* Department of Pharmacology and Toxicology, National Food Safety and Toxicology Center and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824; and
Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-Ku, Tokyo 108-8639, Japan
Received February 11, 2003; accepted March 31, 2003
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ABSTRACT |
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Key Words: liver; inflammation; lipopolysaccharide; monocrotaline; sinusoidal endothelial cells; coagulation; hemorrhage; hyaluronic acid.
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INTRODUCTION |
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Bacterial lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, elicits a potent inflammatory response in mammals (Hewett and Roth, 1993; Holst et al., 1996
). LPS-induced liver injury from large, acutely toxic doses is characterized by neutrophil (polymorphonuclear leukocyte; PMN) infiltration associated with midzonal hepatocellular degeneration, SEC injury, and coagulative necrosis (Hewett and Roth, 1993
; Seto et al., 1998
; Yee et al., 2000
). The complex interaction of numerous soluble mediators and inflammatory cells is critical to the pathogenesis of injury (Brouwer et al., 1995
; Brown et al., 1997
; Hewett and Roth, 1993
; Hewett et al., 1992
; Moulin et al., 1996
).
A more modest, noninjurious inflammatory response in animals results from exposure to smaller doses of LPS. Although overt hepatocellular injury does not develop, the accompanying release of inflammatory mediators has the potential to alter tissue homeostasis (Hewett and Roth, 1993; Michie et al., 1988
; Spitzer and Mayer, 1993
). Such alterations can render tissues more sensitive to toxic chemicals. Accordingly, concurrent exposure to small doses of LPS can act as a determinant of susceptibility to intoxication by a variety of chemicals (Ganey and Roth, 2001
).
Yee et al. (2000) recently demonstrated that a small, noninjurious dose of LPS given to rats 4 h after a small, nonhepatotoxic dose of MCT results in synergistic liver injury that is maximal 18 h after MCT administration. Liver lesions were both centrilobular (CL) and midzonal (MZ), exhibiting characteristics similar to lesions associated with larger, toxic doses of MCT or LPS given separately. MCT-like CL lesions consisted of moderate to marked hepatocellular apoptotic and oncotic necrosis, hemorrhage, and loss of central vein (CV) intima. LPS-like MZ lesions comprised marked but more frequent and well-defined areas of hepatocellular coagulative necrosis accompanied by PMN infiltration, disruption of sinusoidal architecture, and hemorrhage. Vascular injury can be inferred from the loss of central vein intima in the CL lesion and the disruption of sinusoidal architecture and hemorrhage in both the CL and MZ lesions in this model (Yee et al., 2000
).
Injury to endothelial cells in the microvasculature can result in activation of the coagulation system (Hirata et al., 1989; Machovich, 1985
; Ryan, 1986
). Coagulation system activation is critical for the development of hepatic parenchymal cell (HPC) injury in other liver injury models (Arai et al., 1996
; Fujiwara et al. 1988
; Hewett and Roth, 1995
; Perry et al., 1984
; Yamada et al. 1989
). In addition, microcirculatory disturbances from hemorrhage and/or intrasinusoidal fibrin deposition have been postulated to contribute to HPC injury (Ba et al., 2000
; Copple et al., 2002a
,b
; DeLeve et al., 1996
; Saetre et al., 2000
; Shibayama, 1987
). CVEC and SEC injury occurs in the livers of rats after administration of a large, acutely toxic dose of MCT (Copple et al., 2002a
; DeLeve et al., 1996
), leading to coagulation system activation and fibrin deposition (Copple et al., 2002a
). Similar changes at more modest MCT doses in the absence or presence of additional susceptibility factors have not been reported. Accordingly, the present investigation was designed to test the hypothesis that injury to SECs and activation of the coagulation system occur during coexposure to normally nontoxic doses of MCT and LPS.
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MATERIALS AND METHODS |
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Animals.
Male, Sprague-Dawley rats (Crl:CD [SD]IGS BR, Charles River, Portage, MI) weighing 175200 g or 200300 g were used for studies in vitro and in vivo, respectively. Animals were allowed food (Rodent Chow/Tek 8640, Harlan Teklad, Madison, WI) and water ad libitum. They were housed no more than three to a cage on Aspen chip bedding (Northeastern Products Company, Warrenburg, NY) and were maintained on a 12-h light/dark cycle in a controlled temperature (1821°C) and humidity (55 ± 5%) environment for a period of 1 week before use. All procedures on animals followed the guidelines for humane treatment set by the American Association of Laboratory Animal Sciences and the University Laboratory Animal Research Unit at Michigan State University.
Treatment protocol in vivo.
MCT was dissolved in sterile saline minimally acidified by 0.2 M HCl. The pH was brought to 7 by addition of 2 M NaOH, and the volume was adjusted with sterile saline to the appropriate final concentration. Rats were given MCT (100 mg/kg) or an equivalent volume of sterile saline vehicle (Veh), intraperitoneally, followed 4 h later by LPS (7.4 x 106 EU/kg) or its saline Veh via tail vein injection. LPS was administered 4 h after MCT to minimize interference with MCT bioactivation (Allen et al., 1972). No mortality occurred in MCT/LPS-cotreated animals within 12 h after MCT administration, but approximately 20% of rats died by 18 h. No animals that received saline Veh, MCT, or LPS alone died.
Assessment of HPC injury and plasma fibrinogen and HA concentrations.
At the times indicated in the figure legends, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A midline abdominal incision was made, blood was collected from the inferior vena cava into a syringe containing sodium citrate (0.38% final concentration), and animals were euthanized by exsanguination. HPC injury was evaluated by increases in plasma ALT activity. A BBL fibrometer (Becton, Dickinson and Company, Hunt Valley, MD) and a fibrinogen diagnostic kit were used to determine plasma fibrinogen concentration. An ELISA kit was used to measure plasma HA concentration, a biomarker of hepatic SEC injury.
Histopathologic evaluation.
A one-cm3 portion of the liver for immunohistochemical staining was cut from the middle of the left lateral lobe and frozen in isopentane immersed in liquid nitrogen. Further, a second portion of this lobe was snap frozen in liquid nitrogen for evaluation of tissue hemoglobin. The remaining portions of the liver were fixed by immersion in 10% neutral buffered formalin for at least 3 days before being processed for histologic analysis. Serial transverse sections from the left lateral liver lobe were processed for light microscopy. Paraffin-embedded sections were cut at 4 µm, stained with hematoxylin and eosin, and evaluated for lesion size and severity. Tissue sections were analyzed using a light microscope without knowledge of the treatment group.
Additionally, a thin section (12 mm) of the liver from the middle of the left lateral lobe was fixed in 4% glutaraldehyde in 0.1 M phosphate buffer for 24 h, processed, and analyzed by transmission electron microscopy (TEM) using a Phillips 301 transmission electron microscope (FEI Company, Hillsboro, OR).
Immunohistochemistry.
For endothelial cell immunostaining, 8-µm-thick sections of frozen liver were fixed in acetone (4°C) for 5 min. Sections were incubated for 30 min with PBS containing 10% goat serum (i.e., blocking solution) and then with mouse anti-rat RECA-1 diluted (1:20) in blocking solution overnight at 4°C. The RECA-1 antibody binds to rat endothelium but not to other cell types (Duijvestijn et al., 1992). In the liver, RECA-1 antibody stains both SECs and endothelial cells from larger vessels. After incubation with RECA-1 antibody, the sections were incubated for 3 h with goat anti-mouse secondary antibody conjugated to Alexa 594 (1:500) in blocking solution containing 2% rat serum. Sections were washed 3 times for 5 min each with PBS and visualized using fluorescence microscopy.
For fibrin immunostaining, 8-µm-thick sections of frozen tissue were fixed in 10% buffered formalin containing 2% acetic acid for 30 min at room temperature. This fixation protocol solubilizes all fibrinogen and fibrin species except for cross-linked fibrin. Thus, only cross-linked fibrin is stained in liver sections (Schnitt et al., 1993). Sections were incubated for 30 min with PBS containing 10% horse serum (i.e., blocking solution) and then with goat anti-rat fibrinogen diluted (1:1000) in blocking solution overnight at 4°C. Next, the sections were incubated for 3 h in blocking solution with donkey anti-goat secondary antibody conjugated to Alexa 594 (1:1000). Liver sections were washed 3 times for 5 min each with PBS and visualized using fluorescence microscopy.
For both protocols, no staining was observed in the controls in which the primary or secondary antibody was omitted from the staining protocol. All morphometrically compared treatment groups were stained at the same time and evaluated on the same day.
Quantification of liver endothelial cells and fibrin deposition.
Endothelial cells and fibrin deposition in the liver were quantified morphometrically by analyzing the area of immunohistochemical staining for each liver section (Copple et al., 2002a). A decrease in the endothelial cell staining suggests a loss of these cells from the liver. An increase in the area of fibrin staining in the liver indicates fibrin deposition. Fluorescent staining of liver sections was visualized using an Olympus AX-80T microscope (Olympus, Lake Success, NY). For morphometric analysis of total endothelial cell or fibrin deposition in a liver section, digital images of five randomly chosen 100x fields per tissue section were captured using a SPOT II camera and SPOT Advanced Software (Diagnostic Instruments, Sterling Heights, MI). Samples were coded so that the evaluator was not aware of the treatment, and the same exposure time was used for all captured images. Each digital image encompassed a total area of 1.4 mm2 and contained several CL, MZ, and periportal (PP) regions.
The area of immunohistochemical staining (number of pixels) within the CL, MZ, and PP regions was quantified using Scion Image software (Scion Corporation, Frederick, MD). For endothelial cell quantification, a density slice was taken from an inverted, gray-scale digital image of a liver section from a Veh/Veh-treated rat. A density slice allows analysis of pixels in a defined range of gray values (i.e., densities). Next, the threshold was adjusted so that background staining was eliminated from the analysis and only endothelial staining was visualized. For quantification of fibrin deposition, the threshold was selected so that minimal positive staining was present in Veh/Veh-treated controls. The same threshold value was used to analyze digital images from all treatment groups. For quantification of endothelial cells or fibrin, the area of positive staining was measured and divided by the total area of the image. Analysis of endothelial cells or fibrin deposition in the CL, MZ, and PP regions was conducted by drawing a 145-µm-diameter circle around the CV or vessels of the portal triad. The circumference of the circle was about 46 hepatocytes away from the CV or portal triad vessels, and this area was arbitrarily defined as the CL and PP region. The MZ region was defined as the center of area between the CL and PP region using the same circle circumference, without having overlap of these arbitrary circles. The area of endothelial cells or fibrin staining in each region was measured as described above and divided by the total area of the image. Results from the random fields analyzed for each liver section were averaged and counted as a replicate (i.e., each replicate representing a different rat). For the time-course studies, Veh/Veh-treated rats at various times were combined into one group for statistical analysis, since no differences occurred among these groups.
Liver hemoglobin.
Liver hemoglobin concentration was used as a biomarker for hemorrhage (Copple et al., 2002a; Jaeschke et al., 2000
). A 20% homogenate was made from samples of frozen liver in 50 mM sodium phosphate buffer (120 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid). Samples were centrifuged (16,000 x g) for 10 min at 4°C, and 200 µl of the supernatant was diluted in Drabkins solution. Following a 15-minute incubation, the absorbance was measured at 540 nm, and the hemoglobin concentration was determined from a standard curve.
SEC studies in vitro.
Primary SECs were isolated from rat liver as described in Braet et al.(1994). Briefly, the liver from an anaesthetized rat was 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 (NP) fraction was collected. The NP 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 gradients was collected, washed with PBS, and plated. Additionally, a rat liver sinusoidal endothelial cell line, NP-26, was used in studies in vitro. The NP-26 cell line was established from an enriched fraction of liver SECs transfected with SV40 large-T antigen (Maru et al., 1998
).
Scanning electron microscopy.
Primary SECs and NP-26 cells were plated on 12-mm-diameter collagen-coated coverslips and cultured for 24 h in EGM-2. Scanning electron microscopy (SEM) was performed as described in Braet et al.(2002), with minor modifications. Briefly, cells were rinsed twice with PBS and fixed for 12 h with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose (pH 7.4). The fixed cells were subsequently treated with filtered 1% tannic acid in 0.15 M sodium cacodylate buffer (pH 7.4) for 1 h and postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.4) for another hour. The samples were further dehydrated in a graded ethanol series, critical point dried and sputter-coated with gold. The cells were examined using a JSM 6400V scanning electron microscope (JEOL USA Inc., Peabody, MA).
MCT treatment in vitro and Ehrlich assay.
The ability of SECs to convert MCT to pyrrolic metabolite(s) was evaluated in vitro. Isolated primary SECs were routinely greater than 90% pure as determined by morphology; they were contaminated with approximately 10% hepatocytes. To correct for the contribution of these contaminating hepatocytes to MCT metabolism in SEC cultures, MCT metabolism in an equivalent number of hepatocytes was examined. Hepatocytes were isolated by the collagenase perfusion as described previously by Seglen (1973) and Klaunig et al.(1981)
. Primary SECs, NP-26 cells, or hepatocytes were plated on collagen-coated, 12-well plates and cultured for 24 h. MCT was added to the serum-free culture medium to a final concentration of 0, 1, 2 or 4 mM, and cells were incubated for 4 h. Medium was then collected and evaluated for pyrrole concentration by a modified Ehrlich reaction as described previously by Mattocks and White (1970)
. Briefly, one ml of incubation medium was mixed with one ml of 5% ascorbic acid in 80% ethanol. One ml of Ehrlich reagent was added, and the mixture was heated in a waterbath for 1 min. After cooling to room temperature, 0.1 ml of FeCl3 solution was added to inhibit fading of the Ehrlich-pyrrole complex color. Absorbance was measured at 565 nm and compared against a standard curve. The amounts of pyrrolic MCT metabolite(s) formed by primary SECs was corrected for pyrroles produced from contaminating hepatocytes by subtracting the amount of pyrrolic MCT metabolite(s) formed from an equal number of hepatocytes. Results are expressed as µg pyrrole per mg of cellular protein. Protein concentration was evaluated as described by Bradford (1976)
in cells that were solubilized with 1% Triton X-100 and sonicated.
Preparation of microsomal fraction and immunoblotting.
Microsomes were prepared as described by Coffman et al.(1999). Briefly, cultured NP-26 cells or isolated rat hepatocytes were suspended in homogenization buffer (0.25 M sucrose in 5 mM HEPES buffer, pH 7.4). Cells were disrupted by sonication and then centrifuged (9,000 x g) for 20 min. The supernatant was recovered and then centrifuged (100,000 x g) for 1 h. The resulting pellet was resuspended in homogenization buffer. Protein concentrations were measured as described by Bradford (1976)
, and CYP3A2 protein was identified by immunoblotting as described by Sharp et al.(2001)
. Microsomal proteins (0.5 µg from hepatocytes and 5 µg from NP-26 cells) were electrophoresed on a 10% polyacrylamide gel with SDS and then transferred onto a nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk, washed with PBS-Tween, probed with anti-CYP3A2 antibody (1:1000), and subsequently reacted with HRP-conjugated anti-rabbit secondary antibody (1:5000). The protein bands were visualized by ECL.
Statistical analysis.
All results are expressed as mean ± SEM. Data expressed as fractions were arc sine square root-transformed before analysis. When variances were not homogeneous, data were log-transformed before analysis. Homogeneous data were analyzed by one-way or two-way analysis of variance (ANOVA), as appropriate, and group means were compared using Student-Newman-Keuls post hoc test (Steele et al., 1997). The criterion for significance was p ≤ 0.05 for all comparisons.
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RESULTS |
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RECA-1 immunostaining of liver sections has been used to visualize and quantify the loss of endothelial cells in the liver (Copple et al., 2002a). Representative photomicrographs of RECA-1 immunostaining in livers from a Veh/Veh-treated and a MCT/LPS-cotreated rat at 18 h can be seen in Figure 2A
. The liver section from a Veh/Veh-treated rat exhibited panlobular RECA-1 staining of sinusoids and the intima of CVs and vessels of the portal triad. RECA-1 staining decreased in the sinusoids and in CV intima in livers from MCT/LPS-cotreated rats but not in the endothelium in the portal triad. Morphometry conducted on 18-h liver samples (Fig. 2B
) revealed a significant decrease in RECA-1 staining in the CL and MZ regions of livers from MCT/LPS-cotreated animals. No decrease in RECA-1 immunostaining was observed in the livers from Veh, MCT, or LPS treatment groups. A time-dependent decrease in total RECA-1 immunostaining was observed in the livers of MCT/LPS-cotreated animals (Fig. 2C
). This decrease was statistically significant by 12 h and remained at 18 h. Zonal analysis of the liver lobules from MCT/LPS-cotreated animals revealed a significant decrease in RECA-1 immunostaining within the CL region of the liver lobule starting at 6 h and within the MZ region by 12 h (Fig. 2D
).
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After incubation with MCT, primary SECs produced pyrrole in a concentration-dependent manner (Fig. 4A). The amount of pyrrole produced from primary SECs was corrected for the contribution by contaminating hepatocytes. SEM analysis of NP-26 cells revealed fenestrations (Fig. 4C
), a hallmark of SEC morphology (Fig. 4D
). Pyrrole production also occurred in NP-26 cells in a MCT concentration-dependent manner (Fig. 4A
). Immunoblotting of microsomal proteins with an anti-CYP3A2 specific antibody revealed a protein of ~72 kDa in both NP-26 cells and rat hepatocytes (Fig. 4B
).
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To determine if the decrease in plasma fibrinogen was associated with deposition of insoluble fibrin in the liver, hepatic fibrin was examined immunohistochemically. Minimal fibrin immunostaining was observed in the sinusoids of liver sections from Veh/Veh-treated rats at 18 h (Fig. 6A). However, some staining occurred in the intima of larger vessels, which results from fibrin deposition that occurs after animal sacrifice (Copple et al., 2002a
). By contrast, intense fibrin staining occurred in the sinusoids of CL and MZ regions of livers from rats treated with MCT/LPS (Fig. 6A
). Fibrin staining was minimal in the PP regions. Morphometric analysis confirmed a significant increase in fibrin staining at 18 h in the CL and MZ regions in livers of MCT/LPS-cotreated animals (Fig. 6B
). No increase in fibrin staining was observed in the other treatment groups at this time. Furthermore, a time-dependent increase in total fibrin staining occurred in the livers of MCT/LPS-cotreated animals; this was significant at 12 h and remained elevated at 18 h (Fig. 6C
). Zonal analysis of hepatic fibrin staining revealed a time-dependent increase that became significant at 12 h in both CL and MZ regions of livers from MCT/LPS-cotreated animals (Fig. 6D
).
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DISCUSSION |
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RECA-1 immunostaining of liver sections revealed that endothelial cell injury was extensive in MCT/LPS-cotreated animals. Reduction in RECA-1 staining was pronounced within CL regions by 6 h and within MZ regions by 12 h (Fig. 2D). The CL and MZ location of this decrease corresponds to the regions wherein HPC necrosis occurs. The reduction in RECA-1 staining, however, was not observed in the group given LPS alone at 18 h, and there was no reduction in total RECA-1 staining in the MCT/LPS group at 6 h. Since increases in plasma HA occurred under both of these conditions, plasma HA concentration appears to be a more sensitive biomarker for SEC injury than loss of RECA-1 staining. A similar difference in sensitivity was observed in a study of hepatotoxic doses of MCT (Copple et al., 2002a
).
TEM of livers from MCT/LPS-cotreated animals was used to confirm hepatic vascular injury. The sinusoids and CVs from the livers of MCT/LPS-cotreated animals either contained remnants of SECs or CVECs or were devoid of endothelial cells altogether (Fig. 3), and fibrin deposition, hemorrhage, and PMNs were found in sinusoids. Accordingly, endothelial damage, as determined by plasma HA concentration, immunohistochemistry, and TEM, is a pronounced component of synergistic liver injury induced by MCT/LPS cotreatment.
The cause of SEC injury in MCT/LPS-cotreated rats is not fully understood. DeLeve et al.(1996) postulated that SECs in vitro can bioactivate MCT to its toxic metabolite (i.e., MCTP), resulting in SEC injury. Although SECs are known to express some CYPs (DeLeve et al., 1997
; Lester et al., 1993
), it is not known if the particular CYP subfamily (i.e., CYP 3A) responsible for MCT bioactivation is expressed in these cells (Kasahara et al., 1997
; Reid et al., 1998
). However, both isolated primary SECs and the NP-26 rat SEC cell line produced pyrrolic metabolite(s) from MCT in a concentration-dependent manner (Fig. 4A
). In addition, a ~72 kDa protein recognized by the CYP3A2 specific antibody was expressed in the microsomal fractions from NP-26 cells (Fig. 4B
). This confirms that an isozyme from the CYP3A subfamily is present in these cells. Moreover, it has been demonstrated that MCTP causes injury to endothelial cells in vitro. For example, treatment of pulmonary artery endothelial cells with low concentrations of MCTP in vitro results in enhanced cell detachment (i.e., disruption of monolayer integrity), derangement of actin polymerization, and apoptotic necrosis (Reindel and Roth, 1991
; Thomas et al., 1998
; Wilson et al., 1998
). Hence, SECs appear to metabolize MCT, and the consequent production of MCTP has adverse effects on these cells that would explain widespread SEC destruction in vivo.
No SEC injury was observed in vivo when the dose of MCT used in the present study was given by itself. This suggests that the degree of bioactivation by SECs and HPCs combined was insufficient to cause overt injury (Fig. 1B). However, at the small MCT dose used, MCT bioactivation might alter SEC homeostasis to render these cells more susceptible to toxicity from inflammatory factors evoked by concurrent LPS exposure (Jaeschke and Farhood, 1991
; Sarphie et al., 1996
; Springer, 1994
; Ward and Varani, 1990
; Yachida et al., 1998
). Yee et al. (2003a
,b)
demonstrated that Kupffer cell inactivation, TNF-
neutralization, and PMN depletion were each effective in attenuating HPC injury in MCT/LPS-cotreated animals, but these manipulations were only modestly successful in attenuating SEC injury. Hence, it is probable that SEC injury in this model develops in part from the interaction of MCT with LPS or with an unknown inflammatory mediator(s) evoked by LPS. One possible interaction could involve depletion of glutathione (GSH) in endothelial cells (Pan et al., 1993
; Reid et al., 1999
). DeLeve et al.(1996)
showed that MCT is more toxic to SECs than to HPCs in vitro and that toxicity may require profound GSH depletion. It is tempting to speculate that the small dose of MCT used in this model decreases GSH concentration in SECs without resulting in overt injury. Prooxidant inflammatory mediators evoked by LPS might precipitate overt injury to those SECs in which GSH has been decreased by MCT. Further study is required to explore this possibility.
Activation of SECs by LPS or LPS-induced inflammatory mediators (e.g., TNF-) results in SECs becoming procoagulant (Stern et al., 1985
; Takeuchi et al., 1994
). Moreover, SEC injury or destruction can result in the activation of the coagulation system (Copple et al., 2002a
; Hirata et al., 1989
; Machovich, 1985
; Ryan, 1986
). In MCT/LPS-cotreated animals, plasma fibrinogen concentration decreased significantly by 6 h after MCT administration and remained depressed through 18 h (Fig. 5B
). A consequence of coagulation system activation is the formation of insoluble fibrin. Fibrin deposition increased significantly in the livers of MCT/LPS-cotreated animals by 12 h and remained elevated at 18 h (Fig. 6C
). Both CL and MZ regions of the liver lobule experienced fibrin deposition at these times (Fig. 6D
). TEM confirmed the immunohistochemical evidence of fibrin deposition in the sinusoids (Fig. 3B
). The observations that the reduction in plasma fibrinogen concentration preceded HPC damage and that fibrin deposits localized to CL and MZ regions that ultimately experience damage suggest the possibility of a causal relationship between activation of the coagulation system and HPC injury.
Hemorrhage and fibrin deposition caused by injury or loss of SECs can result in the impairment of sinusoidal blood flow (Hirata et al., 1989; Vollmar et al., 1993
; Yachida et al., 1998
). Moreover, activation of SECs by TNF-
or other inflammatory mediators results in adhesion of PMNs to these cells and in SEC swelling that can narrow sinusoidal lumens and contribute to such impairment of blood flow (Springer, 1994
; Vollmar et al., 1996
; Yachida et al., 1998
). It has been postulated that consequent sinusoidal hypoperfusion leads to ischemic/hypoxic injury to HPCs within affected regions (Copple et al., 2002a
; DeLeve et al., 1996
; Fujiwara et al., 1988
; Hirata et al., 1989
; Mochida et al., 1999
; Yee et al., 2000
). Consistent with this hypothesis is the observation in MCT/LPS-treated rats that both SEC injury (Fig. 1B
) and coagulation system activation (Fig. 5B
) precede liver hemorrhage (Fig. 5A
), formation of fibrin deposits, and the onset of HPC injury. In addition, thrombin and other coagulation factors might act on protease-activated receptors to promote liver injury, as occurs after an hepatotoxic dose of LPS (Copple et al., 2003
). Therefore, damage to SECs may lead to HPC injury through activation of the coagulation system resulting in fibrin deposition in the liver and hypoxic injury to HPCs.
Other mechanisms exist by which SEC injury could contribute to HPC injury. For example, SECs perform various functions in the liver, including filtration, endocytosis, and putative regulation of sinusoidal blood flow (Arii and Imamura, 2000; Wisse et al., 1996
). As described above, treatment of endothelial cells with MCTP results in detrimental cytoskeletal, junctional barrier, and permeability changes (Reindel and Roth, 1991
; Thomas et al., 1998
; Wilson et al., 1998
). If similar changes occur in SECs in vivo after MCT/LPS coexposure, such disruption of the SEC barrier could permit access to HPCs of injurious products from activated PMNs and Kupffer cells, resulting in HPC injury (Aria et al., 1993
; Farhood et al., 1995
; Jaeschke et al., 1996
; Yachida et al., 1998
; Yoshidome et al., 2000
). Hence, injury or destruction of SECs might lead to HPC injury through a variety of mechanisms. Accordingly, SECs may be an important initial target in the development of liver injury in this model, as it appears to be in others (Copple et al., 2002a
; Deaciuc and Spitzer, 1996
; DeLeve et al., 1996
; Sarphie et al., 1996
). However, further studies will be needed to determine if SECs have a causal role in the development of HPC injury in MCT/LPS-cotreated animals.
In summary, MCT/LPS cotreatment results in extensive, time-dependent injury to hepatic endothelial cells. Destruction of CVECs is evident by light and electron microscopy as well as in the loss of intimal RECA-1 immunostaining. Similarly, elevated plasma HA concentration, decreased sinusoidal RECA-1 immunostaining, and TEM results all indicate injury to SECs. SEC injury precedes hepatic fibrin deposition, hemorrhage, and HPC injury. Overall, the results indicate that synergistic hepatotoxicity from coexposure to small, noninjurious doses of MCT and LPS involves pronounced SEC dysfunction and injury and activation of the coagulation system.
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ACKNOWLEDGMENTS |
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NOTES |
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