Hepatic microvascular responses to ischemia-reperfusion in low-density lipoprotein receptor knockout mice

Naoharu Mori, Yoshinori Horie, Yuji Nimura, Robert Wolf, and D. Neil Granger

Departments of Molecular and Cellular Physiology and Medicine, Center of Excellence in Arthritis and Rheumatology, Lousiana State University Health Sciences Center, Shreveport, Louisiana 71130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ; 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   Plasma cholesterol levels in wild-type (WT) and low-density lipoprotein receptor-deficient (LDLr-/-) mice. Values were obtained from 6 animals in each group. * P < 0.05 relative to WT.

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.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of accumulation of stationary leukocytes in midzonal (A) and pericentral regions (B) of liver acinus, terminal hepatic venules (THV; C), and total (combined) liver lobule (D) after hepatic ischemia/reperfusion in WT and LDLr-/- mice. Values were obtained from 7 animals in each group. * P < 0.05 relative to WT value at corresponding time.

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Time course of changes in percentage of nonperfused sinusoids in postischemic liver of WT and LDLr-/- mice. Values were obtained from 7 animals in each group. * P < 0.05 compared with WT at corresponding time.

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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of ischemia-reperfusion (I/R) on plasma alanine aminotransferase (ALT) levels in WT and LDLr-/- mice. Nos. of animals in each experimental group were WT controls = 6; WT I/R = 7; LDLr-/- controls = 6; and LDLr-/- I/R = 7. * P < 0.05 vs. corresponding controls; # P < 0.05 vs. corresponding I/R group.

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-alpha 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-alpha , ICAM-1, or P-selectin (Fig. 5D).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of pretreatment with either GdCl3 or antibodies against tumor necrosis factor-alpha (TNF-alpha ), intercellular adhesion molecule-1 (ICAM-1), or P-selectin on number of stationary leukocytes in midzonal (A) and pericentral regions (B) of liver acinus, terminal hepatic venules (C), and total (combined) liver lobule (D) at 60 min after reperfusion in LDLr-/- mice. Nos. of animals in each experimental group were controls = 6, I/R untreated = 7, GdCl3 = 7, anti-TNF-alpha monoclonal antibody (MAb) = 6, anti-ICAM-1 MAb = 6, and anti-P-selectin MAb = 6. * P < 0.05 vs. control; # P < 0.05 vs. I/R untreated group.

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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of pretreatment with either GdCl3 or antibodies against TNF-alpha , ICAM-1, or P-selectin on percentage of nonperfused sinusoids at 60 min after reperfusion in LDLr-/- mice. Nos. of animals in each experimental group were controls = 6, I/R = 7, GdCl3 = 7, anti-TNF-alpha MAb = 6, anti-ICAM-1 MAb = 6, and anti-P-selectin MAb = 6. * P < 0.05 relative to control value.

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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of pretreatment with either GdCl3 or antibodies against TNF-alpha , ICAM-1, or P-selectin on the I/R induced increase in plasma ALT activity in LDLr-/- mice. Nos. of animals in each experimental group were controls = 6, I/R = 7, GdCl3 = 7, anti-TNF-alpha MAb = 6, anti-ICAM-1 MAb = 6, and anti-P-selectin MAb = 6. * P < 0.05 relative to control value.

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.


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 8.   Representative histological section of liver from an LDLr-/- mouse. Hepatocytes of LDLr-/- did not exhibit fatty droplets; sinusoidal spaces and other cellular structures appeared normal. Histological examination of livers in both WT and LDLr-/- groups revealed no differences. Hematoxylin and eosin, ×40.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha 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-alpha 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).


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Brown, WV. Hypercholesterolemia in the United States: how far have we come? Am J Med 102: 1-6, 1997[ISI][Medline].

2.   Carden, DL, and Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol 190: 255-266, 2000[ISI][Medline].

3.   Colletti, LM, Remick DG, Burtch DG, Kunkel SL, Strieter RM, and Campbell DA, Jr. Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J Clin Invest 85: 1936-1943, 1990[ISI][Medline].

4.   Cywes, R, Packham MA, Tietze L, Sanabria JR, Harvey PRC, Phillips MJ, and Strasberg SM. Role of platelets in hepatic allograft preservation injury in rat. Hepatology 18: 635-647, 1993[ISI][Medline].

5.   Farhood, A, McGuire GM, Manning AM, Miyasaka M, Smith CW, and Jaeschke H. Intercellular adhesion molecule-1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver. J Leukoc Biol 57: 368-374, 1995[Abstract].

6.   Golino, P, Maroko PR, and Carew TE. Efficacy of platelet depletion in counteracting the detrimental effect of acute hypercholesterolemia on infarct size and the no-reflow phenomenon in rabbits undergoing coronary artery occlusion-reperfusion. Circulation 76: 173-180, 1987[Abstract].

7.   Granger, DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 6: 167-178, 1999[ISI][Medline].

8.   Granger, DN, and Korthuis RJ. Physiologic mechanisms of postischemic tissue injury. Annu Rev Physiol 57: 311-332, 1995[ISI][Medline].

9.   Guzik, TJ, West NEJ, Black E, McDonald D, Ratnatunga C, Pillai R, and Channon KM. Vascular superoxide production by NAD(P)H oxidase. Association with endothelial dysfunction and clinical risk factors. Circ Res 86: e85-e90, 2000[Abstract/Free Full Text].

10.   Hakamada, K, Sasaki M, Takahashi K, Umehara Y, and Konn M. Sinusoidal flow block after warm ischemia in rats with diet-induced fatty liver. J Surg Res 70: 12-20, 1997[ISI][Medline].

11.   Henninger, DD, Gerritsen ME, and Granger DN. Low-density lipoprotein receptor knockout mice exhibit exaggerated microvascular responses to inflammatory stimuli. Circ Res 81: 274-281, 1997[Abstract/Free Full Text].

12.   Horie, Y, Wolf R, Anderson DC, and Granger DN. Hepatic leukostasis and hypoxic stress in adhesion molecule-deficient mice after gut ischemia-reperfusion. J Clin Invest 99: 781-788, 1997[Abstract/Free Full Text].

13.   Horie, Y, Wolf R, Russell J, Shanley TP, and Granger DN. Role of Kupffer cells in gut ischemia/reperfusion-induced hepatic microvascular dysfunction in mice. Hepatology 26: 1499-1505, 1997[ISI][Medline].

14.   Hui, AM, Kawasaki S, Makuuchi M, Nakayama J, Ikegami T, and Miyagawa S. Liver injury following normothermic ischemia in steatotic rat liver. Hepatology 20: 1287-1293, 1994[ISI][Medline].

15.   Husztik, E, Lazar G, and Parducz A. Electron microscopic study of Kupffer cell phagocytosis blockade induced by gadolinium cloride. Br J Exp Pathol 61: 624-630, 1980[ISI][Medline].

16.   Ishibashi, S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, and Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest 92: 883-893, 1993[ISI][Medline].

17.   Jaeschke, H. Mechanisms of reperfusion injury after warm ischemia of the liver. J Hepatol Pancreat Surg 5: 402-408, 1998.

18.   Jaeschke, H. Is anti-P-selectin therapy effective in hepatic ischemia-reperfusion injury because it inhibits neutrophil recruitment? Shock 12: 233-234, 1999[ISI][Medline].

19.   Jaeschke, H, and Farhood A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver in vivo. Am J Physiol Gastrointest Liver Physiol 260: G355-G362, 1991[Abstract/Free Full Text].

20.   Jaeschke, H, Farhood A, and Smith CW. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J 4: 3355-3359, 1990[Abstract/Free Full Text].

21.   Jaeschke, H, and Smith CW. Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol 61: 647-653, 1997[Abstract].

22.   Kojda, G, and Harrison DD. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res 43: 562-571, 1999[ISI][Medline].

23.   Kurose, I, Argenbright LW, Anderson DC, Tolley J, Miyasaka M, Harris N, and Granger DN. Reperfusion-induced leukocyte adhesion and vascular protein lekage in normal and hypercholesterolemic rats. Am J Physiol Heart Circ Physiol 273: H854-H860, 1997[Abstract/Free Full Text].

24.   Langille, DB, Joffres MR, MacPherson KM, Andreou P, Kirkland SA, and MacLean DR. Prevalence of risk factors for cardiovacular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992. CMAJ 161: S3-S9, 1999[Medline].

25.   Lázár, G, Jr, GLázár Kaszaki J, Oláh J, Kiss I, and Husztik E. Inhibition of anaphylactic shock by gadolinium chloride-induced Kupffer cell blockade. Agents Actions 41: C97-C98, 1994[ISI][Medline].

26.   Liao, L, Starzyk RM, and Granger DN. Molecular determinants of oxidized low-density lipoprotein-induced leukocyte adhesion and microvascular dysfunction. Arterioscler Thromb Vasc Biol 17: 437-444, 1997[Abstract/Free Full Text].

27.   Lorenzl, S, Koedel U, Dirnagl U, Ruckdeschel G, and Phister HW. Imaging of leukocyte-endothelium interaction using in vivo confocal laser scanning microscopy during the early phase of experimental pneumococcal meningitis. J Infect Dis 168: 927-933, 1993[ISI][Medline].

28.   Menger, MD, Richter S, Yamauchi J, and Vollmar B. Role of the microcirculation in hepatic ischemia/reperfusion injury. Hepatogastroenterology 46, Suppl2: 1452-1457, 1999[ISI][Medline].

29.   Mizgerd, JP, Molina RM, Stearns RC, Brain JD, and Warner AE. Gadolinium induces macrophage apoptosis. J Leukoc Biol 59: 189-195, 1996[Abstract].

30.   Mori, N, Horie Y, Gerritsen ME, and Granger DN. Ischemia-reperfusion induced microvascular responses in LDL-receptor -/- mice. Am J Physiol Heart Circ Physiol 276: H1647-H1654, 1999[Abstract/Free Full Text].

31.   Roll, FJ, and Friedman SL. Role of sinusoidal endothelial cells, lipocytes, Kupffer cells, and pit cells in the liver. In: Liver and Biliary Diseases, edited by Kaplowitz N.. Baltimore: Williams & Wilkins, 1992, p. 28-46.

32.   Ross, R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809, 1993[ISI][Medline].

33.   Sawaya, DE, Zibari GB, Minardi A, Bilton B, Burney D, Granger DN, McDonald JC, and Brown M. P-selectin contributes to the initial recruitment of rolling and adherent leukocytes in hepatic venules after ischemia-reperfusion. Shock 12: 227-232, 1999[ISI][Medline].

34.   Suzuki, S, Toledo-Pereyra LH, Rodrigues F, and Lopez F. Role of Kupffer cells in neutrophil activation and infiltration following total hepatic ischemia and reperfusion. Circ Shock 42: 204-209, 1994[ISI][Medline].

35.   Thurman, RG, Gao W, Connor HD, Adachi Y, Stachlewitz RF, Zhong Z, Knecht KT, Bradford BU, Mason RP, and Lemasters JJ. Role of Kupffer in failure of fatty livers following liver transplantation and alcoholic liver injury. J Gastroenterol Hepatol 10, Suppl 1: S24-S30, 1995[ISI][Medline].

36.   Villringer, A, Dirnagl U, Them A, Schürer L, Krombach F, and Einhäupl KM. Imaging of leukocytes within the rat brain cortex in vivo. Microvasc Res 42: 305-315, 1991[ISI][Medline].

37.   Vollmar, B, Glasz J, Menger MD, and Messmer K. Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence. Surgery 117: 195-200, 1995[ISI][Medline].

38.   Zwacka, RM, Zhang Y, Schlossberg JH, Dudus L, and Engelhardt JF. CD4+ T-lymphocytes mediate ischemia-reperfusion induced inflammatory responses in mouse liver. J Clin Invest 100: 279-289, 1997[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 279(6):G1257-G1264
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society