiving both iANP.HD and Mfp (Vector+180 μg Mfp and Vector+90 μg Mfp) showed sustained elevations in plasma ANP for as long as 46 days after pellet implantation. n = 6, for all groups. (B) At 10 weeks after injection (day 0), and before implantation of pellets, no difference in urinary cGMP output was apparent between groups receiving iANP.HD (vector) and groups receiving PBS. Activation of ANP expression by implantation of Mfp pellets in groups receiving iANP.HD (Vector+90 μg Mfp and Vector+180 μg Mfp) resulted in significant elevations in urinary cGMP output lasting as long as 53 days. n = 3, for all groups. *, P < 0.05.


At week 11.5 after injection, ANP expression was induced by s.c. implantation of timed-release, biodegradable pellets containing Mfp (Innovative Research of America). Implantation of 180-μg Mfp pellets into mice infected with iANP.HD resulted in statistically significant increases in average plasma ANP levels observable 4, 18, 32, and 46 days after implantation. Similarly, implantation of 90-μg Mfp pellets into mice infected with iANP.HD resulted in statistically significant increases in average plasma ANP levels 4 and 18 days after implantation; a statistically less significant trend in elevated average plasma ANP levels extended to 32 and 46 days after implantation. Importantly, 4 days after pellet implantation, a statistically significant difference in average plasma ANP levels existed between the groups of mice that had received iANP.HD and either a 180- or 90-μg Mfp pellet. No elevations in plasma ANP levels were seen at any time points in mice receiving PBS by tail-vein injection or by mice that had received iANP.HD but were implanted with a placebo pellet (Fig. 3A).

Data from this study demonstrated the potential of iANP.HD to enable long-term, Mfp-inducible ANP expression and suggested the existence of a dose-dependence relationship between ANP expression levels and dose of Mfp administered. Also, note that Mfp-inducible ANP expression was observed 123 days after a single administration of the iANP.HD vector to mice. This length of expression after vector administration is 74 days longer than any expression that has been reported (8).

ANP Produced from iANP.HD Is Bioactive. Measurement of urinary cGMP output serves as an accurate, noninvasive means of monitoring the bioactivity of plasma ANP (18). To take advantage of this fact, the mice used above to investigate long-term, Mfp-inducible ANP expression were used simultaneously to investigate the bioactivity of Mfp-inducible ANP.

At 11, 25, 39, and 53 days after pellet implantation, statistically significant elevations in urinary cGMP output were observed in mice receiving iANP.HD and a 180-μg Mfp pellet. Similarly, 11, 25, and 39 days after pellet implantation, statistically significant elevations in urinary cGMP output were observed in mice receiving iANP.HD and a 90-μg Mfp pellet. In a manner analogous to that seen with plasma ANP levels, a trend toward dose dependence developed in the urinary cGMP output levels between mice receiving iANP.HD and either a 180- or 90-μg Mfp pellet. Although not statistically significant because of limited sample size and variance, this trend appeared to be maintained throughout the study, days 11–67 (Fig. 3B).

Controlling the Hypotensive Effects of ANP with Mfp. Recognition that uncontrollable expression of a hypotensive gene product could have serious unintentional consequences has led to the recent observation that relevant application of gene therapy to HTN will require a means to control expression of therapeutic gene products (19). The primary reason, then, for incorporating the MIGRS into iANP.HD was to link control of the hypotensive effects of ANP to the administration of Mfp.

To investigate the effects of Mfp-inducible ANP expression on blood pressure, a study was conducted that was designed to complement the studies of long-term, Mfp-inducible ANP expression described above. In the first part of this study, 24 male BPH/2 mice were divided into four groups of six mice. All mice in the first two groups received PBS by tail-vein injection, whereas all mice in the remaining two groups received iANP.HD. As before, 80 days after injection, mice were implanted s.c. with 180-μg Mfp pellets or placebo pellets. At 17 days after pellet implantation, a time point that corresponded to maximum ANP expression levels and maximum urinary cGMP output in the previous study, systolic blood pressure was measured in all mice through carotid artery catheterization. At this time point, a statistically significant decrease in systolic blood pressure was observed between mice receiving iANP.HD and a 180-μg Mfp pellet and all control mice (Fig. 4A).

Fig. 4.
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Fig. 4.

Mfp-induced ANP expression decreases systolic blood pressure. (A) At 17 days after pellet implantation (14 weeks after infection), average systolic blood pressure in mice receiving iANP.HD and a 180-μg Mfp pellet (Vector+180 μg Mfp) was significantly lower than systolic blood pressure in all controls. *, P < 0.05. (B) At 57 days after pellet implantation (19.5 weeks after infection), results were similar to those obtained at 17 days after pellet implantation. *, P < 0.05.


The second part of this blood pressure study was conducted with four additional groups of six BPH/2 mice in a manner nearly identical to the first part of the study. However, blood pressure was measured 57, rather than 17, days after pellet implantation in all four groups. This time point was chosen to correlate with minimum ANP expression levels and minimum urinary cGMP output levels observed in the previous ANP expression study. Results at the day-57 time point were analogous to those seen at the day-17 time point. Average systolic blood pressure in animals receiving iANP.HD and a 180-μg Mfp pellet was significantly lower than average systolic blood pressure in all control groups (Fig. 4B).

Taken together, results from this study indicate that iANP.HD enables long-term, Mfp-inducible control of the hypotensive effects of ANP. Interestingly, whereas ANP expression levels previously demonstrated dose dependence at both 17 and 57 days after pellet implantation, systolic blood pressure did not demonstrate a similar trend. This result is likely to be caused by the potency of ANP, as even picogram quantities of this peptide can significantly decrease systolic blood pressure.

Supraphysiological ANP Expression Decreases the Heart Weight as a Percentage of Body Weight (HW/BW) Ratio. Characterization of the BPH/2 mouse model of genetic HTN by Schlager and Sides (17) revealed an increased HW/BW ratio in this mouse strain when compared with normotensive controls. Using the BPH/2 model, Leckie (20) demonstrated a small decrease in this elevated HW/BW ratio after 7 days of treatment with the angiotensin-converting enzyme inhibitor captopril. Given the importance of addressing hypertensive end-organ disease, such as left-ventricular hypertrophy, by antihypertensive therapy, the effect of Mfp-inducible ANP expression on the HW/BW ratio of BPH/2 mice was investigated. To study this effect, mice in all blood pressure measurement groups were killed 14–16 h after blood pressure measurement and HW/BW was determined.

Analysis of the hearts of these mice revealed decreases in HW/BW ratio with Mfp-induced ANP expression greater than any that have been published to our knowledge. At 17 days after implantation of a 180-μg Mfp pellet into mice that had received iANP.HD, a 15% decrease in HW/BW was evident (Fig. 5A). More impressively, 57 days after activation of ANP expression in mice receiving iANP.HD and a 180-μg Mfp pellet, a 20% decrease in HW/BW was seen (Fig. 5B). Also, histological evaluation of hearts from mice receiving iANP.HD and a 180-μg Mfp pellet for 57 days revealed that cardiac architecture was unchanged, with no evidence of abnormal collagen deposition (data not shown). Histological studies performed on the livers of these mice also indicated no difference between controls and mice receiving iANP.HD in terms of architecture and inflammatory cell infiltrates (data not shown).

Fig. 5.
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Fig. 5.

Mfp-induced ANP expression decreases heart size. (A) At 17 days after pellet implantation, HW/BW was significantly decreased in mice receiving iANP.HD and a 180-μg Mfp pellet (Vector+180 μg Mfp) when compared with all controls. **, P < 0.01. (B) Decreases in HW/BW in mice receiving both iANP.HD and a 180-μg Mfp pellet (Vector+180 μg Mfp) was more pronounced 57 days after pellet implantation than 17 days after implantation. **, P < 0.01.


In a separate but parallel experiment designed to study the functional consequences of this decrease in heart size, 10 BPH/2 male mice at 8 weeks of age were injected with either iANP.HD or PBS, followed by implantation of a 180-μg Mfp pellet. Thirty days later, assessment of cardiac function and left-ventricular dimensions was made through Doppler and 2D-guided mMode echocardiographic studies. Mfp-inducible ANP expression was accompanied by decreases in peak aortic flow velocity and mean and peak aortic flow acceleration. After 30 days of supraphysiological ANP expression, left-ventricular stroke volume was also decreased significantly. These results are likely to be caused by a combination of chronic unloading of the myocardium through ANP-mediated decreases in peripheral vascular resistance and intravascular volume, and direct antihypertrophic, growth-inhibitory effects of ANP on individual cardiomyocytes (21, 22). This demonstration of the remarkable potency of ANP on the myocardium at supraphysiological levels also underscores the importance of control of ANP gene expression in a gene-therapy context.

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Discussion

The strength of ANP as a potential antihypertensive agent arises from the multifactorial nature of its effects on blood pressure homeostasis, fluid-electrolyte balance, and cardiomyocyte hypertrophy. Current antihypertensive therapies such as β-blockers are capable of addressing single mechanistic dysfunctions in the pathophysiology of HTN. ANP, by contrast, has the capacity to address multiple simultaneously dysfunctional systems. In the peripheral vasculature, ANP promotes vasodilation by both relaxing vascular smooth muscle cells (VSMC) and attenuating vascular reactivity to vasoconstrictive agents such as endothelin 1 and angiotensin II (AngII) (23–25). In the kidney, ANP selectively vasodilates afferent glomerular arterioles and inhibits passive and active sodium reabsorption in the renal intramedullary collecting duct. These properties of ANP make it the only currently known agent capable of simultaneously decreasing blood pressure while increasing the glomerular filtration rate (26). In addition to these antihypertensive effects, ANP also directly inhibits the sympathetic nervous system (SNS) and the renin–angiotensin–aldosterone (RAA) axis, two neurohormonal systems strongly implicated in the pathophysiology of HTN. The sympatholytic effects of ANP have been demonstrated in humans at the level of autonomic ganglion transmission and are thought to play a critical role in neuromodulation of both cardiovascular tone and renal SNS activity (21, 27). With respect to the RAA axis, ANP directly inhibits release of renin from the juxtaglomerular apparatus, attenuates the effects of AngII in VSMCs and cardiomyocytes, and directly abrogates the synthesis and release of aldosterone from the zona glomerulosa (26, 28). It is these multisystem effects of ANP that make this peptide hormone a very attractive potential antihypertensive agent.

Practical application of ANP to HTN is limited by a plasma half-life of 30 s and a requirement for intravascular administration. Consequently, gene therapy represents a treatment modality in which ANP could be applied to a chronic disorder such as HTN. ANP gene therapy has been studied in rat models of HTN with positive but limited results (8, 9). Certainly, physiologic results presented here are in accord with results obtained in these previous studies, as significant decreases in systolic blood pressure were seen in mice expressing ANP from iANP.HD. However, our studies were designed to take ANP gene therapy one step closer to practical application. To this end, we focused on addressing both the chronic nature of HTN, as well as the need for control of ANP expression. Our results demonstrate that iANP.HD enabled expression of ANP for 125 days after a single administration of vector. These data represent ANP expression that is 74 days longer than any data that have been reported, to our knowledge. Also, our data convincingly demonstrate that ANP expression from iANP.HD could be controlled by administration of Mfp, and that the physiological effects of ANP are also coupled to Mfp administration.

Given that the true morbidity and mortality from HTN are associated with hypertensive end-organ disease, results from this study showing decreases in the HW/BW ratio with ANP expression are encouraging. The antihypertrophic and growth inhibitory effects of ANP have been well described in vitro and in vivo and involve increases in intracellular cGMP concentrations (5, 22, 29). The decreases in HW/BW ratios reported in this article are similar in many ways to the cardiac changes reported with administration of the PDE5A inhibitor sildenafil, which blocks the breakdown of intracellular cGMP (30). However, whereas PDE5A inhibition increased cardiac functional performance in the context of acute pressure-overload hypertrophy, prolonged ANP expression in our study slightly reduced cardiac performance. This difference was likely because ANP has a potent after-load reduction capability involving decreases in both peripheral vascular resistance and overall circulatory volume. Consequently, it is likely that the significant decreases in HW/BW ratio and the concomitant decreases in cardiac function observed with ANP expression in our experiments arose as a result of both decreased load and a direct antihypertrophic effect of ANP on the cardiomyocytes. These effects were more potent than those that have been described in any antihypertensive gene-therapy experiment of which we are aware, and they underscore the critical importance of regulating the expression of ANP in a gene-therapy context.

The true advantage of iANP.HD as a gene-therapy vector for HTN results from combining an HD-Ad vector with the MIGRS. HD-Ad vectors have significant advantages over their predecessors, including the capacity for up to 36 kbp of foreign DNA, remarkably reduced cytotoxicity and immunogenicity in vivo, and greatly prolonged target gene expression (31, 32). Such characteristics of HD-Ad vectors make them potentially suitable for the gene therapy of HTN, given the chronic nature of this disease. However, it remains unclear whether chronic injection of HD-Ad vectors over years will be tolerated immunologically. Similarly, the MIGRS is well suited to gene-therapy applications and has shown considerable promise when delivered with formulated direct DNA injection (33). Also, Mfp has been approved for use in humans at doses 10- to 100-fold higher than those that are required currently to induce transgene expression from iANP.HD in mice, and it has been well tolerated by patients. By linking ANP gene expression to the administration of Mfp, iANP.HD allows the multisystem effects of this peptide to occur through administration of a single drug, a feat that is impossible with current antihypertensive therapy. Also, incorporating everything into a HD-Ad vector or a formulated direct DNA injection obviates the need for repeated i.v. delivery of synthetic ANP and ensures the persistence of the antihypertensive properties of ANP.

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Acknowledgments

We thank Mark Burcin and Thuy Pham for technical assistance, Ming-Jer Tsai for helpful discussion, Gael Elliston for critical reading of the manuscript, and Valentis for providing plasmid pEP1422. This work was supported by National Institutes of Health Grants HD-7857 (to B.W.O.) and HL-59314 (to L.C.).

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Footnotes

  • ↵ ‡ To whom correspondence should be addressed. E-mail: berto{at}bcm.tmc.edu.

  • Author contributions: K.J.S., S.Y.T., and B.W.O. designed research; K.J.S. performed research; K.J.S., G.E.T., A.K.R., A.J.M., M.L.E., K.O., and L.C. contributed new reagents/analytic tools; K.J.S., S.Y.T., G.E.T., A.K.R., A.J.M., M.L.E., and B.W.O. analyzed data; and K.J.S. wrote the paper.

  • Abbreviations: HTN, hypertension; ANP, atrial natriuretic peptide; Mfp, mifepristone; MIGRS, Mfp-inducible gene-regulatory system; HD-Ad, helper-dependent adenoviral; HW/BW, heart weight as a percentage of body weight.

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  1. Published online before print September 14, 2005, doi: 10.1073/pnas.0506807102 PNAS September 27, 2005 vol. 102 no. 39 13789-13794
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