Endothelial Cell Injury and Coagulation System Activation during Synergistic Hepatotoxicity from Monocrotaline and Bacterial Lipopolysaccharide Coexposure

Steven B. Yee*, Umesh M. Hanumegowda*, Bryan L. Copple*, Masabumi Shibuya{dagger}, Patricia E. Ganey* and Robert A. Roth*,1

* Department of Pharmacology and Toxicology, National Food Safety and Toxicology Center and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824; and {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A small, noninjurious dose of bacterial lipopolysaccharide (LPS; 7.4 x 106 EU/kg) administered 4 h after a small, nontoxic dose of monocrotaline (MCT; 100 mg/kg) produces synergistic hepatotoxicity in rats within 6 to 12 h after MCT exposure. The resulting centrilobular (CL) and midzonal (MZ) liver lesions are characterized by hepatic parenchymal cell (HPC) necrosis. Pronounced hemorrhage, disruption of sinusoidal architecture, and loss of central vein intima suggest that an additional component to injury may be the liver vasculature. In the present investigation, the hypothesis that sinusoidal endothelial cell (SEC) injury and coagulation system activation occur in this model was tested. Plasma hyaluronic acid (HA) concentration, a biomarker for SEC injury, was significantly increased in cotreated animals before the onset of HPC injury and remained elevated through the time of maximal HPC injury (i.e., 18 h). SEC injury was confirmed by immunohistochemistry and electron microscopy. Pyrrolic metabolites were produced from MCT by SECs in vitro, which suggests that MCT may injure SECs directly through the formation of its toxic metabolite, monocrotaline pyrrole. Inasmuch as SEC activation and injury can promote hemostasis, activation of the coagulation system was evaluated. Coagulation system activation, as marked by a decrease in plasma fibrinogen, occurred before the onset of HPC injury. Furthermore, extensive fibrin deposition was observed immunohistochemically within CL and MZ regions after MCT/LPS cotreatment. Taken together, these results suggest that SEC injury and coagulation system activation are components of the synergistic liver injury resulting from MCT and LPS coexposure.

Key Words: liver; inflammation; lipopolysaccharide; monocrotaline; sinusoidal endothelial cells; coagulation; hemorrhage; hyaluronic acid.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that is well known for hepatic and cardiopulmonary toxicity in both animals and humans (Mattocks, 1986Go; Stegelmeier et al., 1999Go). For liver injury to occur, MCT must be bioactivated by cytochrome P450 (CYP) to a toxic metabolite, monocrotaline pyrrole (MCTP; Stegelmeier et al., 1999Go; White and Mattocks, 1972Go). At acutely toxic doses, MCT-induced liver lesions are characterized by centrilobular hepatocellular necrosis, dilated and congested sinusoids, hemorrhage, and injured central venous and sinusoidal endothelial cells (CVECs and SECs, respectively; Copple et al., 2002aGo; DeLeve et al., 1999Go; Schoental and Head, 1955Go; Yee et al., 2000Go).

Bacterial lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, elicits a potent inflammatory response in mammals (Hewett and Roth, 1993Go; Holst et al., 1996Go). 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, 1993Go; Seto et al., 1998Go; Yee et al., 2000Go). The complex interaction of numerous soluble mediators and inflammatory cells is critical to the pathogenesis of injury (Brouwer et al., 1995Go; Brown et al., 1997Go; Hewett and Roth, 1993Go; Hewett et al., 1992Go; Moulin et al., 1996Go).

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, 1993Go; Michie et al., 1988Go; Spitzer and Mayer, 1993Go). 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, 2001Go).

Yee et al. (2000)Go 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., 2000Go).

Injury to endothelial cells in the microvasculature can result in activation of the coagulation system (Hirata et al., 1989Go; Machovich, 1985Go; Ryan, 1986Go). Coagulation system activation is critical for the development of hepatic parenchymal cell (HPC) injury in other liver injury models (Arai et al., 1996Go; Fujiwara et al. 1988Go; Hewett and Roth, 1995Go; Perry et al., 1984Go; Yamada et al. 1989Go). In addition, microcirculatory disturbances from hemorrhage and/or intrasinusoidal fibrin deposition have been postulated to contribute to HPC injury (Ba et al., 2000Go; Copple et al., 2002aGo,bGo; DeLeve et al., 1996Go; Saetre et al., 2000Go; Shibayama, 1987Go). CVEC and SEC injury occurs in the livers of rats after administration of a large, acutely toxic dose of MCT (Copple et al., 2002aGo; DeLeve et al., 1996Go), leading to coagulation system activation and fibrin deposition (Copple et al., 2002aGo). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Isopentane, LPS (Escherichia coli, serotype O128:B12, 1.7 x 106 endotoxin units (EUs)/mg), phosphate buffered saline (PBS), sodium citrate, and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Company (St. Louis, MO). Mouse anti-rat endothelial cell antigen (RECA)-1 was acquired from Serotec, Inc. (Raleigh, NC). Rabbit anti-rat CYP4503A2 was obtained from BD Gentest (Woburn, MA). Horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody was acquired from Santa Cruz Biotech (Santa Cruz, CA). Goat anti-rat fibrinogen was purchased from ICN Pharmaceuticals (Aurora, OH). Goat and horse sera were obtained from Vector Laboratories (Burlingame, CA). Goat anti-mouse secondary antibody conjugated to Alexa 594 and donkey anti-goat secondary antibody conjugated to Alexa 594 were acquired from Molecular Probes (Eugene, OR). Enhanced chemiluminescence (ECL) for immunoblotting was obtained from Amersham Bioscience (Piscataway, NJ). Acrylamide/Bis solution was purchased from Bio-Rad Laboratories (Hercules, CA). Nitrocellulose membrane was procured from Schleicher and Schuell Inc. (Keene, NH). MCT was acquired from Trans World Chemicals (Rockville, MD). Absolute ethanol was purchased from Quantum Chemical Company (Tuscola, IL). Sterile saline was acquired from Abbott Laboratories (North Chicago, IL). Formalin fixative was procured from Surgipath Medical Industries, Inc. (Richmond, IL). Sodium cacodylate buffer was acquired from Electron Microscopy Sciences (Fort Washington, PA). Liver perfusion, liver digestion, and hepatocyte wash media were purchased from Life Technologies, Inc. (Rockville, MD). Endothelial cell medium (EGM-2) was acquired through Biowhittaker, Inc. (Walkersville, MD). Diagnostic kits 59 UV and 886-A for the determination of alanine aminotransferase (ALT) activity and fibrinogen concentration, respectively, and Total Hemoglobin Kits were purchased from Sigma Chemical Company (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kit for hyaluronic acid (HA) was acquired from Corgenix, Inc. (Westminster, CO). All other materials were purchased from Sigma Chemical Company (St. Louis, MO).

Animals.
Male, Sprague-Dawley rats (Crl:CD [SD]IGS BR, Charles River, Portage, MI) weighing 175–200 g or 200–300 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 (18–21°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., 1972Go). 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 (1–2 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., 1992Go). 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., 1993Go). 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., 2002aGo). 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 4–6 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., 2002aGo; Jaeschke et al., 2000Go). 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 Drabkin’s 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)Go. 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., 1998Go).

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)Go, 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)Go and Klaunig et al.(1981)Go. 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)Go. 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)Go 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)Go. 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)Go, and CYP3A2 protein was identified by immunoblotting as described by Sharp et al.(2001)Go. 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., 1997Go). The criterion for significance was p ≤ 0.05 for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver Endothelial Cell Injury in MCT/LPS-Cotreated Animals
As marked by increased ALT activity in plasma, HPC injury from MCT/LPS-coexposure occurred between 6 and 12 h and remained elevated by 18 h after MCT administration (Fig. 1AGo). Yee et al. (2000Go, 2003b)Go have previously demonstrated that HPC injury in the MCT/LPS-induced liver injury model is maximal by 18 h and resolves by 96 h. MCT-like CL and LPS-like MZ liver lesions developed as described by Yee et al.(2000)Go. Although no parenchymal lesions were apparent 6 h after MCT administration, some disruption of central vein intima was observed in MCT/LPS-cotreated animals at this time. Loss of central vein intima, disruption of sinusoidal architecture, and the presence of hemorrhage within lesioned areas at 12 and 18 h suggested vascular injury.



View larger version (24K):
[in this window]
[in a new window]
 
FIG. 1. MCT/LPS-induced liver injury. LPS (7.4 x 106 EU/kg) or saline vehicle (Veh) was administered to rats i.v. 4 h after i.p. administration of MCT (100 mg/kg) or Veh. Rats were killed at 6, 12, or 18 h after administration of MCT or its Veh. HPC injury (A) was measured as increases in plasma ALT activity. SEC injury (B) was estimated as increases in plasma HA concentration. N = 4–7 animals per group at each time. aSignificantly different from all other groups at the same time.

 
Hepatic SECs remove HA from the circulation, and impairment of this function results in increased HA concentration in the plasma (Copple et al., 2002aGo; Deaciuc et al., 1993bGo, 1994Go). Accordingly, to estimate hepatic SEC dysfunction plasma HA concentration was measured. No elevation in plasma HA concentration was observed for Veh/Veh or MCT/Veh treatment groups at any of the times analyzed (Fig. 1BGo). Although no elevation in plasma HA concentration was observed in the Veh/LPS treatment group at 6 and 12 h, a modest but significant increase occurred at 18 h. By 6 h, before the onset of HPC injury, plasma HA concentration was significantly elevated in MCT/LPS-cotreated animals, and this increase became progressively more pronounced with time (Fig. 1BGo). The magnitude of the increase in plasma HA is similar to that reported in animals treated with a large, hepatotoxic dose of MCT (Copple et al., 2002aGo).

RECA-1 immunostaining of liver sections has been used to visualize and quantify the loss of endothelial cells in the liver (Copple et al., 2002aGo). 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 2AGo. 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. 2BGo) 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. 2CGo). 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. 2DGo).



View larger version (60K):
[in this window]
[in a new window]
 
FIG. 2. Immunohistochemical staining of liver with RECA-1. Rats were treated with MCT and/or LPS and killed at the indicated times, as described in Figure 1Go legend. Livers were removed and processed for immunohistochemistry with RECA-1, an antibody selective for endothelial cells. (A) Representative photomicrographs of liver sections from a Veh/Veh- and a MCT/LPS-cotreated rat at 18 h. RECA-1 immunostaining (shown in black) is prominent in the central vein (CV) and sinusoidal endothelium and vessels of the portal triad (PT). Bar = 50 µm. (B) Zonal distribution of RECA-1 immunostaining was evaluated at 18 h for all treatment groups. Time courses for total (C) and zonal distribution (D) of RECA-1 immunostaining in the livers of MCT/LPS-cotreated animals were examined. N = 4–7 animals. For (C) and (D), Veh/Veh-treated animals were combined into one group (N = 12), since no differences occurred among Veh/Veh-treated groups at the various times. aSignificantly different from all other groups in same lobular region. bSignificantly different from Veh/Veh group.

 
Representative TEM photomicrographs from livers of Veh/Veh- and MCT/LPS-cotreated animals at 18 h are shown in Figure 3Go. CVECs as well as SECs in CL and MZ regions of liver lobules from Veh/Veh-treated rats exhibited normal morphology (Fig. 3AGo). CVECs formed a distinct, continuous barrier with well-defined organelles. MZ regions had largely continuous SECs with clearly defined spaces of Disse. HPCs within CL and MZ regions were well defined and had distinct, normal-appearing organelles. By comparison, CVECs, as well as SECs, from both CL and MZ regions of MCT/LPS-cotreated rats were markedly altered or absent (Fig. 3BGo). The CVs in CL regions had highly vacuolated remnants of CVECs without discernible organelles or were completely denuded of CVECs. HPCs contained numerous fat droplets, rounded mitochondria, and indistinct plasma membranes. Within the MZ regions, SECs were indistinct and discontinuous with attenuated and electron-lucent cytoplasm. In other MZ areas, the SECs were completely lost. The sinusoids were narrowed and contained fibrin deposits, red blood cells (RBCs), and PMNs. RBCs and cell fragments were visible in the remaining space of Disse. HPCs had indistinct plasma membranes and disrupted mitochondria. Hence, in the livers of MCT/LPS-cotreated animals, CVECs, SECs, and HPCs were injured by 18 h after MCT administration. Similar but less pronounced changes were also evident at 12 h (data not shown).



View larger version (158K):
[in this window]
[in a new window]
 
FIG. 3. Representative TEM photomicrographs from liver sections of Veh/Veh- and MCT/LPS-cotreated rats. Rats were treated with MCT and LPS as described in Figure 1Go legend. They were killed 18 h after administration of MCT or its Veh, and livers were removed and fixed for TEM as described in Materials and Methods. (A) The central vein (CV) and a midzonal (MZ) sinusoid from a Veh/Veh-treated rat have normal endothelial morphology. (B) The CV and a MZ sinusoid from a MCT/LPS-cotreated rat show a highly vacuolated remnant of a central vein endothelial cell (CVEC) and absence of sinusoidal endothelial cells (SECs), respectively. RBC, red blood cell; PMN, polymorphonuclear leukocyte. Magnification in (A) is x12,180 for CV region and x14,820 for MZ region and in (B) is x19,170 for CV region and x12,150 for MZ region.

 
SECs and NP-26 Cells are Morphologically Similar and Form Pyrrolic Metabolites from MCT
Previously, DeLeve et al.(1996)Go demonstrated that MCT is toxic to SECs in vitro and suggested that the toxicity might be due to the production of a toxic metabolite (i.e., MCTP). To determine if MCT is metabolized by SECs, an Ehrlich assay was used to estimate the formation of pyrrolic metabolite(s) in primary SEC isolates and in a transformed rat SEC line (i.e., NP-26 cells).

After incubation with MCT, primary SECs produced pyrrole in a concentration-dependent manner (Fig. 4AGo). 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. 4CGo), a hallmark of SEC morphology (Fig. 4DGo). Pyrrole production also occurred in NP-26 cells in a MCT concentration-dependent manner (Fig. 4AGo). 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. 4BGo).



View larger version (83K):
[in this window]
[in a new window]
 
FIG. 4. Pyrrole formation from MCT in SECs in vitro. (A) During incubation with various concentrations of MCT, pyrrolic metabolites are formed by primary SECs and NP-26 cells. Pyrrole formation in primary SECs was corrected for the contribution of contaminating hepatocytes (see Materials and Methods). (B) Representative immunoblot revealing CYP3A2 protein in the microsomal fraction of hepatocytes (Lane 1) and NP-26 cells (Lane 2). Representative SEM photomicrographs are depicted for NP-26 cells (C) and primary SECs (D), demonstrating fenestrae on the cell surface (solid black arrows). Bar = 2 µm. N = 3–4 animals. aSignificantly different from Veh control.

 
Liver Hemorrhage
To quantify the degree of hemorrhage in the livers of MCT/LPS-cotreated animals, the concentration of tissue hemoglobin was determined. A time-dependent increase in liver hemoglobin began within 12 h in the MCT/LPS-cotreated group and remained significant at 18 h (Fig. 5AGo). No other treatment group exhibited increased concentration of liver hemoglobin.



View larger version (25K):
[in this window]
[in a new window]
 
FIG. 5. Time course for liver hemoglobin and coagulation system activation in MCT/LPS-coexposed rats. Rats were treated with MCT and/or LPS and killed at the indicated times, as described in Figure 1Go legend. (A) Livers were removed and analyzed for tissue hemoglobin, a biomarker of hemorrhage. (B) Plasma fibrinogen, a biomarker for coagulation system activation, was also analyzed. N = 3–7 animals. aSignificantly different from all other groups at the same time.

 
Coagulation System Activation
During activation of the coagulation system, fibrinogen is converted to fibrin, resulting in a decrease in plasma fibrinogen concentration. Hence, plasma fibrinogen is a biomarker for activation of the coagulation system. No decrease in plasma fibrinogen was observed at any time in groups treated with Veh, LPS, or MCT alone. By contrast, plasma fibrinogen concentration decreased significantly in MCT/LPS-cotreated animals by 6 h (Fig. 5BGo). This decrease occurred before the onset of HPC injury (Fig. 1AGo). Plasma fibrinogen concentration from MCT/LPS-cotreated animals remained significantly depressed at 12 and 18 h.

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. 6AGo). However, some staining occurred in the intima of larger vessels, which results from fibrin deposition that occurs after animal sacrifice (Copple et al., 2002aGo). By contrast, intense fibrin staining occurred in the sinusoids of CL and MZ regions of livers from rats treated with MCT/LPS (Fig. 6AGo). 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. 6BGo). 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. 6CGo). 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. 6DGo).



View larger version (50K):
[in this window]
[in a new window]
 
FIG. 6. Hepatic fibrin deposition. Animals were treated with MCT and/or LPS and killed at the indicated times, as described in Figure 1Go legend. Livers were removed and processed for fibrin immunohistochemistry. (A) Representative photomicrographs of liver sections from a Veh/Veh- and a MCT/LPS-cotreated rat at 18 h immunostained for fibrin deposits, which appear black. CV, central vein; PT, portal triad. Bar = 50 µm. (B) Zonal distribution of fibrin immunostaining was evaluated at 18 h. Time courses for total fibrin immunostaining (C) and zonal fibrin distribution (D) in the livers of MCT/LPS-cotreated animals were determined. N = 4–7 animals. For (C) and (D), Veh/Veh-treated animals were combined into one group (N = 12), since no differences occurred among Veh/Veh-treated groups at the various times. aSignificantly different from all other groups in same lobular region. bSignificantly different from Veh/Veh group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased plasma HA concentration has been used as a biomarker of SEC dysfunction and injury in vivo (Copple et al., 2002aGo; Shimizu et al., 1994Go). A significant increase in plasma HA concentration occurred in MCT/LPS-cotreated animals within 6 h, and the concentration remained elevated until 18 h after MCT administration (Fig. 1BGo). The pronounced rise in plasma HA concentration preceded the onset of HPC injury (Fig. 1AGo), suggesting that SEC dysfunction might be causally involved in HPC injury. Additionally, a modest increase in plasma HA concentration was observed at 18 h in rats given only LPS (Fig. 1BGo). This increase suggests functional impairment of SECs (Deaciuc et al., 1993aGo; Sarphie et al., 1996Go; Takeuchi et al., 1994Go), but in rats treated only with LPS this was not accompanied by HPC injury at 18 h or times thereafter (Yee et al., 2000Go, 2003bGo). Therefore, either the extent of SEC dysfunction that occurred in rats given LPS alone was of insufficient magnitude to produce HPC injury, or HPC injury depended on factors in addition to SEC dysfunction that are absent in animals treated with LPS alone. Alternatively, HPC injury in MCT/LPS-cotreated animals may occur independently of SEC injury.

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. 2DGo). 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., 2002aGo).

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. 3Go), 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)Go 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., 1997Go; Lester et al., 1993Go), 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., 1997Go; Reid et al., 1998Go). 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. 4AGo). In addition, a ~72 kDa protein recognized by the CYP3A2 specific antibody was expressed in the microsomal fractions from NP-26 cells (Fig. 4BGo). 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, 1991Go; Thomas et al., 1998Go; Wilson et al., 1998Go). 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. 1BGo). 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, 1991Go; Sarphie et al., 1996Go; Springer, 1994Go; Ward and Varani, 1990Go; Yachida et al., 1998Go). Yee et al. (2003aGo,b)Go demonstrated that Kupffer cell inactivation, TNF-{alpha} 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., 1993Go; Reid et al., 1999Go). DeLeve et al.(1996)Go 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-{alpha}) results in SECs becoming procoagulant (Stern et al., 1985Go; Takeuchi et al., 1994Go). Moreover, SEC injury or destruction can result in the activation of the coagulation system (Copple et al., 2002aGo; Hirata et al., 1989Go; Machovich, 1985Go; Ryan, 1986Go). In MCT/LPS-cotreated animals, plasma fibrinogen concentration decreased significantly by 6 h after MCT administration and remained depressed through 18 h (Fig. 5BGo). 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. 6CGo). Both CL and MZ regions of the liver lobule experienced fibrin deposition at these times (Fig. 6DGo). TEM confirmed the immunohistochemical evidence of fibrin deposition in the sinusoids (Fig. 3BGo). 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., 1989Go; Vollmar et al., 1993Go; Yachida et al., 1998Go). Moreover, activation of SECs by TNF-{alpha} 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, 1994Go; Vollmar et al., 1996Go; Yachida et al., 1998Go). It has been postulated that consequent sinusoidal hypoperfusion leads to ischemic/hypoxic injury to HPCs within affected regions (Copple et al., 2002aGo; DeLeve et al., 1996Go; Fujiwara et al., 1988Go; Hirata et al., 1989Go; Mochida et al., 1999Go; Yee et al., 2000Go). Consistent with this hypothesis is the observation in MCT/LPS-treated rats that both SEC injury (Fig. 1BGo) and coagulation system activation (Fig. 5BGo) precede liver hemorrhage (Fig. 5AGo), 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., 2003Go). 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, 2000Go; Wisse et al., 1996Go). As described above, treatment of endothelial cells with MCTP results in detrimental cytoskeletal, junctional barrier, and permeability changes (Reindel and Roth, 1991Go; Thomas et al., 1998Go; Wilson et al., 1998Go). 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., 1993Go; Farhood et al., 1995Go; Jaeschke et al., 1996Go; Yachida et al., 1998Go; Yoshidome et al., 2000Go). 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., 2002aGo; Deaciuc and Spitzer, 1996Go; DeLeve et al., 1996Go; Sarphie et al., 1996Go). 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.


    ACKNOWLEDGMENTS
 
The authors are grateful for the technical assistance of Zinah Hong, Yana Itkin, and Stacy Schwartz. The authors would also like to thank Dr. Jack R. Harkema for his assistance with histopathological analysis and Ralph Commons, Donna Craft, and Ewa Danielewicz for their technical assistance with electron microscopy. This study was supported by NIH grant ES 04139. S.B.Y. was supported in part by NIEHS training grant T32 ES 07255.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, B440 Life Sciences Building, Michigan State University, East Lansing, MI 48824. Fax: (517) 353–8915. E-mail: rothr{at}msu.edu Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allen, J. R., Chesney, C. F., and Frazee, W. J. (1972). Modifications of pyrrolizidine alkaloid intoxication resulting from altered hepatic microsomal enzymes. Toxicol. Appl. Pharmacol. 23, 470–479.[ISI][Medline]

Aria, M., Mochida, S., Ohno, A., and Fujiwara, K. (1996). Blood coagulation in the hepatic sinusoids as a contributing factor in liver injury following orthotopic liver transplantation in the rat. Transplantation 62, 1398–1401.[CrossRef][ISI][Medline]

Aria, M., Mochida, S., Ohno, A., Ogata, I., and Fujiwara, K. (1993). Sinusoidal endothelial cell damage by activated macrophages in rat liver necrosis. Gastroenterology 104, 1466–1471.[ISI][Medline]

Arii, S., and Imamura, M. (2000). Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury. J. Hepatobiliary Pancreat. Surg. 7, 40–48.[CrossRef][Medline]

Ba, Z. F., Wang, P., Koo, D. J., Cioffi, W. G., Bland, K. I., and Chaudry, I. H. (2000). Alterations in tissue oxygen consumption and extraction after trauma and hemorrhagic shock. Crit. Care Med. 28, 2837–2842.[CrossRef][ISI][Medline]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.[CrossRef][ISI][Medline]

Braet, F., De Zanger, R., Sasaoki, T., Baekeland, M., Janssens, P., Smedsrød, B., and Wisse, E. (1994). Assessment of a method of isolation, purification and cultivation of rat liver sinusoidal endothelial cells. Lab. Invest. 70, 944–952.[ISI][Medline]

Braet, F., Spector, I., Shochet, N., Crews, P., Higa, T., Menu, E., De Zanger, R., and Wisse, E. (2002). The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells. BMC Cell Biol. 3, 7.[CrossRef][Medline]

Brouwer, A., Parker, S. G., Hendriks, H. F. J., Gibbons, L., and Horan, M. A. (1995). Production of eicosanoids and cytokines by Kupffer cells from young and old rats stimulated by endotoxin. Clin. Sci. 88, 211–217.[ISI][Medline]

Brown, A. P., Harkema, J. R., Schultze, A. E., Roth, R. A., and Ganey, P. E. (1997). Gadolinium chloride pretreatment protects against hepatic injury but predisposes the lung to alveolitis after lipopolysaccharide administration. Shock 7, 186–192.[ISI][Medline]

Coffman, B. L., Rios, G. R., and Tephly, T. R. (1999). Preparation of microsomes from cultured cells. In Current Protocols in Toxicology (M. D. Maines, Ed.), pp. 4.3.1–4.3.15. John Wiley and Sons, New York.

Copple, B. L., Banes, A., Ganey, P. E., and Roth, R. A. (2002a). Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure. Toxicol. Sci. 65, 309–318.[Abstract/Free Full Text]

Copple, B. L., Moulin, F., Hanumegowda, U., Ganey, P. E., and Roth, R. A. (2003). Thrombin and PAR-1 agonists promote lipopolysaccharide-induced hepatocellular injury in perfused livers. J. Pharmacol. Exp. Ther. 305, 417–425.[Abstract/Free Full Text]

Copple, B. L., Woolley, B., Banes, A., Ganey, P. E., and Roth, R. A. (2002b). Anticoagulants prevent monocrotaline-induced hepatic parenchymal cell injury but not endothelial cell injury in the rat. Toxicol. Appl. Pharmacol. 180, 186–196.[CrossRef][ISI][Medline]

Deaciuc, I. V., Bagby, G. J., Lang C. H., Skrepnik, N, and Spitzer, J. J. (1993a). Gram negative bacterial lipopolysaccharide impairs hyaluronan clearance in vivo and its uptake by isolated, perfused rat liver. Hepatology 18, 173–179.[CrossRef][ISI][Medline]

Deaciuc, I. V., Bagby, G. J., Neisman, M. R., Skrepnik, N., and Spitzer, J. J. (1994). Modulation of hepatic sinusoidal endothelial cell function by Kupffer cells: An example of intracellular communication in the liver. Hepatology 19, 464–470.[ISI][Medline]

Deaciuc, I. V., McDonough, K. H., Bagby, G. J., and Spitzer, J. J. (1993b). Alcohol consumption in rats potentiates the deleterious effects of gram-negative sepsis on hepatic hyaluronan uptake. Alcohol Clin. Exp. Res. 17, 1002–1008.[ISI][Medline]

Deaciuc, I. V., and Spitzer, J. J. (1996). Hepatic sinusoidal endothelial cell in alcoholemia and endotoxemia. Alcohol Clin. Exp. Res. 20, 607–614.[ISI][Medline]

DeLeve, L. D., McCuskey, R. S., Wang, X., Hu, L., McCuskey, M. K., Epstein, R. B., and Kanel, G. C. (1999). Characterization of a reproducible rat model of hepatic veno-occlusive disease. Hepatology 29, 1779–1791.[ISI][Medline]

DeLeve, L. D., Wang, X., Kaplowitz, N., Shulman, H. M., Bart, J. A., and van der Hoek, A. (1997). Sinusoidal endothelial cells as a target for acetaminophen toxicity: Direct action versus requirement for hepatocyte activation in different mouse strains. Biochem. Pharmacol. 53, 1339–1345.[CrossRef][ISI][Medline]

DeLeve, L. D., Wang, X., Kuhlenkamp, J. F., and Kaplowitz, N. (1996). Toxicity of aziothioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: The role of glutathione and relevance to hepatic venoocclusive disease. Hepatology 23, 589–599.[ISI][Medline]

Duijvestijn, A. M., van Goor, H., Klatter, F., Majoor, G. D., van Bussel, E., and van Breda Vriesman, P. J. (1992). Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody. Lab. Invest. 66, 459–466.[ISI][Medline]

Farhood, A., McGuire, G. M., Manning, A. M., Miyasaka, M., Smith, C. W., and Jaeschke, H. (1995). Intracellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury. J. Leukoc. Biol. 57, 368–374.[Abstract]

Fujiwara, H., Ogata, I., Ohta, Y., Hirata, K., Oka, Y., Yamada, S., Sato, Y., Masaki, N, and Oka, H. (1988). Intravascular coagulation in acute liver failure in rats and its treatments with anti-thrombin III. Gut 29, 1103–1108.[Abstract]

Ganey, P. E., and Roth, R. A. (2001). Concurrent inflammation as a determinant of susceptibility to toxicity from xenobiotic agents. Toxicology 169, 195–208.[CrossRef][ISI][Medline]

Hewett, J. A., and Roth, R. A. (1993). Hepatic and extrahepatic pathobiology of bacterial lipopolysaccharides. Pharmacol. Rev. 45, 381–411.[ISI]

Hewett, J. A., and Roth, R. A. (1995). The coagulation system, but not circulating fibrin, contributes to liver injury in rats exposed to lipopolysaccharide from gram-negative bacteria. J. Pharmacol. Exp. Ther. 272, 53–62.[Abstract]

Hewett, J. A., Schultze, A. E., VanCise, S., and Roth, R. A. (1992). Neutrophil depletion protects against liver injury from bacterial endotoxin. Lab. Invest. 66, 347–361.[ISI][Medline]

Hirata, K., Ogata, I., Ohta, Y., and Fujiwara, K. (1989). Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats. J. Pathol. 158, 157–165.[ISI][Medline]

Holst, O., Ulmer, A. J., Brade, H., Flad, H.-D., and Rietschel, E. T. (1996). Biochemistry and cell biology of bacterial endotoxins. FEMS Immunol. Med. Microbiol. 16, 83–104.[CrossRef][ISI][Medline]

Jaeschke, H., and Farhood, A. (1991). Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am. J. Physiol. 260, G355–G362.[ISI][Medline]

Jaeschke, H., Farhood, A., Cai, S. X., Tseng, B. L., and Bajt, M. L. (2000). Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice. Toxicol. Appl. Pharmacol. 169, 77–83.[CrossRef][ISI][Medline]

Jaeschke, H., Smith, C. W., Clemens, M. G., Ganey, P. E., and Roth, R. A. (1996). Mechanisms of inflammatory liver injury: Adhesion molecules and cytotoxicity of neutrophils. Toxicol. Appl. Pharmacol. 139, 213–226.[CrossRef][ISI][Medline]

Kasahara, Y., Kiyatake, K., Tatsumi, K., Sugito, K., Kakusaka, I., Yamagata, S.-I., Ohmori, S, Kitada, M., and Kuriyama, T. (1997). Bioactivation of monocrotaline by P-450 3A in rat liver. J. Cardiovasc. Pharmacol. 30, 124–129.[CrossRef][ISI][Medline]

Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Chako, J., and Trump, B. F. (1981). Mouse liver cell culture. I. Hepatocyte isolation. In Vitro 17, 913–925.[ISI][Medline]

Lester, S. M., Braunbeck, T. A., The, S. J., Stegeman, J. J., Miller, M. R., and Hinton, D. E. (1993). Hepatic cellular distribution of cytochrome P-450 IA1 in rainbow trout (Oncorhynchus mykiss): An immunihisto- and cytochemical study. Cancer Res. 53, 3700–3706.[Abstract]

Machovich, R. (1985). Blood coagulation-fibrinolytic system and endothelial cells. Acta Biochim. Biophys. Acad. Sci. Hung. 20, 135–153.[Medline]

Maru, Y., Yamaguchi, S., Takahashi, T., Ueno, H., and Shibuya, M. (1998). Virally activated Ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines. J. Cell, Physiol. 176, 223–234.[CrossRef][ISI][Medline]

Mattocks, A. R. (1986). Chemistry and Toxicology of Pyrrolizidine Alkaloids, pp. 1–393. Academic Press, London.

Mattocks, A. R., and White, I. N. H. (1970). Estimation of metabolites of pyrrolizidine alkaloids in animal tissues. Anal. Biochem. 38, 529–535.[ISI][Medline]

Michie, H. R., Manogue, K. B., Spriggs, D. R., Revhaug, A., O’Dwyer, S., Dinarello, C. A., Cerami, A., Wolff, S. M., and Wilmore, D. W. (1988). Detection of circulating tumor necrosis factor after endotoxin administration. N. Engl. J. Med. 318, 1481–1486.[Abstract]

Mochida, S., Arai, M., Ohno, A., Yamanobe, F., Ishikawa, K., Matsui, A., Maruyama, I., Kato, H., and Fujiwara, K. (1999). Deranged blood coagulation equilibrium as a factor of massive liver necrosis following endotoxin administration in partially hepatectomized rats. Hepatology 29, 1532–1540.[CrossRef][ISI][Medline]

Moulin, F., Pearson, J. M., Schultze, A. E., Scott, M. A., Schwartz, K. A., Davis, J. M., Ganey, P. E., and Roth, R. A. (1996). Thrombin is a distal mediator of lipopolysaccharide-induced liver injury in the rat. J. Surg. Res. 65, 149–158.[CrossRef][ISI][Medline]

Pan, L. C., Wilson, D. W., Lamé, M. W., Jones, A. D., and Segall, H. J. (1993). Cor pulmonale is caused by monocrotaline and dehydromonocrotaline, but not by glutathione or cysteine conjugates of dihydropyrrolizidine. Toxicol. Appl. Pharmacol. 118, 87–97.[CrossRef][ISI][Medline]

Perry, E. W. (1984). A lethal syndrome in mice following administration of carbon tetrachloride and cycloheximide, and its prevention by heparin treatment. J. Comp. Pathol. 94, 505–508.[CrossRef][ISI][Medline]

Reid, M. J., Dunston, S. K., Lamé, M. W., Wilson, D. W., Morin, D., and Segall, H. J. (1999). Effect of monocrotaline metabolites on glutathione levels in human and bovine pulmonary artery endothelial cells. Res. Commun. Mol. Pathol Pharmacol. 99, 53–68.[ISI]

Reid, M. J., Lamé, M. W., Morin, D., Wilson, D. W., and Segall, H. J. (1998). Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14C-monocrotaline in rat liver microsomes. J. Biochem. Mol. Toxicol. 12, 157–166.[CrossRef][Medline]

Reindel, J. F., and Roth, R. A. (1991). The effects of monocrotaline pyrrole on cultured pulmonary artery endothelial cells and smooth muscle cells. Am. J. Pathol. 138, 707–719.[Abstract]

Ryan, U. S. (1986). The endothelial surface and responses to injury. Fed. Proc. 45, 101–108.[ISI][Medline]

Saetre, T., Lindgaard, A. K., and Lyberg, T. (2000). Systemic activation of coagulation and fibrinolysis in a porcine model of serogroup A streptococcal shock. Blood Coagul. Fibrinolysis 11, 433–438.[CrossRef][ISI][Medline]

Sarphie, T. G., D’Souza, N. B., and Deaciuc, I. V. (1996). Kupffer cell inactivation prevented lipopolysaccharide-induced structural changes in the rat liver sinusoid: An electron-microscopic study. Hepatology 23, 788–796.[ISI][Medline]

Schnitt, S. J., Stillman, I. E., Owings, D. V., Kishimoto, C., Dorvak, H. F., and Abelmann, W. H. (1993). Myocardial fibrin deposition in experimental viral myocarditis that progresses to dilated cardiomyopathy. Circ. Res. 72, 914–920.[Abstract]

Schoental, R., and Head, M. A. (1955). Pathological changes in rats as a result of treatment with monocrotaline. Br. J. Cancer 9, 229–237.[ISI][Medline]

Seglen, P. O. (1973). Preparation of rat liver cells. III. Enzymatic requirements for dispersion. Exp. Cell Res. 82, 391–398.[ISI][Medline]

Seto, S., Kaido, T., Yamaoka, S., Yoshikawa, A., Arii, S., Nakamura, T., Niwano, M., and Imamura, M. (1998). Hepatocyte growth factor prevents lipopolysaccharide-induced hepatic sinusoidal endothelial cell injury and intrasinusoidal fibrin deposition in rats. J. Surg. Res. 80, 194–199.[CrossRef][ISI][Medline]

Sharp, J. G., Bishop, M. R., Copple, B., Greiner, T. C., Iversen, P. L., Jackson, J. D., Joshi, S. S., Benner, E. J., Mann, S. L., Rao, A. K., et al. (2001). Oligonucleotide enhanced cytotoxicity of Idarubicin for lymphoma cells. Leuk. Lymphoma 42, 417–427.[ISI][Medline]

Shibayama, Y. (1987). Sinusoidal circulatory disturbance by microthrombosis as a cause of endotoxin-induced hepatic injury. J. Pathol. 151, 315–321.[ISI][Medline]

Shimizu, H., He, W., Guo, P., Dziadkoviec, I., Miyazaki, M, and Falk, R. E. (1994). Serum hyaluronate in the assessment of liver endothelial cell function after orthotopic liver transplantation in the rat. Hepatology 20, 1323–1329.[CrossRef][ISI][Medline]

Spitzer, J. A., and Mayer, A. M. S. (1993). Hepatic neutrophil influx: Eicosanoid and superoxide formation in endotoxemia. J. Surg. Res. 55, 60–67.[CrossRef][ISI][Medline]

Springer, T. A. (1994). Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76, 301–314.[ISI][Medline]

Steele, R. G. D., Torrie, J. H., and Dickey, D. A. (1997). Principles and Procedures in Statistics: A Biometric Approach, 3rd ed., pp. 1–603. McGraw-Hill, New York.

Stegelmeier, B. L., Edgar, J. A., Colegate, S. M., Gardner, D. R., Scloch, T. K., Coulombe, R. A., and Molyneux, R. J. (1999). Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8, 95–116.[ISI][Medline]

Stern, D., Nawroth, P., Handley, D., and Kisiel, W. (1985). An endothelial cell- dependent pathway of coagulation. Proc. Natl. Acad. Sci. U.S.A. 82, 2523–2527.[Abstract]

Takeuchi, M., Nakashima, Y., Miura, Y., Nakagawa, K., Uragoh, K., Iwanaga, S. Hori, Y., and Sueishi, K. (1994). The localization of lipopolysaccharide in an endotoxemic rat liver and its relation to sinusoidal thrombogenesis: Light and electron microscopic studies. Path. Res. Pract. 190, 1123–1133.[ISI][Medline]

Thomas, H. C., Lamé, M. W., Dunston, S. K., Segall, H. J., and Wilson, D. W. (1998). Monocrotaline pyrrole induced apoptosis in pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 151, 236–244.[CrossRef][ISI][Medline]

Vollmar, B., Lang, G., Post, S., Menger, M. D, and Messmer, K. (1993). Microcirculation of the liver in hemorrhagic shock in the rat and its significance for energy metabolism and function. Zentralbl. Chir. 118, 218–225.[ISI][Medline]

Vollmar, B., Richter, S., and Menger, M. D. (1996). Leukocyte stasis in hepatic sinusoids. Am. J. Physiol. 270, G798–G803.[ISI][Medline]

Ward, P. A., and Varani, J. (1990). Mechanisms of neutrophil-mediated killing of endothelial cells. J. Leukoc. Biol. 48, 97–102.[ISI][Medline]

White, I. N. H., and Mattocks, A. R. (1972). Reaction of dihydropyrrolizidines with deoxyribonucleic acids in vitro. Biochem. J. 128, 291–297.[ISI][Medline]

Wilson, D. W., Lamé, M. W., Dunston, S. K., Taylor, D. W., and Segall, H. J. (1998). Monocrotaline pyrrole interacts with actin and increases thrombin-mediated permeability in pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 152, 138–144.[CrossRef][ISI][Medline]

Wisse, E., Braet, F., Lou, D., De Zanger, R., Jans, D., Crabbé, E., and Vermoesen, A. (1996). Structure and function of sinusoidal cells in the liver. Toxicol. Pathol. 24, 100–111.[ISI][Medline]

Yachida, S., Kokudo, Y., Wakabayashi, H., Maeba, T., Kenada, K., and Maeta, H. (1998). Morphological and functional alterations to sinusoidal endothelial cells in the early phase of endotoxin-induced liver failure after partial hepatectomy in rats. Virchows Arch. 433, 173–183.[CrossRef][ISI][Medline]

Yamada, S., Ogata, I., Hirata, K., Mochida, S., Tomiya, T., and Fujiwara, K. (1989). Intravascular coagulation in the development of massive hepatic necrosis induced by Coryebacterium parvum and endotoxin in rats. Scand. J. Gastroenterol. 24, 293–298.[ISI][Medline]

Yee, S. B., Ganey, P. E., and Roth, R. A. (2003a). The role of Kupffer Cells and TNF-{alpha} in monocrotaline and bacterial lipopolysaccharide-induced liver injury. Toxicol. Sci. 71, 124–132.[Abstract/Free Full Text]

Yee, S. B., Hanumegowda, U. M., Hotchkiss, J. A., Ganey, P. E., and Roth, R. A. (2003b). Role of neutrophils in the synergistic liver injury from monocrotaline and bacterial lipopolysaccharide exposure. Toxicol. Sci. 72, 43–56.[Abstract/Free Full Text]

Yee, S. B., Kinser, S., Hill, D. A., Barton, C. C., Hotchkiss, J. A., Harkema, J. R., Ganey, P. E., and Roth, R. A. (2000). Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline. Toxicol. Appl. Pharmacol. 166, 173–185.[CrossRef][ISI][Medline]

Yoshidome, H., Miyazaki, M., Shimizu, H., Ito, H., Nakagawa, K., Ambiru, S., Nakajima, N., Edwards, M. J., and Lentsch, A. B. (2000). Obstructive jaundice impairs hepatic sinusoidal endothelial cell function and renders liver susceptible to hepatic ischemia/reperfusion. J. Hepatol. 33, 59–67.[CrossRef][ISI][Medline]