Gastrointestinal Research Unit and Anesthesia Research Unit, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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Endothelial nitric oxide synthase
(eNOS)-derived nitric oxide (NO) contributes to hepatic vascular
homeostasis. The aim of this study was to examine whether delivery of
an adenoviral vector encoding eNOS gene to liver affects vasomotor
function in vivo and the mechanism of NO production in vitro. Rats were
administered adenoviruses encoding -galactosidase (AdCMVLacZ) or
eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In
animals transduced with AdCMVLacZ,
-galactosidase activity was
increased in the liver, most prominently in hepatocytes. In
AdCMVeNOS-transduced animals, eNOS protein levels and catalytic
activity were significantly increased. Overexpression of eNOS
diminished baseline perfusion pressure and constriction in response to
the
1-agonist methoxamine in the perfused liver.
Transduction of cultured hepatocytes with AdCMVeNOS resulted in the
targeting of recombinant eNOS to a perinuclear distribution and binding
with the NOS-activating protein heat shock protein 90. These events
were associated with increased ionomycin-stimulated NO release. In
summary, this is the first study to demonstrate successful delivery of
the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.
hepatic perfusion; adenovirus vector; -galactosidase
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INTRODUCTION |
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NITRIC OXIDE (NO) modulates numerous physiological processes in the liver circulation (6, 24). The basal production of NO in the hepatic circulation is generated through the catalytic activity of the endothelial NO synthase (eNOS) isoform, localized within liver endothelial cells (LEC) and regulated through physiological stimuli including shear stress (21, 25). Several recent studies (10, 21, 24, 26) suggest that the biological activity of eNOS is diminished in liver diseases associated with intrahepatic vasoconstriction and portal hypertension. Nitrovasodilator agents ameliorate intrahepatic vasoconstriction; however, systemic administration of these compounds is accompanied by excessive peripheral vasodilation (1, 3). Thus the ability to directly modulate the eNOS system selectively within the liver may be of utility in a variety of physiological and pathological circumstances.
The success of in vivo gene delivery in humans has been limited in part
by the inability to selectively target viruses to the desired anatomic
location. In rodents, this technical issue is circumvented when
targeting recombinant genes to the liver because intravenous
administration of adenoviral vectors preferentially targets the liver,
particularly hepatocytes (12, 14). However, in the liver,
eNOS resides exclusively in LEC and undergoes specific posttranslational processing events that are required for NO
production. Additionally, LEC are uniquely situated to transduce
vasodilatory signals to underlying contractile cells. Because
hepatocytes are not a cell type in which eNOS normally resides, it is
not known whether these cells possess the cellular machinery to
posttranslationally process recombinant eNOS protein, produce NO, and
influence vascular tone. Therefore, the goals of this study were to
1) use adenoviral vectors encoding -galactosidase
(
-gal) to determine whether transduction is preferential to
hepatocytes, 2) use adenoviral vectors encoding eNOS to
determine whether systemic delivery of adenovirus to liver results in
functional eNOS catalysis and modulation of hepatic vascular tone, and
3) examine the mechanism of eNOS processing and NO
production in transduced hepatocytes in vitro.
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METHODS |
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Animals and reagents. Animal experiments (Male Fisher rats, Harlan Sprague Dawley Laboratories, Indianapolis, IN) and tissue harvesting were performed in accordance with institutional animal care guidelines. Methoxamine (MTX; Sigma Chemical, St. Louis, MO) was dissolved in distilled water and prepared daily. The equivalent volume of water required for solution of the compound was used as control vehicle. Ca2+ ionophore A-23187 (Sigma Chemical) was dissolved in DMSO, and the equivalent volume of vehicle was used in control experiments.
Adenoviral vectors and viral delivery.
Replication-incompetent serotype 5 adenoviral vectors, driven by the
cytomegalovirus immediate-early promoter, were generated, propagated,
and purified as previously described (5). Viral titers
were determined by plaque assay. Briefly, HEK-293 cells were plated on
60-mm tissue culture plates and allowed to become confluent. Dilutions
(109-10
11 M) of the virus were
incubated with the cells for 1 h. The virus was then aspirated,
and the cells were overlayered with 1% agarose in DMEM and 2% fetal
bovine serum (FBS). Overlayering was repeated every 4-5 days to
maintain the cultures. Viral plaques were counted every other day
starting with day 7 after infection until plaque numbers
stopped increasing. The vectors used in these studies encoded a cDNA
sequence for bovine eNOS (AdCMVeNOS) or, alternatively, Escherichia coli
-gal reporter gene (AdCMVLacZ) in place
of the deleted E1 region of the virus. Viruses were diluted in saline and administered intravenously via the tail vein, at a titer of 1010 plaque-forming units (pfu)/rat, administered in a
volume of 50-100 µl, followed by a 300-µl saline flush. All
rats weighed ~250 g. The viral titer of 1010 was
determined on the basis of preliminary experiments demonstrating inefficient transgene expression at titers of 109 and
5 × 109 pfu/rat. One week after injection of the
virus, animals were killed for biochemical, perfusion, or histochemical
studies. For in vitro transduction, hepatocytes were isolated and
cultured as described in Isolation of hepatocytes and
nonparenchymal cells. After 24 h in culture, cells
were washed with PBS and transduced with PBS and 0.5% albumin
containing 100 multiplicity of infection of AdCMVLacZ or AdCMVeNOS for
1 h. The vector solution was then aspirated, cells were washed
with PBS, and complete culture medium was replaced. At 24 h after
transduction, cells were lysed and prepared for protein
immunoprecipitation, fixed and prepared for indirect immunofluorescence
microscopy, or, alternatively, incubated with
Ca2+-ionophore A-23187 to measure stimulated NO release.
Isolation of hepatocytes and nonparenchymal cells.
Rat hepatocytes and nonparenchymal cell (NPC) fractions were prepared
as previously described (25). Briefly, the liver was digested with a collagenase perfusion. The digest was filtered with a
30-µM silk mesh to remove cell clumps and extraneous tissue, and the
hepatocytes were separated by centrifugation at 250 g for 5 min. The supernatant was centrifuged at 250 g a second time to remove remaining hepatocytes and then centrifuged at 500 g, resulting in a NPC pellet. This pellet consisted of the
nonhepatocyte cell populations within the liver, predominantly
sinusoidal lining cells (4, 13). Exclusion of NPC from the
hepatocyte fraction and hepatocytes from the NPC fraction was
consistently >90%. Freshly isolated hepatocyte and NPC pellets were
examined for -gal activity as described in Measurement of
-gal activity and expression. For in vitro hepatocyte
transduction experiments, the freshly isolated hepatocyte pellet was
suspended in DMEM containing 10% FBS, and cells were plated at a
density of 106/ml culture medium on collagen-coated plastic
culture dishes or glass coverslips and placed in a 37°C, 5%
CO2 incubator.
Measurement of -gal activity and expression.
Transgene expression was assessed by quantification of
-gal activity
using a Galacto-light luminometric assay per the manufacturer's suggested protocol (Tropix, Bedford, MA) as previously described (5). Briefly, 1 wk after virus injection, animals were
killed, and liver, kidney, and mesentery tissues were removed and lysed in a buffer (100 mM KPO4 and 0.2% Triton X-100, pH 7.8).
Alternatively, hepatocytes and NPC were separated as described in
Isolation of hepatocytes and NPC, and cell pellets were
lysed.
-gal levels in samples and purified
-gal standards were
measured in duplicate using a microplate reader (TR717, Tropix).
Cellular localization of transgene expression was determined by
histochemical staining for
-gal. Liver sections were snap-frozen in
optimum cutting temperature compound. Frozen tissue sections were fixed
in 2% paraformaldehyde for 15 min at room temperature. After fixation, sections were stained with X-gal solution [5 mmol/l
K3Fe(CN)6, 5 mmol/l
K4Fe(CN)6 · 3H2O, 1 mM
MgCl2, and 1.5 mg/ml 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) in PBS] at 37°C
for 1-2 h and counterstained with nuclear fast red for 5 min.
Indirect immunofluorescence microscopy. Immunofluorescence was performed as previously described in LEC (25). Briefly, hepatocytes transduced with AdCMVLacZ or AdCMVeNOS were fixed in 2% paraformaldehyde. Fixed cells were incubated in eNOS monoclonal antibody (MAb) (Transduction Laboratories, Lexington, KY), and primary antibody was detected using an FITC-coupled secondary antibody. Washes were performed with PBS and 0.1% BSA after both primary and secondary antibody incubation. Cells were mounted in anti-fade solution (Molecular Probes, Eugene, OR) and visualized using a conventional fluorescence microscope (5100TV, Zeiss).
Immunoprecipitation and Western blotting. Cultured hepatocytes or, alternatively, liver tissue were homogenized in a lysis buffer [50 mM Tris · HCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% (vol/vol) Nonidet P-40, 0.1% SDS, and 0.1% deoxycholate, pH 7.5]. Protein quantification of samples was performed using the Lowry assay. eNOS immunoprecipitation was performed by incubating equal aliquots of detergent-soluble protein lysate from hepatocytes transduced with AdCMVLacZ or AdCMVeNOS, with excess eNOS MAb overnight after preclearing of samples with Pansorbin as previously described (26). Immunocomplexes were bound by incubating protein samples with protein A beads for 1 h at 4°C. Bound proteins were eluted by boiling samples in Laemmli loading buffer. Gel electrophoresis of proteins and Western blotting were performed as previously described (25), using eNOS MAb, inducible NOS (iNOS) polyclonal antibody (Transduction Laboratories), or heat shock protein 90 (HSP 90) MAb (Stressgen). Membranes were stained with Ponceau S to confirm equal transfer, and in some experiments Coomassie blue staining was performed on parallel gels to confirm equal protein loading.
NOS activity assay. The conversion of L-[3H]arginine to L-[3H]citrulline was used to determine NOS activity (26). Briefly, liver tissue was homogenized in a lysis buffer identical to that described for Western blotting. Samples were incubated with a buffer containing 1 mM NADPH, 3 µM tetrahydrobiopterin, 100 nM calmodulin, 2.5 mM CaCl2, 50 mM L-valine, 10 µM L-arginine, and L-[3H]arginine (0.2 µCi) at 37°C. To determine NOS activity, duplicate samples were incubated for 20 min in the presence and absence of 1 mM NG-nitro-L-arginine methyl ester (L-NAME) or vehicle. The reaction was terminated by the addition of 1 ml of cold stop buffer (20 mM HEPES, 2 mM EDTA, and 2 mM EGTA, pH 5.5), and the reaction mix was applied to a Dowex AG 50WX-8 resin column. Radiolabeled counts per minute of L-citrulline generation were measured and used to determine L-NAME-inhibited NOS activity.
Measurement of NO production. After 24 h in culture, hepatocytes were transduced with AdCMVLacZ or AdCMVeNOS. The day after transduction, eNOS-derived NO was stimulated by incubating cells in DMEM with 1 mM L-arginine and 10 µM of the eNOS agonist A-23187 or an equal volume of vehicle in place of A-23187. After 1 h, the medium was collected and analyzed for the stable NO byproduct, nitrite, using NO-specific chemiluminescence (Seivers NOA). Cells were then lysed for protein measurement using the Lowry assay. NO-specific chemiluminescence was performed essentially as previously described (25), except that glacial acetic acid containing sodium iodine was substituted for vanadium/hydrochloric acid as the refluxing agent. A standard curve was generated using known nitrite standards. Samples were examined in duplicate, and nitrite readings were normalized for cellular protein.
Liver perfusion studies. Liver perfusion studies were performed in animals transduced with AdCMVLacZ or AdCMVeNOS, using modifications of previously described methods (9, 26). After laparotomy, ligatures were placed around the portal vein and infrahepatic inferior vena cava (IVC). The portal vein was cannulated and perfused through a 16-gauge angiocath, and the IVC distal to the ligature was ligated, allowing the perfusate to escape. A loose ligature was then placed around the suprahepatic IVC before tying of the prehepatic IVC. The redirected perfusate was allowed to escape through a 16-gauge catheter passed through the right atrium into the suprahepatic IVC. The secured liver preparation was then transferred to an enclosed 37°C perfusion apparatus. The liver was perfused ex vivo through the portal vein with Krebs solution (118.3 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, 11.1 mM glucose, and 2 U/ml heparin, pH 7.4) oxygenated with 95% O2-5% CO2 at 37°C, which was recirculated at 20 ml/min with continuous perfusion pressure monitoring (Sys-200/1 Micro-Med). Global viability of the preparation was assessed by gross appearance of the tissue, stable pH of the perfusate, and stable perfusion pressure for 10 min before administration of compounds.
Statistical analysis. All data are given as means ± SE. Data were analyzed using paired and unpaired Student's t-tests as appropriate.
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RESULTS |
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Adenovirus preferentially targets hepatocytes.
Because of the existence of endogenous eNOS in liver, adenoviral
transgene expression was first assessed by determining expression and
activity of -gal. Studies were performed 1 wk after injection of
virus based on preliminary experiments demonstrating maximum hepatic
transgene expression at 1 wk compared with 3 days or 2 wk after virus
injection (data not shown).
-gal activity was increased severalfold
in liver from animals transduced with AdCMVLacZ compared with AdCMVeNOS
(Fig. 1A). In
contradistinction to the prominent increase in
-gal activity in
liver after viral transduction, there was no increase in
-gal
activity in nearby kidney or mesentery tissues from rats transduced
with AdCMVLacZ (Fig. 1A), suggesting preferential targeting
of systemically administered adenoviral vectors to liver as previously
described (12). We next examined whether transgene
expression within AdCMVLacZ-transduced liver was most prominent in
hepatocytes or sinusoidal lining NPC (4, 13). Liver cells
from AdCMVLacZ-transduced animals were separated into hepatocyte and
NPC fractions and assessed for
-gal activity.
-gal activity was
enriched by nearly threefold in hepatocytes compared with NPC
(P < 0.05; n = 3 animals; Fig.
1B), suggesting that adenovirus preferentially targets
hepatocytes. Next, histochemical staining was performed in liver from
animals transduced with AdCMVLacZ to further examine the cellular
localization of transgene expression. Consistent with the studies in
isolated hepatocytes and NPC, transgene expression was most prominent
in hepatocytes (Fig. 1C, right). No staining was
detected in animals transduced with AdCMVeNOS (Fig. 1C,
left).
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Systemic administration of AdCMVeNOS increases eNOS protein levels
and NOS activity in liver.
To determine whether systemic delivery of recombinant eNOS gene results
in the production of catalytically active eNOS protein in the liver,
animals were transduced with AdCMVLacZ or AdCMVeNOS. As seen in the
representative Western blot (Fig.
2A), eNOS protein levels were
markedly increased in animals transduced with AdCMVeNOS (n = 4; liver lysates from 2 representative animals
transduced with eNOS are depicted in the representative blot) compared
with animals injected with AdCMVLacZ or control rats (n = 4). In contrast, iNOS protein was not detected in any of the
transduced animals but was detected in a positive control lysate
prepared from cultured LEC stimulated with lipopolysaccharide (0.5 µg/ml) for 18 h (Fig. 2B). The catalytic activity of
eNOS in endothelial cells is dependent, in part, on dynamic
interactions of the enzyme with regulatory proteins (7,
26), events that may not be possible in transduced hepatocytes.
Therefore, to determine whether the eNOS protein derived from
recombinant eNOS gene is catalytically active, assays were performed to
measure the conversion of radiolabeled L-arginine to
L-citrulline within liver lysates. As depicted in Fig.
3, NOS catalytic activity was
significantly greater in liver tissue from animals transduced with
AdCMVeNOS compared with animals transduced with AdCMVLacZ
[P < 0.05, eNOS (n = 5) vs. LacZ
(n = 3); preliminary studies had demonstrated that NOS
catalytic activity was not significantly different in control animals
compared with animals transduced with AdCMVLacZ]. These studies
indicate that eNOS protein derived from recombinant eNOS gene is
catalytically active.
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AdCMVeNOS-transduced hepatocytes posttranslationally process
recombinant eNOS and release NO.
eNOS-derived NO production requires extensive posttranslational
processing events that include subcellular targeting to membrane compartments and interactions with activating proteins such as HSP 90 (7, 23, 27). To examine the cellular processing of
heterologously expressed eNOS in the cells that were transduced to the
greatest extent in vivo, hepatocytes were isolated and transduced in
vitro with AdCMVeNOS or AdCMVLacZ and prepared for immunoprecipitation studies, immunofluorescence analysis, or, alternatively, NO release. First, to determine whether recombinant eNOS in transduced hepatocytes binds with the NOS-activating protein HSP 90 (7), we
immunoprecipitated eNOS from lysates prepared from transduced
hepatocytes. As seen in Fig.
5A, recombinant eNOS binds
with the NOS-activating protein HSP 90 in transduced hepatocytes, akin
to that observed in cells expressing endogenous eNOS (7,
27), whereas eNOS is not detected by immunoprecipitation in
cells transduced with AdCMVLacZ. Next, transduced cells were
prepared for immunofluorescence analysis. Recombinant eNOS in
hepatocytes transduced with AdCMVeNOS (Fig. 5B,
right) is detected in a perinuclear distribution, a pattern reminiscent of that observed in LEC expressing endogenous eNOS and
previously demonstrated to colocalize with the Golgi marker mannosidose
II (25). In Fig. 5B, left,
no immunofluorescence signal was detected for eNOS in cells transduced
with AdCMVLacZ. These observations suggest that in hepatocytes,
recombinant eNOS binds with activating proteins and targets to
appropriate membrane compartments. To determine whether these
processing events result in cellular NO release, NO production was
measured in hepatocytes transduced with AdCMVeNOS, using
NO-specific chemiluminescence. As seen in Fig.
6, A-23187-stimulated production of NO
was markedly increased in hepatocytes transduced with AdCMVeNOS
compared with AdCMVLacZ (P 0.05). Western blot analysis
(Fig. 6, inset) demonstrates the prominent increase in eNOS
protein levels in cells transduced with AdCMVeNOS in vitro.
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DISCUSSION |
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In this study we demonstrate, for the first time, in vivo delivery of recombinant eNOS gene to the liver. In vivo, eNOS gene delivery results in the overexpression of catalytically active eNOS protein, which impacts on hepatic vascular tone. Additionally, recombinant eNOS targets and binds regulatory proteins appropriately within hepatocytes, thereby resulting in the cellular release of NO. These experiments suggest that transduction of nonendothelial cell types with genes encoding vasoactive compounds, such as eNOS, may have the potential to impact vascular tone in the liver.
On the basis of our studies, transgene expression is most prominent in hepatocytes, although it is also observed to a lesser degree in NPC, which consist predominantly of sinusoidal lining cells (LEC, Kupffer cells, and hepatic stellate cells) (4, 13). However, eNOS-derived NO production requires distinct posttranslational processing events within the endothelial cell. For example, it has been demonstrated that eNOS targeting to membrane compartments, such as the Golgi apparatus, is necessary for optimal NO production (23). Additionally, binding of eNOS with the molecular chaperone HSP 90 also promotes NOS activity (7, 27). Although hepatocytes do not express eNOS under physiological conditions (21, 26), these studies suggest that when transduced with eNOS, hepatocytes have the capability to carry out appropriate posttranslational processing events, including targeting of eNOS to the Golgi apparatus and binding with HSP 90. We anticipate that these events facilitate eNOS-derived NO production in hepatocytes. Additionally, the changes observed in baseline perfusion pressure and pressure responses to MTX suggest that NO production from hepatocytes may have an impact on vasomotor reactivity in the liver. These observations are reminiscent of previous studies (5) demonstrating that delivery of the eNOS gene to adventitial fibroblasts results in NO production and modulates vasomotor reactivity in the cerebral circulation independent of endothelium. In the transduced liver, NO production from transduced hepatocytes may diffuse toward adjacent effector cells, presumably hepatic stellate cells, thereby modulating vascular responses. Indeed, the unique proximity of parenchymal cells (hepatocytes) to contractile cells (hepatic stellate cells) in the liver may support the kinetics of such a process. However, the lower degree of transduction of NPC that we detected may also be contributing to the vascular changes we observed in vivo.
In rodents, intravenously delivered adenoviral vectors preferentially target liver. Our findings of a lack of transgene expression in kidney and mesentery are consistent with a number of previous studies (12, 14, 28) focused on this issue. For example, Kurata et al. (14) examined transgene expression 7 days after intravenous adenoviral delivery in the mouse and detected a logarithmic increase in transgene expression in liver with minimal transgene detection in 15 other organ beds, including the brain, heart, and lung. However, the liver-specific targeting of adenoviral vectors observed in rodents may not necessarily be relevant for human gene therapy, in which case liver-specific uptake can be problematic. One possible reason for this dichotomy between species may be the low level of expression of Coxsackie adenovirus receptor (CAR) in human liver compared with rodent liver (30), because the degree of CAR expression appears to impact adenoviral uptake (2).
Vascular tone in the liver is determined by a balance of vasodilatory and constrictive compounds, including NO, endothelin, and carbon monoxide (20, 22, 24, 29). Experimental evidence suggests that these vasoactive agents modulate basal tone as well as vascular responses that occur in response to pathophysiological stimuli such as reperfusion injury (17). The premise that eNOS-derived NO contributes to these processes is based on several lines of evidence: 1) pharmacological inhibition of NO biosynthesis increases portal pressure, attenuates NO-dependent vasodilation, and promotes the vasoconstrictive effects of ethanol in the perfused liver (15, 16, 25); 2) eNOS protein is expressed abundantly in the hepatic vascular endothelium and is regulated by hemodynamic stimuli and events such as shear stress and hemorrhagic shock (18, 21, 25); and 3) hepatic stellate cells and hepatic vascular smooth muscle cells relax in response to NO (19, 31). In this study, delivery of the eNOS gene to the liver results in overexpression of hepatic eNOS protein. The diminution of baseline perfusion pressure and lower perfusion pressure in the presence of the constrictor MTX, which we observed in rats overexpressing hepatic eNOS compared with control, provides further evidence for the concept of NO as an important regulator of hepatic vascular tone.
Although hepatic gene therapy has predominantly targeted metabolic disease (11), there is extensive evidence to support a role for gene therapy approaches in the treatment of hepatic vascular conditions based on studies (8) in other circulatory beds. In this regard, recent studies (10, 21, 26) in the liver implicate deficient hepatic eNOS activity and NO production as a putative cause of hepatic vasoconstriction and endothelial dysfunction in experimental models of liver cirrhosis and portal hypertension. These studies, in conjunction with the present findings, suggest the feasibility of utilizing targeted delivery of the eNOS gene to the liver as a potential venue for reversing deficient hepatic NOS activity. Additionally, by demonstrating that transduced hepatocytes produce NO in a functional manner, these studies suggest that eNOS gene delivery may not necessarily need to be targeted to endothelial cells to have an impact on hepatic vascular responses.
In summary, our study shows, for the first time, recombinant eNOS gene delivery to liver in vivo through systemic administration of an adenoviral vector with the subsequent production of catalytically active eNOS protein. These studies provide evidence for the feasibility of using targeted gene delivery approaches to influence vascular tone in the liver and suggest that eNOS gene delivery may not need to be targeted exclusively to the endothelium for potential efficacy.
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NOTE ADDED IN PROOF |
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While this paper was in review, complementary data describing neuronal NOS gene transfer to cirrhotic liver were published (Yu Q, Shao R, Qian HS, George SE, and Rockney DC. Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. J Clin Invest 105: 741-748, 2000).
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ACKNOWLEDGEMENTS |
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We thank David G. Harrison and James M. Wilson for kindly providing the eNOS cDNA and AdCMVLacZ, respectively, and Steve Bronk and Greg Gores for help with hepatocyte cultures.
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FOOTNOTES |
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This work was supported by grants from the National Institutes of Health (DK-02529, V. Shah; HL-53524, Z. S. Katusic) and the Mayo Clinic Foundation.
Present address of A. F. Chen: Dept. of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI 48824.
Address for reprint requests and other correspondence: V. Shah, Gastrointestinal Research Unit, Alfred 2-435, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: shah.vijay{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.
Received 20 September 1999; accepted in final form 9 May 2000.
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