Departments of Molecular and Cellular Physiology and Medicine, Center of Excellence in Arthritis and Rheumatology, Lousiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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The overall objective of
this study was to determine whether genetically induced
hypercholesterolemia alters the inflammatory and microvascular
responses of mouse liver to ischemia-reperfusion (I/R). The
accumulation of rhodamine 6G-labeled leukocytes and the number of
nonperfused sinusoids (NPS) were monitored (by intravital microscopy)
in the liver of wild-type (WT) and low-density lipoprotein receptor-deficient (LDLr/
) mice for 1 h after a
30-min period of normothermic ischemia. Plasma alanine
transaminase (ALT) levels were used to monitor hepatocellular injury.
Microvascular leukostasis as well as increases in NPS and plasma ALT
were observed at 60 min after hepatic I/R in both WT and in
LDLr
/
mice; however, these responses were greatly
exaggerated in LDLr
/
mice. Pretreatment of
LDLr
/
mice with gadolinium chloride, which reduces
Kupffer cell function, attenuated the hepatic leukostasis, NPS, and
hepatocellular injury elicited by I/R. Similar protection against I/R
was observed in LDLr
/
mice pretreated with antibodies
directed against tumor necrosis factor-
, intercellular adhesion
molecule-1 (ICAM-1), or P-selectin. These findings indicate that
chronic hypercholesterolemia predisposes the hepatic microvasculature
to the deleterious effects of I/R. Kupffer cell activation and the
leukocyte adhesion receptors ICAM-1 and P-selectin appear to contribute
to the exaggerated inflammatory responses observed in the postischemic
liver of LDLr
/
mice.
hypercholesterolemia; leukocyte-endothelial cell adhesion; Kupffer cells; intercellular adhesion molecule-1; P-selectin
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INTRODUCTION |
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HEPATIC ISCHEMIA-REPERFUSION (I/R) has been implicated in the pathogenesis of a variety of clinical conditions including trauma, hypovolemic shock with resuscitation, tumor resection, and liver transplantation. The recognition that I/R may contribute to the liver dysfunction and hepatocellular necrosis that are associated with these disease processes has resulted in an intensive effort to define the cellular and molecular events that underlie this injury response. An outgrowth of this effort is the revelation that 1) reperfusion injury in the liver is mainly an inflammatory cell-mediated injury process, and 2) the hepatic microvasculature is particularly vulnerable to the deleterious effects of I/R (17, 21, 28). In addition, it is now widely recognized that reperfusion of the ischemic liver often leads to leukostasis in sinusoids and leukocyte adherence in terminal hepatic venules, activation of intravascular macrophages (Kupffer cells), an increased expression of the endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule-1 (ICAM-1), a reduction in the number of perfused sinusoids, tissue hypoxia, loss of hepatocellular integrity (reflected as an increased plasma level of liver enzymes), and a reduction in bile formation (28). Kupffer cells appear to play a major role in the sinusoidal malperfusion and inflammatory cell infiltration that are associated with hepatic I/R (19, 28). Activated Kupffer cells protrude into the sinusoidal lumen where they come into intimate contact with circulating blood cells and can impede the movement of activated and stiffened leukocytes (28). These resident phagocytic cells can also produce large quantities of oxygen radicals and release inflammatory mediators (e.g., cytokines), which can further amplify the I/R-induced inflammatory cell infiltration by enhancing the expression of endothelial cell adhesion molecules, such as ICAM-1 and P-selectin (13, 31, 34).
Hypercholesterolemia is an important risk factor for the development of
atherosclerosis and a number of diverse diseases. The chronic
inflammatory nature of atherosclerotic lesions has been described as
consisting of inflammatory cell infiltrates, enhanced cytokine
production, and an increased expression of endothelial cell adhesion
molecules (32). Although these inflammatory manifestations of hypercholesterolemia are generally assumed to occur exclusively in
major arterial vessels, the results of recent studies (11, 30) suggest that the inflammatory cell-mediated pathology may also extend to the arterial and venous segments of the
microcirculation. Enhanced microvascular responses to inflammatory
stimuli, such as cytokines (11) and I/R (30),
have been described in skeletal muscle of low-density lipoprotein
receptor-deficient (LDLr/
) mice, a frequently employed
animal model of hypercholesterolemia that closely resembles familial
hypercholesterolemia in humans (16). Given the unique
responsiveness of the hepatic microvasculature to I/R and its
dependence on activated Kupffer cells for developing the
reperfusion-induced inflammatory responses, it is not clear whether the
deleterious effects of hypercholesterolemia that have been described
for some peripheral vascular beds (e.g., skeletal muscle) are also
manifested in the postischemic hepatic microvasculature. This
unresolved issue was addressed in the present study by addressing the
following specific questions: 1) Does genetically induced hypercholesterolemia alter the hepatic microvascular and inflammatory responses to I/R? 2) Are Kupffer cells involved in I/R
responses associated with hypercholesterolemia? 3) Do
P-selectin and ICAM-1, which have been implicated in the pathogenesis
of hepatic I/R in otherwise normal animals (33, 37),
contribute to the hypercholesterolemia-induced responses to I/R? These
issues were addressed by applying the technique of intravital
videomicroscopy to monitor inflammatory and vascular changes in the
hepatic microvasculature after I/R in wild-type (WT) and
LDLr
/
mice.
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MATERIALS AND METHODS |
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Animals.
C57BL mice (background strain for the LDLr/
mice,
n = 13) and LDLr
/
(n = 38) mice were obtained from Jackson Laboratories. The mice were fed a
standard chow and fasted for 18 h before the experiment.
Surgical procedure. After administration of atropine sulfate (0.04 mg/kg body wt im), the mice were anesthetized with ketamine hydrochloride (150 mg/kg body wt im) and xylazine (7.5 mg/kg body wt im). The right carotid artery was cannulated, and systemic arterial pressure was measured with a Statham P23A pressure transducer (Gould, Oxnard, CA) connected to the carotid artery cannula. Systemic blood pressure and heart rate were continuously recorded with a physiological recorder (Grass Instruments, Quincy, MA). The left jugular vein was also cannulated for drug administration. After laparotomy, the blood supply to the hepatic left lateral lobe was occluded for 0 (sham) or 30 min using microvascular clips. During the ischemic period, the abdomen was covered with an abdominal muscle flap and wet gauze. After the ischemic period, the clip was gently removed. The experimental procedures described herein were performed according to the criteria outlined in the National Institutes of Health guidelines and were approved by the Lousiana State University Health Sciences Center Animal Care and Use Committee.
Intravital microscopy. The technique of intravital videomicroscopy was applied to the liver microcirculation as previously described (12, 13). Immediately after the removal of the clip, the mouse was placed on a microscope stage. The left lateral lobe of liver was observed with an inverted intravital microscope (TMD-2S, Diaphot, Nikon, Tokyo, Japan) assisted by a silicon intensified target camera (C-2400-08, Hamamatsu Photonics, Shizuoka, Japan). The liver was placed on an adjustable Plexiglas microscope stage with a nonfluorescent coverslip that allowed for observation of a 2-cm2 segment of tissue. The liver was carefully placed to minimize the influence of respiratory movements. The liver surface was moistened and covered with cotton gauze soaked with saline. Images of the microcirculation near the surface of the liver were observed through a ×40 fluorescent objective lens (Fluor 40/0.85, Nikon, Tokyo, Japan). The microfluorographs were recorded on videotape using a videocassette recorder (NV8950, Panasonic, Tokyo, Japan). A video time-date generator (WJ810, Panasonic) projected the stopwatch function onto the monitor.
Analysis of leukocyte accumulation and sinusoidal perfusion in liver microcirculation. Leukocytes were labeled in vivo with rhodamine 6G (2 mg were dissolved in 5 ml of 0.9% saline) using a previously described method (12, 36). It has recently been shown that rhodamine 6G selectively stains white blood cells and platelets but not endothelial cells (27). Thus the fluorochrome allows for differentiation between adherent leukocytes and endothelial cells. Rhodamine 6G (40 µl/10 g body wt) was injected before reperfusion, with subsequent injections every 30 min. Rhodamine 6G-associated fluorescence was visualized by epi-illumination at 510-560 nm, using a 590-nm emission filter. The number of stationary leukocytes was determined off-line during playback of videotape images. A leukocyte was considered stationary within the microcirculation [sinusoids and terminal hepatic venules (THV)] if it remained stationary for more than 10 s. The sinusoid was considered to be perfused if the labeled white blood cells or platelets were observed moving through it. The percentage of nonperfused sinusoids was calculated as the ratio of the number of nonperfused sinusoids to the total number of sinusoids per viewing field. Stationary leukocytes were quantified in both the midzonal and pericentral regions of the liver lobule and expressed as the number per field of view (8.3 × 104 µm2; Refs. 12, 13).
Experimental protocols.
The blood supply to the hepatic left lateral lobe was occluded with
microvascular clips for 0 (sham) or 30 min. After the normothermic
ischemic period, the clip was gently removed. Leukocyte accumulation
and the number of nonperfused sinusoids were measured 15 min after
reperfusion and every 15 min for 45 min thereafter, i.e., for 60 min
after reperfusion. In some experiments, the resident population of
Kupffer cells were depleted by administering (iv) GdCl3 (10 mg/kg: ICN Biomedicals, Aurora, OH) at 24 h before the experiments, as previously described (13, 25).
Other groups of mice received an antibody directed against tumor
necrosis factor- (TNF-
; 6mg/kg ip; Ref. 13), ICAM-1
monoclonal antibody [(MAb) YN-1, 4 mg/kg iv], or P-selectin (MAb
RB40.34, 2 mg/kg iv; Refs. 13, 33), and the
same protocol as described previously was followed. The antibodies were
administered 10-60 min before the induction of ischemia.
Enzyme assay. Blood samples for plasma alanine transaminase (ALT) activity were collected from the carotid artery immediately after obtaining the 60-min reperfusion measurements. ALT activity was determined from these samples using a spectrophotometric assay obtained as a commercial kit (Sigma, St. Louis, MO).
Statistics. The data were analyzed using standard statistical analyses, i.e., one-way ANOVA and Scheffé's (post hoc) test. All values are reported as means ± SE, with at least 6 mice per group. Statistical significance was set at P < 0.05.
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RESULTS |
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Figure 1 presents the plasma
cholesterol levels that were measured in WT and LDLr/
mice fed normal rodent chow. Plasma cholesterol concentration was 3.1 times higher in LDLr
/
than in WT mice. Circulating
leukocyte counts (at 60 min after reperfusion) for the WT and
LDLr
/
mice were 4,157 ± 447 and 3,957 ± 452 per mm3 , respectively. The values obtained for all other
(treatment) groups did not differ significantly from either the WT or
LDLr
/
(untreated) groups.
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Figure 2 summarizes the time course of
changes in leukocyte accumulation in the midzonal (Fig. 2A)
and pericentral (Fig. 2B) regions of the liver
microcirculation and in THV (Fig. 2C) and the total number
of accumulated leukocytes per viewing field (Fig. 2D) after
30 min of ischemia and 60 min of reperfusion in WT and LDLr/
mice. In sham-operated animals with nonischemic
livers, there were no significant differences noted for any of the
measured variables between WT and LDLr
/
mice. However,
after I/R, highly significant differences were noted between
LDLr
/
and WT mice, with a substantially greater
accumulation of leukocytes in all vascular segments of
LDLr
/
mice.
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Figure 3 illustrates the effects of I/R
on the number of nonperfused liver sinusoids in WT and
LDLr/
mice. Hepatic I/R elicited progressive increases
in the number of nonperfused sinusoids in both WT and
LDLr
/
mice. However, the magnitude of the no-reflow
phenomenon in LDLr
/
mice was significantly greater than
that observed in WT mice, such that twice as many sinusoids were closed
to blood perfusion after I/R in livers of LDLr
/
mice.
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Figure 4 summarizes the changes in serum
ALT levels (an index of hepatocellular injury) detected at 60 min after
I/R in both WT and LDLr/
mice. No significant
differences in plasma ALT levels were noted between sham-operated WT
and LDLr
/
mice. In both groups of mice, I/R elicited a
significant increase in serum ALT level above control values. However,
plasma ALT increased to a significantly higher level after hepatic I/R
in LDLr
/
mice.
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Figure 5 summarizes the effects of
various mechanistic interventions on I/R-induced leukocyte recruitment
in the midzonal (Fig. 5A) and pericentral (Fig.
5B) regions of the hepatic acinus and THV (Fig.
5C) and the total number of accumulated leukocytes (Fig.
5D) in LDLr/
mice. The leukostasis response
elicited by I/R in both the midzonal and pericentral regions was
attenuated by pretreatment with either GdCl3 or antibodies
directed against TNF-
or ICAM-1 (Fig. 5, A and
B). A P-selectin specific MAb did not significantly
influence this response in the sinusoids of either acinar region. A
similar pattern of protection was noted for the total number of
stationary leukocytes (Fig. 5D). In contrast, the
I/R-induced recruitment of adherent leukocytes in THV was largely
abolished by all interventions, i.e., GdCl3, and antibodies
against TNF-
, ICAM-1, or P-selectin (Fig. 5D).
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Figure 6 illustrates how the different
mechanistic interventions (GdCl3 and antibodies) influenced
the increase in nonperfused sinusoids induced by I/R in
LDLr/
mice. Although a significant increase in the
percentage of nonperfused sinusoids was elicited by hepatic I/R in
untreated animals, none of the various treatment groups exhibited a
significant increase in nonperfused sinusoids.
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The changes in plasma ALT levels observed in LDLr/
mice
exposed to I/R are summarized in Fig. 7.
Although a significant increase in plasma ALT was noted after I/R in
untreated LDLr
/
mice, those animals pretreated with
either GdCl3 or antibodies directed against TNF, ICAM-1, or
P-selectin did not exhibit a significant increase in plasma ALT after
I/R.
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Figure 8 is a representative histological
section (hematoxylin and eosin) of nonischemic liver from an
LDLr/
mouse. The hepatocytes of LDLr
/
did not exhibit fatty droplets; the sinusoidal spaces and other cellular structures appeared normal. Histological examination of livers
in both WT and LDLr
/
groups revealed no differences.
|
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DISCUSSION |
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Over the past two decades, hundreds of reports have been published that deal with the pathogenesis of hepatic I/R injury in conditions such as hemorrhage and resuscitation, organ preservation and transplantation, and reconstructive vascular surgery. Virtually all of these studies have been performed in experimental animals that are otherwise healthy and do not exhibit any chronic pathology that may influence the quality and/or severity of the tissue responses to I/R. This contrasts with research performed on other vascular beds, where there is growing emphasis given to defining the effects of different risk factors for cardiovascular disease on the severity and mechanisms of I/R injury (7). Hypercholesterolemia, which is estimated to occur in up to 20-30% of the adult population in North America (1, 24), has been shown to significantly worsen the inflammatory cell infiltration and microvascular dysfunction that is elicited by I/R in skeletal muscle (30), heart (6), and mesentery (23). These exaggerated responses to I/R in hypercholesterolemic animals appear to occur before the development of atherosclerotic lesions in major blood vessels. In the present study, we sought to determine if and how hypercholesterolemia alters the microvascular and inflammatory responses to I/R and to identify mechanisms that may underlie any altered responses.
The results of this study indicate that genetically induced
hypercholesterolemia significantly enhances the inflammatory responses normally elicited by hepatic I/R, which includes leukostasis in both
the midzonal and pericentral regions of the liver sinusoid and
leukocyte adhesion to endothelial cells in THV. The exaggerated I/R-induced leukostasis in the sinusoids of LDLr/
mice
is associated with a more profound reduction in the number of perfused
sinusoids and a corresponding enhancement of I/R-induced hepatocellular
injury, as judged by the larger increment in plasma ALT levels detected
after I/R in LDLr
/
mice. These findings indicate that
hypercholesterolemia renders the liver more vulnerable to the
deleterious inflammatory and microvascular effects of normothermic
ischemia and reperfusion.
A number of cell types and chemical mediators have been implicated in the pathogenesis of hepatic I/R in otherwise healthy animals (17, 21, 28). Indeed, the literature suggests that platelets (4), T lymphocytes (38), neutrophils (20), and Kupffer cells (34) all contribute significantly to the microcirculatory failure and organ dysfunction that is associated with reperfusion of the ischemic liver. Kupffer cells have received much attention in this regard because these intravascular cells are capable both of releasing factors that are directly toxic to endothelial cells and hepatocytes (e.g., oxygen radicals and proteases) and of generating substances that promote the recruitment and activation of other cytotoxic inflammatory cells (31).
The role of Kupffer cells in mediating hepatocellular injury after I/R
has been previously addressed in otherwise healthy animals using
GdCl3 (13). This rare earth metal is avidly
phagocytosed by Kupffer cells and consequently blocks further
phagocytosis (15). GdCl3 also eliminates
Kupffer cells from the liver (for up to 4 days) after a single
intravenous injection, possibly by enhancing the rate of macrophage
apoptosis (29). The use of GdCl3 in the
present study revealed that Kupffer cell activation may repesent a
major contributor to the exaggerated inflammatory and microvascular
responses to I/R that are observed in the liver of
LDLr/
mice. GdCl3 treatment of
LDLr
/
mice effectively prevented the exaggerated
leukostasis in sinusoids, leukocyte adhesion in THV, recruitment of
nonperfused sinusoids, and hepatocellular injury (increased plasma ALT)
induced by I/R.
Although activated Kupffer cells release a variety of inflammatory
mediators (e.g., cytokines, leukotrienes) and cytotoxic agents that
could account for the exaggerated responses to hepatic I/R in
LDLr/
mice, our results suggest that the cytokine
TNF-
may be the primary Kupffer cell-derived mediator of these
responses. Previous studies (3, 13) on otherwise healthy
animals have demonstrated that hepatic I/R is associated with profound
increases in circulating TNF levels within 60 min after reperfusion.
Furthermore, it has been shown that immunoneutralization of this
circulating TNF effectively blunts the local and distant inflammatory
responses elicited by hepatic I/R as well as the elevated liver enzyme
levels that signal hepatocellular injury (3). Our finding
that a blocking antibody against mouse TNF-
is as effective as
GdCl3 treatment in ablating the microvascular and
inflammatory responses to hepatic I/R supports the possibility that
Kupffer cell-derived TNF-
mediates the exaggerated responses to I/R
in livers of LDLr
/
mice.
Leukocyte-endothelial cell adhesion has been implicated as a critical
determinant of the microvascular dysfunction and parenchymal cell
injury caused by I/R in a number of tissues, including liver (8,
28). The expression of ICAM-1 (5) and P-selectin
(33) is increased in the postischemic liver. Previous
studies in otherwise healthy animals have shown that blocking MAb
directed against either P-selectin (33) or ICAM-1
(37) reduces the leukocyte adhesion in THV and elevates
the circulating level of liver enzymes that is normally elicited by
hepatic I/R. A similar protective effect has been reported for
postischemic livers of P-selectin-deficient mice (33). The
results of the present study also implicate the adhesion molecules
ICAM-1 and P-selectin as contributors to the exaggerated microvascular
and inflammatory responses to hepatic I/R in chronically
hypercholesterolemic mice. Pretreatment with an ICAM-1-specific MAb
effectively reduced all of the measued responses to hepatic I/R in
LDLr/
. Similar results were obtained with the
P-selectin MAb; however, it did not significantly alter the sinusoidal
leukostasis seen in untreated LDLr
/
mice. Although the
protective effects of the ICAM-1 MAb are likely due to
immunoneutralization of endothelial cell ICAM-1, we cannot exclude the
possibility that some of the protective actions of the P-selectin MAb
reflect an effect on P-selectin expressed on the surface of activated
platelets (18).
An interesting aspect of the exaggerated responses of the liver
to I/R in hypercholesterolemic animals is the similarity to some of the
responses previously reported for livers with massive fatty
infiltration, i.e., steatotic livers. Diet-induced fatty livers exhibit
an exaggerated malperfusion of sinusoids after hepatic I/R, with
enhanced inflammatory cell infiltration and increased hepatocellular
injury (10, 14, 35). These exaggerated responses of the
fatty liver to I/R also appear to involve activated Kupffer cells and
ICAM-1, because both GdCl3 and an anti-ICAM-1 MAb are
effective in blunting the I/R-induced responses. Although the livers of
LDLr/
mice do not exhibit the histologically
demonstrable lipid deposition seen in steatotic livers, it appears
likely that cholesterol levels are elevated within membranes of
different resident and recruited cells of the liver in
LDLr
/
mice. Whether such deposition of cholesterol can
explain the exaggerated responses of LDLr
/
mice to I/R
remains to be determined.
Although the mechanism that accounts for the exaggerated inflammatory responses to I/R in hypercholesterolemic animals has not been precisely defined, there is a large body of evidence that supports a role for enhanced oxidant production as a key initiating event. Cholesterol oxidation products, such as those found in oxidized low-density lipoprotein cholesterol, appear to cause endothelial dysfunction and leukocyte chemoattraction in both large vessels and the microcirculation (26). Increased oxidant stress, resulting from both increased oxygen free radical production and decreased nitric oxide generation, also appears to play an important role in the chronic inflammatory responses to hypercholesterolemia and atherosclerosis (22). Furthermore, a recent study (9) demonstrated a signficant association between vascular superoxide production by NAD(P)H oxidase and the endothelial dysfunction that accompanies hypercholesterolemia in human blood vessels. Irrespective of the enzymatic source of the oxidants generated during hypercholesterolemia, there is ample published evidence that such elevated fluxes of oxidants could result in an exaggerated inflammatory response by virtue of the ability of oxidants to 1) increase the expression of adhesion molecules on vascular endothelium; 2) enhance the production of leukocyte-activating substances (e.g., platelet-activating factor); and 3) promote the rolling, firm adhesion, and emigration of leukocytes in the vasculature (2).
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the National Heart, Lung, and Blood Institute (HL-26441) and the Ministry of Education, Science, Sports and Culture of Japan (C,11671228).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. N. Granger, Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (E-mail: dgrang{at}lsuhsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 April 2000; accepted in final form 20 July 2000.
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