1 Departments of Medicine and 2 Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Adenovirus-mediated
gene transfer has become an important tool with which to introduce
genetic material into cells. Available data emphasize efficient
adenoviral transduction of parenchymal liver cells (i.e., hepatocytes)
in both in vitro and in vivo model systems, typically in normal cells.
The aim of this study was to evaluate gene transfer to nonparenchymal
(and parenchymal) cells of the normal and injured rat liver.
Hepatocytes, stellate cells, and endothelial cells were isolated by
standard methods. Liver injury was induced by bile duct ligation or
carbon tetrachloride administration. Cells were transduced in vitro
with an adenovirus encoding -galactosidase (Ad.
-gal) over a range
of viral titers, and transduced cells were identified by detection of
X-gal. In vivo transduction efficiency was studied in normal and
injured livers using cell isolation techniques. Nonparenchymal cells
were transduced with greater frequency than hepatocytes at all
adenoviral titers tested, both in vitro and in vivo. After liver
injury, adenoviral transduction was reduced for all liver cell types
compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that,
to achieve comparable gene expression in the injured liver, higher
adenoviral titers may be required, an important consideration as gene
therapy in disease states is considered.
cirrhosis; bile duct ligation; carbon tetrachloride; stellate cell; sinusoidal endothelial cell
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INTRODUCTION |
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OVER THE PAST 30 YEARS, gene therapy has transitioned from a concept to human clinical trials (8). Furthermore, the scope and application of gene therapy has expanded greatly over the last several years. In addition to the possibility of correcting inherited defects of metabolism, gene therapy approaches are being used or considered to treat cancer, acquired immunodeficiency syndrome, and other acquired diseases. Among the major issues in the area of gene therapy are the following: specific vector systems (i.e., RNA and DNA viruses, modified viruses), the cellular targets of vector systems, and the efficiency of cellular transduction.
Replication deficient-recombinant adenovirus represents one of the most advanced and best-studied vector systems (40). It is highly efficient at transferring genes to a wide variety of target cells both in vitro and in vivo; furthermore, it is capable of expressing exogenous genes at a high level (40). The best characterized adenoviruses are types 2 and 5. These adenoviruses have substantial tropism for epithelial cells from the respiratory tract as well as the liver. In fact, when administered intravenously at high titer, over 95% of hepatocytes have been shown to be transduced with adenovirus (14, 24).
Liver is made up of multiple cellular components including parenchymal (epithelial) and nonparenchymal (stellate cells, sinusoidal endothelial cells, Kupffer cells, and others) elements. Although the epithelial compartment has received the greatest attention, endothelial and stellate cells (as well as Kupffer cells, and resident lymphocyte populations) are important constituents of the liver (41). Quantitatively, nonparenchymal cells make up 15-20% of the cells found in the liver and have been implicated in a wide variety of diseases (41). For example, work over the past two decades indicates that hepatic stellate cells are the primary effectors of fibrogenesis after liver injury, thus mediating the wounding response to injury (5). Thus because manipulating gene expression in nonparenchymal cells is likely to have important biological relevance for many diseases, one of the aims of this study was to examine gene transfer to nonparenchymal cells.
Because nonparenchymal cells play important roles not only in normal liver physiology but also in a number of liver diseases, a further critical area when considering gene therapy in disease states is transduction efficiency in the injured liver. Most forms of liver injury result in a wound healing process that is characterized by increased fibrogenesis and wound contraction (4, 39). In addition, vascular abnormalities are prominent in chronic liver injury (32), raising the possibility that access to specific hepatic cellular compartments may be compromised. As gene therapy is considered for various liver diseases, it will become necessary to understand characteristics of cellular transduction in the injured liver. In this study, we have further postulated that liver injury is likely to compromise the transduction efficiency of liver cells by prominent vectors such as adenovirus, and therefore we have examined adenoviral-mediated gene transfer after liver injury in nonparenchymal cells.
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MATERIALS AND METHODS |
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Animals and liver injury. Hepatic injury was induced in male retired breeder Sprague-Dawley rats (450-550 g) by bile duct ligation for 9 days or by intragastric administration of carbon tetrachloride (CCl4 was given at a concentration of 1 ml/kg once per week for 10 wk), each as previously described (21, 33). All animals received humane care according to National Institutes of Health guidelines; the studies were performed after approval by the Duke University Medical Center Institutional Animal Care and Use Committee.
Cell isolation and culture. Hepatocytes, stellate cells, and sinusoidal endothelial cells were isolated as described previously. For nonparenchymal cells, after in situ perfusion of the liver with 20 mg %pronase (Boehringer Mannheim, Indianapolis, IN) followed by collagenase (Crescent Chemical, Hauppauge, NY), dispersed cell suspensions were layered on a discontinuous density gradient of 8.2 and 15.6% Accudenz (Accurate Chemical and Scientific, Westbury, NY). The resulting upper layer consisted of >95% stellate cells. Endothelial cells in the lower layer were further purified by centrifugal elutriation (18 ml/min flow). Hepatocytes isolated after collagenase perfusion (as above) were placed in modified medium 199 OR containing 5% fetal bovine serum (GIBCO, Grand Island, NY); nonparenchymal cells were grown in the same medium containing 20% serum (10% horse/10% calf; Flow Laboratories, Naperville, IL). The viability of all cells was verified by phase contrast microscopy as well as the ability to exclude propidium iodide. Cell viability of cultures utilized for study was >95%.
Adenovirus preparation.
A vector containing -galactosidase (Ad.
-gal) was derived from the
in340 mutant strain of Ad5, containing a nuclear-targeted
-gal
transgene and expression cassette in the E1 region as previously described (6). Briefly, virus was purified from infected
293 cells by lysis in virus storage buffer consisting of (in mM/l) 20 Tris (pH 7.4), 150 NaCl, 5 KCl, and 1 MgCl2, followed by
ultracentrifugation of the lysate on a 1.3-1.4 g/ml CsCl step
gradient at 100,000 g for 2.5 h at 4°C. Virus was harvested, and
residual cellular RNA was removed by incubation with RNase A (100 µg/ml, Sigma) for 30 min at room temperature. Pooled virus was
ultracentrifuged overnight on a second CsCl gradient at 4°C. Pure
virus was harvested and desalted by serial gel filtration on Sepharose
CL-6B spin columns (Pharmacia) in virus storage buffer. Gel-filtered
virus was diluted 1:1 with 80% normal rabbit serum/20% glycerol
(virus storage medium) and immediately frozen in aliquots. Viral
concentration was initially estimated spectrophotometrically by
absorbance at 260 nm, and the infectious titer of all viral stocks was
determined by at least three independent plaque assays on 293 cells
using standard techniques. In brief, dishes of 293 cells were grown to
90% confluence in six-well plates and infected with serial dilutions
of Ad.
-gal for 1 h. The cell monolayer was then overlaid with
2% serum-containing medium containing 0.8% agarose and allowed to
gel. After 7 days, plaques were visualized by adding neutral red
(Sigma) and 5 ml of 40 mg/ml X-gal (Sigma) in agarose; after counting blue plaques, viral titers were calculated.
X-gal staining and histochemistry.
X-gal staining was utilized to determine transduction efficiency after
Ad.-gal exposure. In brief, cells were washed and fixed in 2%
paraformaldehyde for 10 min and incubated in a solution of (in mM) 50 Tris, pH 8.0, 2 MgCl2, 15 NaCl, 2 K3Fe(CN)6, and 2 K4Fe(CN)6 · 3H2O, with 12.5 mg/ml X-gal in DMSO for 1-2 h. Mock-infected cells were stained in
parallel for the same period of time. Cellular transduction efficiency
was determined by cell counting. For the purposes of quantitation,
cells in which the nucleus was blue, or in which the entire cytoplasm
was blue, were considered transduced. Cells in which only portions of
the cytoplasm were transduced were not considered transduced.
Quantitative data for cells transduced in vitro were generated by
blinded counting of cells in 10 random fields; the average of this
number was used as a single data point. For in vivo experiments, livers
were embedded in optimal cutting temperature compound (Miles
Scientific), frozen in liquid nitrogen, and sectioned (8-µm sections)
onto silane-coated glass microscope slides. Liver tissue was thawed,
dried, and stained for
-gal by incubation at room temperature in the
above solution for 4 h. Sections were counterstained with 1%
Scott's solution (Sigma) for 40 s, viewed, and photographed with
a Nikon TE 300 photomicroscope (Nikon, Tokyo, Japan) equipped with a
Nikon DXM 1200 digital camera.
Immunoblot. Lysates were prepared by homogenizing freshly isolated cells in a solution containing 20 mM Tris · HCl buffer (pH 7.4), 5 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 20 µM pepstatin A, and 20 µM leupeptin. Protein content in the lysate was quantitated (Bio-Rad), and equal quantities of protein (50 µg) were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (Bio-Rad). Blots were incubated with monoclonal anti-adenovirus 2/5 receptor (anti-CAR) antibody (a kind gift of Dr. Masato Yamamoto, University of Alabama at Birmingham). Membranes were washed with PBS, and primary antibody was detected with horseradish peroxidase-linked secondary antibodies. Specific signals were detected using enhanced chemiluminescence (Amersham), and data were linearity collected over a narrow range of X-ray film (Eastman Kodak, Rochester, NY).
Statistics. Analysis of variance or Student's t-test was used for statistical comparisons. Cells from different animals were used in each experiment. For calculation of mean values and statistical variation, n indicates the number of separate experiments. Error bars depict the SE; absence of error bars indicates that the SE was <1% of the mean, unless stated otherwise.
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RESULTS |
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Adenoviral gene transfer in nonparenchymal liver cells.
In initial experiments, we tested the ability of recombinant adenoviral
vectors to transduce sinusoidal endothelial and stellate cells in
vitro; hepatocytes, which have been transduced extensively in previous
studies, were additionally examined as a reference point. In
preliminary experiments, liver cells were isolated, cultured, and
exposed to relatively low adenoviral titers (1-5 pfu/cell).
Although transduction of hepatocytes was readily apparent at these
viral concentrations, the transduction rate for stellate and
endothelial cells appeared to be uniformly greater. Given the apparent
high transduction efficiency of nonparenchymal cells compared with
hepatocytes, we asked whether the same phenomena held true over a range
of adenovirus concentrations. Figure
1A shows the dose- response
effect of increasing adenovirus concentrations on adenovirus-mediated
gene transfer in liver cells isolated from normal livers. After
isolation, cells were allowed to adhere in culture dishes for 48 h
and then transfected with Ad.-gal over a range of concentrations
(i.e., 0.1-20 pfu/cell). As predicted, transduction efficiency
increased with increasing titer; additionally, the level of hepatocyte
transduction appeared to be in the same range as that reported
previously (18). At all adenovirus concentrations, stellate and endothelial cells were transduced at a slightly higher frequency than hepatocytes.
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Transduction efficiency in injured liver cells. Because gene delivery to injured cells is important from a therapeutic standpoint, we then asked to what extent liver injury affected the transduction efficiency of nonparenchymal cells and hepatocytes. Liver injury induced by bile duct ligation resulted in periportal expansion of biliary duct cells with concomitant periductular and lobular matrix deposition; this model also reliably resulted in portal hypertension, typical of severe wound healing. Repeated administration of carbon tetrachloride (10 doses over 70 days) led to extensive portal-portal, portal-central, and central-central bridging fibrosis and portal hypertension (portal venous dilation with portal pressure >10 cm H2O), also typical of severe hepatic wounding. In initial experiments, liver cells were isolated from rats after bile duct ligation (Fig. 1B). Transduction efficiency of adenovirus was reduced in all cell types isolated from injured livers compared with that for cells from normal livers, at all adenovirus titers. Interestingly, transduction efficiency remained greater for nonparenchymal cells than for hepatocytes, similar to cells from normal livers. In cells freshly isolated from livers after injury induced by CCl4, transduction was similarly reduced at all adenovirus titers (data not shown).
In vivo transduction efficiency of cells from normal and injured
livers.
We then examined the transduction efficiency of liver cells from normal
and injured livers in vivo. In these experiments, Ad.-gal or control
vector only was administered via the femoral vein to normal rats or to
those after induction of liver injury (bile duct ligation and
CCl4 as in MATERIALS AND METHODS) at multiple titers. To explore the issue of transduction in liver injury, adenovirus administration was timed to optimize the relationship between injury and exposure to adenovirus; adenovirus was introduced only after the induction of liver injury (for bile duct ligation, adenovirus was administered 2 days after bile duct ligation; for CCl4, adenovirus was administered 2 days after the 10th and
final dose of CCl4). Livers were harvested 7 days after
administration of adenovirus. In initial experiments, we examined gene
transfer by staining of whole livers. In normal liver, a significant
number of cells were clearly transduced, and moreover, using this
visual method, it appeared that hepatocytes were the cell population most prominently transduced, although nonparenchymal cells were also
seen to be transduced. The relative level of transduction was
substantial, given the 7-day interval between adenovirus administration and liver harvest. In the bile duct ligation model, it was readily evident that the global transduction rate (i.e., transduction to all
liver cells) of Ad.
-gal in the injured livers was less than that in
normal livers (Fig. 2). Likewise, in the
CCl4 model, injury resulted in reduced cellular
transduction as visualized by whole liver X-gal staining (data not
shown). We more precisely examined cellular transduction efficiency in
vivo by isolating cells from each normal and injured rat liver and
quantitating expression of
-gal (Table
1). Although, as predicted, cellular transduction was titer dependent in vivo, transduction
efficiency was clearly reduced for each cell type after liver injury.
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Transduction efficiency in quiescent and cultured activated
stellate cells.
We noted that the transduction efficiency of adenoviral vectors was
particularly high in stellate cells transduced after prolonged periods
of culture or after passage. Because this phenomenon could have
important implications for those wishing to introduce genes into
cultured cells, we examined adenoviral transduction efficiency in
stellate cells after different periods of time in culture. Quiescent
cells were those isolated from normal livers and examined within
24 h, whereas culture-activated stellate cells were grown in the
presence of serum-containing medium for 7 days. At an Ad.-gal titer
of 7.5 pfu/cell, X-gal staining was readily detected in each quiescent
and activated cell, but culture-activated stellate cells were
transduced with greater frequency than quiescent stellate cells (Fig.
3 B, D, and
E). X-gal staining was absent in stellate cells transduced
with control vector (Fig. 3A, C, and
E).
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CAR expression in liver cells.
In an attempt to begin to understand the cellular mechanism of
adenovirus uptake in liver cells, we explored expression of the common
Coxsackie B virus and adenovirus 2/5 receptor (also known as CAR) on
liver cells. The CAR appears to be critical for adenovirus type 2 and 5 gene transfer (2). By immunoblot, this 365 amino acid
transmembrane protein immunoprecipitates at 46 kDa but can be
identified by immunoblot analysis at other, typically smaller, masses (personal communication, M. Yamamoto). We found that all liver cell types studied expressed CAR (Fig.
4). Interestingly, hepatocytes appeared
to express a lower molecular mass and the 46-kDa form, whereas stellate
cells from normal livers expressed only the lower molecular mass form.
Notably, the lower molecular mass version found in normal stellate
cells appeared to transition to the 46-kDa version after
culture-induced activation. Whether this finding might be related to
the enhanced transduction efficiency seen after culture-induced
activation is unknown. We also queried whether changes in expression of
CAR occurred after liver injury, but in preliminary experiments, we
were unable to demonstrate significant variation in its expression by
immunoblot (data not shown).
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DISCUSSION |
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In this study, we have demonstrated that each endothelial and stellate cell (both hepatic nonparenchymal cells) is transduced with high efficiency and, under the conditions tested here, with greater efficiency than are hepatocytes. Additionally, we have shown that liver injury compromises adenovirus-mediated transduction efficiency in liver cells, not only in vivo but also in cells after their isolation. This feature was consistent in two mechanistically different models of liver injury and across all cell types at all adenovirus titers studied. It was also noteworthy that, at low adenovirus concentrations, gene transfer efficiency in injured livers (i.e., compared with that in normal livers) was particularly poor.
We were somewhat surprised by the exceptionally high transduction efficiency of nonparenchymal liver cells in both cultured cells as well as those in vivo. Although we report a hepatocyte transduction rate in culture studies that might be considered slightly lower than that commonly accepted, the transduction efficiency was consistent with previous work (18). However, it is important to emphasize that numerous variables (i.e., virus type, dose, route of administration, interval between administration of virus and detection of transduction) must be taken into account when comparing gene transduction among different constructs and in different systems. Indeed, we took care to maintain consistency across experiments in terms of virus, cell culture technique, etc.; thus our data represent a concordant and reproducible internal data set.
On the other hand, the finding that adenoviral-mediated transduction of stellate and endothelial cells was high may not be surprising given the physical characteristics of the hepatic vascular space (i.e., sinusoid). Substances circulating within the vascular compartment presumably come into contact with the endothelium before hepatocytes. Additionally, stellate cells, which reside in close proximity to the endothelium (i.e., in the sinusoidal space of Disse), are also likely to readily come into contact with blood-borne particles before hepatocytes. Data revealing that nonparenchymal cells express cell surface receptors responsible for adenovirus uptake (Fig. 4) are likewise highly consistent with adenoviral localization to these cells.
A seeming paradox raised by our studies is that liver endothelial cells and stellate cells are efficiently transduced by adenoviruses; however, parallel cell types in the peripheral vasculature do not appear to be equivalently targeted. It is notable that there are several critical differences between liver and other organ systems (especially the vasculature), perhaps the most critical of which is that the liver is a low-flow and low-pressure system. This contrasts with the particularly high-flow systems in arterial and even in venous vascular beds. We speculate that the low-pressure system found in the liver is more likely to be conducive to adenoviral attachment and particle uptake. A further major difference between liver and other vascular beds is that liver endothelial cells are fenestrated, whereas typical vascular endothelial cells are not. Whereas fenestrae clearly facilitate egress of adenovirus to cell layers beyond the endothelium, it is unknown whether they play a specific role in liver endothelial cell transduction efficiency.
An important consideration regarding cellular uptake of adenovirus revolves around the fact that this complicated process involves attachment of the virus to specific cell surface receptors, such as the 46-kDa Coxsackie virus and adenovirus receptor (also known as CAR) (1, 2, 7, 26). The importance of CAR as a mediator of viral attachment and as a critical component of the gene transfer process is emphasized by a number of studies (9, 16, 20, 25, 31, 35, 43) that have modified CAR, altered its expression, or suggested that its levels vary. Although the data vary depending on the biological system studied, they suggest that CAR expression is necessary but not sufficient for vector transfer after systemic injection. Anatomic compartments, in particular the endothelium, appear to be important physical barriers to gene transfer. Likewise, we speculate that similar considerations are important for transduction of liver cells.
A second interaction between the viral penton base and cell surface
integrins facilitates virus internalization (20, 27, 37).
In particular, the family of v-integrins appears to be required for adenovirus uptake (12, 36, 38). For example, the specific level of integrin
v
5 may
predict whether certain cell types can be transduced effectively. Thus,
although we have not examined these cell surface receptors on hepatic
cells, an attractive postulate is that differences in expression of
these ligands could contribute to the differential uptake of adenovirus in liver cells.
Our data are consistent with other work that has shown that
nonparenchymal cells, in particular stellate cells, can be efficiently transduced by adenovirus (15, 22). These studies have
focused largely on culture-activated stellate cells, having highlighted efficient gene expression by adenoviral vectors. We found that the
transduction efficiency of culture-activated stellate cells was greater
than that of stellate cells freshly isolated from normal
livers. The basis for this finding is unknown, although the
phenomenon was highly reproducible, being demonstrable at multiple
different viral titers. It is known that stellate cell activation is
associated with multiple cellular changes, including increased
expression of a number of receptors, including platelet-derived growth
factor (42), transforming growth factor-
(10), and endothelin receptors (23). In
addition, stellate cell activation is associated with a number of
culture-based, artifactual changes in cell surface receptor expression.
For example,
2
1 receptors become
upregulated after culture-induced activation, whereas their levels do
not change in vivo after stellate cell activation (34). Thus we speculate that culture-induced stellate cell activation could
be associated with changes in cell surface receptors that favor uptake
of adenovirus.
The mechanism by which cellular transduction efficiency in injured,
compared with normal, liver is reduced is unknown but predicted to be
multifactorial. Liver injury is associated with a number of processes
occurring at the cellular level. Processes of major importance include
endothelial injury, hepatocyte injury, and often activation of hepatic
stellate cells (3, 13, 29, 41). Endothelial injury, which
is prominent in many forms of liver injury (3, 11, 17,
19), has been emphasized as a critical aspect of adenovirus
uptake in other systems (9). Endothelial injury may not
only be important in adenovirus uptake by endothelial cells themselves
but may also play a role in adenovirus uptake by other cells in the
injured liver. Additionally, vascular abnormalities are frequent after
liver injury (28, 44) and may impair access of adenovirus
to liver cells. Finally, it is known that liver injury induces changes
in cell surface protein expression; changes in integrin expression
patterns after other forms of injury have been demonstrated
(31), raising the possibility that similar changes occur
in the liver. Although beyond the scope of this study, our data suggest
that an in-depth examination of cell surface receptor (i.e., CAR and/or
5 integrin) modulation during liver injury will be of interest.
There are several important implications of the finding that liver injury leads to reduced transduction efficiency of liver cells in vivo. This information is important in the context of currently available data that indicate that transgene expression is also dependent on other factors such as the dose of virus administered, the promoter chosen to drive expression of the recombinant gene, the innate immune response to the vector (and transgene), and cytotoxicity caused by expression of viral genes. One of the more important implications of reduced transfection efficiency after liver injury is that, to achieve a certain level of gene expression, higher adenovirus concentrations are likely to be required. This would be particularly critical in situations in which certain absolute quantities of proteins are required or in situations in which large populations of liver cells must be targeted. This, in turn, raises the specter of adenovirus-mediated hepatotoxicity in the injured liver. Although we did not detect notable hepatotoxicity in rats exposed to as much as 1012 pfu/kg adenovirus, it has been our experience that higher concentrations of this adenovirus lead to hepatocellular (and perhaps nonparenchymal cell) injury. Alternatively, newer adenoviral constructs that lack viral coding sequences or those that contain specific promoters may help circumvent dose-dependent toxicity (30, 40).
We have not addressed the efficiency of gene therapy over time in the current study, nor have we addressed the implications of liver injury on long-term gene transduction. It is well established that adenovirus induces high level gene expression at 3-5 days after intravenous administration of adenovirus (40) and that gene expression subsequently decreases over time. Rather, we chose to study a later time point and examine gene expression across cell types and after liver injury. Although we have not studied the kinetics of gene transfer over an entire time course, given the known kinetics of adenovirus-mediated gene transfer, it is highly unlikely that any of our conclusions would be different from those made on the basis of the current experiments. We are unable to make firm conclusions about long-term (i.e., weeks to months) gene transfer in injured liver, although on the basis of the current work, we would speculate that it is reduced compared with normal liver. Furthermore, we have not studied vectors other than adenovirus such as adeno-associated virus or retrovirus and cannot state with certainty whether transfer of genes using these vectors is altered by liver injury. However, we would speculate that injury-induced changes to liver cells and overall hepatic structure/function (in particular vascular derangement) would similarly impair the in vivo transduction efficiency of these vectors as well.
In summary, we have raised several important issues in this study. The first is that gene transfer to hepatic nonparenchymal cells using adenovirus is highly efficient, potentially more so than to hepatocytes. This indicates that gene transfer to these important cell populations is readily feasible. Additionally, we have demonstrated that liver injury significantly impairs adenovirus-mediated transduction to liver cells. This finding implies that when gene transfer to cells in the injured liver is being considered, modifications in dosage, timing, or perhaps even in viral construct may be necessary.
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ACKNOWLEDGEMENTS |
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We thank Fred Noubani for expert technical assistance and Masato Yamamoto for the gift of anti-CAR monoclonal antibody.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-50574 and R01-DK-54038 (to D. C. Rockey) and the Burroughs Wellcome Fund (to D. C. Rockey).
This work was presented, in part, at the 50th Annual Meeting of the American Association for the Study of Liver Diseases, Dallas, TX, 1999.
Address for reprint requests and other correspondence: D. C. Rockey, Duke Liver Center, Rm. 336, Sands Bldg., Box 3083, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: dcrockey{at}acpub.duke.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.
10.1152/ajpgi.00512.2000
Received 13 December 2000; accepted in final form 24 October 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bergelson, JM.
Receptors mediating adenovirus attachment and internalization.
Biochem Pharmacol
57:
975-979,
1999[ISI][Medline].
2.
Bergelson, JM,
Cunningham JA,
Droguett G,
Kurt-Jones EA,
Krithivas A,
Hong JS,
Horwitz MS,
Crowell RL,
and
Finberg RW.
Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.
Science
275:
1320-1323,
1997
3.
Bhunchet, E,
and
Fujieda K.
Capillarization and venularization of hepatic sinusoids in porcine serum-induced rat liver fibrosis: a mechanism to maintain liver blood flow.
Hepatology
18:
1450-1458,
1993[ISI][Medline].
4.
Bissell, DM.
Hepatic fibrosis as wound repair: a progress report.
J Gastroenterol
33:
295-302,
1998[ISI][Medline].
5.
Bissell, DM,
Friedman SL,
Maher JJ,
and
Roll FJ.
Connective tissue biology and hepatic fibrosis: report of a conference.
Hepatology
11:
488-498,
1990[ISI][Medline].
6.
Channon, KM,
Blazing MA,
Shetty GA,
Potts KE,
and
George SE.
Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells.
Cardiovasc Res
32:
962-972,
1996[ISI][Medline].
7.
Cohen, CJ,
Gaetz J,
Ohman T,
and
Bergelson JM.
Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting.
J Biol Chem
276:
25392-25398,
2001
8.
Crystal, RG.
Transfer of genes to humans: early lessons and obstacles to success.
Science
270:
404-410,
1995[Abstract].
9.
Fechner, H,
Haack A,
Wang H,
Wang X,
Eizema K,
Pauschinger M,
Schoemaker R,
Veghel R,
Houtsmuller A,
Schultheiss HP,
Lamers J,
and
Poller W.
Expression of Coxsackie adenovirus receptor and v-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers.
Gene Ther
6:
1520-1535,
1999[ISI][Medline].
10.
Friedman, SL,
Yamasaki G,
and
Wong L.
Modulation of transforming growth factor receptors of rat lipocytes during the hepatic wound healing response. Enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo.
J Biol Chem
269:
10551-10558,
1994
11.
Gatmaitan, Z,
Varticovski L,
Ling L,
Mikkelsen R,
Steffan AM,
and
Arias IM.
Studies on fenestral contraction in rat liver endothelial cells in culture.
Am J Pathol
148:
2027-2041,
1996[Abstract].
12.
Goldman, MJ,
and
Wilson JM.
Expression of v
5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway.
J Virol
69:
5951-5958,
1995[Abstract].
13.
Gressner, AM.
The cell biology of liver fibrogenesisan imbalance of proliferation, growth arrest and apoptosis of myofibroblasts.
Cell Tissue Res
292:
447-452,
1998[ISI][Medline].
14.
Guo, ZS,
Wang LH,
Eisensmith RC,
and
Woo SL.
Evaluation of promoter strength for hepatic gene expression in vivo after adenovirus-mediated gene transfer.
Gene Ther
3:
802-810,
1996[ISI][Medline].
15.
Hellerbrand, C,
Jobin C,
Iimuro Y,
Licato L,
Sartor RB,
and
Brenner DA.
Inhibition of NFB in activated rat hepatic stellate cells by proteasome inhibitors and an I
B super-repressor.
Hepatology
27:
1285-1295,
1998[ISI][Medline].
16.
Hidaka, C,
Milano E,
Leopold PL,
Bergelson JM,
Hackett NR,
Finberg RW,
Wickham TJ,
Kovesdi I,
Roelvink P,
and
Crystal RG.
CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts.
J Clin Invest
103:
579-587,
1999
17.
Irving, MG,
Roll FJ,
Huang S,
and
Bissell DM.
Characterization and culture of sinusoidal endothelium from normal rat liver: lipoprotein uptake and collagen phenotype.
Gastroenterology
87:
1233-1247,
1984[ISI][Medline].
18.
Jaffe, HA,
Danel C,
Longenecker G,
Metzger M,
Setoguchi Y,
Rosenfeld MA,
Gant TW,
Thorgeirsson SS,
Stratford-Perricaudet LD,
Perricaudet M,
Pavirani A,
Lecocq JP,
and
Crystal RG.
Adenovirus-mediated in vivo gene transfer and expression in normal rat liver.
Nat Genet
1:
372-378,
1992[ISI][Medline].
19.
Jarnagin, WR,
Rockey DC,
Koteliansky VE,
Wang SS,
and
Bissell DM.
Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis.
J Cell Biol
127:
2037-2048,
1994[Abstract].
20.
Kirby, I,
Davison E,
Beavil AJ,
Soh CP,
Wickham TJ,
Roelvink PW,
Kovesdi I,
Sutton BJ,
and
Santis G.
Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein.
J Virol
74:
2804-2813,
2000
21.
Kountouras, J,
Billing BH,
and
Scheuer PJ.
Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat.
Br J Exp Pathol
65:
305-311,
1984[ISI][Medline].
22.
Lang, A,
Schoonhoven R,
Tuvia S,
Brenner DA,
and
Rippe RA.
Nuclear factor kappa B in proliferation, activation, and apoptosis in rat hepatic stellate cells.
J Hepatol
33:
49-58,
2000[ISI][Medline].
23.
Leivas, A,
Jimenez W,
Bruix J,
Boix L,
Bosch J,
Arroyo V,
Rivera F,
and
Rodes J.
Gene expression of endothelin-1 and ET(A) and ET(B) receptors in human cirrhosis: relationship with hepatic hemodynamics.
J Vasc Res
35:
186-193,
1998[ISI][Medline].
24.
Li, Q,
Kay MA,
Finegold M,
Stratford-Perricaudet LD,
and
Woo SL.
Assessment of recombinant adenoviral vectors for hepatic gene therapy.
Hum Gene Ther
4:
403-409,
1993[ISI][Medline].
25.
Li, Y,
Pong RC,
Bergelson JM,
Hall MC,
Sagalowsky AI,
Tseng CP,
Wang Z,
and
Hsieh JT.
Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy.
Cancer Res
59:
325-330,
1999
26.
Lortat-Jacob, H,
Chouin E,
Cusack S,
and
van Raaij MJ.
Kinetic analysis of adenovirus fiber binding to its receptor reveals an avidity mechanism for trimeric receptor-ligand interactions.
J Biol Chem
276:
9009-9015,
2001
27.
Nemerow, GR,
and
Stewart PL.
Role of (v) integrins in adenovirus cell entry and gene delivery.
Microbiol Mol Biol Rev
63:
725-734,
1999
28.
Nishida, J,
McCuskey RS,
McDonnell D,
and
Fox ES.
Protective role of NO in hepatic microcirculatory dysfunction during endotoxemia.
Am J Physiol Gastrointest Liver Physiol
267:
G1135-G1141,
1994
29.
Oda, M,
Azuma T,
and
Watanabe N.
Regulatory mechanism of hepatic microcirculation: involvement of the contraction and dilation of sinusoids and sinusoidal fenestrae.
Prog Appl Microcirc
127:
103-128,
1990.
30.
Pastore, L,
Morral N,
Zhou H,
Garcia R,
Parks RJ,
Kochanek S,
Graham FL,
Lee B,
and
Beaudet AL.
Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors.
Hum Gene Ther
10:
1773-1781,
1999[ISI][Medline].
31.
Pearson, AS,
Koch PE,
Atkinson N,
Xiong M,
Finberg RW,
Roth JA,
and
Fang B.
Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines.
Clin Cancer Res
5:
4208-4213,
1999
32.
Pinzani, M,
and
Gentilini P.
Biology of hepatic stellate cells and their possible relevance in the pathogenesis of portal hypertension in cirrhosis.
Semin Liver Dis
19:
397-410,
1999[ISI][Medline].
33.
Proctor, E,
and
Chatamra K.
High yield micronodular cirrhosis in the rat.
Gastroenterology
83:
1183-1190,
1982[ISI][Medline].
34.
Racine-Samson, L,
Rockey DC,
and
Bissell DM.
The role of 1
1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture.
J Biol Chem
272:
30911-30917,
1997
35.
Roelvink, PW,
Mi Lee G,
Einfeld DA,
Kovesdi I,
and
Wickham TJ.
Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae.
Science
286:
1568-1571,
1999
36.
Takayama, K,
Ueno H,
Pei XH,
Nakanishi Y,
Yatsunami J,
and
Hara N.
The levels of integrin v
5 may predict the susceptibility to adenovirus-mediated gene transfer in human lung cancer cells.
Gene Ther
5:
361-368,
1998[ISI][Medline].
37.
Wang, K,
Guan T,
Cheresh DA,
and
Nemerow GR.
Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin 5.
J Virol
74:
2731-2739,
2000
38.
Wickham, TJ,
Mathias P,
Cheresh DA,
and
Nemerow GR.
Integrins v
3 and
v
5 promote adenovirus internalization but not virus attachment.
Cell
73:
309-319,
1993[ISI][Medline].
39.
Williams, EJ,
and
Iredale JP.
Liver cirrhosis.
Postgrad Med J
74:
193-202,
1998[Abstract].
40.
Wilson, JM.
Adenoviruses as gene-delivery vehicles.
N Engl J Med
334:
1185-1187,
1996
41.
Wisse, E,
Braet F,
Luo D,
De Zanger R,
Jans D,
Crabbe E,
and
Vermoesen A.
Structure and function of sinusoidal lining cells in the liver.
Toxicol Pathol
24:
100-111,
1996[ISI][Medline].
42.
Wong, L,
Yamasaki G,
Johnson RJ,
and
Friedman SL.
Induction of -platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture.
J Clin Invest
94:
1563-1569,
1994[ISI][Medline].
43.
You, Z,
Fischer DC,
Tong X,
Hasenburg A,
Aguilar-Cordova E,
and
Kieback DG.
Coxsackievirus-adenovirus receptor expression in ovarian cancer cell lines is associated with increased adenovirus transduction efficiency and transgene expression.
Cancer Gene Ther
8:
168-175,
2001[ISI][Medline].
44.
Zimmermann, A,
Zhao D,
and
Reichen J.
Myofibroblasts in the cirrhotic rat liver reflect hepatic remodeling and correlate with fibrosis and sinusoidal capillarization.
J Hepatol
30:
646-652,
1999[ISI][Medline].