Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, Arizona, 85724-5044
1 To whom correspondence should be addressed at Department of Cell Biology and Anatomy, College of Medicine, P.O. Box 245044, University of Arizona, Tucson, AZ, 85724-5044. Fax: (520) 626-2097. E-mail: mccuskey{at}email.arizona.edu
Received August 11, 2004; accepted September 24, 2004
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ABSTRACT |
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Key Words: microcirculation; sinusoidal endothelial cells; hemorrhage; hepatotoxicity.
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INTRODUCTION |
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In addition to direct hepatocellular damage through metabolic activation of APAP, hepatic microcirculatory dysfunction has been shown to participate in liver injury elicited by APAP overdose. The APAP-induced hepatic necrosis is preceded by centrilobular microvascular congestion and the infiltration of blood elements into the space of Disse through the large gaps formed in the endothelium (McCuksey et al., 2001; Walker et al., 1983
). APAP is directly toxic to sinusoidal endothelial cells (SECs) isolated from mice through profound reduction of their levels of GSH (Deleve et al., 1997
). However, others (Gerard-Moninier et al., 1992; Redegeld et al., 1992
) have suggested that GSH depletion by itself appears not to be responsible for toxicity elicited by diethylmaleate. In vivo microscopic studies (Ito et al., 2003
) suggest that rounding-up or swelling of SECs is the initiating morphological alteration within 0.5 h after APAP, and that infiltration of red blood cells (RBCs) in the space of Disse is evident as early as 2 h after APAP gavage, when the levels of transaminase remain unchanged. Parallel to the rising hepatic transaminase levels, the extrasinusoidal area occupied by RBCs progressively is increased to maximal levels 6 h after APAP treatment. The findings that hepatic microvascular injury precedes hepatic necrosis suggest that hepatic microvascular injury is likely to be an early result of APAP toxicity.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that participate in degradation of extracellular matrix (Visse and Nagase, 2003). Increased activity of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) is involved in SECs injury as evidenced by large gap formation or their dehiscence in monocrataline-induced veno-occlusive disease in rats (Deleve et al., 2003b
; Hanumegowda et al., 2003
) as well as in cold preservation injury of the liver in humans and in rats (Upadhya et al., 1997
). The similar pattern of SEC injury to monocrotaline and cold preservation suggests that MMPs are responsible for hepatic microvascular injury in response to toxic doses of APAP, and that inhibition of MMPs might attenuate APAP-induced liver injury. As a result, the present study was conducted to examine the effect of an MMP inhibitor on hepatic microcirculation after APAP administration using in vivo microscopic methods (McCuskey, 1986
).
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MATERIALS AND METHODS |
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In vivo microscopy. Animals were anesthetized with urethane (2 mg/g b.w., subcutaneously). The hepatic microvascular responses were examined using established high resolution in vivo microscopic methods (McCuskey, 1986). Briefly, a compound binocular microscope (Leitz, Wetzlar, Germany) adapted for in vivo microscopy was equipped to provide either transillumination or epi-illumination, as well as video microscopy using a charge-coupled device (CCD) camera (MTI, Michigan City, IN). The liver was exteriorized through a left subcostal incision and positioned over a window of optical grade mica in a specially designed tray mounted on a microscopic stage. The tray provided for the drainage of irrigating fluids, and the window overlaid a long working distance condenser. The liver was covered by a piece of Saran Wrap (Dow Chemical, Midland, MI), which held it in position and limited movement. Homeostasis was insured by a constant suffusion of the organ with Ringer's solution maintained at body temperature. With the x80/1.0 numerical aperture water immersion objective (Leitz 80x/1.00, Wetzlar, Germany) employed for these studies, the methods permitted visualization of the cells comprising the sinusoidal lining, the formed elements of the blood, the nuclei, nucleoli, mitochondria, and fat droplets in hepatic cells, and bile canaliculi. Under optimal conditions, the resolution was 0.30.5 µm. Microvascular events were observed and recorded for at least 30 s for subsequent off-line analysis using a Sony Betacam video tape recorder (Sony Medical Electronics, Park Ridge, NJ).
Kupffer cell function was assessed by measuring the phagocytosis of fluorescent 1.0-µm latex particles (Fluoresbrite-fluorescent monodispersed polystyrene microspheres; Polysciences, Warrington, PA) by individual cells. The latex was diluted 1:10 with sterile saline and injected in 0.1-ml volume through a mesenteric vein using a 30-gauge lymphangiography needle (Becton Dickinson and Co., Franklin Lakes, NJ). The distribution and relative number of phagocytic Kupffer cells was measured by counting the number of cells that phagocytosed latex particles in a standardized microscopic field 15 min after each mice had received the injection of latex. To assess regional distribution, the number of phagocytic Kupffer cells per microscopic field was counted in ten periportal and ten centrilobular regions in each animal. The relative adequacy of blood perfusion through the sinusoids was evaluated by counting the number of perfused sinusoids in the same ten microscopic fields in which the numbers of phagocytic Kupffer cells were determined. Because reduced perfusion of individual sinusoids can limit the delivery of the latex particles to Kupffer cells in these vessels, the ratio of Kupffer cells that phagocytosed latex particles to perfused sinusoids was used as an overall index of Kupffer cell phagocytic activity.
To examine the interaction of leukocytes with the sinusoidal wall, quantification of leukocytes adhering to the endothelial lining of sinusoids was calculated by counting the number of leukocytes per unit length of sinusoid (adherent leukocytes/100 µm) in the same microscopic fields. A leukocyte was defined as adhering to the sinusoidal wall if it remained stationary for at least 30 s. The results were averaged, and the data were represented as the average number in each animal.
To quantify the extent of hemorrhage elicited by APAP gavage, the area occupied by extrasinusoidal red blood cell (RBC)s was measured in the same microscopic fields using a computer-assisted digital imaging processor (Scion Image, Scion Corp., Frederick, MD). The results were expressed as extrasinusoidal area occupied by RBCs (µm2/10 centrilobular regions).
Sampling and ALT assay. In a separate set of experimental animals, blood was collected from the inferior vena cava, and the samples were separated by centrifugation at 13,000 x g for 5 min at 4°C. The samples were stored at 70°C until assays were performed. The serum activities of alanine aminotransferase (ALT) were measured by enzymatic procedures using a diagnostic kit (Sigma Chemical Co., St. Louis, MO).
Histology. The hepatic tissues were immediately fixed with 10% formalin. After fixation, the tissues were dehydrated with a graded series of ethanol solutions and embedded in paraffin. The sections (5 µm) from the paraffin-embedded tissues were stained with hematoxylin and eosin (H&E).
MMPs activity assay. The liver was perfused with phosphate buffered saline (PBS) and was removed from the animal. Then the left lobe of the liver was sectioned into 0.5 cm blocks, snap frozen in liquid nitrogen, and stored at 70°C until assays were performed. The levels of MMP-2 and MMP-9 were measured by a commercially available ELISA kit (Amersham Biosciences, Piscataway, NJ). Briefly, 25 mg of the liver tissue was homogenized in 50 mM Tris-HCL containing 1 mM monothioglycerol. The homogenized samples were centrifuged at 13,000 x g for 10 min. Following the manufacture's instructions, 100 µl of either standard or experimental sample(s) was added to the designated wells and incubated overnight at 4°C. After incubation, the plate was washed four times and blotted dry. Aminophenylmericuric acetate (AMPA) was added to all wells, and the plate was incubated at 37°C. Then the plate was read at a wavelength of 405 nm using an ELISA plate reader. Total MMP-2 and MMP-9 activity was calculated using the standard curve for this assay. The results were expressed as ng/g liver tissue.
Semiquantitative analysis of MMP-2 and -9 mRNA. The liver was perfused with PBS and was removed from the animal. Part of the left lobe of the liver was cut into 0.1-cm blocks and placed into a cryovial containing 1.5 ml of RNALater (Qiagen Valencia, CA) and equilibrated at 4°C overnight then stored at 70°C until assays were performed. Total RNA was extracted from the tissue using a commercially available kit (Promega, Madison, WI). Total RNA was quantitated using a spectrophotometer 260/280 program and stored at 70°C. Semiquantitative RT-PCR was performed using a PCR kit (USB, Cleveland, Ohio). To standardize the reactions, the primers for murine ß-actin (Chemicon, Temecula, CA), MMP-2, and MMP-9 (BioMol, Plymouth Meeting, PA) were added to each reaction tube and all three cDNAs were amplified. From preliminary experiments, we determined 28 cycles to be optimal. Briefly, 10 µl of PCR product and standards were added to designated wells. Denaturing solution was added to all wells and incubated for 10 min. After incubation, hybridization buffer was added to each well and incubated for 30 min at 37°C. The plate was then washed three times and blotted dry. The Strep-HRP conjugate was added to all wells, and the plate was incubated for 30 min. The Strep-HRP conjugate was aspirated off, and the plate was washed three times. Finally 100 µl of substrate was added, and the plate was incubated for 10 min at 37°C. After incubation 100 µl of stop solution was added to each well. The plate was immediately read on an ELISA plate reader at 450 nm. MMP-2 and -9 levels were calculated from the standard curve generated in this assay.
Measurement of hepatic total glutathione. Hepatic total glutathione (GSH) was determined colorimetrically using a commercially available kit (Oxford Biomedical Research, Oxford, MI) as described previously (Ito et al., 2004).
Statistical analysis. All data were expressed as means ± SE. Multiple comparisons were performed using one-way analysis of variance (ANOVA) with a post-hoc Fisher's test. Differences were considered to be significant for p values less than 0.05.
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RESULTS |
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To determine whether inhibition of MMPs attenuates liver injury elicited by APAP, 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (0.5 and 5 mg/kg) or vehicle (1% DMSO in PBS) was given. Figure 2A shows the effect of 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid on ALT levels 6 h after APAP administration. Oral gavage with APAP caused a significant increase in ALT levels (41.1-fold) when compared with water-gavaged mice. Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid decreased ALT levels by 71.3% in a dose-dependent manner. Oral gavage with APAP exhibited centrilobular necrosis associated with hemorrhage and congestion (Fig. 2B). 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid reduced hemorrhagic centrilobular necrosis; however, it failed to eliminate the necrosis completely (Fig. 2C). Based on these results, we selected a dose of 5 mg/kg 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid for remaining studies.
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DISCUSSION |
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Oral gavage with APAP induced hepatic mRNA expression of MMP-2 and MMP-9 as early as 1 h after APAP. At the same time, MMP-2 in the liver was activated 2 h after APAP, and MMP-9 activation was shown at 6 h after APAP. The reasons for the disconcordance between mRNA levels and enzyme activity for MMP-9 within 6 h after APAP gavage remain unclear; however, the induced levels of MMP-9 mRNA from 1 to 4 h after APAP gavage were not sufficient for MMP-9 activation. The levels of mRNA expression of MMP-2 and MMP-9 following APAP gavage were the same, though the activity of MMP-2 was significantly higher than that of MMP-9. These results indicate that increased hepatic expression and activity of MMP-2 and, to a lesser degree, of MMP-9 correlated with liver injury elicited by APAP. Recently, Gardner et al. (2002) have reported that MMP-9 mRNA expression is increased 18 h after an intraperitoneal administration of 300 mg/kg APAP to mice, but not at 6 h after. The different results between others and us may be due to different doses and routes of APAP administration. Late induction of MMP-9 may be related to regression of hepatic necrotic tissue following acute injury.
It is known that Kupffer cells and hepatic stellate cells have the capacity to secrete MMPs (Arthur et al., 1989; Knittel et al., 1999
; Theret et al., 1999
; Winwood et al., 1995
). Recently, isolated SECs from rat liver are suggested to be another source of MMPs (Deleve et al., 2003; Upadhya and Strasberg, 1999
). In addition, increased activity of MMPs including MMP-2 and MMP-9 is demonstrated in isolated SECs from the rat liver exposed to cold preservation (Upadhya et al., 1997
) and to monocrotaline (Deleve et al., 2003b
). However, major sources of MMPs in APAP hepatotoxicity remained to be elucidated.
Induction of MMP-2 and MMP-9 during APAP hepatotoxicity indicates that MMPs play a role in the injury. As a result, mice were treated with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor, prior to APAP gavage. The pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid reduced ALT levels, an indicator of liver injury, by inhibiting activities of MMP-2 and MMP-9. However, its inhibitory effect on ALT levels was partial, as evidenced by sustained relatively high values of ALT, which was consistent with histological findings of remaining necrosis in centrilobular hepatocytes. Early induction of MMP-2 and MMP-9 also was associated with occurrence of the infiltration of RBCs in Disse space at 2 h after APAP gavage, when the levels of transaminase remain unchanged (Ito et al., 2003). 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid eliminated the infiltration of RBCs during the early phase of APAP toxicity. In addition, the administration of 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated APAP-induced hepatic microcirculatory dysfunction, including impaired sinusoidal perfusion and infiltration of RBCs in Disse space at 6 h after APAP treatment. Histological studies also revealed that MMP inhibitor eliminated hemorrhage. These findings suggest that inhibition of MMPs attenuated SEC injury to APAP, as indicated by elimination of infiltrated RBCs in Disse space, and restored sinusoidal blood flow. Alternatively, induction of MMPs may be involved in gap formation in SECs of the liver treated with toxic doses of APAP, leading to penetration of RBCs in the space of Disse and to dehiscence of SEC from the space of Disse. Although the inhibitory effects of 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid on enzyme activities for MMP-2 and MMP-9 are partial (approximately 30% decrease), prevention of accumulation of RBCs from the early stage of APAP toxicity would minimize ischemic parenchymal cell injury following a toxic does of APAP gavage and thereby suppress raised ALT values after APAP gavage.
It has been suggested that cold preservation of SECs and treatment of SECs with monocrotaline cause disassembly of filamentous (F)-actin, which in turn mediates the secretion of MMPs from SECs (Deleve et al., 2003b; Upadhya and Strasberg, 1999
). The initial morphological changes in SECs during cold preservation injury and monocrotaline-induced sinusoidal obstruction syndrome (hepatic veno-occlusive disease) include rounding up and an appearance of gap formation in SECs (Deleve et al., 1999
, 2003a
; Momii and Koga, 1990
). The actin cytoskeleton play a role in maintaining cell shape, and depolymerization of F-actin results in the structural changes in cellular cytoskeleton characterized by rounding up of SECs and gap formation in SECs. Release of MMPs as a result of F-actin disassembly also contribute to digestion of component of tissue matrix such as fibronectin and type IV collagen in the space of Disse, leading to the Disse space enlargement (Walker et al., 1983
), and to detachment of SECs from the Disse space. However, the mechanisms by which APAP injures SECs are unknown.
The bioactivation of APAP, resulting in the formation of the reactive metabolite, presumably NAPQI, depletes intracellular sources of GSH (Mitchell et al., 1973b). To determine whether the protective effect of MMP-2/MMP-9 inhibitor was through inhibition of metabolic activation of APAP, hepatic GSH was measured in APAP-gavaged mice receiving the inhibitor. The results showed that MMP inhibitor did not suppressed the initial dropped hepatic total GSH levels following APAP gavage, suggesting that inhibitory effect of 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid on liver injury is not mediated through a decrease in bioactivation of APAP.
In summary, increased MMPs during APAP intoxication are associated with hepatocellular damage and with hepatic microcirculatory dysfunction including impaired sinusoidal perfusion and infiltration of RBCs in the Disse space. Inhibition of MMPs attenuates APAP-induced liver injury and may provide a useful therapy for the prevention of APAP hepatotoxicity.
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ACKNOWLEDGMENTS |
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