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Am J Physiol Heart Circ Physiol 286: H2213-H2218, 2004. First published February 12, 2004; doi:10.1152/ajpheart.00465.2003
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Gene transfer of a novel vasoactive natriuretic peptide stimulates cGMP and lowers blood pressure in mice

Shuchong Pan,1,3 Rajiv Gulati,1,3 Cheryl S. Mueske,1,3 Tyra A. Witt,1,3 Amir Lerman,1 John C. Burnett, Jr.,1 and Robert D. Simari1,2,3

1Division of Cardiovascular Diseases and Internal Medicine, 2Department of Biochemistry and Molecular Biology, and 3Molecular Medicine Program, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Submitted 22 May 2003 ; accepted in final form 26 January 2004


   ABSTRACT
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METHODS
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Dendroaspis natriuretic peptide (DNP) is a recently described peptide produced by Dendroaspis angusticeps with structural and functional similarities to mammalian natriuretic peptides. These similarities suggest a potential role for DNP in cardiovascular therapeutics. To determine the physiological effects of chronic delivery of DNP, a gene transfer approach using first generation adenoviral vectors was utilized. Although the gene for DNP has not been cloned in any species, the peptide sequence in the snake is known. Preferred mammalian codons for snake DNP were cloned downstream of either the leader sequence (referred to as pBDNP-1) or prepropeptide sequence of human brain natriuretic peptide (BNP) cDNA (referred to as pBDNP-2). Transfections with pBDNP-1 or pBDNP-2 resulted in expected forms of chimeric DNP (cDNP) in cell lysates and conditioned media. Functional studies demonstrated the ability of both forms of cDNP within conditioned media to stimulate cGMP production in human vascular smooth muscle cells (hVSMC). Expressed cDNP inhibited hVSMC proliferation and stimulated vasorelaxation in a similar fashion. To investigate the chronic physiological effects of administration of cDNP, an adenoviral vector expressing cDNP (Ad-BDNP) was generated. Intravenous delivery of Ad-BDNP in mice resulted in dose-dependent systemic expression of cDNP. The highest level of expression was associated with consistent elevation of its presumed second messenger (cGMP) for 21 days but with transient lowering of systolic blood pressure in normotensive mice. This study demonstrates the biological features of the expression of the xenogenic peptide DNP.

dendroaspis natriuretic peptide; adenoviral; gene delivery

DENDROASPIS NATRIURETIC PEPTIDE (DNP), which can be isolated from the venom of the green mamba (Dendroaspis angusticeps) snake, has been shown to have structural as well as biological similarities to other natriuretic peptide family members including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (18). Recent studies have demonstrated the existence of DNP-like immunoreactivity in myocardial tissue and serum from humans with normal or failing left ventricles (LVs) (9, 10, 17). In addition, DNP-like immunoreactivity has been demonstrated in rat aortic vascular smooth muscle cells (VSMCs) (22). Despite this immunoreactivity, it remains unknown whether a gene encoding for DNP or a homolog is present in mammals (16). Furthermore, although the amino acid sequence of the peptide in the snake is known (presumed to be a processed form based on homology with mammalian natriuretic peptides), the gene encoding this peptide has not been cloned in any species.

Irrespective of the lack of a confirmed native DNP gene, the function of synthesized DNP has been studied in vitro and in vivo. DNP has been shown to have potent natriuretic and diuretic effects (9), stimulate vasorelaxation (2, 3), and unload the LV in acute animal studies (7). The major objective of this study was to use gene transfer to express high levels of this potent peptide and to determine the physiological effects of this expression in normal mice. To do so, constructs were developed to express DNP in mammalian cells. Chimeric sequences were developed using the coding sequence for the amino terminus of BNP to generate a prepropeptide hormone similar to other natriuretic peptide family members. These constructs were used to determine whether chronic expression of DNP could result in stimulation of plasma cGMP and have physiological effects in normotensive mice.


   METHODS
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All animal experiments were approved by the Mayo Foundation Instsitutional Animal Care and Use Committee.

Vector construction. A DNA sequence was generated using human preferential codons for the amino acid sequence of snake DNP (14). To express chimeric DNP (cDNP), two expression plasmids were generated (Fig. 1). The first, pBDNP-1, contained only the "humanized" cDNA for DNP downstream of the signal peptide from human BNP (BDNP-1), whereas pBDNP-2 contained the coding sequence for the leader sequence and entire propeptide from human BNP cDNA and the "humanized" codon sequence for DNP (BDNP-2).



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  		Fig. 1. Schematic representation of human brain natriuretic peptide (BNP) cDNA and the chimeric constructs generated. BDNP-1 contains the signal peptide sequence of BNP upstream of the "humanized" dendroaspis natriuretic peptide (DNP) coding sequence. BDNP-2 contains the entire prepropeptide sequence from BNP upstream of the "humanized" DNP coding sequence. Each was subcloned into an eukaryotic expression plasmid containing the cytomegalovirus promoter/enhancer.

 
To generate pBDNP-1 to express the mature form of DNP with a signal peptide, two oligonucleotides were synthesized as templates for overlap PCR. The first 146-mer sense oligonucleotide included an EcoRV site and 30 amino acids of NH2-terminal human BNP, which are 26 amino acids of signal peptide, 4 additional following amino acids of human BNP, and 14 amino acids from NH2-terminal DNP. The second 127-mer antisense oligonucleotide included 38 amino acids of DNP and a BglII site. These two oligonucleotides were used as templates for overlap PCR to generate the BDNP-1 DNA coding sequence using BNP-5' (5'-TGCAGATATCCATGGATCCCCAGACAGCAC-3') and DNP-3' (5'-GAAGATCTTCTTAGGCGCTGGTGCTGGGGGCG-3') as primers.

To generate pBDNP-2 to express DNP with a signal peptide and the propeptide form of BNP, the 106-amino acid fragment of the NH2 terminus of BNP containing the signal peptide and propeptide was generated by PCR using BNP cDNA as a template and oligonucleotides BNP-5' and hBNPmid (5'-CATCTTGGGGCTTCGTGGTGCCCG-3') as primers. The 175-mer antisense oligonucleotide, including 16 amino acids from the COOH terminus of the human BNP fragment and 38 amino acids from DNP, and a BglII site was synthesized. These two fragments were used as templates in overlapping PCR with BNP-5' and DNP-3' as primers to generate pBDNP-2 DNA. Each cDNP gene was cloned into a mammalian expression vector containing the cytomegalovirus (CMV) promoter/enhancer (19). A similar construct encoding for full-length human BNP cDNA (pBNP) was also generated for comparison.

Cell culture and Western blotting. NIH3T3, 293, and HepG2 cells were grown to 70% confluence in 100-mm tissue culture dishes and transfected for 2 h with lipofectamine using standard methods with pBDNP-1 or pBDNP-2. Thirty-six hours after transfection, the conditioned media were collected. Cells were washed twice in cold PBS and flash frozen in 500 µl of lysis buffer in an ethanol-dry ice bath, followed by scraping and a 2-s sonication to achieve a homogeneous solution. Sample protein concentrations were determined by the Bradford technique (Bio-Rad). To characterize cDNP in protein levels in whole cell lysates and conditioned media, 20 µg of total protein or 20 µl of conditioned media were size fractionated by 20% SDS-PAGE, transferred to nitrocellulose membranes, and stained with Ponceau-S stain to ensure equal protein loading. For Western blot analysis, membranes were incubated for 1 h in a blocking solution containing 5% dry milk in PBS + Tween 20, followed by overnight incubation with rabbit anti-DNP antibodies (1:2,000, Phoenix Pharmaceuticals) or rabbit anti-actin antibodies (1:1,000, Sigma; St. Louis, MO). Nitrocellulose membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000, Calbiochem; San Diego, CA) and visualized using enhanced chemiluminescence detection (Pierce; Rockford, IL). Rabbit anti-actin antibody was used to probe the same membrane to confirm equal loading of cell lysates.

BNP radioimmunoassay. The concentration of secreted recombinant human BNP (rhBNP) in conditioned media from pBNP-transfected cells culture was determined by a standard method using a Shionoria BNP Kit (Shionogi; Osaka, Japan) (23).

DNP radioimmunoassay. The concentration of cDNP in conditioned media was measured by a modified radioimmunoassay method previously described (17). Briefly, 1 ml conditioned media was applied to the prepared C-8 Bond-Elut cartridge, and 2 ml of 90% methanol in 1% trifluoroacetic acid were used to elute the bound material from cartridge. The eluate was dried and reconstituted in 300 µl of assay buffer and ready for radioimmunoassay using the kit from Phoenix Pharmaceuticals (Mountain View, CA). Samples and standards were incubated with anti-DNP antibody at 4°C for 24 h. 125I-labeled DNP was added and incubated for 24 h. Free and bound fractions were separated by the addition of a second antibody and normal rabbit serum and centrifuged. Radioactivity of the bound fraction was measured with a gamma counter. A standard curve was generated and used to calculate the concentrations of the unknown samples (reported in pg/ml).

Proliferation assays. Cell proliferation was quantified using a colorimetric immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis (Cell Proliferation ELISA, Boehringer Mannheim). Briefly, human VSMCs (hVSMCs; passages 4–6) were grown in SmBM media with supplements (Clonetic). Cells were plated into a 96-well plate with 105 cells/ml and serum deprived for 24 h. Cells were then treated with 10% fetal calf serum-SmBM media plus various dilutions of conditioned media from pBDNP-2-transfected HepG2 cells (cDNP concentration in media were 10–12–10–9 M) and nontransfected media for an additional 24 h. BrdU was then added into the culture media to obtain a final concentration of 10 µM and cells were then incubated an additional 5 h. After incubation, the labeling medium was washed off, the cells were fixed for 30 min, and wells were incubated with anti-BrdU conjugate followed by substrate reaction. Final absorbency at 450 nm was detected using an ELISA plate reader, and experimental conditions were compared with serum-free conditions.

Measurements of intracellular cGMP. The intracellular cGMP levels were measured using a BIOTRAK cGMP enzyme immunoassay system kit (Amersham). hVSMCs were plated in 96-well Falcon plates and incubated 24 h to reach 80% confluence. Cells were then stimulated with 200 µl of various diluted conditioned media from transfected cells for 30 min at 37°C with 5% CO2, and cells were subjected to lysis following the instructions of the BIOTRAK kit. The amount of cGMP was calculated according the standard curve that was generated from parallel reactions within the same experiment.

Measurements of cGMP in tissue. The changes of the cGMP level in cDNP-treated vascular rings were measured by using the same kit as above. Rabbit carotid artery rings were isolated and weighed. These rings were then transferred to minimal essential medium with 1% fetal calf serum as described previously (2). The tissue was incubated for 2 h at 37°C (5% CO2). Indomethacin and IBMX were added into the medium after that at final concentrations of 1.1 x 10–4 and 1.3 x 10–3 M, respectively. The tissue was incubated for an additional 30 min. The conditioned medium from the transfected cell culture was then added into the tissue at various dilutions for 10 min, and the tissue was removed from medium and frozen in liquid nitrogen. The tissue was homogenized in cold 6% (wt/vol) trichloroacetic acid on ice and centrifuged at 4°C. The supernatant was washed four times with water-saturated ether and lyophilized. Dried extract was dissolve in assay buffer and measured by a cGMP enzyme immunoassay system kit.

Arterial vasoreactivity. Carotid arteries were excised from New Zealand White rabbits immediately after euthanization with pentobarbital and immersed in cold Krebs solution [composed of (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.0026 calcium sodium EDTA, and 11.1 glucose] for studies of vasoactivity (2). Arterial rings ~3 mm long were generated by careful dissection, connected to isometric force displacement transducers, and suspended in organ chambers filled with 25 ml of oxygenated Krebs solution (94% O2-6% CO2). The rings were left to equilibrate for 1 h at 37°C and then stretched to 3 g in 1-g increments. Viability and maximum contraction was determined with 60 mM KCl. After 3 washes with Krebs solution and further equilibration, arteries were precontracted with phenylephrine in a titrated fashion to achieve ~80% stable maximal contraction. To study vasoresponsiveness, conditioned media containing cDNP were added to the organ chambers (n = 8) in a cumulative fashion (final concentration: 10–13.5–10–10.5 M). Control media (obtained from HepG2 cells transfected with empty vector) had no vasoactive effect. To determine the effect of the nitric oxide pathway, NG-monomethyl-L-arginine (L-NMMA; Sigma), a nitric oxide synthase inhibitor, was added to additional rings (final concentration: 10 –4 M, n = 8) 20 min before the addition of phenylephrine as previously described (16), followed by cumulative concentrations of cDNP. After three further washes and equilibration, arteries were recontracted with phenylephrine, and the viability of all rings was confirmed by the assessment of endothelium-dependent responses to incremental doses of acetylcholine.

Adenovirus generation and delivery. The pBDNP-2 cassette containing a CMV promoter/enhancer was cloned into a shuttle plasmid containing LoxP sites and recombined with a cosmid containing the Ad5 genome in the presence of Cre recombinase (1). This solution was then transfected into cells, resulting in recombinant adenoviral generation. Final preparations were purified using standard double-cesium chloride banding and titered. An adenovirus expressing Escherichia coli {beta}-galactosidase from the CMV promoter/enhancer (Ad-LacZ) was used as a control construct. A dose response was performed. One hundred microliters of the viral solution containing 1.33 x 1010, 1.33 x 109, or 1.33 x 108 plaque-forming units (pfu)/ml of Ad-BDNP-2 were injected into the tail vein of immunocompetent BALB/c mice. An adenoviral vector expressing {beta}-galactosidase (Ad-LacZ) was used as a control at the highest dose.

Measurement of blood pressure. Tail systolic blood pressure was measured using a BP-200 Blood Pressure Analysis System (Visitech Systems; Apex, NC). Mice were acclimated to the system for 1 wk before the injection. Blood pressure was measured at the same time daily under controlled room light and temperature conditions.

Statistical analysis. Results are reported as means ± SE. Western blots were scanned to yield arbitrary densitometric units. Differences between groups were made using unpaired t-tests. Statistical analysis was performed with the SAS software package. Physiological parameters in study groups were compared with repeated-measures ANOVA. The normal distribution was tested with univariate analysis. A P value <0.05 was considered significant.


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Expression of cDNP. Transfection of multiple cell types (293, NIH3T3, and HepG2 cells) with pBDNP-1 and pBDNP-2 resulted in expression of the predicted forms of cDNP (Fig. 2). Transfection of pBDNP-1 resulted in a 4-kDa immunoreactive band in cell lysates and conditioned media. Transfection of pBDNP-2 resulted in an 18-kDa immunoreactive band in cell lysates and two bands representing the larger form and a smaller (4 kDa) band. Thus transfection of the humanized cDNA resulted in expression of the predicted forms of cDNP in multiple cell types. In conditioned media, a processed form of the longer (pBDNP-2) form was identified, confirming processing. pBDNP-2 was selected for further study due to its inclusion of a prepropeptide sequence.



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  		Fig. 2. Expression of chimeric DNP (cDNP) in vitro. Western blot analysis using conditioned media (A) and cell lysates (B) from 293, 3T3, and HepG2 cells after transfection with pBDNP-1 or pBDNP-2 or from nontransfected cells (NT) is shown. Transfection with pBDNP-1 results in a 4-kDa form in cells and conditioned media. Transfection with pBDNP-2 results in a larger (18 kDa) form in cell lysates and conditioned media and a short, processed form only in conditioned media. Immunoblotting to actin (bottom) confirmed equal loading and intact transfer of samples.

 
Function of cDNP in vitro. One of the paracrine effects of natriuretic peptide function is regulation of VSMC proliferation. To determine whether expression of cDNP would inhibit serum-induced proliferation of VSMCs, primary hVSMCs were incubated in the presence or absence of 10% FBS and conditioned media from HepG2 cells transfected with pBDNP-2 or empty vector. Serum induced a threefold increase in BrdU labeling, which was abrogated by conditioned media containing cDNP from pBDNP-2-transfected cells in a dose-dependent manner (Fig. 3A). This attenuation of BrdU labeling was associated with increased intracellular cGMP (Fig. 3B). Thus cDNP expressed from pBDNP-2 is capable of stimulating its second messenger, cGMP, and inhibiting hVSMC proliferation.



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  		Fig. 3. Inhibition of serum-induced proliferation of human vascular smooth muscle cells (hVSMC) by cDNP. Conditioned media containing cDNP from cells transfected with pBDNP-2 vs. control transfected media decreased the bromodeoxyuridine (BrdU) incorporation in hVSMCs stimulated by 10% fetal bovine serum (A). The increased levels of cGMP in hVSMCs under the same treatment were correlated with the cDNP concentrations (B). Values are means ± SE of 3 independent experiments. *Significant difference relative to no cDNP-treated control (P < 0.02).

 
Vasoactive effects of cDNP. An important endocrine function of natriuretic peptides is vasorelaxation. To determine the effect of cDNP on vascular tone, excised rabbit carotid arteries were studied in an organ chamber. Suspended arterial rings were equilibrated in Krebs solution and submaximally precontracted with phenylephrine. Conditioned media from cells transfected with either pBDNP-2 or empty vector (control) were then added to the organ bath (n = 8 each). cDNP-containing media provoked arterial relaxation in a dose-dependent manner (Fig. 4A), whereas control media produced no detectable change in vessel tone (not shown). Inhibition of nitric oxide synthase by preincubation of rings with L-NMMA (n = 8) did not affect cDNP-mediated relaxation (Fig. 4A). Additional studies were performed to determine the effect of cDNP on the generation of cGMP by excised carotid rings. The addition of cDNP-containing media produced dose-dependent generation of cGMP. Rings treated with cDNP resulted in a dose-dependent increase in cGMP compared with those treated with control media (Fig. 4B). Collectively, these data suggest that cDNP provokes vasodilation of contracted arterial segments in an endothelium-independent manner and stimulates significant tissue generation of cGMP.



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  		Fig. 4. Effects of cDNP on vascular tone. Conditioned media containing cDNP from pBDNP-2-transfected cells provoked immediate arterial relaxation after precontraction with phenylephrine (PE). The addition of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase, did not attenuate cDNP-mediated arterial relaxation (A). cDNP stimulated cGMP production in arterial rings treated with conditioned media (B). Values are means ± SE of 4 independent experiments. *Significant difference relative to untreated control (P < 0.02).

 
Function of cDNP in vivo. To determine the function of cDNP expressed in vivo, an adenoviral vector was generated using the expression cassette from pBDNP-2. One hundred microliters of the viral solution containing 1.33 x 108–1.33 x 1010 pfu/ml of Ad-BDNP-2 were injected into the tail vein of BALB/c mice (n = 6 each for 3 concentrations). Ad-LacZ (1.33 x 1010 pfu/ml) was used as a control for viral infection (n = 6). Expression after the tail vein adenoviral delivery in mice is primarily in the liver (5). A dose-dependent increase in plasma levels of DNP was demonstrated in the highest dose (Fig. 5A) for the 3 wk of measurement (P < 0.05) but only at 3 and 7 days in the 109 group. To determine whether chronic elevation of DNP would affect plasma cGMP levels, additional mice were studied (n = 6 mice/group). Plasma cGMP levels were increased at the highest dose throughout the study period (Fig. 5C). Plasma cGMP levels were not elevated at 21 days in the lower two doses. To determine whether this increase in DNP and cGMP would affect systolic blood pressure, a similar study was performed in mice acclimated to an automatic tail blood pressure cuff. Mice were acclimated over 10 days before injection. After injection, blood pressure was decreased transiently at the highest dose (Fig. 5D) but not the lower two doses (data not shown) following injection of Ad-BDNP-2 compared with Ad-LacZ (P < 0.05 on days 1 and 2). No changes in heart rate were seen between the groups. Thus the delivery of Ad-BDNP-2 was associated with dose-dependent expression of DNP. The highest levels of expression were associated with elevated levels of cGMP and a transient decrease in blood pressure.



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  		Fig. 5. Effects of expression of cDNP in normotensive mice. Intravenous delivery of increasing doses of Ad-BNP-2 resulted in sustained different elevated circulating levels of cDNP for at least 21 days at the highest dose (A) and transiently at a lower dose (B). Intravenous delivery of Ad-BDNP-2 with the dose of 1.33 x 1010 plaque-forming units/ml resulted in sustained elevated levels of cGMP for at least 21 days but not at lower doses (C). Intravenous delivery of Ad-BDNP-2 resulted in lowering systolic blood pressure transiently in normotensive mice (D). *Significant difference relative to LacZ control (P < 0.05).

 

   DISCUSSION
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Multiple properties of the natriuretic peptides make them ideal candidates for pharmacological therapy of cardiovascular disease. This family of peptides has important effects on diuresis, natriuresis, vasorelaxation, and mitogenesis. Indeed, the use of BNP as a therapeutic in congestive heart failure has added a new class of agents in the treatment of congestive heart failure (4). In the search for novel natriuretic peptides, a peptide with homology to the natriuretic peptide family was identified in the venom of the green mamba snake (18). This 38-amino acid peptide, named DNP, has the characteristic 17-amino acid ring and shares 12 residues with human ANP and 14 residues with human BNP. DNP was shown to have similar receptor and functional characteristics of ANP and BNP, including stimulation of the second messenger cGMP. Despite this discovery 10 years ago, no gene sequence in any species for DNP has been identified. To begin to determine the functional effects of DNP expression in vivo, a novel chimeric expression system was generated.

The structure of the natriuretic peptides is notable for a signal peptide and a propeptide that are cleaved to generate a mature peptide containing a 17-amino acid ring structure and carboxy terminal tail. With only the amino acid sequence of the mature snake peptide known, two chimeric cDNA for DNP using the cDNA for the signal peptide (pBDNP-1) and the signal peptide and propeptide (pBDNP-2) from human BNP cDNA were generated using the preferred human codon sequence for the mature snake peptide. When either construct transfected into multiple cell types matures, processed DNP was secreted. This form was able to stimulate cGMP in VSMCs and arterial rings inhibiting VSMC proliferation and inducing vasorelaxation, two known properties of natriuretic peptides.

In vivo, expression of the longer form in an adenoviral vector at the highest doses tested resulted in high levels of DNP and its presumed second messenger, cGMP, in blood and a transient but pronounced decrease in systolic blood pressure. Levels of circulating DNP were ~3-log fold normal levels for ANP or BNP in normal humans or animals. Lower levels of expression failed to increase plasma cGMP or affect blood pressure. The lack of a sustained decrease in blood pressure is not due to a decrease in gene expression or lack of sustained elevation of cGMP. The transient nature of the effect on blood pressure may be due to counterregulatory systems stimulated by the high circulating levels and the original 30- to 40-mmHg drop in blood pressure. Alternatively, unique effects of cDNP may be responsible for this effect. The autonomic nervous system and renin-angiotensin system might counteract such a significant drop in blood pressure over short periods of time. This response was not due to a blunting of the effect of DNP on cGMP. Whether the effects would be as transient in a hypertensive model remain to be seen. It is also unknown whether other potential physiological effects of DNP expression such as renal or cardiac effects might have a similar transient duration. Recognizing the antiproliferative properties of DNP in cultured VSMCs, it will be important in further in vivo studies to determine whether this antiproliferative property persists in the absence of sustained decreases in blood pressure.

Gene transfer of the natriuretic peptides was first demonstrated by Morishita and colleagues (13). They showed that ANP expressed in a heterologous manner had potent autocrine and paracrine effects that included stimulation of cGMP. In animals, expression of ANP and CNP have demonstrated antiproliferative and antihypertensive effects associated with increased local or urinary cGMP levels (8, 12). These studies have led to great interest in the gene transfer of natriuretic peptides in the treatment of hypertension and congestive heart failure (15). The present study is the first to express DNP in any animal model. For this approach to be clinically useful, a vector system with potential for long-term expression such as adeno-associated viruses or lentiviruses would be required (6, 11, 20).

Chimeric natriuretic peptides have been developed to have unique properties (21). Unlike other attempts that had chimerized mature forms, the present approach differed from these by using the cDNA sequence for the prepropeptide NH2-terminal portion of BNP and the humanized coding sequence for the DNP peptide sequence. The processed form was the same as the snake peptide. The creation of the humanized chimeric cDNA allowed for the generation of vectors capable of expressing high levels of recombinant peptide. In vitro, DNP is a potent stimulator of cGMP and inhibitor of proliferation. However, despite generating elevated levels in mice for up to 21 days, blood pressure was lowered transiently in this normotensive model. Further studies are now warranted in models of cardiovascular disease to assess the potential of chronic DNP elevation by gene transfer technology to positively impact altered cardiovascular function or outcome.


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We acknowledge the support of the Mayo Foundation and the Miami Heart Research Institute. R. D. Simari is an Established Investigator of the American Heart Association.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical expertise of Denise Heublein and the secretarial support of Traci Paulson.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. D. Simari, Mayo Clinic College of Medicine, 200 First St., SW, Rochester, MN 55905 (E-mail: simari.robert{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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