* Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824;
Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Institute of Medical Biochemistry and Molecular Biology, Karl-Franzens University, Graz, Austria
Received June 10, 2003; accepted August 12, 2003
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ABSTRACT |
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Key Words: sinusoidal endothelial cells; NP-26 cells; collagen IV; PMNs; myeloperoxidasehydrogen peroxidehalide system.
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INTRODUCTION |
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Endothelial cell injury can be the result of a variety of factors, among which is the loss of attachment following the degradation of components of basement membrane, such as collagen IV, by matrix metalloproteinases (MMPs), specifically MMP-9 and MMP-2 (for review see Visse and Nagase, 2003). Loss of basement membrane occurs in pathological conditions of lungs (Torii et al., 1997
; Van de Louw et al., 2002
), kidneys (Davies et al., 1992
; Zaoui et al., 2000
), and liver (Benyon and Arthur, 2001
; Oyaizu et al., 1997
; Ueno et al., 1996
; Upadhya and Strasberg, 1999
).
MMPs are a family of Zn-dependent endopeptidases involved in degradation of extracellular matrix (Brinckerhoff and Matrisian, 2002). They play a critical role in a variety of physiological and pathological conditions such as organogenesis, tumor metastasis, wound repair, angiogenesis, and arthritis (Kleiner and Stetler-Stevenson, 1993
; Massova et al., 1998
; Mignatti and Rifkin, 1996
). MMP-2 (gelatinase A) and MMP-9 (gelatinase B) degrade basement membrane collagen IV and, hence, are known as type IV collagenases. Collagenases are synthesized, stored, and secreted by a variety of cell types such as inflammatory cells, endothelial cells, and tumor cells (Borregaard and Cowland, 1997
). Stored MMPs are activated intracellularly or at the plasma membrane and then secreted (Miyamori et al., 2001
; Pei and Weiss, 1995
). Stored MMPs are also secreted as inactive proenzymes that are later activated by proteolytic cleavage (Itoh and Nagase, 1995
).
In the liver, subendothelial basement membrane is discontinuous and comprises collagen IV, heparan sulfate proteoglycan (perlecan), fibronectin, and laminin (Griffiths et al., 1991; Takahashi et al., 1994
; Tovari et al., 1997
). In comparison with subendothelial matrix of other microvascular structures, the amount of basement membrane matrix in the liver is minimal. We hypothesized that collagenase activation and disruption of basement membrane occur after MCT exposure and are associated with the loss of SECs. Accordingly, the distribution of basement membrane collagen IV and collagenase activity in the livers of MCT-treated rats were examined. In addition, the contribution of polymorphonuclear leukocyte (PMN) collagenase to SEC loss and the possibility that SECs themselves release MMPs in response to MCT was evaluated.
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MATERIALS AND METHODS |
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Animals, treatment, and tissue collection.
Male Sprague-Dawley rats (Crl:CD [SD]IGS BR, Charles River, Portage, MI) weighing 100150 g were used in the study. Animals were allowed food (Rodent Chow/Tek 8640, Harlan Teklad, Madison, WI) and water ad libitum and maintained in the experimental animal housing facility under standard conditions. All procedures on animals followed the guidelines for humane treatment set by the American Association of Laboratory Animal Sciences and the Michigan State University Laboratory Animal Research Unit. MCT was dissolved in sterile saline by minimal acidification and then the pH was adjusted to 7. Rats fasted for 24 h were given MCT at a dose of 300 mg/kg body weight or an equal volume of its saline vehicle by intaperitoneal (ip) injection. Food was returned to the rats after MCT administration. At this dose, hepatic parenchymal cell injury begins by 12 h after MCT administration (Copple et al., 2002a). At 8, 12, or 18 h, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip). Blood was collected in sodium citrate (0.38% final concentration) from the abdominal aorta for measurement of plasma alanine aminotransferase activity and HA concentration. For immunofluorescence analyses, a portion of the liver (1 cm3) was cut from the middle of the left lateral lobe and frozen in isopentane immersed in liquid nitrogen. In addition, portions of several lobes were snap-frozen in liquid nitrogen for MMP and MPO activity assays.
For PMN-depletion studies, rats received 0.75 ml (ip) of rabbit antirat PMN antiserum or normal rabbit serum (control serum) followed by a second injection of PMN antiserum (0.5 ml, ip) or control serum (0.5 ml, ip) 18 h later. Rats then received MCT or saline vehicle 6 h after the second serum treatment and were killed 18 h later. This treatment with PMN antiserum depletes circulating PMN numbers below 1% of normal by 12 h, and depletion persists for several days (Snipes et al., 1995).
Cell culture and treatment.
Primary SECs were isolated from rat liver as per the procedure of Braet et al. (1994). Briefly, the livers from anesthetized rats were digested by in situ sequential perfusion with liver perfusion and liver digestion media. Cells were released by gentle scraping and suspended in hepatocyte wash medium. The cell suspension was centrifuged (100 x g) for 5 min, and the supernatant containing the nonparenchymal cell fraction was collected. This fraction was layered on top of a two-layer Percoll gradient (50% and 25%) and centrifuged (900 x g) for 20 min. The SEC-enriched zone between the Percoll layers was collected, washed with PBS, and plated. Additionally, the rat liver sinusoidal endothelial cell line NP-26 was used. The NP-26 cell line was established from an enriched fraction of liver SECs transfected with SV40 large-T antigen (Maru et al., 1998
). Primary SECs and NP-26 cells were plated at a density of 0.25 x 106/ ml in 12-well plates and cultured in EGM-2. When cells reached ~70% confluency, MCT was added to the serum-free culture medium to a final concentration ranging from 0 to 4 mM. Medium was collected after 6 h of treatment and used for gelatin zymography or LDH assays. Cells were lysed with 1% Triton X-100 and sonication and then centrifuged. LDH activity in the supernatant was determined.
Immunofluorescence and morphometry.
For immunostaining, either frozen or formaldehyde-fixed sections were used; 8 µm thick frozen sections of liver were fixed in ice-cold acetone for 5 min and rehydrated in PBS for 10 min. Formaldehyde-fixed, paraffin-embedded sections were deparaffinized and rehydrated. Sections were incubated in blocking solution containing 10% goat serum in PBS and then with one of the primary antibodies, anticollagen IV (1:50), antiheparan sulfate proteoglycan (1:50), or clone 2D10G9 (1:2), in blocking solution for 1 to 2 h at room temperature. Incubation with secondary Alexa Fluorconjugated antibodies (1:500) in blocking solution was carried out for 3 h at room temperature. Sections were then washed three times for 5 min each in PBS and visualized using fluorescence microscopy and appropriate filters (Olympic AX-80T, Olympus, Lake Success, NY). For morphometric analysis, digital images from 35 randomly chosen, 100x fields per tissue section were captured using a SPOT II camera and associated SPOT software (Diagnostic Instruments, Sterling Heights, MI). The areas of total immunostaining or immunostaining within the designated centrilobular or periportal regions were quantified using Scion image software (Scion Corp., Frederick, MD), as detailed by Copple et al. (2002a).
Gelatin zymography.
MMP activities were identified by gelatin zymography according to the procedure of Ricke et al. (2002). Briefly, 100 mg frozen liver tissue were homogenized in 50 mM Tris/HCl buffer (pH 7.4) containing 10 mM CaCl2 and 0.25% Triton X-100 using a polytron homogenizer. The homogenate was centrifuged at 9000 x g for 30 min at 4°C, and the supernatant was collected. Cell culture media were concentrated 20-fold using a centrifugal concentrator with a molecular size limit of 10 kDa (Microcon YM-10; Millipore Corp., Bedford, MA). Homogenates containing 1 mg tissue or equal volumes of concentrated cell culture media were mixed with equal volumes of 2X Laemmli sample buffer lacking ß-mercaptoethanol, and the proteins were subjected to electrophoresis in 10% SDS polyacrylamide gels containing 1 mg/ml bovine skin gelatin. After electrophoresis, SDS was removed from the gels by two washes of 20 min each with 2.5% Triton X-100. Subsequently, the gels were incubated in 50 mM Tris buffer (pH 7.4) containing 10 mM CaCl2 and 1 µM ZnSO4 for 24 h at 37°C without shaking. Gels were stained with Coomassie stain (Bio-Rad Laboratories, Hercules, CA). MMPs were identified by their ability to digest gelatin (clear bands) and by their molecular weights. The gels were scanned and densitometric evaluation of the bands was performed by Scion image software.
Collagenase activity assay.
Collagenolytic activity of MMPs was measured using a fluorescein-conjugated collagen type IV substrate, DQ collagen type IV, by a modified method of Ricke et al. (2002). Briefly, in a 96-well plate liver homogenates (10 mg wet weight) were incubated with 10 µg DQ collagen type IV in the presence or absence of 100 µM EDTA in a reaction buffer (50 mM Tris, pH 7.4, containing 10 mM CaCl2 and 1 µM ZnSO4). The reaction was allowed to run for 24 h at room temperature in the dark. Samples were then shaken for 1 min and fluorescence was recorded in a fluorescence plate reader at excitation and emission wavelengths of 495 and 515 nm, respectively. Collagenase activity in the tissue homogenates was calculated by subtracting the fluorescence measurement of samples with EDTA from that of samples without EDTA.
MPO and LDH activity assays.
MPO activity in the liver was measured according to the method of Harada et al. (2000). Briefly, 100 mg frozen liver tissue were weighed and suspended in 50 mM phosphate buffer (pH 6.0) containing 1% hexadecyltrimethylammonium bromide. The samples were homogenized using a polytron homogenizer, and the homogenate was sonicated, freeze-thawed, and then centrifuged at 4500 x g for 15 min at 4°C. MPO activity in the supernatant (corresponding to 1 mg wet tissue weight) was determined in a reaction mixture containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide in 50 mM phosphate buffer (pH 6.0). The change in absorbance at 460 nm over 3 min was measured in a 96-well plate reader. MPO activity was expressed as change in absorbance per minute per mg of tissue. LDH activities in the cell culture medium and cell lysate were measured according to the method of Bergmeyer and Bernt (1974)
. LDH release was expressed as the percentage of total cellular LDH released into the medium.
Hyaluronic acid assay.
HA concentration in the plasma samples was measured with an ELISA kit, as per the instructions of the manufacturer.
Statistical analysis.
All experiments were carried out with n = 48. Results are expressed as mean ± SEM. Comparisons were made with one-way ANOVA and Student-Newman-Keuls post hoc test. The criterion for significance was p 0.05 for all comparisons.
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RESULTS |
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DISCUSSION |
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Loss of basement membrane collagen IV is often due to the activity of the matrix-degrading collagenases MMP-9 and MMP-2. Increased activity of collagenases has been reported in acute conditions such as hepatic ischemia/reperfusion, cold preservation of livers (Upadhya and Strasberg, 1999; Yang et al., 2002
), and the early phase of chronic liver injury (Arthur, 2000
; Haruta et al., 1999
; Knittel et al., 2000
). Increased MMP activity in acute injury usually results from the release of a preformed pool of MMPs from endothelial cells or from influx and activation of inflammatory cells, unlike chronic injury or tissue remodeling in which increased de novo synthesis of MMPs plays a major role (Benyon and Arthur, 2001
).
Type IV collagenase activity in MCT-treated rat livers was significantly increased by 12 h after the administration of MCT (Fig. 3A). Increased MMP-9 was detected by gelatin zymography at the same time (Figs. 3B
and 3C
). MMP-9 is the form of type IV collagenase found in inflammatory cells such as PMNs, and it also occurs in SECs in the liver (Borregaard and Cowland, 1997
; Upadhya et al., 1997
). MMP-9 in PMNs is stored in tertiary granules and secreted in response to chemotactic stimuli (Borregaard and Cowland, 1997
). Increased PMN-derived MMP activity has been reported in myocardial ischemia/reperfusion injury, endotoxin-induced uveitis, and endotoxin-induced liver injury (Cuello et al., 2002
; Lindsey et al., 2001
; Solorzano et al., 1997
).
The respiratory burst of activated phagocytes both in vivo and in vitro results in the generation of superoxide radical and hydrogen peroxide, and degranulation of activated PMNs entails the release of the heme enzyme MPO. This enzyme catalyzes the reaction of hydrogen peroxide with chloride ions to yield the potent oxidant HOCl. HOCl reacts with numerous biomolecules. Brown et al. (2001) demonstrated the presence of HOCl-modified proteins in cases of acute liver injury and cirrhosis. MPO activity in liver increased as early as 8 h after the administration of MCT (Fig. 4A
), suggesting an influx of PMNs at this time. Immunolocalization of HOCl-modified proteins revealed pronounced staining in livers of MCT-treated rats (Fig. 4
). The presence of HOCl-modified proteins suggests activation (i.e., degranulation) of PMNs, as described previously in kidney tissue (Malle et al., 1997
). The staining tended to occur outside of the lesioned centrilobular areas (Fig. 4
), suggesting that PMN activation and degranulation are not required for MCT hepatotoxicity. This interpretation is consistent with the earlier observation that prior PMN depletion does not afford protection from the hepatocellular necrosis that occurs in MCT-exposed rats (Copple et al., 2003
).
During the degranulation process, MMPs would be expected to be released along with MPO. However, staining for HOCl-modified proteins was not restricted to centrilobular regions where injury was observed, casting doubt on whether PMN-derived collagenases are sufficient for the centrilobular endothelial cell loss. Consistent with this, depletion of PMNs did not offer protection against MCT-induced SEC injury (Fig. 5C).
Endothelial cells are another source of MMPs (Partridge et al., 1997; Taraboletti et al., 2002
). SECs store both MMP-9 and MMP-2 (Fig. 6A
; Upadhya and Strasberg, 1999
; Yang et al., 2002
). We recently demonstrated that both primary SECs and NP-26 cells in culture metabolize MCT to a toxic pyrrolic metabolite and contain cytochrome P450 3A, an isozyme responsible for the conversion of MCT to its pyrrole metabolite, MCTP (Kasahara et al., 1997
; Yee et al., 2003
). MCTP has been shown to interact with cellular macromolecules, including actin in endothelial cells, thereby affecting their function (Wilson et al., 1998
). Actin disassembly can lead to release of MMPs from SECs (Upadhya and Strasberg, 1999
). Thus, it is possible that MCTP-actin interaction results in MMP release. Accordingly, depolymerization of F-actin and subsequent release of MMPs on the basolateral side of SECs might be a factor for the degradation of extracellular matrix in the space of Disse in livers from MCT-treated rats (DeLeve et al., 2003
). Supporting this contention, NP-26 cells exposed to MCT for 6 h released MMPs. MMP release was dependent on MCT concentration (Figs. 6B
and 6C
) and occurred in the absence of cytotoxicity (Fig. 6D
). MCT at the concentration tested did not affect SEC membrane integrity (i.e., LDH release), suggesting that SEC loss might occur from basement membrane degradation by the released MMPs and consequent cell detachment.
In vivo, MMPs can be released by SECs in a precursor form that requires activation. After activation, these SEC-derived MMPs are available in the immediate vicinity of the basement membrane for their collagenolytic activity (Partridge et al., 1997). The oxidant HOCl can activate pro-MMPs to their proteolytically active forms (Weiss et al., 1985
). MMPs are also activated by proteases released from stimulated neutrophils (Okada et al., 1992
) by thrombin generated during activation of the coagulation system (Nguyen et al., 2001
) or by membrane typeMMP. Since PMNs do not seem to play a critical role in liver pathogenesis in this model (Copple et al., 2003
), these cells are probably not the sole source of these activating proteases. However, an activated coagulation system does seem to be required for hepatocellular injury (Copple et al., 2002b
), raising the possibility that coagulation system proteases such as thrombin might participate in MMP activation in MCT-treated rats. Anticoagulants, however, did not prevent MCT-induced endothelial cell injury, suggesting that thrombin alone may not be sufficient for MMP activation (Copple et al., 2002b
). MMPs can also be derived from other cell types such as Kupffer cells, stellate cells, and platelets (Sawicki et al., 1997
; Theret et al., 1999
; Winwood et al., 1995
). The contribution of these cells in MCT-induced liver injury remains to be studied.
Collagen IV, along with other components of the basement membrane, communicates with integrins, adhesion receptors on the cell surface (Maru et al., 1998; Sacca and Moroder, 2002
). Such focal adhesions transduce signal via a focal adhesion kinase. Loss of collagen IV and consequent loss of adhesion results in focal adhesion kinase dephosphorylation and its cleavage and inactivation by caspases. This process is associated with apoptosis (van de Water et al., 1999
). MCTP has been reported to alter adhesion molecules and to induce fragmentation of focal adhesion kinase in cultured endothelial cells from pulmonary artery (Taylor et al., 2003
). Weakening of cell adhesion to the basement membrane matrix and subsequent activation of cell death pathways might contribute to the observed loss of SECs from sinusoids in MCT-treated rats (Copple et al., 2002a
).
Protection against SEC loss by MMP inhibition has not been demonstrated after MCT exposure. Doxycycline, a well-known MMP inhibitor, also inhibits hepatic MCT bioactivation (data not shown) and, therefore, was deemed not useful for supporting a role for MMPs in this model. Definitive proof for the role of these collagenases in basement membrane degradation in vivo awaits widely available, specific inhibitors of MMPs that can be used in vivo.
In summary, MCT causes loss of basement membrane components in sinusoids of rat liver, including collagen IV in the centrilobular regions. Loss of basement membrane is associated with an increase in MMP-9 and MMP-2. Neutrophils and their products do not appear to contribute to the loss of endothelial cells. However, NP-26 cells release MMPs in response to MCT, suggesting that MMPs from SECs and perhaps other cell types might contribute to the destruction of basement membrane and loss of SECs in vivo.
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ACKNOWLEDGMENTS |
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NOTES |
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