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J Biol Chem, Vol. 275, Issue 17, 12743-12751, April 28, 2000


Successful Targeting to Rat Hepatic Stellate Cells Using Albumin Modified with Cyclic Peptides That Recognize the Collagen Type VI Receptor*

Leonie BeljaarsDagger §, Grietje MolemaDagger , Detlef Schuppan, Albert Geerts||, Pieter J. De Bleser||, Betty WeertDagger , Dirk K. F. MeijerDagger , and Klaas PoelstraDagger

From the Dagger  Groningen University Institute for Drug Exploration (GUIDE), Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, 9713 AV Groningen, The Netherlands, the  Med Klinik I, Unversität Erlangen-Nuernberg, 90154 Erlangen, Germany, and the || Laboratory for Cell Biology and Histology, Free University of Brussels, 1090 Brussels, Belgium

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The key pathogenic event in liver fibrosis is the activation of hepatic stellate cells (HSC). Consequently, new antifibrotic therapies are directed toward an inhibition of HSC activities. The aim of the present study was to develop a drug carrier to HSC, which would allow cell-specific delivery of antifibrotic drugs thus enhancing their effectiveness in vivo. We modified human serum albumin (HSA) with 10 cyclic peptide moieties recognizing collagen type VI receptors (C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) yielding pCVI-HSA. In vivo experiments showed preferential distribution of pCVI-HSA to both fibrotic and normal rat livers (respectively, 62 ± 6 and 75 ± 16% of the dose at 10 min after intravenous injection). Immunohistochemical analysis demonstrated that pCVI-HSA predominantly bound to HSC in fibrotic livers (73 ± 14%). In contrast, endothelial cells contributed mostly to the total liver accumulation in normal rats. In vitro studies showed that pCVI-HSA specifically bound to rat HSC, in particular to the activated cells, and showed internalization of pCVI-HSA by these cells. In conclusion, pCVI-HSA may be applied as a carrier to deliver antifibrotic agents to HSC, which may strongly enhance the effectiveness and tissue selectivity of these drugs. This approach has the additional benefit that such carriers may block receptors that play a putative role in the pathogenesis of liver fibrosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Liver fibrosis is characterized by an increased deposition of extracellular matrix constituents, resulting from an enhanced de novo synthesis of matrix proteins (fibrogenesis) and their compromised removal by matrix degrading enzymes (fibrolysis). The major cell type responsible for hepatic fibrogenesis is the activated hepatic stellate cell (HSC)1 (1-5). Therefore, this cell type is an important target for antifibrotic therapies. However, contrary to in vitro data, in vivo studies indicate that most antifibrogenic agents are not efficiently taken up by HSC. To attain HSC-specific uptake, drugs can be coupled to carrier molecules that are designed for selective uptake by target cells. To date, drug carriers to hepatocytes, Kupffer, and liver sinusoidal endothelial cells have been described extensively (6-8). Recently, we reported about a neoglycoprotein, albumin modified with mannose 6-phosphate moieties (M6P28-HSA), which preferentially distributed to HSC in fibrotic rat livers (9). We now choose a different approach for the development of drug carriers. This approach is based on the use of peptide moieties as a homing device. These (cyclic) peptides are designed to recognize specific (pathological) receptors on target cells. If successful, this may have implications for the design of other receptor-specific carriers.

Specific interactions between cells and extracellular matrix components are mediated by transmembrane proteins, in particular the heterodimeric integrins. Cell-matrix interactions guide or modulate many cellular activities, such as adhesion, migration, differentiation, proliferation, and apoptosis (10-12). Many integrins recognize and bind the amino acid sequence Arg-Gly-Asp (RGD) present in various matrix proteins. Collagen type VI is a major matrix protein involved in the adhesion of cells to the surrounding matrix. In addition, it interacts with a number of other matrix molecules such as collagens type I and III, decorin, and hyaluronic acid (12). The main cellular sources of collagen type VI in normal and fibrotic livers are HSC (13). In normal livers, collagen type VI is localized in the portal areas and at the plasma membranes of hepatocytes, endothelial cells, and HSC within the lobule. The hepatic deposition of this type of collagen is increased during liver fibrosis, in particular in the fibrous septa that invade the lobule (14, 15). Serum levels of collagen type VI allowed the non-invasive differentiation of children with liver fibrosis from those without fibrosis approaching a 100% accuracy (16). It could be demonstrated that the core fragment of collagen type VI has potent growth factor activities stimulating DNA synthesis of mesenchymal cells severalfold via activation of erk2 and tyrosine phosphorylation of several proteins (17, 18). Binding of cells to collagen type VI by integrins is RGD-dependent. Although several RGD sequences are found in collagen type VI, Marcellino and McDevitt (19) showed that the cyclic peptide C*GRGDSPC* specifically inhibited the attachment of collagen type VI to cells, while the RGD-dependent binding of fibronectin to these cells was not inhibited. Furthermore, the linear analogue of this peptide failed to inhibit the cellular attachment of collagen type VI (19), indicating that a non-integrin collagen type VI receptor was antagonized by this peptide.

Therefore, we studied whether C*GRGDSPC*, which selectively interferes with collagen type VI-mediated cell adhesion, can be used as a homing device to target to HSC in fibrotic livers. In this study, we present evidence that human serum albumin (HSA) modified with this cyclic peptide binds to HSC in vivo and in vitro and is applicable as a drug carrier to this crucial cell type in liver fibrosis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthesis and Characterization of pCVI-HSA

Reagents-- The cyclic peptide C*GRGDSPC* was prepared by Ansynth Service BV (Roosendaal, The Netherlands). HSA was obtained from the Central Laboratory of Blood Transfusion Services (Amsterdam, The Netherlands) and consisted mainly of monomeric protein. All other reagents were of the highest purity grade available.

Synthesis-- The cyclic octapeptide C*GRGDSPC* was covalently coupled to HSA as illustrated in Fig. 1. First, a sulfhydryl group was introduced into the cyclic peptide. Therefore, C*GRGDSPC* was dissolved in dimethyl formamide and reacted with N-succinimidyl S-acetyl thioacetate (Pierce) for 2 h in the presence of diisopropylethylamine. Primary epsilon -NH2 groups of lysine moieties in HSA were derivatized with maleimido-hexanoyl-N-hydroxysuccinimide ester in phosphate-buffered saline, pH 7.2. Subsequently, the cyclic peptide was added to HSA. The molar ratio peptide to HSA determined the degree of substitution. Hydroxylamine was added to remove the protecting acetate group from the sulfhydryl group of the cyclic peptide. The mixture was stirred for 2 h. The obtained product was purified with Sephadex G-25SF gel chromatography (Amersham Pharmacia Biotech), lyophilized, and stored at -20 °C until use.


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  		Fig. 1.   Covalent coupling of the cyclic peptide moiety Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (=C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) to HSA. I, first, a sulfhydryl group is introduced to the cyclic peptide by reaction with succinimide-acetyl thioacetate (SATA). II, the epsilon -NH2 groups of lysine in HSA are derivatized with maleimide-hexanoyl-N-hydroxysuccinimide ester (MHS). III, subsequently, the cyclic peptide is coupled to HSA. In this latter reaction, hydroxyl amine is used to remove the protecting acetate group from the sulfhydryl group of the cyclic peptide.

Characterization-- The molecular weight of pCVI-HSA and HSA was determined by a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast protein liquid chromatography analysis. The fast protein liquid chromatography system was equipped with a Superdex-200 column (Amersham Pharmacia Biotech) and an UV detector (280 nm). The proteins (0.2 mg) were eluted with phosphate-buffered saline at a flow rate of 0.4 ml/min.

The amount of alpha -helices and beta -sheets in pCVI-HSA was determined by recording circular dichroism (CD) spectra in the 197-250 nm range with an circular dichroism spectrometer model 62A DS (AVIV Instruments, Lakewood, NY) (20). The spectra were the average of 5 scans (1 nm band width, 1-nm step width, 1 s averaging time per point) recorded at constant temperature (20 °C). The spectrum of pCVI-HSA (0.2 mg/ml phosphate-buffered saline) was compared with the spectra of native HSA and HSA treated with dithiothreitol (Sigma), which induces linearization of proteins by cleavage of disulfide bridges (21).

Radiolabeling

pCVI-HSA and HSA were labeled with 125I as described by Mather and Ward (22) to a specific activity of 2.5 × 106 cpm/µg for in vivo and in vitro studies. Before each experiment, protein bound radioactivity was measured after precipitation with 10% trichloroacetic acid. The preparations were dialyzed against phosphate-buffered saline if the solution contained of >5% free 125I.

Animal Model of Liver Fibrosis

Male Wistar rats (±250 g, obtained from Harlan, Zeist, The Netherlands) were housed in 12-h light/dark cycles at constant temperature and humidity and maintained on standard lab chow and tap water. The study as presented was approved by the Local Committee for Care and Use of Laboratory Animals and was performed according to strict governmental and international guidelines on animal experimentation.

To induce liver fibrosis, the rats were submitted to common bile duct ligation (BDL) under fluothane/O2 anesthesia (23). Three weeks after bile duct ligation (BDL3), these fibrotic rats were used for in vivo experiments. Previous studies showed that the livers of these rats displayed all signs of fibrosis, that is abundant HSC proliferation and excessive matrix deposition (7).

In Vivo Experiments

Organ Distribution Studies with Radioactivity Detection-- Distribution studies were performed as described (9). The distributions of 125I-pCVI-HSA and 125I-HSA were assessed at 10, 30, or 180 min after intravenous injection of tracer doses in the penis vene in normal and BDL3 rats.

In Vivo Localization of pCVI-HSA with Immunohistochemical Techniques-- Anesthetized normal and BDL3 rats received an intravenous dose of 10 mg/kg pCVI-HSA (via the penis vene). Ten minutes after administration, specimens of liver, spleen, lungs, kidneys, and muscles were frozen in isopentane (-80 °C) for immunohistochemical analysis. Since technical problems hampered the preparation of bone cryostat sections, acetone fixed specimens of the hind leg bone were embedded in glycol methacrylate plastic after dehydration in an alcohol series according to standard procedures (24).

Immunohistochemical Analysis

Acetone-fixed cryostat sections (4 µm) of liver, spleen, lung, and kidneys were stained for the presence of pCVI-HSA with a rabbit polyclonal antibody directed against HSA (Cappel, Organon Teknika, Turnhout, Belgium) according to standard indirect immunoperoxidase methods using aminoethyl carbazole (25). The polyclonal antibody displayed no cross-reactivity with rat serum albumin.

Double Immunostaining-- To assess whether the modified albumins were taken up by HSC, Kupffer cells, or endothelial cells, the liver sections were double stained with HSA antibodies and a cell type-specific marker (9). HSC were demonstrated with two mouse monoclonal primary antibodies directed against desmin (Cappel) and glial fibrillary acidic protein (Biogenex, San Ramon, CA). Kupffer cells and endothelial cells were detected with the monoclonal antibodies ED2 and HIS52, respectively (both Serotec, Oxford, United Kingdom). The sections stained for the modified albumin and the hepatic cell markers were quantitatively evaluated in a double-blind manner by two independent observers as described before (9). The number of double positive cells was related to the total number of HSA-positive cells yielding a relative accumulation of pCVI-HSA by each cell type. The double staining techniques were also used to examine spleen, lung, and kidneys for the co-localization of pCVI-HSA with fibroblast markers (with antibodies against desmin, alpha -smooth muscle actin (Sigma), and vimentin (DAKO, Glostrup, Denmark)), or with the monocyte/macrophage marker ED1 (DAKO).

Immunohistochemical Analysis of Plastic Sections-- The presence of pCVI-HSA in plastic embedded bone was studied by immunohistochemical staining of 0.1% trypsine-treated sections (2 µm) and anti-HSA IgG (9, 24). The staining in bone was detected after amplification of the signal with peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG. Glycol methacrylate-plastic embedded liver samples containing HSA were prepared and treated similarly and served as positive controls.

In Vitro Experiments

Isolation of HSC-- The livers of male Wistar rats (450-550 g) were perfused with Gey's balanced salt solution (GBSS) containing collagenase P (Roche Molecular Biochemicals, Mannheim, Germany), Pronase (Merck, Darmstadt, Germany), and DNase (Roche Molecular Biochemicals). The HSC were separated from the other hepatic cells by density-gradient centrifugation and collected at the top of an 11% Nycodenz solution (Nyegaard, Oslo, Norway). The cells were then cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Paisley, Scotland) containing 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (26). After 2 days, cell debris and nonadherent cells were removed by washing and the medium was changed every 2 or 3 days thereafter. Cells cultured for 2 days after isolation represented quiescent HSC, whereas those cultured for 10 days after isolation represented activated HSC (26).

Binding Experiments of pCVI-HSA with HSC-- The cellular handling of 125I-pCVI-HSA was assessed in cultures of quiescent HSC and activated HSC and compared with 125I-HSA. Before each experiment, HSC were trypsinized and seeded in plastic 35-mm dishes (Falcon, Becton Dickinson, Lincoln Park, NJ) at a density of approximately 70,000 HSC per dish (2,000/cm2) and cultured overnight at 37 °C. Then the number of adherent cells (nonconfluent cell layer) was counted using a phase-contrast microscope. The cells were blocked with 1% bovine serum albumin/DMEM for 15 min at 4 °C and subsequently incubated with 50,000 cpm of 125I-pCVI-HSA or 125I-HSA per dish in 0.2% bovine serum albumin/DMEM for 0.5 to 24 h at 4 and 37 °C. The medium was removed after the incubation period and analyzed for the presence of degradation products by precipitation of the protein bound radioactivity with a 10% trichloroacetic acid solution. Subsequently, the dishes were washed three times with cold GBSS, and the cells were harvested with a cell scraper (Costar, Cambridge, MA) to assess the total amount of cell bound radioactivity.

We performed inhibition experiments to determine the specificity of binding and uptake of 125I-pCVI-HSA to HSC. We added an excess of unlabeled pCVI-HSA (30 µM dissolved in GBSS) to the incubation media at 4 °C. At 37 °C, 2 µM monensin (Sigma, dissolved in ethanol), an inhibitor of endocytosis, was added to the incubation media.

Immunohistochemical Analysis-- HSC (10 days old) were seeded on glass Lab-tek chamber slides (Nalge Nunc International, Naperville, IL) and recultured overnight in cDMEM. Adherent cells were incubated with 1 mg/ml pCVI-HSA or HSA for 2 h at 37 °C. Subsequently, the wells were washed three times with DMEM and fixed with acetone/methanol (1:1, v/v). To detect modified albumins bound to activated HSC, the slides were stained with anti-HSA antibodies as described above. Incubations of cells and pCVI-HSA were also performed in the presence of 1 µg/ml soluble collagen type VI (18) or the polyanion suramin (200 µM, obtained from ICN Biomedicals, Aurora, OH).

Slice Experiments-- To determine the degradation of pCVI-HSA and HSA, we used precision-cut liver slices, containing all hepatic cells in their native state. Fresh slices (±12 mg wet weight) were prepared from BDL rat livers with a Krumdieck slicer and stored in ice-cold UW until the start of the experiments (27, 28). The slices were incubated with 125I-pCVI-HSA or 125I-HSA (150,000 cpm/well), as described (9), for 2-24 h at 37 °C. After the incubation period, the medium was used for the determination of released degradation products by precipitation of the protein bound radioactivity with 10% trichloroacetic acid.

Adhesion of pCVI-HSA to Matrix Proteins-- To assess the binding of pCVI-HSA to matrix proteins, we coated 0.15 mg of collagen S (=90% collagen type I, 10% collagen type III, and others, obtained from Roche Molecular Biochemicals) overnight at 37 °C in 35-mm dishes. In addition, the individual matrix proteins collagen type I, III, IV, and VI (10 µg/dish, collagen type I, III, and IV were obtained from Sigma) were coated to dishes. These dishes were washed twice with ice-cold GBSS, blocked with 1% bovine serum albumin/DMEM for 15 min at 4 °C, and subsequently incubated with 50,000 cpm of 125I-pCVI-HSA or 125I-HSA/dish in 0.2% bovine serum albumin/DMEM for 2 h at 4 °C. After the incubation period, the dishes were washed three times with cold GBSS and the total amount of collagen bound radioactivity was harvested with a cell scraper (Costar) and measured with a gamma -counter.

Statistical Analysis-- Results were expressed as the mean ± S.D. Statistical analysis of the data was performed with an unpaired Student's t test. The differences were considered significant at p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of pCVI-HSA-- After modification of albumin with the cyclic peptide C*GRGDSPC*, the molecular weight of the pCVI-HSA complex was demonstrated to be higher than that of unmodified HSA (Fig. 2A). The molecular weight of one cyclic peptide moiety was 92,000. From the increase in molecular weight, we estimated that pCVI-HSA contained an average of 10 cyclic peptide moieties per HSA molecule. Only monomeric protein was detected after Coomassie Brilliant Blue staining of the gel, while no residual unmodified HSA was found in the pCVI-HSA preparation. In addition, fast protein liquid chromatography-Superdex analysis was performed with pCVI-HSA and HSA. The chromatograms of pCVI-HSA and HSA were similar as depicted in Fig. 2B, indicating minor polymerization of the protein after the chemical procedures. The major part of the pCVI-HSA preparation consisted of monomeric protein (75%). The decrease in the retention time from 34.5 min (HSA) to 33.0 min (pCVI-HSA) reflects the increased molecular weight of the modified albumin.


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  		Fig. 2.   Characterization of the modified albumin pCVI-HSA. A, SDS-polyacrylamide gel (7.5%) electrophoresis displays an increased molecular weight for pCVI-HSA (lane 2) as compared with HSA (lane 3). A molecular weight marker is shown in lane 1. B, fast protein liquid chromatography-Superdex analysis of pCVI-HSA (straight line) and HSA (dotted line). The major part of the pCVI-HSA and HSA preparations is in a monomeric form, respectively, 75 and 88%, at retention times of 33.06 and 34.54 min. This shift in retention time indicates a higher molecular weight for pCVI-HSA.

Attachment of cyclic peptide moieties to albumin might induce conformational changes in the HSA molecule. Therefore, we assessed the secondary structure of pCVI-HSA using circular dichroism analysis and compared the obtained spectrum to that of HSA. The CD spectra of both pCVI-HSA and HSA were alike (Fig. 3), which indicates that the amount of alpha -helices and beta -sheets are comparable in pCVI-HSA and HSA. HSA treated with dithiothreitol served as a positive control. The cleavage of disulfide bridges by this reducing agent resulted in unfolding of HSA and destruction of its secondary structure, which was reflected by a change in the CD spectrum (Fig. 3).


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  		Fig. 3.   Circular dichroism analysis of pCVI-HSA () and HSA (open circle ). The obtained spectra demonstrate that the secondary structure of the peptide modified albumin was not altered as compared with that of unmodified HSA. Note the strong change in the spectrum of HSA treated with dithiothreitol (down-triangle), a compound that cleaves disulfide bridges in HSA.

Organ Distribution Studies of pCVI-HSA-- The organ distribution of 125I-pCVI-HSA was studied in normal rats and in rats with advanced liver fibrosis (BDL3). After intravenous administration of 125I-pCVI-HSA, this drug carrier was rapidly cleared from the blood (Fig. 4, A and B). Already at 10 min after injection, only 15 ± 4% (normal rats) and 17 ± 4% (BDL3 rats) of the pCVI-HSA dose was present in the blood. In contrast, the complete dose of 125I-HSA was recovered in blood at that time point without significant distribution to any other organ (Fig. 4C). No significant differences were found between the radioactivity detected in blood and in plasma indicating that pCVI-HSA did not bind to blood cells.


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  		Fig. 4.   Organ distribution of 125I-pCVI-HSA (tracer) in normal rats (A) and BDL3 rats (B). The protein was intravenously administered and 10 min (closed bars), 30 min (hatched bars), or 180 min (open bars) after injection the in vivo distribution of pCVI-HSA was determined. C, organ distribution of 125I-HSA in normal (open bars) and BDL3 rats (closed bars) at 10 min after intravenous injection. The results are expressed as the mean ± S.D. (n >=  3).

The organ responsible for most of the uptake of 125I-pCVI-HSA was the liver. After 10 min 75 ± 16 and 62 ± 6% of the dose distributed to the livers of normal and BDL3 rats, respectively. The hepatic content of pCVI-HSA gradually declined with time and 125I-pCVI-HSA and/or degradation products redistributed to urine, and to a minor degree to the intestinal tract, muscles, and skin. The radioactivity excreted by the kidneys into urine consisted of non-protein bound radioactivity as assessed after trichloroacetic acid precipitation. Minor amounts of the dose of 125I-pCVI-HSA were taken up by the spleen (4.9 ± 1.2 and 7.3 ± 4.2% for normal and BDL3 rats, respectively) and kidneys (2.6 ± 0.5 and 1.8 ± 0.5% for normal and BDL3 rats, respectively). Also, radioactivity was recovered in bone, muscle, and skin of the rats. The accumulation of 125I-pCVI-HSA in all organs and tissues was not significantly different between diseased and non-diseased rats at any time point tested.

Immunohistochemical analysis of organ sections confirmed the major distribution of pCVI-HSA to the liver in both normal and BDL3 rats. Also distribution to the spleen was detected immunohistochemically. The localization of pCVI-HSA in the spleen corresponded with vimentin staining, which outlined the venous sinusoids in the spleen. It did not correspond with desmin, alpha -smooth muscle actin, or ED1 (macrophage) staining. In BDL3 rats also, cells of the lung, identified as macrophages, stained positive for pCVI-HSA. In contrast to the staining found in the plastic-embedded livers, the staining in bone could only be detected after amplification of the peroxidase signal. The staining of bone was identified as nonspecific precipitated material in blood vessels and no cell associated staining was found. None of the other tissues stained positive for anti-HSA IgG at 10 min after administration of pCVI-HSA.

Intrahepatic Distribution of pCVI-HSA-- Fig. 5A shows the localization of pCVI-HSA in BDL3 livers as detected immunohistochemically. In BDL3 as well as normal livers, the carrier pCVI-HSA distributed to the nonparenchymal cells, whereas no accumulation of this modified albumin could be found in hepatocytes or bile duct epithelial cells. pCVI-HSA did not bind to cells in the portal areas of BDL3 rat livers 10 min after intravenous injection.


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  		Fig. 5.   Hepatic localization of pCVI-HSA in rats with liver fibrosis (BDL3). A, immunohistochemical detection with an antibody directed against HSA showed the distribution to nonparenchymal cells (original magnification ×160). B, double immunohistochemical staining of BDL3 liver sections for pCVI-HSA (red) and for HSC, identified with the cell markers desmin and glial fibrillary acidic protein (blue). Double positive cells are indicated with arrows (original magnification ×250).

Double-staining of liver sections was performed to identify the cell type(s) responsible for hepatic accumulation of pCVI-HSA. In fibrotic rat livers, pCVI-HSA staining corresponded mainly with the localization of desmin and glial fibrillary acidic protein (Fig. 5B), whereas in normal rat livers pCVI-HSA distributed predominantly to HIS52-positive endothelial cells. Quantitative evaluation of the liver sections confirmed these observations (Table I). In normal rat livers, 70 ± 7% of total hepatic pCVI-HSA uptake was attributable to endothelial cells, whereas in BDL3 rat livers the major part of HSA positive cells (73 ± 14%) were identified as HSC. This increase in HSC uptake in fibrotic livers was paralleled by a concomitantly decreased endothelial uptake (30 ± 10%). Minor accumulation was found in Kupffer cells. Attempts to double label the transformed HSC (myofibroblasts) failed due to the insensitivity of the alpha -smooth muscle actin antibody with alkaline phosphatase detection, but lack of pCVI-HSA staining in the portal areas where actin-positive cells are most abundant indicates no uptake by the cells in these areas.

                              
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Table I
Relative accumulation of pCVI-HSA in the hepatic cell types in normal and fibrotic (BDL3) rat livers
The contribution of hepatic stellate cells (HSC), Kupffer cells (KC), and endothelial cells (EC) in the total liver uptake of the neoglycoprotein was determined by counting the number of cells positive for both pCVI-HSA and the respective cell type marker, and the total number of pCVI-HSA positive cells in the same microscopic field. Values are expressed as the percentage double positive cells of total pCVI-HSA positive cells (±S.D.).

Binding and Internalization of pCVI-HSA by HSC in Vitro-- Cultured rat HSC were used to study the handling of pCVI-HSA by this cell type. First, we determined the binding and uptake of 125I-pCVI-HSA to quiescent and activated HSC. The results were compared with data obtained by incubation of the cells with 125I-HSA. As shown in Fig. 6, significantly higher amounts of 125I-pCVI-HSA bound to activated HSC at both 4 and 37 °C as compared with 125I-HSA. In contrast, quiescent HSC did not bind significant amounts of the modified albumin. Cell-associated radioactivity was significantly higher at 37 °C than at 4 °C, indicating cellular uptake of the protein. Since pCVI-HSA specifically bound to activated HSC, all other in vitro experiments were performed with activated cell cultures. Fig. 7 depicts the time-dependent association of 125I-pCVI-HSA to activated HSC at 4 °C and 37 °C.


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  		Fig. 6.   The cell-bound radioactivity of 125I-pCVI-HSA (A) and 125I-HSA (B) after incubation with primary cultures of quiescent rat HSC (qHSC) and activated rat HSC (aHSC) measured after 2 h of incubation at 4 °C (open bars) and 37 °C (closed bars). The difference between 125I-pCVI-HSA and 125I-HSA binding is indicated with an asterisk (*) (p < 0.05). The difference between 4 and 37 °C is indicated by the double asterisk (**) (p < 0.01). The difference between qHSC and aHSC is indicated by a circle (open circle ) (p < 0.01). Results are expressed as the mean ± S.D. (n >=  3).


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  		Fig. 7.   Time-dependent binding of 125I-pCVI-HSA (circles) and 125I-HSA (triangles) at 4 °C (open symbols) and 37 °C (closed symbols) to activated HSC. Results are expressed as the mean ± S.D. (n >=  3).

Prolonged incubation of pCVI-HSA with HSC up to 24 h resulted in an increased cellular binding of pCVI-HSA as compared with the binding at 2 h (p < 0.001). After 24 h 6907 ± 1009 cpm/105 HSC were found after incubation of HSC with pCVI-HSA, whereas 977 ± 301 cpm/105 HSC bound when incubations were performed with HSA. Incubation of HSC cultures with pCVI-HSA did not result in a change of cell morphology as inspected at the light-microscopical level.

To examine the specificity of the binding of pCVI-HSA to activated HSC, we incubated this modified albumin in the presence of excess unlabeled pCVI-HSA, which reduced the amount of 125I-pCVI-HSA bound to HSC by 65% (p < 0.01, Fig. 8A). To assess whether pCVI-HSA is taken up in HSC via receptor-mediated endocytosis, we incubated pCVI-HSA at 37 °C in the presence of 2 µM monensin. Monensin inhibits acidification of endosomes and lysosomes and thus blocks dissociation of the ligand from the receptor and consequently further cellular uptake of this ligand. In addition, monensin prevents the release of receptor-ligand containing vesicles from the microtubuli (29, 30). Incubation of HSC with monensin under our experimental conditions did not affect the viability of HSC as assessed with trypan blue exclusion tests. Fig. 8B shows that total radioactivity incorporated in HSC declined to approximately 50% with monensin (p < 0.001), indicating internalization of pCVI-HSA via an endosomal and lysosomal pathway. Uptake of mannose 6-phosphate-modified albumin, known to be endocytosed by cells (31, 32), was also inhibited by monensin, by 63%.2 Despite uptake of the modified albumin by HSC was found, only minor degradation products (non-trichloroacetic acid precipitable radioactivity) were detected in the media following incubations for 2 h. To determine cellular degradation of pCVI-HSA, we incubated 125I-pCVI-HSA and 125I-HSA with slices prepared from fibrotic livers. As can be derived from Fig. 9, incubation of these slices with pCVI-HSA resulted in an increased formation of degradation products in time, whereas native HSA was not degraded by these hepatic cells.


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  		Fig. 8.   A, the binding of 125I-pCVI-HSA to activated HSC after 2 h of incubation at 4 °C in the absence and presence of an excess unlabeled pCVI-HSA (*, p < 0.01). B, effect of monensin on the uptake of 125I-pCVI-HSA in activated HSC after 2 h of incubation at 37 °C (*, p < 0.001). Results are expressed as the mean ± S.D. (n = 3).


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  		Fig. 9.   Time-dependent degradation of 125I-pCVI-HSA (circles) and HSA (triangles) at 37 °C in liver slices prepared from BDL3 rat livers. No degradation products of 125I-HSA were found at all time points studied in contrast to incubations with pCVI-HSA (*, p < 0.001). The results are expressed as the mean value ± S.D. (n = 3).

To confirm the data of the in vitro binding studies with radiolabeled proteins, primary cultures of activated HSC were incubated with unlabeled pCVI-HSA for immunohistochemical analysis. We could clearly demonstrate the binding of pCVI-HSA to activated HSC, while unmodified HSA showed no association with the cells. Furthermore, the binding of pCVI-HSA to HSC could be inhibited by the presence of excess collagen type VI, while a polyanion such as suramin did not prevent binding of pCVI-HSA to the HSC in vitro (Fig. 10).


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  		Fig. 10.   Immunohistochemical demonstration of HSC binding of pCVI-HSA (A), HSA (B), and pCVI-HSA (C) in the presence of collagen type VI and or suramin (D). After incubating the (modified) albumins with activated HSC, the albumins were detected with polyclonal anti-HSA IgG (original magnification ×160).

Finally, we studied the in vitro binding of 125I-pCVI-HSA to different collagen types I, III, IV, and VI. These matrix proteins are abundantly present in fibrotic livers. As can be seen in Fig. 11, pCVI-HSA displayed a higher binding to collagen S, a mixture of collagen type I (95%) and collagen type III (5%), as compared with 125I-HSA. The binding to collagen was also found in experiments with purified forms of collagen. pCVI-HSA displayed a higher binding for collagen type VI and interstitial collagens type III and I than for type IV collagen. In all cases, significantly higher amounts of pCVI-HSA were associated with collagen proteins as compared with HSA (p < 0.01).


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  		Fig. 11.   The binding of 125I-pCVI-HSA to matrix proteins. Dishes were coated with collagen S (0.15 mg) and purified collagens types VI, I, III, and IV (10 µg). The amount of collagen-bound 125I-pCVI-HSA (closed bars) was significantly higher (p < 0.01) than 125I-HSA binding to the matrix components (open bars). Results are expressed as the mean ± S.D. (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To increase the cell-specific uptake of antifibrotic drugs, we developed drug carriers to HSC. Receptors expressed on (activated) HSC may serve as targets for such carriers. Activated HSC avidly bind collagen type VI, which is an important matrix protein involved in the binding of cells to extracellular matrix proteins. Since the cyclic RGD-peptide, C*GRGDSPC*, specifically binds to mesenchymal cells via collagen type VI receptors (19), it was used to design a specific carrier for HSC in fibrotic livers. A construct of approximately 10 cyclic peptides attached to human albumin was employed to study the liver selectivity of pCVI-HSA in normal rats and in rats with liver fibrosis and to study the cellular handling by HSC.

Quality assessment of the pCVI-HSA preparations led to the conclusion that the product was mostly monomeric. Attachment of cyclic peptides to the lysine groups of albumin might result in conformational changes of the albumin backbone due to electrostatic interactions between the carrier and the attached peptide moiety or the reaction conditions used for the coupling of cyclic peptides to HSA may cause a denaturation or refolding of the albumin molecule. This may lead to immunogenic responses or nonspecific removal by Kupffer cells or other macrophages in vivo (33). The identical CD spectra of pCVI-HSA and HSA, however, indicated that the conformation of the albumin molecule was not affected by the introduction of the 10 C*GRGDSPC* groups to albumin.

In vivo experiments demonstrated that pCVI-HSA strongly accumulated in rat livers. Ten minutes after intravenous injection, about 75% (normal livers) and 62% (fibrotic livers) of the dose pCVI-HSA was distributed to the livers, whereas unmodified HSA completely remained in the blood. The majority of hepatic cells in BDL3 rats that bound pCVI-HSA were identified as HSC, whereas in normal rat livers pCVI-HSA was mainly distributed to endothelial cells. This binding to endothelial cells may be explained by the ability of endothelial cells to bind matrix molecules (34), but also by the presence of other protein removal systems (35). pCVI-HSA displayed only minor binding to Kupffer cells, the hepatic cell type involved in the removal of large molecular weight molecules and foreign particles (33). Therefore, although the total distribution of pCVI-HSA to normal and fibrotic rat livers is almost identical, the intrahepatic distribution is completely different. This may be due to disease-induced changes in the sinusoidal endothelial cells or to the increased number of activated HSC in fibrotic livers in combination with increased numbers of collagen type VI receptors on these cells.

Immunohistochemical staining of fibrotic liver sections showed a distribution of pCVI-HSA to the non-parenchymal cells in zones II and III, but not to cells in the periportal areas where most alpha -smooth muscle actin-positive cells (activated HSC) reside. To study whether pCVI-HSA is able to bind to activated HSC, we performed binding studies with cultured rat HSC. These in vitro studies demonstrated that pCVI-HSA specifically bound to the activated HSC compared with minimal binding to quiescent HSC. These results could be confirmed by immunohistochemical studies demonstrating the preferential binding of pCVI-HSA to cultured activated HSC. Inhibition of the cellular binding of pCVI-HSA in the presence of collagen type VI but not in the presence of the polyanion suramin further indicated that the binding to these cells was specific. In addition, different types of purified collagens (I, III, VI, and IV) interfered with 125I-pCVI-HSA binding to HSC (data not shown). There are several reasons for the discrepancy between the in vitro studies showing binding to activated HSC and the results obtained in vivo that demonstrate lack of binding in periportal areas where activated HSC reside. Since the accessibility of hepatic cells in the portal area may be hampered during fibrosis due to increased collagen deposition, pCVI-HSA may not have reached the portal areas in vivo as early as 10 min after intravenous injection. Also, a competition between the modified albumins and endogenous substrates, such as collagen type VI (14, 15) present at these particular sites in fibrotic livers, may occur in diseased livers. Therefore, the binding to these cells may be enhanced by prolonged exposure to the carrier. Accordingly, we showed in vitro that an increase in the incubation time to 24 h resulted in a 10-fold higher binding of pCVI-HSA to HSC than incubation for 2 h. Binding of pCVI-HSA to HSC in vivo in zones II and III may also indicate that HSC in these areas of the fibrotic livers express more collagen type VI receptors than those in zone I.

In addition to the activated HSC specificity of the carrier, any extrahepatic uptake of the carrier must be considered to anticipate possible side effects of drugs. However, organ distribution studies showed that the extrahepatic uptake of pCVI-HSA in fibrotic rats was low (less than 5% was present in spleen, kidneys, and other organs). Although the radioactive studies indicated distribution of pCVI-HSA to bone, muscles, and skin, immunohistochemical analysis of these organs could not confirm its cell associated presence in these tissues. The gradually increasing amounts of pCVI-HSA in skin and muscles in time probably represented redistribution of parts of 125I-pCVI-HSA degraded in the liver. Therefore, although some extrahepatic uptake of pCVI-HSA was found, the major part of the intravenous injected pCVI-HSA distributed to the (fibrotic) livers. In vitro studies showed enhanced binding of pCVI-HSA to different types of collagen (I, III, IV, and VI) as compared with HSA. The immunohistochemically based organ distribution studies performed in vivo, however, did not show any association with the extracellular matrix. The significance of the matrix binding properties of the carrier has to be considered, but relative to the binding of pCVI-HSA to HSC it will only play a minor role in vivo.

In the present study, we showed that pCVI-HSA was internalized by HSC. This is in accordance with studies performed by Everts et al. (36) demonstrating uptake and degradation of collagen type VI in the lysosomes of fibroblasts. Cellular uptake of pCVI-HSA allows the delivery of antifibrotic drugs that interfere with intracellular processes. Among the drugs of interest are the inhibitors of collagen synthesis such as prolyl-4-hydroxylase inhibitors (37, 38), activators of collagenase expression/activation, like pentoxifylline (39), or inhibitors of HSC activation (NFkappa B inhibitors or histone deacetylase inhibitors (40-42)).

The modified albumin pCVI-HSA is not only endocytosed but also degraded by the liver cells. The latter was assessed in vitro (rat liver slices) as well as in vivo. The in vivo studies showed a decreased liver content of pCVI-HSA in time together with a recovery of degradation products (free 125I) in the urine of these rats. In vitro studies confirmed the in vivo data: degradation products were released by slices incubated with pCVI-HSA. The biological degradation has implications for the drug targeting concept since accumulation of a compound within the cell will cause toxicity of the compound and moreover, release of active compounds within the cell will be limited if the carrier would not be degraded. If necessary, release characteristics of the drug could be improved by coupling of the drug to the carrier via, for instance, a spacer, which will cause release of drugs in the endosomes (43).

pCVI-HSA may also have intrinsic activities, that is, a direct influence on the fibrotic process itself (44). It is known that integrin-mediated cell attachment in general influences and regulates cell migration, growth, differentiation, and apoptosis of cells (10). Collagen type VI potently regulates mesenchymal cell proliferation and activation in vitro (17, 18). Like single chain collagen type VI, the peptide-modified albumin pCVI-HSA may interfere with processes of HSC activation by blocking cellular binding sites of native collagen type VI. RGD-containing peptides were reported to interfere with liver fibrogenesis and other wound healing processes (45-47).

Previously, we reported about the specific distribution of albumin modified with mannose 6-phosphate groups (M6P28-HSA) to HSC (9). The selectivity of the distribution of this M6P28-HSA to HSC was similar to that of pCVI-HSA. The specificity of pCVI-HSA, however, may be further optimized by varying the amount of cyclic peptides or by variations in the amino acids flanking the RGD motif (10, 48, 49). Whether neoglycoproteins or peptide-modified albumins represent the most optimal carrier remains to be established.

Targeting receptors by substituting albumin with (cyclic) peptide motifs is a new approach in the design of drug targeting preparations. Due to structural stabilization, cyclization of peptide motifs is associated with a higher affinity for the receptors compared with their linear analogues (18, 48, 50). The design of carrier molecules described in this study may also be applicable for other receptors, such as cytokine or growth factor receptors (48, 51-53). Studies on drug carriers to the platelet-derived growth factor receptor are in progress. The targeting to receptors that are up-regulated on the activated fibrogenic cells in pathological processes may also be applicable for targeting of therapeutic agents to other fibrogenic processes, such as atherosclerosis or glomerulosclerosis (54).

Drug targeting also offers possibilities in more pathological or fundamental research areas. Targeting of pharmacological active agents to an individual cell type offers the advantage of selective elimination of one cell type or blockade of a single process within this cell type. After specific inhibition of a process, the implications for the development of a particular disease can be studied. In this way, drug targeting allows us to gain more insight in the molecular basis of diseases in vivo. As a consequence, drug targeting preparations may create new leads for novel therapeutic interventions.

In conclusion, we showed that HSA modified with cyclic peptides, that contain RGD sequences, represents a new type of carrier. pCVI-HSA is preferentially taken up by activated rat HSC. Application of this novel carrier may strongly increase the tissue selectivity and efficacy of antifibrotic drugs. Since the core amino acids (RGD) responsible for receptor binding are identical in rats and humans, pCVI-HSA may also be applied in cirrhotic patients.

    ACKNOWLEDGEMENTS

Circular dichroism analysis was performed with the help of M. L. de Vocht (Dept. of Biochemistry, University of Groningen, The Netherlands). Part of this study was performed in cooperation with the Human Liver Group Groningen. We thank Dr. P. Olinga (Dept. of Pharmacokinetics and Drug Delivery, University of Groningen, The Netherlands) for the preparation of liver slices and Prof. Dr. M. J. H. Slooff (Dept. of Surgery, University Hospital of Groningen, The Netherlands) for providing human liver tissue.

    FOOTNOTES

* This work was supported by a grant from the Foundation of Technical Sciences (STW), which is part of the Dutch Organization for Scientific Research (NWO).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.

§ To whom correspondence should be addressed: Dept. of Pharmacokinetics and Drug Delivery, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands. Tel.: 00-31-50-3633276; Fax: 00-31-50-3633247; E-mail: L.Beljaars@farm.rug.nl.

2 L. Beljaars, P. Olinga, P. J. De Bleser, A. Geerts, G. Molema, G. M. M. Groothuis, D. K. F. Meijer, and K. Poelstra, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HSC, hepatic stellate cells; HSA, human serum albumin; pCVI-HSA, HSA modified with cyclic peptide C*GRGDSPC*; RGD, Arg-Gly-Asp; BDL, bile duct ligation; BDL3, 3 weeks after BDL; DMEM, Dulbecco's modified Eagle's medium; GBSS, Gey's balanced salt solution.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ramadori, G., Knittel, T., and Saile, B. (1998) Digestion 59, 372-375[CrossRef][Medline] [Order article via Infotrieve]
2. Arthur, M. J. P. (1998) Digestion 59, 376-380[CrossRef][Medline] [Order article via Infotrieve]
3. Alcolado, R., Arthur, M. J. P., and Iredale, J. P. (1997) Clin. Sci. 92, 103-112[Medline] [Order article via Infotrieve]
4. Friedman, S. L. (1990) Semin. Liver Dis. 10, 20-29[Medline] [Order article via Infotrieve]
5. Pinzani, M. (1995) Pharmacol. Ther. 66, 387-412[CrossRef][Medline] [Order article via Infotrieve]
6. Meijer, D. K. F., and Molema, G. (1995) Semin. Liver Dis. 15, 202-256[Medline] [Order article via Infotrieve]
7. Beljaars, L., Poelstra, K., Molema, G., and Meijer, D. K. F. (1998) J. Hepatol. 29, 579-588[CrossRef][Medline] [Order article via Infotrieve]
8. Albrecht, C., Meijer, D. K. F., Lebbe, C., Sägesser, H., Melgert, B. N., Poelstra, K., and Reichen, J. (1997) Hepatology 26, 1553-1559[CrossRef][Medline] [Order article via Infotrieve]
9. Beljaars, L., Molema, G., Weert, B., Bonnema, H., Olinga, P., Groothuis, G. M. M., Meijer, D. K. F., and Poelstra, K. (1999) Hepatology 29, 1486-1493[CrossRef][Medline] [Order article via Infotrieve]
10. Ruoslahti, E. (1996) Annu. Rev. Cell Dev. Biol. 12, 697-715[CrossRef][Medline] [Order article via Infotrieve]
11. Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
12. Aumailley, M., and Gayraud, B. (1998) J. Mol. Med. 76, 253-265[CrossRef][Medline] [Order article via Infotrieve]
13. Loreal, O., Clement, B., Schuppan, D., Rescan, P. Y., Rissel, M., and Guillouzo, A. (1992) Gastroenterology 102, 980-987[Medline] [Order article via Infotrieve]
14. Schuppan, D., Ruhlmann, T., and Hahn, E. G. (1985) Anal. Biochem. 149, 238-247[CrossRef][Medline] [Order article via Infotrieve]
15. Takahara, T., Sollberg, S., Muona, P., and Uitto, J. (1995) Liver 15, 78-86[Medline] [Order article via Infotrieve]
16. Gerling, B., Becker, M., Staab, D., and Schuppan, D. (1997) N. Engl. J. Med. 336, 1611-1612[Free Full Text]
17. Ruhl, M., Johannson, M., Atkinson, J., Manski, D., Sahin, E., Somasundaram, R., Riecken, E. O., and Schuppan, D. (1999) Exp. Cell Res. 250, 548-557[CrossRef][Medline] [Order article via Infotrieve]
18. Atkinson, J. C., Ruhl, M., Becker, J., Ackermann, R., and Schuppan, D. (1996) Exp. Cell Res. 228, 283-291[CrossRef][Medline] [Order article via Infotrieve]
19. Marcelino, J., and McDevitt, C. A. (1995) Biochim. Biophys. Acta 1249, 180-188[CrossRef][Medline] [Order article via Infotrieve]
20. Provencher, S. W., and Glockner, J. (1981) Biochemistry 20, 33-37[CrossRef][Medline] [Order article via Infotrieve]
21. Pless, D. P., and Lennarz, W. J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 134-138[Abstract/Free Full Text]
22. Mather, S. J., and Ward, B. C. (1987) J. Nucl. Med. 28, 1034-1036[Abstract/Free Full Text]
23. Kountouras, J., Billing, B. H., and Scheuer, P. J. (1984) Br. J. Exp. Pathol. 65, 305-311[Medline] [Order article via Infotrieve]
24. Van Goor, H., Gerrits, P. O., and Hardonk, M. J. (1989) J. Histochem. Cytochem. 37, 399-403[Abstract]
25. Harms, G., Dijkstra, C. D., Lee, Y. C., and Hardonk, M. J. (1990) Cell Tissue Res. 262, 35-40[CrossRef][Medline] [Order article via Infotrieve]
26. Geerts, A., Niki, T., Hellemans, K., De Craemer, D., Van den Berg, K., Lazou, J.-M., Stange, G., Van de Winkel, M., and De Bleser, P. J. (1998) Hepatology 27, 590-598[CrossRef][Medline] [Order article via Infotrieve]
27. Olinga, P., Groen, K., Hof, I. H., de Kanter, R., Koster, H. J., Leeman, W. R., Rutten, A. A. J. J. L., van Twillert, K., and Groothuis, G. M. M. (1997) J. Pharmacol. Toxicol. Methods 38, 59-69[CrossRef][Medline] [Order article via Infotrieve]
28. Olinga, P., Merema, M., Hof, I. H., de Jong, K. P., Slooff, M. J. H., Meijer, D. K. F., and Groothuis, G. M. M. (1998) Drug Metab. Dispos. 26, 5-11[Abstract/Free Full Text]
29. Goltz, J. S., Wolkoff, A. W., Novikoff, P. M., Stockert, R. J., and Satir, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7026-7030[Abstract/Free Full Text]
30. Eskild, W., and Berg, T. (1988) Biochim. Biophys. Acta 968, 143-150[Medline] [Order article via Infotrieve]
31. Roche, A. C., Midoux, P., Bouchard, P., and Monsigny, M. (1985) FEBS Lett. 193, 63-68[CrossRef][Medline] [Order article via Infotrieve]
32. Braulke, T., Tippmer, S., Chao, H. J., and Von Figura, K. (1990) J. Biol. Chem. 265, 6650-6655[Abstract/Free Full Text]
33. Toth, C. A., and Thomas, P. (1992) Hepatology 16, 255-266[CrossRef][Medline] [Order article via Infotrieve]
34. Adler, S., and Eng, B. (1993) Kidney Int. 44, 278-284[Medline] [Order article via Infotrieve]
35. Meijer, D. K. F., and Ziegler, K. (1993) in Biological Barriers to Protein Delivery (Audus, K. L. , and Raub, T. J., eds) , pp. 339-408, Plenum Press, New York
36. Everts, V., Korper, W., Niehof, A., Jansen, I., and Beertsen, W. (1995) Matrix Biol. 14, 665-676[CrossRef][Medline] [Order article via Infotrieve]
37. Bickel, M., Baringhaus, K.-H., Gerl, M., Günzler, V., Kanta, J., Schmidts, L., Stapf, M., Tschank, G., Weidmann, K., Werner, U., Baringhaus, K. H., and Gunzler, V. (1998) Hepatology 28, 404-411[CrossRef][Medline] [Order article via Infotrieve]
38. Schuppan, D., Strobel, D., and Hahn, E. G. (1998) Digestion 59, 385-390[CrossRef][Medline] [Order article via Infotrieve]
39. Windmeier, C., and Gressner, A. M. (1997) Gen. Pharmacol. 29, 181-196[Medline] [Order article via Infotrieve]
40. Lee, K. S., Buck, M., Houglum, K., and Chojkier, M. (1995) J. Clin. Invest. 96, 2461-2468
41. Lee, J. I., and Burckart, G. J. (1998) J. Clin. Pharmacol. 38, 981-993[Abstract/Free Full Text]
42. Niki, T., Rombouts, K., De Bleser, P. J., de Smet, K., Rogiers, V., Schuppan, D., Yoshida, M., Gabbiani, G., and Geerts, A. (1999) Hepatology 29, 858-867[CrossRef][Medline] [Order article via Infotrieve]
43. Soyez, H., Schacht, E., and Van Der Kerken, S. (1996) Adv. Drug Deliv. Rev. 21, 81-106[CrossRef]
44. Meijer, D. K. F., Molema, G., Moolenaar, F., De Zeeuw, D., and Swart, P. J. (1996) J. Contr. Rel. 39, 163-172[CrossRef]
45. Bruck, R., Hershkoviz, R., Lider, O., Shirin, H., Aeed, H., and Halpern, Z. (1998) Yale J. Biol. Med. 70, 391-402
46. Iwamoto, H., Sakai, H., Kotoh, K., Nakamuta, M., and Nawata, H. (1999) Dig. Dis. Sci. 44, 1038-1045[CrossRef][Medline] [Order article via Infotrieve]
47. Pierschbacher, M. D., Polarek, J. W., Craig, W. S., Tschopp, J. F., Sipes, N. J., and Harper, J. R. (1994) J. Cell Biol. 56, 150-154
48. Koivunen, E., Wang, B., and Ruoslathi, E. (1995) Bio/Technology 13, 265-270[CrossRef][Medline] [Order article via Infotrieve]
49. Danilov, Y. N., and Juliano, R. L. (1989) Exp. Cell Res. 182, 186-196[CrossRef][Medline] [Order article via Infotrieve]
50. Hart, S. L., Knight, A. M., Harbottle, R. P., Mistry, A., Hunger, H. D., Cutler, D. F., Williamson, R., and Coutelle, C. (1994) J. Biol Chem. 269, 12468-12474[Abstract/Free Full Text]
51. Dower, W. J. (1998) Curr. Opin. Chem. Biol. 2, 238-334
52. Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J. (1996) Science 273, 458-463[Abstract]
53. Yanofsky, S. D., Baldwin, D. N., Butler, J. H., Holden, F. R., Jacobs, J. W., Balasubramanian, P., Chinn, J. P., Cwirla, S. E., Peters-Bhatt, E., Whitehorn, E. A., Tate, E. H., Akeson, A., Bowlin, T. L., Dower, W. J., and Barrett, R. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7381-7386[Abstract/Free Full Text]
54. Horton, M. A. (1999) Exp. Nephrol. 7, 178-184[CrossRef][Medline] [Order article via Infotrieve]

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