(Circulation. 1998;98:1905-1911.)
© 1998 American Heart Association, Inc.
Basic Science Reports |
In Vivo Gene Transfer of Nitric Oxide Synthase Enhances Vasomotor Function in Carotid Arteries From Normal and Cholesterol-Fed Rabbits
From the Divisions of Cardiology (K.M.C., H.Q., V.N., M.A.B., E.O., G.A.S., S.A.Y., J.S.S., S.E.G.) and Pulmonology (J.P., T.M., J.S.S.), Departments of Medicine and Pharmacology (S.E.G.), Howard Hughes Medical Institute (J.S.S.), Duke University Medical Center, Durham, NC.
Correspondence to Samuel E. George, MD, Box 3060, Division of Cardiology, Duke University Medical Center, Durham, NC 27710. E-mail segeorge{at}duke.edu
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Abstract |
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Background—The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit.
Methods and Results—We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 µmol/L acetylcholine, Ad.nNOS-treated arteries showed an 86±4% reduction in precontracted tension, whereas control CA showed a 47±6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad.ßGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer.
Conclusions—In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.
Key Words: genes • adenovirus • endothelium • nitric oxide • atherosclerosis
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Introduction |
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The free radical second messenger nitric oxide (NO) and related bioactive molecules1 may have a significant role in atherogenesis. Reduced endothelium-dependent vascular relaxation is an early event in atherosclerosis2 3 ; in addition, NO inhibits many key atherogenic processes, including platelet adhesion and aggregation,4 5 adhesion molecule and chemokine expression,6 7 and smooth muscle cell migration and proliferation.8 9 Thus, early endothelial NO deficiency may promote progression to more advanced vascular lesions.
Because of their potential importance in vascular diseases, the NO synthase (NOS) isoforms represent attractive targets for vascular gene transfer. Plasmid-liposome gene transfer of the endothelial isoform of NOS (eNOS) restored NO production and inhibited intimal hyperplasia in balloon-injured rat carotid arteries.10 More recently, ex vivo11 and adventitial12 deliveries of eNOS-expressing adenoviruses were shown to favorably affect vasomotor function. However, no study to date has shown that gene transfer can reverse hyperlipidemia-induced vascular dysfunction.
We recently developed an adenoviral vector for gene transfer of the neuronal isoform of NOS (Ad.nNOS).13 This vector generates high-level nNOS protein expression and augments agonist-stimulated NO production in cultured vascular smooth muscle and endothelial cells. In the present study, we use Ad.nNOS (with some of its control elements modified to enhance in vivo expression) for carotid artery gene transfer in normal and cholesterol-fed rabbits. We demonstrate in vivo expression of functional recombinant nNOS in the vessel wall, predominantly in endothelial cells. More importantly, we show that Ad.nNOS gene transfer enhances endothelium-dependent vasomotor function in the normal CA and substantially restores the vasomotor deficit in CA from cholesterol-fed rabbits.
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Methods |
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Construction and Purification of Recombinant Adenoviruses
A recombinant adenovirus encoding ß-galactosidase (ß-Gal), Ad.PacßGal, served as a control virus. We generated Ad.nNOS, containing a cDNA encoding the rat neuronal NOS isoform, driven by the cytomegalovirus immediate-early enhancer/promoter, as previously described.13 For in vivo studies, Ad.nNOS was modified by removing the 5'-untranslated region from the nNOS cDNA and replacing the Simian virus 40 polyadenylation signal (pA) with a bovine growth hormone pA derived from the plasmid pcDNA3 (InVitrogen), which enhanced in vivo expression.
In Vivo Carotid Artery Gene Transfer
All animal care and procedures were approved by the Duke
University Institutional Animal Care and Use Committee and complied
with the "Guide for the Care and Use of Laboratory Animals" (NIH
publication No. 80–23, revised 1985). Male New Zealand White rabbits
(weight, 2 to 2.5 kg) were maintained on a normal diet or were fed a
1% cholesterol diet for 12 weeks before surgery. Vascular
surgery, gene transfer to the carotid arteries, and vessel harvest were
performed essentially as described
previously.14
NOS Protein Analysis by Western Immunoblotting
Freshly harvested vessels were immediately frozen at -80°C
for storage. For analysis, thawed vessels were weighed and then
sonicated in 3 vol of ice-cold lysis buffer (20 mmol/L Tris, pH
7.4, 0.1% Triton X-100, containing 0.2 mg/mL phenylmethylsulfonyl
fluoride and 0.5 µg/mL leupeptin). Tissue debris was pelleted
at 14 000g, and total protein concentration in the lysate
was determined. In addition to analysis of NOS protein in the
crude lysate, NOS protein was enriched by binding the crude lysate for
1 hour at 4°C with 25 µL/mL of 2',5'-ADP–agarose (Sigma Chemical).
After binding, the beads were pelleted, washed in lysis buffer to
remove residual unbound protein, and then boiled in SDS-PAGE loading
buffer to release the bound proteins. Samples were analyzed by
Western blotting using an isoform-specific mouse monoclonal anti-NOS
peptide antibody (Transduction Laboratories).
Immunohistochemistry and Staining for NADPH-Diaphorase
Activity
Vessel segments were briefly equilibrated in 30% sucrose
in PBS at 4°C, embedded in optimal cutting temperature compound (OCT;
Miles Scientific), frozen in liquid nitrogen, and sectioned (6-µm
sections) onto silane-coated glass microscope slides.
Immunohistochemistry to identify nNOS protein expression was performed
using a monoclonal anti-nNOS peptide antibody (Transduction
Laboratories). Immunostaining of smooth muscle cells
(HHF 35; DAKO) and endothelial cells (anti–von
Willebrand factor; Atlantic Antibodies) also was performed.
Immune complexes were localized with the use of chromogenic
alkaline phosphatase substrate Vector red (Vector Laboratories), and
sections were lightly counterstained with hematoxylin.
Vessels also were stained for NADPH-diaphorase
activity.13 This stain demonstrates the presence
of functional NOS protein but is not isoform
specific.15 Briefly, vessel cryosections were
fixed for staining in 4% paraformaldehyde for 30
minutes; permeabilized for 30 minutes in 0.1 mol/L
Tris, pH 7.2, and 0.2% Triton X-100; and then stained with 1
mmol/L NADPH and 0.2 mmol/L nitroblue tetrazolium in the same
buffer at 37°C for 
20 minutes, until the development of
blue-purple staining was observed. Staining intensity was quantified
using an image analysis system (Olympus IX70 inverted
microscope, Optronics DEI-750 image-capturing hardware; PowerTowerPro
180 CPU). Images were captured using Adobe Premiere and quantified
using NIH Image software.
Determination of Vascular NOS Activity
The conversion of [3H]arginine to
[3H]citrulline was used to measure NO
production from intact vessel rings according to modifications
of existing methods.13 Briefly, freshly harvested
vessels were weighed, cut into 2-mm rings, equilibrated for 1 hour in 1
mL of Krebs-Henseleit buffer (KHB) (containing [in mmol/L] NaCl
120, KCl 4.7, CaCl2 2.5,
MgSO4 1.2,
KH2PO4 1.2,
NaHCO3 25 mm, glucose 5.5 mm, pH 7.4), and
gassed with 95% O2/5% CO2
in miniature organ baths maintained at 37°C. After equilibration, KHB
was replaced with 0.6 mL of fresh, warm KHB containing
[3H]arginine (5 µCi/mL, 63 Ci/mmol; New
England Nuclear) and 1 µmol/L calcium ionophore (A23187; Sigma).
Incubation was continued for 4 hours at 37°C, with 95%
O2/5% CO2 gassing. The
conditioned KHB (0.6 mL) was added to 1 mL of NOS assay stop solution
(20 mmol/L HEPES, pH 5.5, 2 mmol/L EDTA), and the organ
chambers were washed out with 0.4 mL of fresh stop solution.
[3H]citrulline was separated from
[3H]arginine by ion-exchange
chromatography of the pooled KHB/stop solution over a
1-mL column of AG 50w-X8 resin (Na form; Bio-Rad), prewashed with 2 mL
of stop solution, and quantified by liquid scintillation counting. NO
production was calculated from the proportion of total arginine
counts converted to citrulline, with the background counts having been
subtracted in a blank sample, and from the known specific activity of
the [3H]arginine.
Vasomotor Studies
The vasomotor studies were conducted as described
previously.14 Briefly, freshly harvested vessel
rings (5 mm) were maintained in oxygenated KHB at
37°C; cumulative dose-response curves to phenylephrine
(PE; 10-9 to 10-4 mol/L)
were established; the vessels were submaximally precontracted with PE
(typically 1x10-6 mol/L); and
endothelial function was evaluated by vascular
relaxation to acetylcholine (ACh; 10-8 to
3x10-5 mol/L).
Endothelium-independent relaxation responses to sodium
nitroprusside (SNP; 10-8 to
10-4 mol/L) also were determined. Statistical
significance was assessed by ANOVA.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In Vivo Adenoviral Gene Transfer Augments Endothelial NOS Activity
In Western immunoblots (Figure 1
), no nNOS protein was seen in either
native carotid arteries or arteries after ß-Gal gene transfer. In
contrast, nNOS protein was clearly detectable in crude vessel lysate
after Ad.nNOS infection and was greatly enriched by 2',5'-ADP affinity
purification.
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To investigate the distribution of NOS activity, frozen tissue sections
were stained for NADPH-diaphorase activity (Figure 2). Because development of the blue color
requires active NOS, NADPH-diaphorase staining accurately
reflects the distribution of functional NOS
enzyme.15 Control carotid arteries (both
Ad.ßGal infected, shown in Figure 2, and sham-infected, not shown)
had moderate diaphorase staining confined to
endothelial cells, reflecting native eNOS activity. In
arteries infected with Ad.nNOS, however, endothelial
diaphorase staining was clearly more intense; in addition,
significant blue staining was observed in the adventitia. To confirm
the NADPH-diaphorase findings and to determine the extent
of nNOS expression, we also performed immunohistochemical staining for
nNOS protein (Figure 2). The areas of nNOS expression corresponded to
the areas of enhanced NADPH-diaphorase activity. In control
arteries, no nNOS immunostaining was observed.
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We quantified the degree of endothelial
NADPH-diaphorase staining by computer-assisted image
analysis (Figure 3, left). This
revealed that the endothelial layer of Ad.nNOS-treated
vessels had 3-fold higher diaphorase staining intensity
than sham- or Ad.ßGal-infected vessels. We next assessed NOS activity
in vessels stimulated with 1 µmol/L A23187. Mean NOS activity
after nNOS gene transfer was increased by 3.4-fold compared with
control arteries (Figure 3; P<0.001). Thus, Ad.nNOS gene
transfer produces a quantitatively similar increase in
NADPH-diaphorase staining intensity and vascular NO
production (a relationship that is also present across a
broad range of infecting titers in cultured vascular cells; H.S. Qian,
V. Neplioueva, and S.E. George, unpublished data).
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In Vivo NOS Gene Transfer Enhances Endothelial
Vasomotor Function
The responses of arteries to vasoactive agonists were
determined using isometric tension studies. Responses to cumulative
doses of PE, ACh, and SNP were recorded (Figure 4). All vessels contracted similarly in
response to PE. In Ad.ßGal- or sham-infected arteries, acetylcholine
reduced tension in PE-precontracted arteries to 25% of
precontracted values. Ad.nNOS gene transfer significantly augmented
ACh-dependent relaxation. Relaxation at every concentration of ACh was
greater than that in controls (P<0.05), and maximal
relaxation was greatly increased, to almost complete relaxation (5% of
precontracted tension). Finally, Ad.nNOS-infected arteries showed a
20-fold increase in sensitivity to ACh relative to sham-infected
arteries, as judged by reduction in EC50 value
(0.22x10-6 mol/L versus
4.5x10-6 mol/L; P<0.01). The
difference in ACh-induced relaxation was not due to differences in
sensitivity to NO because all vessels relaxed in a similar manner to
that in response to SNP (Figure 4C).
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In Vivo NOS Gene Transfer Substantially Reverses Vasomotor
Impairment in Cholesterol-Fed Rabbits
We next sought to determine whether NOS gene transfer could
reverse the vasomotor impairment known to occur in
cholesterol-fed rabbits. Ten male New Zealand White rabbits
(weight, 2 to 2.5 kg) were fed a 1% cholesterol diet for
11 to 12 weeks. Serum cholesterol levels were monitored at
4 and 8 weeks and at the time of gene transfer, and levels were
markedly elevated in all rabbits (mean±SEM cholesterol
levels in mg/dL: baseline, 67±8; 4 weeks, 1260±259; 8 weeks,
1141±334; 12 weeks, 2279±320). The rabbits underwent carotid artery
gene transfer with either Ad.nNOS (10 arteries) or Ad.PacßGal (4
arteries) or were mock-transferred (6 arteries) according to the
protocol used in normal rabbits. At harvest, segments of the arteries
were stained for NADPH-diaphorase activity, and the
staining intensity was quantified by image analysis (Figure 5). Arteries receiving Ad.nNOS showed
>2-fold higher diaphorase staining intensity than either
Ad.PacßGal or sham-transfected arteries, directly reflecting higher
NOS activity in the Ad.nNOS-treated arteries.
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As expected, ACh-induced relaxation was significantly impaired in
arteries from cholesterol-fed rabbits. In sham-infected
carotid arteries, ACh reduced tension to 69±5% of precontracted
values (Figure 5C
) (compare with values for normal carotids in Figure 4, where ACh relaxes normal carotids to 25% of precontracted tension).
Ad.ßGal-infected arteries yielded similar results. In contrast,
Ad.nNOS substantially reversed the vasomotor impairment associated with
cholesterol feeding; Ad.nNOS-treated vessels relaxed to
43±5% of precontracted tension (P<0.01 versus sham- and
Ad.ßGal-infected arteries). The degree of contraction in response to
PE and relaxation in response to SNP were not affected by Ad.nNOS,
Ad.PacßGal, or sham infection (data not shown). In summary, the
results in cholesterol-fed rabbits show that Ad.nNOS gene
transfer approximately doubles the amount of functional NOS in the
vascular wall and substantially reverses the vasomotor deficit
associated with cholesterol feeding.
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Discussion |
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Although eNOS is the only NOS isoform found in the normal vessel wall, other NOS isoforms may be effective for vascular gene therapy in diseased or injured vessels. Our studies establish that Ad.nNOS expresses a functional enzyme that augments vascular NO production and enhances endothelial vasomotor function in arteries from both normal and cholesterol-fed rabbits. This adds to recent evidence suggesting that the calcium/calmodulin–regulated NOSs can functionally complement each other.16 In support of this view, Shears et al17 recently showed that an adenovirus expressing the immunological isoform of NOS (iNOS) reduced vasculopathy in rat aortic allografts. Thus, all three major NOS isoforms appear to be reasonable candidates for vascular gene therapy applications.
In this study, we sought to avoid the confounding effects of
virus-induced inflammation. First-generation, E1-deleted adenoviral
vectors have a well known propensity to provoke a chronic inflammatory
response in infected tissues.18 In the
vasculature, this response characterized by a viral antigen–specific
cytotoxic T lymphocyte response beginning 7 days after infection,
with loss of transgene expression and intimal hyperplasia in the
ensuing days and weeks.19 Accordingly, we chose
to study the vessels at an early time point, 3 days after infection,
when adenovirus-mediated transgene expression is near-maximal but no
chronic inflammatory response has developed. In addition, adenoviral
infection can cause a dose-dependent acute inflammatory injury that can
impair endothelial vasomotor
function.14 To avoid this, we conducted our
studies at a relatively low infectious titer
(3x109 pfu/mL), a dose that we have shown yields
maximal transgene expression without apparent vascular injury. Although
others have reported medial inflammation and contractile dysfunction
after higher-titer infection rabbit arteries,20
we observed no such impairment here. The difference is likely
attributable to the fact that we used a 10-fold lower adenovirus
titer.
Prior published studies have shown that eNOS gene transfer has favorable vascular effects in normal and balloon-injured arteries.
First, Von der Leyen et al10 used liposome–Sendai virus hemagglutinin protein complexes to deliver an eNOS-expressing plasmid to balloon-denuded rat carotids. This approach restored NOS activity to near-normal levels and significantly limited the subsequent development of neointimal hyperplasia.
Second, Janssens et al21 delivered recombinant adenovirus expressing eNOS (Ad.eNOS) to rat lungs via aerosol; these experiments showed that Ad.eNOS significantly attenuated the rise in pulmonary artery pressure induced by acute hypoxia.
Third, Kullo et al12 showed that the adventitial
delivery of an eNOS-expressing adenovirus markedly enhanced vascular
NOS enzymatic activity and basal vascular cGMP levels in rabbit carotid
arteries. The effects on vasomotor function, however, were more modest:
reduced contractile force in response to PE and 2-fold enhanced
sensitivity to A23187 and ACh. Although comparisons should be made with
caution, we observe substantially greater enhancement of
endothelium-dependent relaxation: for example,
Ad.nNOS-infected arteries have 20-fold increased sensitivity to ACh.
One possible explanation is that endovascular delivery is more
effective than adventitial delivery; the somewhat higher maximal
velocity and specific activity of the nNOS isoform relative to eNOS
also may contribute.22 23 However, the precise
reason for the differences cannot be stated in the absence of direct
experimental comparisons.
NOS Gene Transfer Restores Vasomotor Function in
Cholesterol-Fed Rabbits
Although normal arteries represent a significant proving
ground for NOS gene transfer, an important next step is to determine
whether NOS gene transfer can reverse the vasomotor deficit known to
occur in arteries from hypercholesterolemic rabbits.
Reduced NO bioavailability occurs early in the atherosclerotic process
in animal models; for example,
hypercholesterolemia impairs
endothelium-dependent relaxation in
rabbits,24 pigs,25 and
primates.26 Similarly, a number of studies in
humans document decreased vasomotor responsiveness in
preatherosclerotic conditions such as
hypercholesterolemia,27
hypertension,28 and cigarette
smoking.29
The mechanism for the NO deficit has not been firmly established in any model of atherogenesis. Possibilities include (1) impaired NO production due to reduced NOS expression, reduced substrate availability (eg, L-arginine), reduced cofactor availability (eg, tetrahydrobiopterin),30 or an effect of endogenous inhibitors (eg, asymmetric dimethylargine)31 ; (2) enhanced NO destruction, due to increased production of reactive oxygen species such as superoxide radical32 ; and (3) deleterious effects on downstream mediators of NO activity. The effect of NOS gene transfer in atherosclerosis models would depend on the underlying mechanism. To the extent that NO deficit is due to decreased expression, forced overexpression of NOS should have a favorable impact. However, in cholesterol-fed rabbits, eNOS expression appears to be well preserved, and the NO deficit appears to be due to increased production of reactive oxygen species.33 In this circumstance, nNOS overexpression might increase NO bioavailability, but it also might increase the production of other reactive nitrogen species with an uncertain effect on vasomotor function.34 However, if the observed deficit is due to NOS substrate or cofactor deficiency, nNOS overexpression should have a limited impact on NO production and indeed may merely enhance the production of reactive oxygen species.35 36 That Ad.nNOS gene transfer substantially improved vasomotor function in arteries from cholesterol-fed rabbits is compatible with NOS deficiency, enhanced NO destruction, or impaired downstream signaling, but it suggests that NOS substrate or cofactor deficiency is not the dominant mechanism in this model.
The impact of NOS gene transfer on other indexes of atherosclerosis remains to be investigated. Ad.nNOS roughly doubles NO bioavailability in arteries from hypercholesterolemic rabbits, which may have favorable effects on arterial platelet deposition,4 expression of chemokines such as monocyte chemoattractant protein-1,7 expression of adhesion molecules,37 and inflammatory cell accumulation. These possibilities are currently under investigation.
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Acknowledgments |
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This work was supported by an American Heart Association, North Carolina Affiliate, Grant-in-Aid (to Dr Channon) and US Public Health Service Grant HL-48662 (to Dr George). Dr Channon is a British Heart Foundation Clinical Scientist Fellow. Dr Blazing is a Clinician-Scientist and Dr George is an Established Investigator of the American Heart Association.
Received March 16, 1998; revision received June 1, 1998; accepted June 3, 1998.
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