Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver

Qing Yu1,2, Loretta G. Que1, and Don C. Rockey1,2

1 Departments of Medicine and 2 Cell Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -galactosidase (Ad.beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -galactosidase (Ad.beta -gal) was derived from the in340 mutant strain of Ad5, containing a nuclear-targeted beta -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.beta -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.

For cellular transduction with adenovirus, isolated cells were plated at a density of 1 ×106 cells/ml in 35 or 60 mm plastic or collagen-coated (stellate cells or hepatocytes and sinusoidal endothelial cells, respectively) culture dishes and exposed to the indicated concentrations of adenovirus for 2 h at 37°C. Subsequently, adenovirus-containing medium was exchanged for standard medium. For in vivo experiments, rats were anesthetized and Ad.beta -gal was administered via the femoral vein at the indicated titer. In the bile duct ligation model, adenovirus was administered 2 days after bile duct ligation; for the carbon tetrachloride model, adenovirus was administered 2 days after the 10th and final dose of carbon tetrachloride. Cells were routinely harvested (i.e., perfusion performed) 7 days after adenovirus administration.

X-gal staining and histochemistry. X-gal staining was utilized to determine transduction efficiency after Ad.beta -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 beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.beta -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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Fig. 1.   Adenovirus-mediated gene transfer is reduced after liver injury and is titer dependent. Hepatocytes, sinusoidal endothelial cells, and stellate cells were isolated from normal rat livers (A) and livers after bile duct ligation (B) as described in MATERIALS AND METHODS. After adherence for 48 h, cells were transfected with Ad.beta -gal at the indicated titers (expressed in pfu/cell). Transduction efficiency was determined by X-gal staining and cell counting as described in MATERIALS AND METHODS. Experiments in normal and injured livers were performed using identical viral stocks under identical conditions. *P < 0.05 compared with hepatocytes (n = 3-6). In addition, differences in transduction efficiency for all 3 cell types between cells from normal livers and those from injured livers were significant (*P < 0.05) at viral titers of >= 10 pfu/cell.

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.beta -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.beta -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 beta -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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Fig. 2.   Adenovirus-mediated gene transfer in normal and injured livers. Liver injury was induced by bile duct ligation as described in MATERIALS AND METHODS. After liver injury, Ad.beta -gal and control vectors were administered via the femoral vein at 5 × 1011 pfu/kg. For the bile duct ligation model, adenovirus was administered 2 days after bile duct ligation. Seven days after administration of adenovirus, livers were snap frozen in liquid nitrogen and sectioned (8-µm sections) onto silane-coated glass microscope slides. Liver sections were thawed, dried, and then stained for beta -gal by incubation at room temperature in X-gal solution for 4 h as described in MATERIALS AND METHODS, and slides were lightly counterstained with Scott's solution. A: a normal liver exposed to Ad.beta -gal. B: a liver after bile duct ligation. In A the arrow points to a portal triad; in B, the arrow points to an expanded portal triad, containing proliferating bile ducts. Livers exposed to control vectors exhibited no blue staining (not shown).


                              
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Table 1.   In vivo transfection efficiency in normal and injured livers

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.beta -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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Fig. 3.   Enhanced adenoviral transduction efficiency in culture-activated stellate cells. Stellate cells were isolated from normal rat livers and grown for 2 or 7 days on plastic substrata in the presence of medium containing 20% serum. A and C: cells exposed to an adenovirus encoding neuronal nitric oxide synthase (Ad.nNOS) (used as a control vector). B and D: cells exposed to Ad.beta -gal (7.5 pfu/cell) for 2 h at 37°C. Cells were washed and, after a subsequent 24 h, were washed and fixed, and beta -galactosidase expression was detected as described in MATERIALS AND METHODS. A and B: photomicrographs of cells after culture for 2 days. C and D: photomicrographs of cells after culture for 7 days. E: the proportion of transduced quiescent and activated cells. *P < 0.05 compared with quiescent stellate cells (n = 6). The photomicrographs shown are representative of 6 different experiments.

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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Fig. 4.   Adenovirus 2/5 receptor (CAR) expression in liver cells. Liver cells were isolated from normal rats, and cellular lysates were prepared (50 µg total cellular lysate was loaded) and subjected to immunoblotting as described in MATERIALS AND METHODS. The immunoblot depicted is representative of 3 other experiments. H, hepatocyte; E, endothelial cell; S, stellate cell; S-A, culture-activated stellate cell.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha v-integrins appears to be required for adenovirus uptake (12, 36, 38). For example, the specific level of integrin alpha vbeta 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-beta (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, alpha 2beta 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 alpha 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.


    ACKNOWLEDGEMENTS

We thank Fred Noubani for expert technical assistance and Masato Yamamoto for the gift of anti-CAR monoclonal antibody.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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