1Gastrointestinal Research Unit, Department of Physiology and Tumor Biology Program, Mayo Clinic, Rochester, Minnesota 55905;2Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510; and3Laboratorio de Hormonal, Hospital Clinic, 08036 Barcelona, Spain
Submitted 26 March 2003 ; accepted in final form 26 May 2003
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ABSTRACT |
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endothelial nitric oxide synthase; caveolin; bile duct ligated rat; portal hypertension; hepatic vasculature
The mechanism of eNOS-derived NO generation requires diverse posttranslational events, and recent studies indicate that the phosphorylation of the serine residue at amino acid (AA) 1179 on eNOS is one of the important steps in the process of eNOS activation (5, 6). In this regard, mutation of AA1179 in eNOS from a serine to aspartate modifies the calcium activation characteristics of the enzyme, resulting in a constitutively active form of eNOS (S1179DeNOS) that produces excess levels of NO compared with the wild-type eNOS (11, 18). Alternatively, mutation of serine 1179 to alanine in eNOS (S1179AeNOS) mimics a nonphosphorylated form of eNOS (11).
In this study, we analyzed the function of S1179DeNOS in liver cells in vitro and in vivo through the use of an adenoviral vector encoding S1179DeNOS (AdS1179DeNOS). We demonstrated that overexpression of S1179DeNOS in both isolated liver cell populations and in sham liver stimulates NO generation and vasodilatory responses, respectively, but does not improve vasodilatory responses in cirrhotic rats, despite similar hepatic transgene expression within the two groups. Immunoelectron microscopy studies detect the eNOS inhibitory protein caveolin in sinusoidal lining cells in the bile duct-ligated (BDL) liver, including hepatic stellate cells, a prominent target of adenoviral vectors in vivo. In vitro studies in the LX2 hepatic stellate cell line indicate that caveolin binds recombinant S1179DeNOS and that overexpression of caveolin further potentiates binding of the two proteins from cell lysates in conjunction with reducing the activity of recombinant S1179DeNOS. These studies thereby suggest that the lack of vasodilatory effect of recombinant S1179DeNOS in BDL liver may be mediated through caveolin upregulation and ensuing S1179DeNOS inhibition in sinusoidal lining cells, including hepatic stellate cells.
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MATERIALS AND METHODS |
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Adenoviral gene delivery in vitro and in vivo. For in vitro
transduction, hepatocytes and nonparenchymal cells (NPC) were isolated and
cultured as described previously
(20,
22) using a collagenase
perfusion technique. With the use of this approach, exclusion of NPC from the
hepatocyte fraction and hepatocytes from the NPC fraction was consistently
>95%. For in vitro NPC transduction, the freshly isolated NPC pellet was
suspended in DMEM containing 10% FBS, and cells were plated at a density of
106/ml culture media on 60-mm collagen-coated plastic tissue
culture dishes or glass coverslips and placed in a 37°;C, 5%
CO2 incubator. Four hours after plating, cells were washed with PBS
and transduced with PBS/0.1% albumin containing 50 multiplicity of infection
(MOI) of AdS1179D or AdGFP for 1 h. The vector solution was then aspirated,
and after being washed with PBS, complete culture medium was replenished.
Twenty-four hours after transduction, culture media were collected to analyze
basal NO accumulation, and cells were lysed and prepared for protein analysis,
fixed, and prepared for epifluorescence microscopy to detect GFP or
alternatively prepared for measurement of A23187
[GenBank]
-stimulated NO release. Each
of these techniques is described below. Transduction and media collection from
hepatocytes was performed in an analogous manner using the vectors described
above as well as an adenoviral vector encoding an eNOS construct, which mimics
a nonphosphorylated form of eNOS (mutation of serine 1179 to alanine),
AdS1179AeNOS (11).
Cotransduction experiments were performed in LX2 cells, a well-characterized
hepatic stellate cell line
(27,
30), which were transduced
with both AdS1179DeNOS and AdCav or with one of the above in conjunction with
an empty vector using procedures and viral titers identical to that described
above. For in vivo studies, adenoviruses were diluted in saline and
administered intravenously via the tail vein 4 wk after surgery at a
titer of 1010 plaque-forming units (pfu)/rat administered in a
volume of 50-100 µl followed by 100 µl saline flush. All rats weighed
250 g. This titer was selected based on previous studies
(20) performed to optimize in
vivo transduction, which indicated that lower titers of adenovirus resulted in
suboptimal transduction, whereas higher titers were associated with
histological evidence of inflammatory cell infiltration in liver of some
animals. One week after injection of the virus, animals were killed for
biochemical and pharmacological studies as described below.
Fluorimetry from liver lysates. Liver tissue GFP levels were measured from transduced animals to confirm adenoviral-mediated overexpression of transgene. Liver tissues were homogenized in a lysis buffer [50 mM Tris · HCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM leupeptin, 1 mM PMSF, 1% (vol/vol) Nonidet P-40 (NP-40), 0.1% SDS, 0.1% deoxycholate, pH 7.5] (22), and protein quantification of samples was performed using the Lowry assay. GFP was quantified by measuring fluorescence intensity from aliquots of liver lysates in each of the experimental groups using a fluorimeter (Versa-Fluor Flurometer, BioRad) (24). Samples were analyzed in microcuvettes at excitation of 490 nm and emission of 520 nm with duplicate readings from duplicate wells and normalized for cellular protein content. The negative control sample included normal rat liver lysate, whereas the positive control sample included liver lysate derived from a transgenic mouse that constitutively expresses GFP in liver (17).
Measurement of NO production. Hepatocytes and NPC were transduced with adenoviral vectors as described above. Basal NO production was assessed from NPC by measuring NO accumulation in the supernatant during the 24 h immediately following adenoviral transduction by collecting the media, and assessing levels of the stable NO byproduct nitrite by the Greiss reaction. Subsequently, agonist-stimulated NO production was assessed by incubating transduced cells with calcium ionophore A23187 [GenBank] (10 µM). After 1 h, supernatant was collected and analyzed for nitrite using NO-specific chemiluminescence (Seivers NOA, Boulder, Colorado). NO-specific chemiluminescence and Greiss reaction were performed as previously described (1, 20, 21). For both methods, a standard curve generated using known nitrite standards was performed in parallel with samples, which were analyzed in duplicate and then normalized for cellular protein.
Liver perfusion studies. Analysis of hepatic vascular responses was performed 1 wk after in vivo adenoviral transduction experiments using the isolated perfused rat liver preparation (20, 23). 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-g 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 the prehepatic IVC. The redirected perfusate was allowed to escape through a 16-g 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 [in mM: 118.3 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, and 11.1 glucose, with 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 m before administration of compounds. Perfusion pressure changes in response to a vasoconstrictor were assessed by cumulative addition of MTX to the perfusate (10-6 to 10-4 M) (20). Perfusion pressure changes in response to flow were performed as previously described (23) by increasing the flow rate from 20 to 40 ml/min in increments of 10 ml/min.
Fluorescence microscopy. Fluorescence microscopy for detection of GFP protein was performed as previously described (1). In brief, NPC transduced with AdGFP were fixed in 2% paraformaldehyde, mounted in Anti-fade (Molecular Probes, Eugene, OR), and GFP was detected using a conventional fluorescence microscope (5100TV; Zeiss, Germany).
Transmission electron microscopy and immunogold analysis. For morphological examination of sham and BDL liver, tissues were fixed in Trump's fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2). Tissue was then rinsed for 30 min in three changes of 0.1 M phosphate buffer, pH 7.2, followed by a 1-h postfix in phosphate-buffered 1% OsO4. After being rinsed in three changes of distilled water for 30 min, the tissue was en bloc stained with 2% uranyl acetate for 30 min at 60°;C. After being en bloc stained, the tissue was rinsed in three changes of distilled water, dehydrated in progressive concentrations of ethanol and 100% propylene oxide, and embedded in Spurr's resin. Thin (90 nm) sections were cut on a Reichert Ultracut E ultramicrotome, placed on 200 mesh copper grids, and stained with lead citrate. Samples for immunogold analysis were prepared as previously described (9) with minor modifications. In brief, the specimens were fixed in 4% formaldehyde and 0.2% gluaraldehyde in phosphate buffer and rinsed in phosphate buffer. Dehydration was done in a series of ethyl alcohols ranging from 60 to 80%, while progressively lowering the temperature to -20°;C. After two changes in 100% LR White resin, the specimen was embedded and the resin was polymerized at 55°;C. Thin sections were mounted on nickel grids and dried overnight. Nonspecific antigen sites were blocked first in 1% glycine then in PBS and 0.05% Tween 20 (PBST) with 2% acetylated bovine serum albumin. Caveolin polyclonal antibody (PAb) (Transduction Laboratories, Lexington KY) was diluted 1:100 in PBST, and sections were incubated in primary antibody for 2 h at room temperature. Lung tissue, which is highly enriched in caveolin, was used as a positive control, whereas negative control samples were examined in the absence of PAb. Grids were rinsed in PBST and incubated for 60 min in secondary antibody (goat anti-rabbit) conjugated to 5 nm colloidal gold. The grids were rinsed sequentially in PBST and water and then silver-enhanced for 20 min. The sections were then stained with uranyl and lead. Micrographs were taken on a JEOL 1200 EXII operating at 60 KV at magnification ranging from x5,000 to x50,000.
Immunoprecipitation and Western blotting. Twenty-four hours after adenoviral transduction, LX2 cells were homogenized in a lysis buffer [in mM: 50 Tris · HCl, 0.1 EGTA, 0.1 EDTA, 100 leupeptin, and 1 PMSF, with 1% (vol/vol) NP-40 and 0.1% deoxycholic acid (pH 7.5)] (23). Protein quantification of samples was performed using the Lowry assay. Immunoprecipitation was performed by incubating equal aliquots of detergent-soluble protein lysate from whole liver with excess eNOS MAb or caveolin PAb (Transduction Laboratories) overnight, after preclearing of samples with Pansorbin (23). Immunocomplexes were bound by incubating protein samples with Protein A beads for 1 h at 4°;C. Complexes were washed three times in the lysis buffer described above in the absence of detergents. Bound proteins were eluted by boiling samples in Laemmli loading buffer. Immunoprecipitated proteins or, alternatively, detergent-soluble protein lysates, were separated by SDS-PAGE on a 12% acrylamide gel, and proteins were electroblotted onto nitro-cellulose membranes. The membranes were washed in Tris-buffered saline with 0.1% Tween, blocked in 5% milk, and incubated with eNOS MAb and caveolin PAb. Membranes were stained with Ponceau S to confirm equal protein loading and transfer between lanes.
NOS activity assay. LX2 cells were lysed in a buffer identical to
that described above for preparation of Western blot lysates, then prepared
for NOS activity by measuring the conversion of
L-[3H]arginine to
L-[3H]citrulline. Briefly, detergent soluble lysates
were incubated for 20 min with a buffer containing 1 mM NADPH, 3 µM
tetrahydrobiopterin, 10 nM calmodulin, 0.25 mM CaCl2, 10 µM
L-arginine, and L-[3H]arginine (0.2 µCi)
at 37°;C. It is important to note that the concentrations of calmodulin
and CaCl2 were lower in the current study than in prior studies
(23) so as to facilitate
detection of the inhibitory influence of caveolin on activity of S1179DeNOS,
which has lower calcium/calmodulin activation requirements than the wild-type
enzyme (11). Samples were run
in duplicate in the presence and absence of
N-nitro-L-arginine methyl ester
(L-NAME; 1 mM) or vehicle. The reaction was terminated by the
addition of 1 ml of cold stop buffer (20 mM HEPES, 2 mM EDTA, 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.
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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S1179DeNOS improves vasodilatory responses in sham rats, but not BDL rats after in vivo transduction
As AdS1179DeNOS transduction of liver cells potentiated NO production, we
next sought to examine the influence of S1179DeNOS gene delivery on hepatic
vascular responses in the intact rat liver after in vivo transduction. Sham or
BDL rats were transduced with AdS1179DeNOS or AdGFP (1010 pfu into
tail vein) 4 wk after surgery. One week later, animals were killed and
vascular responses were examined in the isolated perfused liver in response to
increasing concentrations of the vasoconstrictor MTX
(10-6 to 10-4 M), an agent that
increases both presinusoidal and sinusoidal/postsinusoidal pressure in the
perfused rat liver preparation
(10). In sham animals,
S1179DeNOS overexpression was associated with significantly diminished
constriction in response to incremental doses of MTX
(Fig. 2A), similar to
prior studies performed with wild-type eNOS overexpression
(20). However, surprisingly,
BDL rats transduced with AdS1179DeNOS did not demonstrate improved
vasodilatory responses as evidenced by similar flow-dependent pressure
increases as that observed in BDL rats transduced with AdGFP
(Fig. 2B). Similarly,
a diminution in constriction in response to incremental doses of MTX was also
not detected in BDL rats transduced with AdS1179DeNOS compared with BDL rats
transduced with AdGFP (data not shown). Owing to the extensive portasystemic
shunting observed after BDL as well as the previously documented difficulties
in transfecting liver cells in the injured/cirrhotic liver
(32), we next sought to assess
the adequacy of transgene expression in livers after in vivo delivery of
adenoviral vectors to exclude the possibility that the lack of vasodilatory
effect of AdS1179DeNOS in BDL rats may be due to inadequate transgene
expression. However, as seen in Fig.
2C, GFP transgene levels were similar in all experimental
groups including BDL animals transduced with AdS1179DeNOS and AdGFP as well as
sham animals transduced with both these vectors, as assessed by fluorimetric
quantification from respective liver lysates. Thus S1179DeNOS gene delivery
augments vasodilatory tendencies in sham animals but does not augment
vasodilatory responses in BDL animals, despite adequate transgene
expression.
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Caveolin-1 is expressed in stellate cells in BDL liver and binds S1179DeNOS. We and others have previously demonstrated that increased expression and eNOS binding of caveolin-1 in liver endothelial cells may contribute to deficient eNOS function in the BDL rat (22) and in human cirrhosis (3, 31). Because hepatic stellate cells constitute an important target for the vasoregulatory effects of NOS-encoding adenoviral vectors (33), we wondered whether inhibitory signaling events in stellate cells may contribute to deficient function of heterologously expressed recombinant S1179DeNOS. We have previously demonstrated that caveolin is detected in stellate cells in normal liver (22). To detect for caveolin expression in stellate cells in intact tissue, BDL or sham liver sections were prepared for morphological and immunogold examination using transmission electron microscopy. The lowmagnification electron micrograph of the sinusoids in Fig. 3, A and B, depicts the cells of interest, the sinusoidal endothelial cell in Fig. 3A and the hepatic stellate cell in Fig. 3B. As seen in the higher magnification image in Fig. 3C, analysis of caveolin immunogold particles in sham liver sinusoids did not reveal very prominent detection of gold particles, consistent with our prior light microscopic immunohistochemical analyses for caveolin (22, 23). As seen in a high-magnification electron micrograph in Fig. 3D, caveolin immunogold particles were more prominent in endothelial cells lining larger vascular structures such as venules as depicted by the gold labeling of nonclathrin-coated endothelial cell vesicles. Caveolin immunogold detection was also more prominent in BDL liver that was analyzed, as evidenced in the higher magnification images in Fig. 3, E and F, which depict caveolin immunogold particles prominently visible on sinusoidal endothelial cells and hepatic stellate cells, respectively.
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Next, to determine whether caveolin expression from hepatic stellate cells, which do not normally express eNOS (16, 21), could influence the function of recombinantly expressed S1179DeNOS, we performed in vitro studies in the LX2 hepatic stellate cell line. Cells were cotransfected with AdS1179DeNOS, AdCav, empty vector, or combinations of these vectors and prepared for immunoprecipitation analysis using eNOS MAb or, alternatively, caveolin PAb. As seen in Fig. 4A, top, immunoprecipitation of caveolin coprecipitates S1179DeNOS (lane 3) from cells cotransduced with AdCav and S1179DeNOS but does not coprecipitate eNOS in cells transduced with only AdCav (lane 2) or only empty vectors (lane 1). Figure 4A, bottom, displays the corresponding Western blot analysis from total cell lysates from each of the experimental groups demonstrating overexpression of myc-tagged caveolin (larger molecular weight band in lanes 2 and 3) and overexpression of S1179DeNOS (lane 3). Furthermore, as seen in Fig. 4B, immunoprecipitation of eNOS in cells transduced with AdS1179DeNOS coprecipitates small amounts of caveolin (lane 2), and caveolin coprecipitation is further enhanced upon cotransduction of these cells with AdCav (lane 3). Figure 4B, bottom, displays the corresponding Western blot analysis from total cell lysates from each of the experimental groups demonstrating overexpression of myc-tagged caveolin (larger molecular weight band in lane 3) and overexpression of S1179DeNOS (lanes 2 and 3). To examine whether caveolin-1 overexpression and enhanced binding with S1179DeNOS influences NOS function, NOS activity was assayed in LX2 cells transfected with S1179DeNOS alone or with S1179DeNOS in conjunction with caveolin-1. As seen in Fig. 4C, caveolin overexpression resulted in a significant reduction in the catalytic activity of S1179DeNOS. These studies indicate that heterologously expressed recombinant S1179DeNOS interacts with the eNOS inhibitory protein caveolin, endogenously expressed in LX2 cells, and furthermore that increases in caveolin levels, as observed after BDL (22) and as mimicked in these studies by recombinant caveolin-1 overexpression, promote further binding of caveolin with S1179DeNOS in these cells and diminish NOS activity.
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DISCUSSION |
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Interestingly, a vasodilatory influence of S1179DeNOS in cirrhotic liver was not evident from the current study. The lack of functional effect of S1179DeNOS in cirrhotic liver suggests that the post-translational processing defects of endogenous hepatic eNOS protein evident in prior studies (16, 22) also abrogates activity of exogenously delivered S1179DeNOS protein. This hypothesis is supported by the demonstration that in hepatic stellate cells, which are a key target cell for systemically delivered adenoviral vectors (32, 33), the NOS inhibitory protein caveolin binds to exogenously delivered S1179DeNOS and that increases in cellular caveolin levels in these cells via adenoviral-based overexpression result in even greater binding interactions. These studies highlight the dependence of eNOS function on appropriate posttranslational processing rather than on absolute levels of the protein (7) and are consistent with prior studies that have failed to demonstrate a deficiency of eNOS protein levels in this model of portal hypertension despite deficient NO generation (16, 23). It has been recently demonstrated that overexpression of an adenoviral vector encoding wild-type eNOS protein improves portal hypertension in a chemical model of experimental cirrhosis (29), and, consistent with the aforementioned considerations, the model used in that study was, by those authors, characterized by a deficiency of eNOS protein levels within the cirrhotic liver. The deficiency in eNOS protein levels may thereby account for the beneficial influence of eNOS gene delivery in that study (29), although alternative explanations such as differences in viral vector or method of delivery may also account for the differences in results between that study and our study. Interestingly, prior studies (33) have demonstrated that delivery of the neuronal NOS gene to cirrhotic liver via adenoviral vector also partially corrects deficient NO production and NO-deficient hepatic vasodilatory responses that characterize the cirrhotic liver. The contrasting effects of eNOS and nNOS gene delivery in cirrhosis highlight the prominent distinctions of the activation processes of these two NOS isoforms. In this context, future studies examining the influence of gene delivery of the high NO-generating inducible NOS isoform on hepatic vascular responses in cirrhotic liver will be of interest.
It is possible that resistance to efficient cell transduction of AdS1179DeNOS in the BDL liver (32) or inadequate viral titering due to portasystemic shunting may partially account for the difficulty in promoting vasodilatory responses in the BDL liver. However, the detection of similar levels of transgene overexpression in BDL liver compared with sham liver reduces this likelihood in our study. Analysis of effect with higher viral titers would be desirable; however, this option is limited by excess inflammatory responses induced by excess adenoviral vector titering. Thus future studies using alternative vector systems with higher and longer duration of overexpression might result in a greater vasodilatory response in cirrhotic liver transduced with eNOS gene than that which we observed using traditional adenoviral vectors and are worthy of pursuit.
In rodents, systemic delivery of adenovirus results in prominent hepatic delivery with transduction of multiple liver cell types to varying degrees, including hepatic stellate cells (20, 33). Transduction of these cells is particularly intriguing, because these cells contain the downstream signaling targets, including soluble guanylate cyclase, necessary to influence vascular tone in response to NO as well as other vasodilator molecules such as CO (14, 15, 19, 26, 28). We and others (3, 22, 23, 31) have previously demonstrated that caveolin-1 protein abundance is increased in cholestatic and noncholestatic models of chronic liver injury and cirrhosis within liver endothelial cells and that this may contribute to deficient hepatic eNOS activity in these models. The mechanism of caveolin upregulation remains uncertain but does not appear to be secondary to bile acids, at least as evidenced by a lack of bile acid-mediated stimulation of caveolin promoter activity as assessed by a reporter assay in transfected cells (S. Cao and V. Shah, unpublished data). The current studies demonstrate, by immunoelectron microscopy, that caveolin expression also occurs in stellate cells in the BDL liver. This is not entirely surprising, because caveolin has been previously demonstrated in pericytes and smooth muscle cells in other organs (2). As stellate cells are readily transduced with intravenously administered adenoviral vectors and may be the target cell by which NOS gene delivery imparts beneficial vasodilatory actions (32, 33), we anticipate that increases in caveolin, not only in liver endothelial cells but also in other perisinusoidal cells including hepatic stellate cells, may contribute to the inability of S1179DeNOS to promote vasodilatory responses in the transduced BDL liver. This hypothesis is supported by our in vitro studies in LX2 hepatic stellate cells in which, overexpression of caveolin-1 is associated with increased association of caveolin with recombinant S1179DeNOS and diminished NOS activity. These studies suggest that potentiation of eNOS-derived NO generation in the cirrhotic liver may be more likely achieved by modulating the signaling of existing eNOS protein through approaches that dissociate the inhibitory eNOS-caveolin interaction or that potentiate the phosphorylation of existing eNOS protein, rather than by delivering excesses of eNOS protein, and recent studies (12a) using gene delivery of activated Akt in liver cirrhosis support this concept. Alternatively, molecular or pharmacological targets downstream of NOS generation, such as activation of guanlyate cyclase, remain an area of further investigation.
In summary, the present study provides important insights into the function of hepatic eNOS in the cirrhotic liver by demonstrating that 1) overexpression of a constitutively active form of eNOS potentiates NO production from both hepatocytes and NPC and promotes hepatic vasodilatory responses when injected in vivo, and 2) constitutively active recombinant eNOS does not function optimally in the context of the cirrhotic liver, perhaps, in part, because of the inhibitory influence of excess caveolin protein levels within adenovirally transduced cells, including hepatic stellate cells.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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