(Hypertension. 1997;30:307.)
© 1997 American Heart Association, Inc.
Articles |
Prolonged Reduction of High Blood Pressure With Human Nitric Oxide Synthase Gene Delivery
From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston.
Correspondence to Julie Chao, PhD, or Lee Chao, PhD, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.
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Abstract |
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Abstract Endothelium-derived nitric oxide (NO) in peripheral vessels has been shown to modulate vascular resistance and blood pressure. We explored the effect of a continuous supply of human endothelial NO synthase (eNOS) on the blood pressure of spontaneously hypertensive rats (SHR) by somatic gene delivery. A DNA construct containing the human eNOS gene fused to the cytomegalovirus promoter/enhancer was injected into SHR through the tail vein. A single injection of the naked eNOS plasmid DNA caused a significant reduction of systemic blood pressure for 5 to 6 weeks in SHR, and the effect continued for up to 10 to 12 weeks after a second injection. The differences were significant from 2 to 12 weeks postinjections (n=6, P<.01). In a separate experiment, L-arginine, the substrate of eNOS, was supplied in drinking water at a concentration of 7.5 g/L for 11 weeks after eNOS gene delivery. A maximal blood pressure reduction of 21 mm Hg in SHR was observed with eNOS DNA compared with that of control SHR injected with vector DNA (181.9±1.46 versus 202.7±2.79 mm Hg, mean±SEM, n=6, P<.01). Human eNOS gene delivery induces significant increases in urinary and aortic cGMP levels and urinary and serum nitrite/nitrate content (P<.05), while no significant differences in body weight, heart rate, water intake, food consumption, or urine excretion were observed. These results indicate that somatic delivery of the human eNOS gene induces a prolonged reduction of high blood pressure and raises the potential of using eNOS gene therapy for hypertension and cardiovascular diseases.
Key Words: nitric oxide synthase • inbred SHR • somatic gene therapy • blood pressure • hypertension
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Introduction |
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Nitric oxide, a potent regulator of vascular tone, is produced in endothelial cells by NOS, which converts L-arginine to L-citrulline and NO.1 2 3 4 NO is thought to be the EDRF that mediates vascular relaxation in response to acetylcholine, bradykinin, and substance P in vascular beds.4 5 6 7 Continuous production of endothelium-derived NO in peripheral vessels has been shown to modulate vascular resistance and blood pressure.4 Mice lacking the gene for eNOS are hypertensive.8 NOS activity in the renal cortex is not different between normotensive WKY and SHR; however, the medulla of the SHR displays a significantly higher NOS activity than that of WKY.9 Moreover, no differences in renal eNOS activity were noted between Dahl salt-sensitive and salt-resistant rats.10 In recent years, the role of L-arginine, the substrate of NOS, in the regulation of blood pressure in vivo was studied extensively.11 12 13 14 The acute infusion of L-arginine caused a rapid onset of hypotension in normotensive healthy volunteers and in patients with essential hypertension.11 Chronic L-arginine administration attenuates cardiac hypertrophy in SHR.12 L-Arginine increased production of NO and prevented salt-sensitive hypertension in Dahl/Rapp rats.13 Exogenous L-arginine administration produces a vasodilatory effect by increasing NO production and modulates the release of neuroendocrine hormones in hypertensive subjects.14 Wistar rats given chronic administration of an NOS inhibitor, NG-nitro-L-arginine methyl ester, developed time- and dose-dependent hypertension without cardiac hypertrophy.15 Also, long-term inhibition of NO synthesis caused coronary microvascular remodeling and cardiac hypertrophy in WKY in vivo by a mechanism other than arterial hypertension.16 In vivo gene transfer of bovine eNOS inhibits neointima formation in Sprague-Dawley rats.17
To investigate the role of constitutive eNOS in blood pressure regulation in vivo, we delivered the human eNOS gene linked with the CMV promoter/enhancer in SHR that were given either tap water or L-arginine in drinking water for 11 weeks. In this study, the results revealed that delivery of the human eNOS gene into SHR via two IV injections led to a sustained lowering of blood pressure for up to 12 weeks postinjection. However, chronic L-arginine administration did not cause a further reduction of blood pressure in SHR injected with human eNOS plasmid DNA or the control vector DNA. These findings indicate that the feasibility of NOS gene therapy for treating human hypertensive or vascular diseases should be studied.
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Methods |
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Experiment I
Twelve male SHR (7 weeks old, Harlan Sprague Dawley, Indianapolis, Ind) were used in this study. These rats received tap water throughout the experimental period.
Experiment II
Eighteen male SHR (9 weeks old, Harlan Sprague Dawley,
Indianapolis, Ind) were divided into three groups, one receiving tap
water, and the other two receiving L-arginine hydrochloride
(pH 6.5 to 7.0, Sigma Chemical Co) in drinking water (35.6
mmol/L, resulting in a daily intake of 5.93
mmol/kg). Each group in two separate experiments consisted of 6
age-matched rats. Throughout the study period, all animals were housed
at a constant room temperature with a 12-hour light/dark cycle and had
free access to tap water (or L-arginine) and rat chow
(Harlan Teklad) that consisted of 191 mmol of sodium, 248
mmol of potassium, and 80 mmol of arginine per kilogram. All
procedures complied with the standards for care and use of animal
subjects as stated in the Guide for the Care and Use of
Laboratory Animals (Institute of Laboratory Resources, National
Academy of Sciences, Bethesda, Md). The eukaryotic
expression vector pcDNA3 was purchased from Invitrogen.
CMV-eNOS Plasmid DNA Preparation
The CMV-eNOS plasmid construct was kindly provided by Dr James
K. Liao at Harvard Medical School, Boston, Mass. The 4.0-kb human eNOS
cDNA containing the entire coding sequence (nucleotides 1
through 3612) was cloned in the CMV promoter-directed
vector,18 which contains a bovine growth hormone
polyadenylation sequence. A neomycin resistance gene, under the control
of an SV40 promoter with an SV40 polyadenylation signal sequence, has
the same orientation in the transcription unit of the eNOS gene (Fig 1). The plasmid DNAs (CMV-eNOS and pcDNA3
constructs) were purified with a plasmid purification kit (Qiagen)
according to the manufacturer’s instructions.
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Transient Transfection of CMV-eNOS Plasmid DNA in BPAECs
BPAECs were purchased from ATCC. BPAECs were maintained in
RPMI-1640 medium (GIBCO-BRL) with 20% fetal calf serum and 200
mmol/L L-glutamine (both from Sigma Chemical Co) in
a 37°C incubator supplied with 5% CO2. BPAECs at 80%
confluence were transfected according to the manufacturer’s
instructions with 6 µg of plasmid CMV-eNOS DNA and pcDNA3 by
Lipofectamine reagent (Bethesda Research Labs). Eighty-four hours
posttransfection, the transfected BPAECs were washed with 1x PBS and
harvested. BPAECs were homogenized in 0.1 mol/L HCl.
The cGMP levels in cell lysates and media were assayed by a cGMP RIA.
RIA for cGMP
The procedure for assay of cGMP was conducted according to the
general procedure of Brooker et al19 and Harper and
Brooker,20 as modified by Gettys et al.21 22
The iodination was performed by adding 20 µL of 50 mmol/L
phosphate buffer to 25 µg (in 10 µL of 0.5 mol/L potassium
phosphate buffer, pH 7.4) of 2'-O-monosuccinylguanosine
3':5'-cyclic monophosphate tyrosyl methyl ester (cGMP-TME, Sigma
Chemical Co), followed by 5 µL of Na125I (0.5 mCi).
Twenty microliters of 0.01% chloramine T (Sigma Chemical Co) solution
was added to the mixture and incubated for 30 seconds. The reaction was
stopped by adding 50 µL of 25% acetic acid. The resultant mixture
was subjected to C-18 reverse-phase high-performance liquid
chromatography to separate the iodinated
cGMP-TME from free iodine. Standards (10, 5, 2.5, 1.25, 0.63, 0.32,
0.16, and 0.08 nmol/L) and samples were acetylated by
adding 20 µL of triethylamine and 10 µL of acetic anhydride to each
tube. Aliquots (50 µL) of each acetylated standard and
sample, 25 µL of diluted cGMP antiserum (1:10 000), and 25 µL of
iodinated cGMP (15 000 cpm) were mixed in the assay tubes
and incubated overnight (16 hours) at 4°C. The assay was stopped by
adding 50 µL of the 5x diluted human plasma containing 4
mmol/L EDTA, followed by 1 mL of cold 12% PEG. The tubes were
vortexed and incubated at 4°C for 1 hour before spinning for 20
minutes at 1000g at 4°C. The supernatant was aspirated and
another 1 mL of 12% PEG was added gently to each tube. Tubes were
centrifuged as before, the supernatants aspirated, and the
tubes counted in a gamma counter.
IV DNA Delivery
In experiment I, six SHR of each group were injected IV with
either CMV-eNOS plasmid DNA or pcDNA3 DNA. In experiment II, six SHR of
the group drinking tap water were injected IV with pcDNA3 DNA. The
other two groups (six SHR per group) with L-arginine
administration received either CMV-eNOS plasmid DNA or pcDNA3 DNA
injections. Plasmid DNA constructs were diluted in PBS, and 1 mg of DNA
was injected into the tail vein of SHR as previously
described.23
Blood Pressure Measurement
Systolic blood pressure of SHR was measured with a
manometer-tachometer (Nastume KN-210; Nastume Seisakusho Co Ltd) using
the tail-cuff method.24 Unanesthetized rats were
placed on a plastic holder mounted on a thermostatically controlled
warm plate that was maintained at 37°C during measurement. An average
of 10 readings were taken for each animal after they became acclimated
to the environment.
Urine Collection and Analysis of Physiological
Parameters
Twenty-four-hour urine of rats was collected using
metabolic cages 5 weeks postinjection. Controls and
eNOS-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 and centrifuged in a microfuge at 1000g to
remove particles. The volume of the supernatant was measured and stored
at -80°C for analysis.
Tissue Preparation
At the end of the experimental protocol, all rats were
anesthetized IP with pentobarbital at a dose of 50 mg/kg
body weight. Blood samples were collected by direct cardiac puncture
and chilled at 4°C overnight. These samples were centrifuged
at 1000g for 20 minutes and sera were removed and frozen at
-20°C. At the same time, rats were perfused with normal saline
(0.9% NaCl) from the heart, and the thoracic aorta was rapidly
excised, rinsed in cold normal saline, frozen in liquid nitrogen, and
stored at -80°C. The heart was also excised and rinsed in cold
normal saline. The left ventricle was separated from the right
ventricle and atria and all were weighed.
Determination of Urinary and Aortic cGMP
Concentrations
The thoracic aorta was thawed and homogenized in 10
vol 0.1 mol/L HCl with an all-glass homogenizer
at 4°C. The homogenates were centrifuged at
15 000g for 30 minutes, and aliquots of the supernatants
were stored at -20°C until assay. One of the aliquots was used to
determine the protein concentration by the method of Lowry et
al,25 with bovine serum albumin as the standard.
Urinary and aortic cGMP levels were measured by a cGMP RIA as described
above.
Measurements of Serum and Urinary NOx Content
Serum and urine samples were sent to the New York Medical
College for measurements of NOx content. Serum and urinary NOx content
were measured by a calorimetric assay based on the Griess
reaction.26
Statistical Analysis
Data were analyzed using standard statistical
methods. Repeated blood pressure measurements at each time point were
taken after gene delivery for comparison between control and
experimental groups, and data in the
Table were analyzed with the use
of either unpaired Student’s t test or ANOVA and Fisher’s
protected least significant differences. Group data are expressed as
mean±SEM. Values were considered significantly different at a value of
P<.05.
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Results |
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Increase of cGMP Levels in BPAECs Transfected with CMV-eNOS DNA
The human eNOS cDNA construct (pCMV-eNOS) that was linked to a CMV promoter/enhancer is shown in Fig 1. The human eNOS cDNA construct (9.4 kb) was transiently transfected into BPAECs. Eighty-four hours after transfection, the intracellular cGMP level in NOS-transfected cells increased by about 100% (Fig 2) compared with the basal level in the BPAECs transfected with pcDNA3 plasmid DNA (276.5±47.8 versus 136.9±48.3 fmol/mg protein, n=4, P<.05).
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Experiment 1
Effects of NOS Gene Delivery on Blood Pressure of SHR
The effect of human eNOS DNA on the systolic blood
pressure of SHR (7 weeks old) was monitored weekly for 12 weeks after
IV injections. The plasmid DNA of pcDNA3 vector was injected IV as a
control. The results show that a single injection of the naked human
eNOS plasmid DNA caused a significant reduction in blood pressure for 6
weeks in SHR and the effect continued for up to 12 weeks after a second
injection (Fig 3). The differences were
significant at 2 to 12 weeks after injections (n=6, P<.01),
while a second injection was given to rats 6 weeks after the first
injection. A maximal blood pressure reduction of 22 mm Hg in SHR
was observed 2 weeks after the second injection with eNOS DNA
(177.4±2.27 mm Hg, mean±SEM, n=6) compared with SHR injected
with vector DNA alone (199.3±2.55 mm Hg, mean±SEM, n=6,
P<.01). The effects of NOS gene delivery on the blood
pressure of SHR were observed in two separate experiments.
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P<.01, CMV-eNOS vs vector DNA control group.
Increase of Urinary cGMP Levels After NOS Gene Delivery
At 5 weeks post–NOS gene delivery, the 24-hour rat urine was
collected and the cGMP level determined by RIA. The extracellular cGMP
level in NOS-injected rat urine increased by 45% (Fig 4) compared with that of control rats
receiving pcDNA3 plasmid DNA (22.38±3.31 versus 14.77±2.92 nmol per
rat per day, n=6, P<.05).
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Experiment 2
Effects of NOS Gene Delivery on Blood Pressure of SHR With
L-Arginine Administration
During the experimental period, two groups (n=6), one receiving
the CMV-eNOS DNA injection and one injected with the pcDNA3 vector as a
control, were additionally supplied with L-arginine in
drinking water at a concentration of 7.5 g/L. One group
receiving pcDNA3 DNA injection was given tap water as another control.
The effect of human eNOS DNA on the blood pressure of SHR (9 weeks old)
with L-arginine administration was monitored weekly for 10
weeks after IV injections (Fig 5).
Similarly, the differences were significant at 1 to 10 weeks after
injections (n=6, P<.01), while a second injection was given
to rats 5 weeks after the first injection. A maximal blood pressure
reduction of 21 mm Hg in SHR receiving the L-arginine
treatment was observed 2 weeks after the second injection with eNOS DNA
(181.9±1.46 mm Hg, mean±SEM, n=6) compared with SHR injected
with vector DNA alone (202.7±2.79 mm Hg, mean±SEM, n=6,
P<.01). In addition, L-arginine administration
did not cause further reduction of blood pressure in vehicle-injected
rats throughout the experimental period compared with the group
drinking tap water (Fig 5).
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Effects of Human NOS Gene Delivery on the
Physiological Parameters in
SHR
The Table shows the results of physiological
measurements performed on SHR injected with CMV-eNOS or vehicle DNA 5
weeks postinjection with L-arginine administration. At the
time of urine collection, effects of blood pressure reduction were
observed from 195.5±1.19 mm Hg in the control group to
181.5±1.90 mm Hg in the NOS group (n=6, P<.01).
After NOS gene delivery, the aortic cGMP level in the NOS group
increased by 41% (Fig 6) compared with
that of control rats receiving pcDNA3 plasmid DNA (28.34±3.58 versus
20.15±2.24 pmol/mg protein, n=6, P<.05), while
urinary and serum NOx content increased 25-fold and 2-fold in the NOS
group, respectively, compared with control rats (3.03±1.91 versus
0.12±0.20 µmol per rat per day urine and 9.76±1.47 versus
4.55±0.94 nmol/mL serum, n=6, P<.05). However, no
significant changes in heart rate, body weight, water intake, urine
volume, or food consumption were observed between these two groups,
which were given L-arginine in the drinking water. The
administration of L-arginine triggered a dipsogenic
response and diuresis compared with control rats injected with
pcDNA3 and provided tap water for drinking (19.32±1.81 versus
6.89±1.35 mL/100 g body weight per day and 15.20±1.77 versus
6.04±0.90 mL/100 g body weight per day, n=6, P<.05). Also,
aortic cGMP levels were significantly increased by
L-arginine treatment (20.15±2.24 versus 12.96±0.75
pmol/mg protein, n=6, P<.05).
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Discussion |
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The present study shows that somatic gene delivery of naked human eNOS DNA results in a prolonged reduction in the systolic blood pressure of SHR (7 to 9 weeks old). The maximal reduction of blood pressure was 21 to 22 mm Hg after NOS gene delivery with or without L-arginine administration, respectively, compared with control rats receiving vehicle DNA injection. Reduction of blood pressure in SHR lasts for 10 to 12 weeks after two injections of human eNOS plasmid DNA. In SHR injected with the human eNOS gene construct, there were no obvious changes in body weight, heart rate, or activity of rats. These findings suggest that eNOS gene delivery may compensate for the dysfunction in the L-arginine–NO pathway in patients with essential hypertension and could be a potentially effective and feasible alternative for treating human hypertensive or vascular diseases.
Previous studies suggest that continuous production of endothelium-derived NO in peripheral vessels has been shown to modulate vascular resistance and blood pressure.4 Furthermore, a recent study has shown that transgenic mice lacking the gene for eNOS are hypertensive.8 The present study shows that a continuous supply of constitutive eNOS by somatic gene delivery has a prolonged effect on blood pressure reduction, despite no further blood pressure–lowering effect on those rats receiving the L-arginine treatment. Collectively, these findings are consistent with our recent studies using other vasodilators such as tissue kallikrein, ANP, and adrenomedullin. Previously, we have shown that transgenic mice overexpressing human tissue kallikrein are hypotensive24 and somatic gene delivery of human tissue kallikrein into SHR by IV portal vein and IM injections induces a prolonged reduction of high blood pressure in these rats.27 28 29 IV delivery of the human ANP gene causes a sustained reduction of hypertension in SHR for several weeks.23 Like ANP and kallikrein gene therapies, eNOS gene delivery could potentially be used to alleviate complex hypertensive diseases.
Rats receiving IV injections of human eNOS DNA have significant increases in cGMP levels in urine and aorta and NOx content in serum and urine. The results indicate that the blood pressure reduction was likely due to increased NO and intracellular cGMP production, which mediates the relaxation of vascular smooth muscle. There are significant increases of urine excretion and water consumption in L-arginine–treated SHR receiving either vehicle or eNOS DNA injection compared with those rats drinking tap water. This finding that thirst caused by L-arginine administration induced a dipsogenic response and diuresis in rats is consistent with the report by Nakaki et al.11 Surprisingly, the continuous supply of L-arginine in excess did not produce further reductions of blood pressure in SHR given eNOS gene injection, although acute infusion of L-arginine caused a rapid onset of hypotension in both normotensive and hypertensive human subjects.11 This observation raises the possibility that L-arginine is not the limiting factor in the L-arginine–NO pathway of SHR. The Km of EDRF synthase for L-arginine, partly purified from the rat cerebellum, is less than 10 µmol/L.30 Since the intracellular concentration of L-arginine has been estimated to be about 100 µmol/L,31 the enzyme might be expected to be saturated by endogenous L-arginine. However, Kilbourn and Belloni32 have shown that endothelial production of nitrite, an indicator of NO formation, is not saturated at 2.5 mmol/L L-arginine in in vitro studies. This finding suggests that endothelial NOS may not be saturated by endogenous L-arginine and that L-arginine administration should result in greater hypotensive effect via more NO and cGMP production, but the consequence is not what we expected.
Some evidence suggests that SHR have intrinsic abnormalities or defects of the L-arginine–NO axis. Endothelium-dependent vasodilatory responses to various stimuli are impaired.33 An impairment of L-arginine metabolism after stress has been elicited in SHR.34 Moreover, SHR have lower levels of cardiac cGMP and cGMP-dependent protein kinase than do normotensive rats.35 In our study, chronic L-arginine administration did not reduce blood pressure in vehicle-injected rats, a result similar to the finding reported by Matsuoka et al.12 In this study, oral L-arginine administration did not attenuate cardiac hypertrophy in either control or NOS-injected SHR (data not shown). This finding is different from the report by Matsuoka et al12 but similar to a study showing that L-arginine administration did not attenuate hypertrophy in stroke-prone SHR.36 The reasons for the different effects of L-arginine on cardiac hypertrophy between SHR in our study and SHR in the study of Matsuoka and coworkers may be attributed to different sodium concentrations of rat chow used. In our study, we used a 0.44% sodium diet instead of 0.25% as in their study.12 It is unknown whether, like the renin-angiotensin or natriuretic peptide system, other mechanisms may be involved in the development of cardiac hypertrophy of SHR at an earlier stage, probably from 6 to 10 weeks old. This is considered the most critical period of SHR in blood pressure changes.37
The possibility is that the benefit of the excessive L-arginine supply and the exogenous expression of human eNOS in 9-week-old SHR with the existing dysfunction of the L-arginine–NO axis may be unable to reflect on the cardiac protective effect on account of systemic homeostasis of L-arginine metabolism, reduced intracellular availability of L-arginine, or other compensatory mechanisms involving the renin-angiotensin system. After eNOS gene delivery, the hypotensive effect observed in this study could be eliminated by acute infusion of NG-nitro-L-arginine methyl ester via cannulation (data not shown), indicating that the exogenous NOS expression and NO production in SHR contributed to the reduction of blood pressure. While the exogenous eNOS may compensate for defects in the L-arginine–NO pathway in SHR, continuous NO production relaxed the vascular smooth muscle via a cGMP-coupled pathway and further resulted in sustained blood pressure reduction in NOS-injected rats.
Somatic gene transfer techniques using various constructs and vectors have been developed extensively in recent years.38 39 40 41 In this study, the gene transfer method using a simple injection of naked plasmid DNA produced highly effective and prolonged systemic effects; the possible mechanism for the advantages of this technique is that the exogenously delivered nucleic acid in the body is considered a poor antigen and fails to generate antibody against injected plasmid DNA (data not shown). Therefore, naked plasmid DNA administered by IV injection was randomly taken into cells and extrachromosomally expressed until it was degraded. Concerning low and limited efficiency of naked gene delivery, liposome-mediated and adenovirus-mediated gene transfer methods would serve as better vehicles in future gene therapy studies. From our recent studies, the blood pressure–lowering effect via adenovirus-mediated gene transfer results in high efficiency of gene expression, but it lasts for 4 weeks,42 which is shorter than the 6 to 12 weeks observed in this study. Improvements in both the efficiency of foreign gene expression and duration of the effect after somatic gene delivery will be evaluated in our further studies.
In conclusion, significant findings in this study suggest that eNOS gene delivery using an IV injection technique may compensate for the dysfunction in the L-arginine– NO pathway in patients with essential hypertension. This approach could be a potentially effective and feasible alternative for treating human hypertensive or vascular diseases.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL 29397, HL 44083, and HL 56683. We thank Dr Thomas H. Hintze at New York Medical College for help in the measurement of NOx content and Dr James K. Liao at Harvard Medical School for kindly providing the human CMV-eNOS construct used in this study.
Received December 4, 1996; first decision January 16, 1997; accepted February 14, 1997.
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