Hepatic fibronectin matrix turnover in rats: involvement of the asialoglycoprotein receptor

Robert F. Rotundo, Peter A. Vincent, Paula J. McKeown-Longo, Frank A. Blumenstock, and Thomas M. Saba

Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibronectin (Fn) is a major adhesive protein found in the hepatic extracellular matrix (ECM). In adult rats, the in vivo turnover of plasma Fn (pFn) incorporated into the liver ECM is relatively rapid, i.e., <24 h, but the regulation of its turnover has not been defined. We previously reported that cellular Fn (cFn) and enzymatically desialylated plasma Fn (aFn), both of which have a high density of exposed terminal galactose residues, rapidly interact with hepatic asialoglycoprotein receptors (ASGP-R) in association with their plasma clearance after intravenous infusion. With the use of adult male rats (250-350 g) and measurement of the deoxycholate (DOC)-insoluble 125I-labeled Fn in the liver, we determined whether the ASGP-R system can also influence the hepatic matrix retention of various forms of Fn. There was a rapid deposition of 125I-pFn, 125I-aFn, and 125I-cFn into the liver ECM after their intravenous injection. Although 125I-pFn was slowly lost from the liver matrix over 24 h, more than 90% of the incorporated 125I-aFn and 125I-cFn was cleared within 4 h (P < 0.01). Intravenous infusion of excess nonlabeled asialofetuin to competitively inhibit the hepatic ASGP-R delayed the rapid turnover of both aFn and cFn already incorporated within the ECM of the liver. ECM retention of both 125I-aFn and 125I-cFn was also less than 125I-pFn (P < 0.01) as determined in vitro using liver slices preloaded in vivo with either tracer form of Fn. The hepatic ASGP-R appears to participate in the turnover of aFn and cFn within the liver ECM, whereas a non-ASGP-R-associated endocytic pathway apparently influences the removal of normal pFn incorporated within the hepatic ECM, unless it becomes locally desialylated.

extracellular matrix proteins; hepatocyte receptors; liver; galactose


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PLASMA FIBRONECTIN (pFn) consists of two subunits of ~220 kDa, linked by disulfide bonds located in the carboxy-terminal region of the molecule (26, 37). It is a glycoprotein with ~5% of its molecular weight represented as carbohydrate molecules. Most of the carbohydrate side chains found in human pFn are biantennary N-linked carbohydrates containing terminal sialic acid residues linked primarily in the 2-6 position to beta -galactose, although a few O-linked carbohydrate chains have been identified (28, 45, 55).

pFn can be incorporated into the extracellular matrix (ECM) of a wide variety of tissues and blood vessels, where it is believed to influence wound healing, cell adhesion, and vascular integrity (26, 37). In vitro studies by McKeown-Longo and Mosher (23-25) and Wheatley et al. (52, 53) documented the incorporation of soluble pFn into the ECM of fibroblasts and endothelial cells after its interaction with cell-associated matrix assembly sites. Immunofluorescent and radioactive tracer studies in mice, rats, and sheep have also confirmed the incorporation of circulating soluble Fn into the ECM of many tissues, including the liver, lungs, kidneys, spleen, and various blood vessels (6, 7, 10, 16, 27, 34), with the liver ECM apparently being the primary in vivo site for deposition of Fn from the plasma (10, 34). In addition, the initial in vitro observations on cultured layers (24, 25), which demonstrated that soluble pFn must first bind to cell-associated matrix assembly sites before being incorporated into the ECM, have now been verified in vivo by the comparative analysis of the ECM incorporation of normal plasma Fn vs. N-ethylmaleimide (NEM)-alkylated plasma Fn, which cannot bind to matrix assembly sites (24, 25, 34).

Increased levels of insoluble Fn in the ECM of various diseased organs have been observed with hepatic fibrosis (4, 5, 18, 22) and chronic lung injury (13). Changes in the Fn content of the ECM seen in various disease states, especially within the liver, could be due to alterations in either the rate of Fn assembly into the matrix or the rate of Fn loss or turnover from the matrix. Rebres et al. (34) calculated that the half-life for the turnover of pFn already incorporated within liver ECM was <24 h. This estimate is consistent with the two-compartment mathematical model formulated by Deno et al. (10) to account for the kinetics of pFn in rats after its initial distribution between the vascular and extravascular compartments.

Liver parenchymal cells, also called hepatocytes, express receptors known as asialoglycoprotein receptors (ASGP-R), which can mediate the binding and endocytosis of experimentally desialylated plasma proteins injected into the blood (2, 3). The ASGP-R is an endocytic receptor expressed in high density, especially on the sinusoidal surface of hepatocytes (40) adjacent to the extracellular space of Disse in the liver, which contains a matrix rich in Fn. The ASGP-R is believed to mediate the removal of plasma glycoproteins that have their terminal galactose or N-acetyl-D-galactosamine residues exposed (2, 3), although this pathway has yet to be fully verified in vivo. We recently reported that pFn that has been experimentally desialylated with neuraminidase to expose penultimate galactose residues (called aFn) as well as cellular Fn (cFn), which naturally has a large density of terminal galactose residues, are both rapidly removed from the blood by a ASGP-R-linked pathway in the liver (35).

The current study was designed to determine whether the hepatic ASGP-R could also influence the retention of various forms of Fn within in the hepatic ECM. To accomplish this goal, we quantified the matrix incorporation of purified radiolabeled pFn, cFn, and aFn by analysis of their levels in the deoxycholate (DOC) detergent-insoluble fraction within the liver, because this fraction contains Fn covalently cross-linked into the insoluble ECM (24, 25, 34). Selective inhibition of the ASGP-R in the liver was accomplished by the intravenous infusion of purified asialofetuin, a known inhibitor of the ASGP-R, to further determine the importance of the ASGP-R in the turnover of Fn by the liver.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Materials and reagents. cFn was purchased from Upstate Biomedical (Lake Placid, NY). Neuraminidase (sialidase) derived from Clostridium perfringens was purchased from Oxford Glycosystems (Rosedale, NY). Gelatin-Sepharose and G-25 gel filtration columns were purchased from Pharmacia (Uppsala, Sweden). NEM was purchased from Pierce Chemical (Rockford, IL). DMEM used for culture medium as well as streptomycin and penicillin were obtained from GIBCO (Grand Island, NY). FBS used in the culture medium was obtained from HyClone (Logan, UT). Prolong Anti Fade was purchased from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma.

Purification and preparation of pFn and aFn. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY; 250-350 g) were used in all experiments. pFn was rapidly purified from fresh rat plasma or human cryoprecipitate by gelatin-Sepharose affinity chromatography as previously described (10, 16, 26, 34, 35). Removal of the terminal sialic acid residues from purified human and rat pFn was done with neuraminidase (35). Normal nondesialylated pFn was handled the same as aFn except for lack of exposure to neuraminidase. The removal of terminal sialic acid residues and exposure of penultimate galactose residues were previously confirmed by comparative lectin dot-blot analysis of all purified pFn, cFn, and aFn preparations (35).

Iodination of purified Fn and asialofetuin. Purified Fn and asialofetuin were iodinated using a chloramine T method (34, 35). Reduced and nonreduced samples (0.5 µg/ml) were applied to 4-15% gradient SDS-PAGE gels and analyzed by autoradiography to verify the integrity of each Fn preparation both before and after iodination.

Human fibroblast monolayers. To verify the ability of the various forms of Fn to incorporate within the ECM, we used cultured cell layers of A1-F human foreskin fibroblasts (a gift from Dr. Lynn Allen-Hoffman, University of Wisconsin, Madison, WI) in a matrix assembly assay as previously described (1, 23-25, 34). Briefly, fibroblasts were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded at a density of 5 × 104 cells/ml in 12-well tissue culture plates using a volume of 1 ml/well. Cell layers were used at 3 days postconfluence. For immunofluorescent experiments, the cells were seeded onto 12-mm coverslips in separate 12-well culture plates at a density of 5 × 104 cells/ml.

Immunofluorescent analysis of Fn incorporated in the ECM of cultured fibroblasts. Fibroblast cell layers at 3 days postconfluence were washed with PBS before adding 1 ml of DMEM supplemented with 10% Fn-depleted FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml of purified rat pFn or rat aFn. Fn-depleted FBS was produced by passage over a gelatin-Sepharose column. The fibroblast layers were incubated for 24 h, after which time the cells were washed with PBS, fixed for 15 min with 3% formaldehyde, and permeabilized with 0.5% Triton X-100 in HEPES for 5 min on ice. The fixed cells were blocked for 1 h with PBS containing 2% BSA, 50 mM glycine, and 0.2% Triton X-100. The primary antibody used was rabbit anti-rat pFn purchased from Calbiochem (San Diego, CA), which had no cross-reactivity with human pFn. It was added to the fixed cells at a 1:100 dilution in PBS for 1 h. Rhodamine-conjugated goat anti-rabbit secondary antibody (Cappel, Durham, NC) was added for 1 h at a dilution of 1:50 in PBS. Coverslips were mounted using Prolong Anti Fade and viewed at a magnification of ×40 using an Olympus BX60 fluorescent scope and photographed with a Diagnostic Instruments digital camera.

Isotopic analysis of Fn incorporated in the ECM of cultured human fibroblasts. 125I-Fn deposited in the DOC-insoluble fraction has been used to quantify matrix deposition of Fn in vitro (23, 24, 52) and in vivo (33, 34). Fibroblasts at 3 days postconfluence were washed with PBS and supplemented with 1 ml/well of DMEM containing 10% Fn-depleted FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml of either rat 125I-pFn or rat 125I-aFn. Cell layers were incubated for 1, 3, 6, or 24 h, after which time they were washed with PBS and extracted with 1 ml of DOC extraction buffer (1% DOC in 20 mM Tris at pH 8.3 containing 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM iodoacetic acid, and 2 mM NEM). The 125I-Fn in both the DOC-soluble and the DOC-insoluble fractions was quantified using a gamma counter. The DOC-soluble pool represents reversibly bound or internalized Fn, whereas the DOC-insoluble pool of 125I-Fn represents Fn covalently incorporated into the ECM (24, 25, 33, 34). DOC-insoluble fractions of cell layers incubated with 125I-pFn or 125I-aFn were also separated by SDS-PAGE using a 6% gradient gel with a 4% stacker to confirm the existence of high molecular weight Fn multimers by autoradiographic analysis.

In vivo analysis of ECM incorporation of various forms of 125I-Fn. The plasma removal and extravascular deposition of the various intravenously injected 125I-Fn preparations within the DOC-insoluble pool in the liver were done as recently described (34). Rats were injected intravenously with either 125I-pFn, 125I-aFn, or 125I-cFn at a dose of 3 µg Fn/100 g body wt. Serial 250-µl blood samples were collected via tail vein puncture into an anticoagulant solution (100 mM EDTA, 40.5 mM iodoacetate, and 40 mM benzamidine) and centrifuged, and 25-µl aliquots were analyzed. For analysis of the liver tissue, the rats were first anesthetized and perfused intravenously with 150 ml sterile lactated Ringer's solution, which removes residual blood from the tissues and vascular space (10, 34). Perfusion continued until the hematocrit was <2% (10). Harvested livers were rapidly rinsed in 4°C saline and weighed. Livers were rapidly sectioned to about 200-300 mg (0.3-0.5 mm) with a Stadie-Riggs tissue slicer, and the slices were rinsed in 4°C sterile saline, blotted dry, and weighed. Each tissue slice was oscillated on a rocker with 5 ml of DOC extraction buffer for 3 h at 4°C and then centrifuged at 35,000 g for 30 min. The 125I-Fn in both the DOC-soluble fraction and the DOC-insoluble fraction were quantified.

In vitro turnover of Fn by incubated liver slices preloaded in vivo with various forms of 125I-Fn. The turnover of various 125I-Fn preparations in preloaded liver slices, which were harvested from rats after intravenous injection of either 125I-pFn, 125I-aFn, 125I-cFn, or 125I-asialofetuin, was studied using a dose of 12 µg 125I-Fn/100 g body wt. At either 15 min or 4 h after tracer injection, rats were anesthetized and perfused intravenously with 150 ml sterile lactated Ringer solution to remove residual tracer from both the liver and vascular compartment (10, 34). The liver was rapidly harvested, rinsed in 4°C saline, and finely sectioned into 0.3-0.5 mm liver slices (200-300 mg) with a Stadie-Riggs tissue slicer. Liver slices were incubated in a Dubnoff metabolic shaker with slow oscillation in 3 ml DMEM containing 20% rat serum, 10 µg/ml bovine insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml of noniodinated Fn. Incubation was conducted at 37°C under a gas mixture of 95% O2 and 5% CO2. Lactate dehydrogenase activity in the medium (data not shown) was analyzed as an index of tissue integrity. After incubation (0-8 h), the liver slices were washed in 4°C saline, blotted dry, and weighed. Liver slices were then oscillated on a rocker with 5 ml DOC extraction buffer for 3 h at 4°C and centrifuged at 35,000 g for 30 min. 125I activity in both the DOC-soluble and the DOC-insoluble (ECM) fractions was measured. Free 125I in the medium was separated from protein-bound 125I by precipitation with 12% TCA.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ability of aFn to incorporate into the ECM of human A1-F fibroblasts. The autograph in Fig. 1 validates the integrity of the radiolabeled purified rat 125I-pFn and 125I-aFn preparations. Nonreduced Fn samples retained their molecular mass of ~440 kDa, whereas samples reduced with beta -mercaptoethanol showed bands at 220 kDa. Samples displayed neither degradation nor multimerization. To verify that the desialylation of pFn with neuraminidase did not impair the ability of aFn to be incorporated into the ECM, the comparative deposition of pFn and aFn into the ECM of the fibroblast cell layers was studied over 24 h. Fibroblasts were used because, unlike hepatocytes, which express the ASGP-R, fibroblasts do not significantly degrade Fn in culture, thus allowing Fn deposition within the DOC-insoluble pool or ECM to be readily detected. Plasma Fn can be incorporated into the ECM of cultured fibroblasts as DOC-insoluble multimers, which are reducible on SDS-PAGE gels (23-25, 34). As shown in Fig. 2, rat 125I-aFn and 125I-pFn showed similar levels of binding to the A1-F fibroblast cell layers as represented by the DOC-soluble fraction. More importantly, analysis of the DOC-insoluble fraction revealed marked ECM incorporation of both pFn and aFn, with the ECM incorporation of 125I-aFn being greater (P < 0.01) than 125I-pFn at 24 h. An autoradiographic analysis of both rat 125I-pFn and rat 125I-aFn in the DOC-insoluble fraction of the cultured human A1-F fibroblasts was performed after its separation on a 6% gradient gel with a 4% stacker. As shown in Fig. 3, in the nonreduced samples, both 125I-pFn and 125I-aFn appeared as high molecular weight multimers (HMWM), which remained in the stacker (Fig. 3, left). These multimers were reducible with beta -mercaptoethanol to ~220 kDa, consistent with the monomeric form of pFn (Fig. 3, right).


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Fig. 1.   Autoradiograph of purified rat 125I-labeled plasma fibronectin (pFn) and purified rat 125I-labeled desialyated plasma fibronectin (aFn). Samples of both nonreduced and reduced rat 125I-pFn (first and third lanes) and rat 125I-aFn (second and fourth lanes) were loaded onto an SDS-PAGE 4-15% gradient gel at 0.5 µg/well. Samples were analyzed by autoradiography to confirm presence of intact dimer at ~440 kDa and monomer at ~220 kDa after reduction using beta -mercaptoethanol.



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Fig. 2.   Incorporation of both 125I-pFn (A) and 125I-aFn (B) into deoxycholate (DOC)-insoluble extracellular matrix (ECM) fraction of cell layer of human A1-F fibroblasts. Three-day-old confluent A1-F human fibroblasts were incubated for varying intervals up to 24 h at 37°C with 1 µg/ml of either 125I-pFn or 125I-aFn in a total volume of 1 ml of DMEM containing 10% Fn-deficient FBS. After specific time intervals, cell layers were extracted in DOC detergent for 3 h and then centrifuged to separate DOC-soluble and DOC-insoluble fractions. Values are means ± SE (n = 12 per group). 125I-aFn was capable of incorporating into DOC-insoluble pool at a greater level than 125I-pFn at 24 h (P < 0.01).



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Fig. 3.   Formation of reducible high molecular weight multimers (HMWM) of Fn in the ECM of human A1-F fibroblasts. Fibroblasts were cultured in 12-well plates and incubated for 24 h at 37°C with 10 µg/ml of either 125I-pFn or 125I-aFn in a total volume of 1 ml of DMEM containing 10% Fn-deficient FBS. At 24 h, cell layers were extracted in DOC detergent for 3 h and then centrifuged to separate the DOC-soluble and DOC-insoluble fractions. Equal counts per minute of the DOC-insoluble rat 125I-pFn- or 125I-aFn-containing fractions were run in duplicate on a 6% SDS-PAGE gel with a 4% stacker. DOC-insoluble fractions of both 125I-pFn and 125I-aFn gels contained similar levels of HMWM, which were reducible to ~220 kDa by beta -mercaptoethanol.

To further validate the ECM assembly of both rat pFn and rat aFn, the human fibroblast cell layers were examined by immunofluorescence using an antibody that reacted specifically with the exogenously added rat Fn but not with human Fn produced endogenously by the cultured fibroblasts. As shown in Fig. 4, a similar fibrillar Fn staining pattern for both rat pFn (Fig. 4A) and rat aFn (Fig. 4B) was observed, further validating their ECM incorporation in agreement with the analysis of the 125I activity in the DOC-insoluble fraction (Fig. 2) and HMWM formation (Fig. 3). Control cell layers treated with only secondary antibody (negative control) showed no extracellular fibrillar staining for Fn (data not shown). Thus aFn is able to incorporate into the ECM.


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Fig. 4.   Fibrillar ECM deposition of both rat pFn and rat aFn added to culture medium of human A1-F fibroblast cell layers. Fibroblasts were cultured in 12-well plates and incubated for 24 h at 37°C with 100 µg/ml of either rat pFn (A) or rat aFn (B) in a total volume of 1 ml of DMEM containing 10% Fn-deficient FBS. Cell layers were washed with PBS and fixed with 3% formaldehyde for 15 min. Cell layers were again washed, permeabilized on ice with 0.5% Triton X-100, washed, and then blocked with BSA. Rabbit anti-rat Fn polyclonal antibody, which does not react with endogenous human Fn, was used (1:100 in PBS) to selectively detect staining pattern for newly incorporated rat Fn. Secondary antibody was rhodamine-conjugated goat anti-rabbit polyclonal antibody (1:50 in PBS). Fibrillar staining of both pFn and aFn is present in these fibroblast monolayers.

Incorporation of 125I-aFn into liver ECM in vivo. It was next determined whether the aFn could also deposit in vivo within the liver DOC-insoluble pool (ECM). The time course for its subsequent loss from the liver was also studied. Figure 5 shows the comparative content of both 125I-pFn and 125I-aFn in the DOC-insoluble fraction of the liver over a 24-h interval after intravenous injection and depicts the liver DOC-insoluble 125I activity as a percentage of the initial level measured at 15 min postinjection (Fig. 5, inset). The DOC-insoluble fraction in the liver at 15 min contained ~70 ng/g tissue 125I-pFn, with >30% lost by 4 h (P < 0.01) and >90% lost by 24 h (P < 0.01) compared with the 15 min level (Fig. 5). In contrast, even though the in vivo liver DOC-insoluble pool of 125I-aFn was 30-35% greater than 125I-pFn at 15 min (P < 0.01), the 125I-aFn was rapidly turned over, with >90% removed by 4 h (Fig. 5). When analyzed by SDS-PAGE and autoradiography, we observed that both 125I-pFn and 125I-aFn were rapidly incorporated in the liver as HMWM complexes, which were readily reducible to 200 kDa (micrographs not shown).


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Fig. 5.   Deposition and turnover of pFn and aFn from the rat hepatic ECM. Rats were injected intravenously with either rat 125I-pFn or rat 125I-aFn at a dose of 3 µg/100 g body wt. At indicated times, rats were anesthetized and rapidly perfused intravenously with 150 ml lactated Ringer solution and their livers were rapidly harvested. Livers were sliced, weighed, and treated with DOC extraction buffer. DOC-soluble and DOC-insoluble (ECM-incorporated) fractions were quantified for 125I content. Fn deposition is expressed as nanograms 125I-Fn per gram wet weight tissue. Values are means ± SE of 3 rats. Tissue analysis was done on triplicate samples harvested from each liver. Inset: residual tissue levels relative to the early 15-min postinjection interval. Liver levels of both 125I-pFn and 125I-aFn at 4 and 24 h were significantly lower than 15-min values (P < 0.05), and the loss of 125I-aFn from liver was faster than that of 125I-pFn (P < 0.01).

Inhibition of the ASGP-R allows accumulation of 125I-aFn in the hepatic DOC-insoluble pool. To determine if the rapid loss of aFn from the liver ECM was influenced by the hepatic ASGP-R, purified asialofetuin was injected intravenously into normal rats at a dose known to saturate and inhibit the ASGP-R. The asialofetuin was given 5 min before either 125I-pFn or 125I-aFn, was injected. Rats were killed at 15 min, 1 h, and 4 h, and collected livers were rapidly processed and analyzed after DOC extraction. Inhibiting the ASGP-R with asialofetuin infusion did not alter either the plasma retention of 125I-pFn or its subsequent rate of loss from the DOC-insoluble fraction in the liver once it had incorporated (data not shown). In contrast, as shown in Fig. 6, the rapid clearance of 125I-aFn from the blood as well as its rapid turnover from the liver matrix was significantly (P < 0.01) prevented by prior intravenous infusion of asialofetuin to block the ASGP-R.


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Fig. 6.   Effect of inhibition of the asialoglycoprotein receptor (ASGP-R) by asialofetuin on turnover of 125I-aFn already deposited in rat liver at 15 min. Rats were injected intravenously with 1 ml asialofetuin in saline (3 mg/ml) or with saline alone 5 min before intravenous injection of rat 125I-aFn at a dose of 3 µg/100 g body wt. A: to maintain the blood level of asialofetuin, rats also received 3 mg asialofetuin at 30 min and at 1, 2, and 3 h. B: rats were perfused with 150 ml of lactated Ringer solution at the times noted, and livers were harvested, sliced, and treated with DOC extraction buffer before analysis of 125I-activity in both the DOC-soluble and insoluble fractions. Fn deposition was calculated as nanograms 125I-Fn per gram wet weight tissue and plotted as percent of 15-min baseline level. Values are means ± SE of 3 rats. Tissue analysis was done on triplicate random samples from livers harvested from each rat. Asialofetuin inhibited loss of 125I-aFn from liver DOC-insoluble fraction.

Rapid turnover of 125I-cFn already incorporated into liver ECM in vivo. Human fibroblast-derived cFn, which naturally contains a large density of terminal galactose residues, rapidly interacts in vivo with the ASGP-R (35). This suggested that cFn already incorporated into the hepatic ECM may also be rapidly lost from the DOC-insoluble pool, similar to aFn. To test this concept, both 125I-pFn and 125I-cFn were injected intravenously into rats, and their comparative early deposition and subsequent loss from the liver was studied over 24 h. Because human cFn was used for this experiment (rat cFn is not available), its loss from the liver was compared with that of human pFn. Similar to the loss seen for 125I-aFn (Fig. 5), the loss of 125I-cFn from liver ECM was also very rapid compared with 125I-pFn (Fig. 7), with about 90% of the 15-min baseline content removed by 4 h (Fig. 7, inset). Thus the turnover of both cFn and aFn from the hepatic ECM is likely mediated by a process involving the ASGP-R.


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Fig. 7.   Deposition and turnover of pFn and cFn from rat hepatic ECM. Rats were injected intravenously with either human 125I-pFn or human 125I-cFn at a dose of 3 µg Fn/100 g body wt. At indicated times, rats were anesthetized and rapidly perfused intravenously with 150 ml lactated Ringer solution and their livers were rapidly harvested. Livers were sliced, weighed, and treated with DOC extraction buffer. DOC-soluble and DOC-insoluble (ECM-incorporated) fractions were quantified for 125I content. Fn deposition is expressed as nanograms 125I-Fn per gram wet weight tissue. Values are means ± SE of 3 rats. Tissue analysis was done on triplicate samples harvested from each liver. Inset: residual tissue levels relative to early 15-min postinjection interval. Liver levels of both 125I-pFn and 125I-cFn at 4 and 24 h were significantly lower than 15-min values (P < 0.05), and loss of 125I-cFn from the liver was faster than 125I-pFn (P < 0.01).

Inhibition of the ASGP-R allows accumulation of 125I-cFn in the hepatic DOC-insoluble pool. If the terminal galactose residues on cFn were the basis for its interaction with the ASGP-R, we predicted that, like aFn, the rapid loss of cFn from the liver ECM would also be delayed by blocking the ASGP-R with intravenous asialofetuin injection. Accordingly, 125I-cFn was injected intravenously into rats, which were again pretreated with either the saline diluent or an inhibitory intravenous dose of asialofetuin. As predicted (Fig. 8), selective blocking of the ASGP-R with asialofetuin delayed the rapid plasma removal of 125I-cFn and attenuated the rapid turnover of cFn already deposited within the liver DOC-insoluble fraction, thus indicating that the turnover of cFn from the liver matrix was also influenced by the hepatocyte ASGP-R system.


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Fig. 8.   Effect of inhibition of the ASGP-R by asialofetuin on turnover of 125I-cFn already deposited in rat liver at 15 min. Rats were injected intravenously with 1 ml asialofetuin in saline (3 mg/ml) or saline as a control 5 min before intravenous injection of human 125I-cFn at a dose of 3 µg/100 g body wt. A: to maintain blood level of asialofetuin, rats also received 3 mg asialofetuin at 30 min and at 1, 2, and 3 h. B: rats were perfused with 150 ml lactated Ringer solution at times noted, and the livers were harvested, sliced, and treated with DOC extraction buffer before analysis of 125I activity in both DOC-soluble and DOC-insoluble fractions. Fn deposition was calculated as nanograms of 125I-Fn per gram wet weight tissue and plotted as percent of the 15-min baseline level. Values are means ± SE of 3 rats. Tissue analysis was done on triplicate random samples from livers harvested from each rat. Asialofetuin inhibited loss of 125I-cFn from liver DOC-insoluble fraction.

Comparative turnover of 125I-aFn, 125I-cFn, and 125I-pFn by preloaded liver slices. In theory, the rapid decrease of 125I-aFn and 125I-cFn activity in the liver ECM could be mediated, in part, by a reduced tracer input from the plasma as their blood concentrations rapidly declined after intravenous injection. This low level of aFn and cFn in the plasma by 15 min after intravenous injection can be seen in Figs. 6 and 8, respectively. To study this possibility, we used an in vitro liver slice technique to control the extracellular concentration of Fn and also allow us to measure the release of 125I-pFn from the liver slices as degraded protein (TCA-soluble 125I). Figure 9 illustrates the in vitro turnover of 125I-aFn and 125I-cFn by liver slices harvested 15 min after in vivo intravenous injection of the 125I tracer protein to preload the liver. As a positive control, 125I-asialofetuin-injected rats were also studied, because 125I-asialofetuin is selectively degraded via the hepatic ASGP-R pathway (2, 3). Our measurements at 0, 3, and 6 h confirmed that 125I-pFn, which initially bound to the liver slices, had continued to incorporate into the DOC-insoluble (ECM) fraction (Fig. 9A) with little release of TCA-soluble 125I into the medium (Fig. 9B). In contrast, 125I-aFn, 125I-cFn, and 125I-asialofetuin levels in the DOC-insoluble (ECM) fraction declined significantly over 6 h (P < 0.05) with the release of TCA-soluble 125I activity into the medium (Fig. 9B).


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Fig. 9.   Changes in 125I activity in both DOC-insoluble fraction of incubated liver slices (A) and the TCA-soluble fraction in incubation medium (B) using liver slices preloaded in vivo. Rats were injected intravenously with either 125I-pFn, 125I-aFn, 125I-cFn, or 125I-asialofetuin at a dose of 12 µg/100 g body wt. At 15 min, rats were perfused intravenously with 150 ml cold Ringer lactate, and liver slices prepared from rapidly harvested livers were placed in DMEM containing 20% rat serum, 10 µg/ml bovine insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Slices were incubated at 37°C for 0, 3, and 6 h, washed with cold PBS, extracted in DOC buffer, and assayed for 125I in DOC-insoluble fraction. Values are means ± SE (n = 12 per group). Values for DOC-soluble 125I in the liver and TCA-precipitable 125I in medium are not shown, but sum of all 4 pools equaled 100% of total 125I activity.

However, when saturating doses of "cold" nonlabeled asialofetuin were again infused in vivo to inhibit the hepatic ASGP-R, before the intravenous injection of the tracer 125I-aFn, the 125I-aFn continued to incorporate into the ECM of harvested liver slices over the 6 h in vitro incubation (Fig. 10A) similar to 125I-pFn (Fig. 9). In addition, inhibition of the hepatic ASGP-R by asialofetuin treatment markedly attenuated the release of TCA-soluble 125I into the medium (P < 0.01; Fig. 10B).


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Fig. 10.   Ability of asialofetuin to inhibit loss of DOC-insoluble 125I-aFn from liver slices preloaded in vivo. Rats were injected intravenously with 1 ml bovine asialofetuin in saline (3 mg/ml) or with saline alone, followed 5 min later with an intravenous injection of 12 µg/100 g body wt 125I-aFn. A higher tracer dose was used to facilitate detection of turnover by liver slices. Rats were perfused at 15 min with 150 ml Ringer lactate, and slices prepared from the harvested livers were placed in DMEM containing 20% rat serum, 10 µg/ml bovine insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Slices were incubated at 37°C for 0, 3, or 6 h and then washed with cold PBS, blotted dry, weighed, and assayed for 125I. Values are means ± SE (n = 12 per group). Inhibition of ASGP-R activity by intravenous infusion of asialofetuin significantly blocked the rapid loss or turnover of aFn from the DOC-insoluble fraction (P < 0.01; A) and lowered the TCA-soluble 125I detected in the medium (B).

Effect of chloroquine on the turnover of 125I-pFn by preloaded liver slices. Although no loss of pFn from the DOC-insoluble pool could be measured using the in vitro slice assay represented in Fig. 9, a modified liver slice assay was utilized that could detect pFn loss from this DOC-insoluble fraction. To perform this experiment, liver slices were harvested 4 h after intravenous tracer injection (not 15 min) and the incubation interval was lengthened from 4 to 8 h, because the liver turnover of pFn is much slower than either aFn or cFn. With this extended incubation interval, we observed that ~30% of the original liver slice content of 125I-pFn was released into the TCA-soluble fraction of the medium by 8 h (0 µM chloroquine concentration; Fig. 11). To determine if this release of 125I might reflect lysosomal degradation of the 125I-pFn, chloroquine was added to the incubation medium at a concentration documented to prevent proteolysis of endocytosed proteins in rat liver liposomes (54). As shown in Fig. 11, the addition of chloroquine reduced the TCA-soluble 125I detected in the medium in a dose-dependent manner (P < 0.05). In parallel, the protein-bound 125I activity in the DOC-insoluble ECM fraction of the liver slices increased (P < 0.05) in the presence of 500 µM chloroquine (Fig. 11, inset).


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Fig. 11.   Effect of chloroquine on hepatic degradation of pFn by liver slices. Rats were injected intravenously with 12 µg/100 g body wt 125I-pFn. A higher tracer dose was used to facilitate detection of turnover by liver slices. At 4 h, rats were perfused with 150 ml Ringer lactate and slices prepared from harvested livers were placed in DMEM containing 20% rat serum, 10 µg/ml bovine insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, and increasing concentrations of chloroquine. Slices were incubated at 37°C for 8 h, washed with cold PBS, blotted dry, weighed, and assayed for 125I. Extended in vivo (4 h) and in vitro (8 h) intervals were used to allow adequate turnover of pFn to begin to take place. DOC-soluble and TCA-precipitable medium pools were not significantly altered by chloroquine (data not shown). Values are means ± SE (n = 6 per group).

Alternative endocytic pathway for the removal of the pFn from the hepatic ECM. To determine if the ASGP-R system was also involved in the turnover of 125I-pFn from the DOC-insoluble pool within these preloaded liver slices, asialofetuin was again added to the medium to inhibit the ASGP-R in the liver slices. After the end of the 8-h incubation, control liver slices had about 45% of the total 125I activity in the DOC-insoluble fraction (ECM), with ~25% released as TCA-soluble 125I in the medium (Fig. 12). Addition of asialofetuin did not inhibit the release of TCA-soluble 125I, nor did it cause a greater incorporation of 125I-pFn into the ECM of the liver slice over the 8 h (Fig. 12). In contrast, chloroquine increased the amount of 125I-pFn in the ECM as measured by the DOC-insoluble fraction (P < 0.05) and also inhibited the release of TCA-soluble 125I into the medium (P < 0.05). Thus pFn already incorporated within the liver ECM may be removed by a lysosomal degradative pathway independent of the ASGP-R system, unless it can become locally desialylated within the liver ECM.


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Fig. 12.   Comparative effect of chloroquine and asialofetuin on both retention and degradation of 125I-pFn from DOC-insoluble fraction in liver. Rats were injected intravenously with 12 µg/100 g body wt 125I-pFn. At 4 h, rats were perfused with 150 ml Ringer lactate, and slices prepared from rapidly harvested livers were placed in DMEM containing 20% rat serum, 10 µg/ml bovine insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Medium was supplemented with either 500 µM chloroquine or 10 µM asialofetuin. Slices were incubated at 37°C for 8 h, washed with cold PBS, blotted dry, weighed, and assayed for 125I. Accumulation of 125I-pFn in DOC-insoluble fraction of the liver slice (A) over 8-h incubation interval was increased with chloroquine (P < 0.05) but not altered by asialofetuin (P > 0.05). In parallel, degradation of 125I-pFn with release of 125I into the medium (B) was reduced by chloroquine (P < 0.05) but not altered by asialofetuin. DOC-soluble and TCA-precipitable counts were not significantly altered by chloroquine (P > 0.05; data not shown). Values are means ± SE (n = 6 per group).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The liver matrix is a major site for the normal deposition of soluble Fn from the plasma in both animals and humans (27, 34, 48), perhaps due in part to its large size, high blood flow, and fenestrated vascular barrier, which would allow easy access of plasma Fn to matrix assembly receptors and the liver interstitial matrix in the space of Disse (36). The liver may also provide an important pathway for the turnover of Fn from both the liver ECM and the blood. Indeed, the two-compartment model for pFn kinetics developed by Deno et al. (10) documents that loss or degradation of Fn directly from the tissue compartment must exist, especially within the liver, to account for its known plasma concentration, rate of synthesis, rate of ECM deposition, and rate of in vivo turnover from the blood. The hepatocyte-expressed ASGP-R system is believed to mediate the removal of blood-borne desialylated plasma proteins on the basis of data using proteins that were experimentally desialylated before intravenous injection (2, 3). The ASGP-R system consists of heterooligomeric endocytic receptors expressed in high density, especially on the sinusoidal surface of hepatocytes (40) adjacent to the extracellular space of Disse, which is rich in Fn (16, 22, 34). Our current study indicates that the ASGP-R system likely modulates both the deposition and retention of aFn and cFn in the hepatic ECM. These findings also suggest that pFn already incorporated into the hepatic ECM would first have to be desialylated within the liver before it could utilize the ASGP-R system for its pathway of turnover.

We observed that all three forms of Fn (125I-pFn, 125I-aFn, and 125I-cFn) began to deposit in the liver DOC-insoluble pool within 15 min after intravenous injection. Although DOC-insoluble levels of both 125I-aFn and 125I-cFn declined markedly within 4 h, much of the pFn was retained in the liver ECM for at least 24 h. Rat liver slices, preloaded in vivo with 125I-pFn, 125I-aFn, or 125I-cFn, showed a similar retention of pFn in the DOC-insoluble pool for an extended interval, whereas both 125I-aFn and 125I-cFn rapidly disappeared from the DOC-insoluble pool in the matrix. This loss was likely related to lysosomal degradation in view of the rapid appearance of 125I activity in the TCA-soluble fraction of the medium. This conclusion is consistent with recent in vivo kinetic studies from our lab (35) that document the appearance of both free 125I and iodinated Fn fragments in the bile during the turnover of 125I-Fn deposited in the liver.

In the current study, selective inhibition of the ASGP-R by infusion of excess nonlabeled asialofetuin caused retention of both 125I-aFn and 125I-cFn in the liver ECM at a level similar to that, normally seen with 125I-pFn. Thus it appears that, when the ASGP-R is functional, aFn and cFn, both of which have a high density of exposed galactose residues, are rapidly lost from the liver ECM; however, when the ASGP-R is inhibited or possibly downregulated, then both aFn and cFn will be retained in the ECM for extended intervals. We initially concluded that the rapid in vivo loss of 125I-aFn from the liver ECM may have been due to limited continuous gradient-dependent input into the liver matrix, perhaps as a result of declining tracer levels of the 125I-aFn in the blood. However, this explanation was soon discounted because liver slices preloaded in vivo with 125I-aFn and then incubated in medium containing a relatively constant concentration of 125I-aFn also displayed a very rapid turnover of 125I-aFn from the matrix.

Although the removal of pFn from the hepatic ECM also appears to occur via an endocytic pathway(s) as defined by inhibition with chloroquine, our data suggested that this removal mechanism may not be directly linked to the ASGP-R system. For example, liver slices preloaded with 125I-pFn in vivo were shown to release free 125I from their ECM over an 8-h incubation period, but this degradation or release was not inhibited by asialofetuin, which selectively blocks the ASGP-R. Thus clearance of normal nondesialylated pFn from the hepatic ECM may be accomplished by an endocytic degradative pathway independent of the ASGP-R, unless the pFn is desialylated within the liver. However, we cannot rule out the possibility that our asialofetuin inhibitor did not have access to a compartment involved in the pFn turnover. Two other receptors in the liver besides the ASGP-R are known to interact with pFn and may be involved in its turnover. These are the integrin alpha 5beta 1-Fn receptor as well as the nonintegrin AGp110 receptor, both of which are found on hepatocytes (41, 42). However, a role for these receptors in the removal of Fn from the liver ECM can only be speculated.

With respect to the participation of ASGP-R in the turnover of pFn already incorporated into the hepatic ECM, it would appear that such ECM-bound pFn must first be desialylated to expose its galactose residues (35). Removal of sialic acid from Fn could potentially be accomplished by liver sialidase activity (15, 31, 46, 47, 49). For example, hepatic endothelial cells are believed to bind transferrin or ceruloplasmin via specific receptors on their luminal face and then transcytose such proteins internally, where they are desialylated and released into the space of Disse before being directly cleared by ASGP-R (15, 46, 47). Desialylation of proteins may also take place in Kupffer cells. For example, carcinoembryonic antigen is endocytosed by Kupffer cells and then desialylated internally before being released into the interstitial space of Disse, where it can be removed by hepatocytes using the ASGP-R (3, 49). The liver also contains other extracellular sialidases as reviewed by Sweeley (44), which further supports the concept that matrix-localized Fn could be locally desialylated within the liver.

Turner (50) observed that a common characteristic of the carbohydrate moieties of many proteins in the plasma of patients with hepatic fibrosis is their high degree of desialylation compared with plasma proteins from healthy individuals. If such proteins could competitively inhibit the hepatocyte ASGP-R clearance process, similar to the effect we observed in rats with asialofetuin, then elevations in the plasma level of asialoglycoproteins could delay the normal turnover of matrix-localized Fn, including locally synthesized cFn, resulting in their accumulation in the matrix. In support of this concept are the findings that multiple injections of D-galactosamine into rats will cause hepatic inflammation and the accumulation of both Fn and collagen in the liver ECM similar to that observed with fibrosis in humans with alcoholic liver disease (18). Indeed, even a single dose of D-galactosamine infused intravenously can significantly downregulate ASGP-R activity (39) and increase the liver ECM content of Fn as detected by immunofluorescence (17). In the liver, galactosamine can cause a depletion in uridine nucleotides, which results in a decrease in RNA synthesis and a decrease in the biosynthesis of glycoproteins and lipoproteins, eventually contributing to hepatic necrosis (8, 9, 10a, 11, 19, 30, 32). Theoretically, a direct competitive inhibition of the ASGP-R system by D-galactosamine and/or the improper expression of such asialoglycoprotein receptors caused by such uridine nucleotide depletion could reduce the cell surface function of the ASGP-R system. Galactosamine infusion has been shown to inhibit the binding of iodinated asialoorosomucoid to the ASGP-R in the liver (29), whereas other sugars manifested little or no inhibitory activity. Thus galactosamine in the blood has the potential to interact with the ASGP-R and inhibit the removal of circulating Fn.

The normal liver sinusoid is a discontinuous endothelial barrier that allows rapid flux of blood-borne macromolecules to the interstitial space of Disse. As shown by Martinez-Hernandez (21), Fn in the liver is localized to the interstitial space of Disse and the walls of central and portal blood vessels, as well as the collagen-rich liver capsule. Cultured hepatocytes can also produce a well-defined ECM that consists of laminin, Fn, and collagen (20). Hepatocytes can incorporate plasma-derived or locally synthesized Fn into their ECM (12, 14, 20, 43) via a matrix assembly site-dependent process (34).

Matrix-associated plasma-derived Fn in the rat liver ECM has been shown to have a half-life of <24 h (34, 35). In addition, the synthetic rate of pFn in adult rats is about 0.50-0.54 mg/h, which correlates well with the fact that, in adult rats, at least 50% of the plasma pool is also replaced every 24 h (10, 51). Such results suggest that the assembly, and subsequent turnover, of Fn in the liver ECM is a dynamic process, tightly coordinated with the synthesis of pFn, which also appears to take place mainly in the liver (10). From this perspective, small changes in either Fn assembly or removal from the liver ECM would have profound effects on the Fn content in the liver ECM.

In summary, the current study suggests that the ASGP-R system is capable of influencing the retention of Fn in the ECM of the liver, specifically those forms of Fn which have a large density of exposed or terminal galactose residues, such as cFn and aFn. Disturbances of the ASGP-R system in the liver during disease states, such as alcoholic liver disease, may influence the content of Fn in the hepatic ECM and perhaps the accumulation of other matrix proteins. Patients with alcoholic liver disease often have a high content of Fn in the liver ECM, and they also manifest a disturbance in their ASGP-R system.


    ACKNOWLEDGEMENTS

We thank Eshin Cho and Edward Lewis for technical assistance and Debbie Moran and Wendy Ward for secretarial assistance.


    FOOTNOTES

This study was primarily supported by National Institute of General Medical Sciences Grant GM-21447 (T. M. Saba) and in part by National Cancer Institute Grant CA-69612 (P. J. McKeown-Longo). R. F. Rotundo was a postdoctoral research fellow supported by National Institutes of Health Postdoctoral Training Grants T32-GM-07033 and T32-HL-07529.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. M. Saba, Dept. of Physiology & Cell Biology (MC-134), Albany Medical College, 47 New Scotland Ave., Albany, New York 12208 (E-mail: thomas_saba{at}ccgateway.amc.edu).

Received 19 March 1999; accepted in final form 11 August 1999.


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Am J Physiol Gastroint Liver Physiol 277(6):G1189-G1199
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