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,
,,1
Departments of
* Anesthesiology and Intensive Care, Lund University Hospital, and
Surgery, Malmö University Hospital, Lund University, Malmö, Sweden; and
Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany
1Correspondence: Department of Surgery, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden. E-mail: henrik.thorlacius{at}med.lu.se
ABSTRACT
Rho-kinase signaling regulates important features of inflammatory reactions. Herein, we investigated the effect and mechanisms of action of the Rho-kinase inhibitor fasudil in endotoxemic liver injury. C57/BL/6 mice were challenged with lipopolysaccharide (LPS) and D-galactosamine, with or without pretreatment with the Rho-kinase inhibitor fasudil. Six hours after endotoxin challenge, leukocyte-endothelium interactions in the hepatic microvasculature were studied by use of intravital fluorescence microscopy and tumor necrosis factor 
(TNF-); CXC chemokines as well as liver enzymes and apoptosis were determined. Administration of fasudil reduced LPS-induced leukocyte adhesion in postsinusoidal venules and sequestration in sinusoids. Moreover, we found that fasudil abolished extravascular infiltration of leukocytes as well as production of TNF- and CXC chemokines in the liver of endotoxemic mice. Liver enzymes and hepatocellular apoptosis were markedly reduced, and sinusoidal perfusion was improved significantly in endotoxemic mice pretreated with fasudil. Our novel data document that fasudil is a potent inhibitor of endotoxin-induced expression of TNF- and CXC chemokines as well as leukocyte infiltration and hepatocellular apoptosis in the liver. Based on the present findings, it is suggested that inhibition of the Rho-kinase signaling pathway may be a useful target in the treatment of septic liver injury.
Key Words: adhesion • endotoxin • intravital microscopy • sepsis
INTRODUCTION
Lipopolysaccharide (LPS) constitutes the major portion of the outer membrane of most clinically relevant, gram-negative bacteria found in human infections [1
]. Gram-negative infection and subsequent multiple organ failure are still major causes of morbidity and mortality in Intensive Care Units despite substantial efforts to improve surgical treatment, antimicrobial therapies, and immunomodulatory regimes [2
, 3
]. Deterioration of liver function is an insidious feature of septic patients, and several reports have shown that leukocyte recruitment is a rate-limiting step in endotoxin-induced liver damage [4
5
6
]. Extravascular recruitment of leukocytes is a multistep process, comprising initial tethering and rolling along the microvascular endothelium, followed by subsequent firm leukocyte adhesion and transendothelial migration [7
, 8
]. The host response to endotoxin challenge is associated with enhanced production of proinflammatory cytokines, such as tumor necrosis factor- (TNF-) and interleukin (IL)-1ß, which in turn, up-regulate endothelial cell adhesion molecules and stimulate chemokine expression. Indeed, it has been shown that P-selectin-dependent leukocyte rolling is a precondition for subsequent lymphocyte function antigen-1 (LFA-1)-mediated firm adhesion of leukocytes in postsinusoidal venules in the liver [9
, 10
]. Moreover, extravascular accumulation of leukocytes in the liver is critically dependent on the formation and action of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant {keratinocyte-derived chemokine (KC) [11
]}. Considered together, the role of specific adhesion molecules and chemoattractants in endotoxin-induced leukocyte infiltration in the liver is relatively well described, whereas the understanding of the signaling pathways orchestrating these endotoxin-provoked, proinflammatory actions in the liver is limited.
LPS binds to the cell-surface receptor CD14 and activates Toll-like receptor-4 [12
], which initiates intracellular signaling cascades that converge on specific transcription factors regulating gene expression of proinflammatory mediators [13
]. Small (
21 kDa) guanosine triphosphatases (GTPases) of the Ras-homologus (Rho) family and one of their effectors, Rho-kinase, are known to act as molecular switches controlling several critical cellular functions, such as actin cytoskeleton organization, cell adhesion, migration, reactive oxygen species (ROS) formation and apoptosis, as well as cytokinesis and oncogenic transformation [14
, 15
]. Moreover, Rho-kinase inhibitors have been shown to be effective against reperfusion injury in the liver [16
] and heart [17
], tissue fibrosis [18
], cerebral ischemia [19
], and pulmonary hypertension [20
]. It is interesting that two in vitro studies have reported that GTPases of the Rho family may be involved in LPS-induced expression of IL-8 in monocytes [21
] and endothelial cells [22
]. However, the role of the Rho-kinase signaling pathway in endotoxin-provoked liver injury has not been elucidated yet. In the present study, we therefore examined the effects of fasudil, a Rho-kinase inhibitor, on chemokine formation, leukocyte recruitment, and apoptotic cell death in the liver of endotoxemic mice.
MATERIALS AND METHODS
Animals
Adult male C57/BL/6 mice (22–27 g) were kept on a 12–12-h light-dark cycle with free access to food and tap water. Animals were anesthetized by intraperitoneal (i.p.) administration of 7.5 mg ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 2.5 mg xylazine (Janssen Pharmaceutica, Beerse, Belgium) per 100 mg body weight. The right jugular vein was cannulated with a polyethylene catheter for intravenous administration of test substances, fluorescent dyes, and additional anesthesia. The local ethics committee at Lund University (Malmö, Sweden) approved all the experiments of this study.
Experimental protocol
To delineate the role of Rho-kinase in endotoxin-induced leukocyte recruitment and liver injury, specific Rho-kinase inhibitors, fasudil (Sigma Chemical Co., St. Louis, MO) and Y-27632 (Sigma Chemical Co.), were administered i.p. 15 min prior to endotoxin challenge. It has been demonstrated in in vitro assays that fasudil and Y-27632 are specific inhibitors of Rho-kinase [23
24
25
]. For example, fasudil and Y-27632 have been shown to inhibit Rho-kinase more than 100-fold more potently than protein kinase C and myosin light-chain kinase [23
, 25
26
27
]. In one set of experiments, the effect of fasudil (40 mg/kg) was compared with phosphate-buffered saline (PBS) in endotoxin-treated mice. One group of animals received fasudil (40 mg/kg) alone without endotoxin challenge. In another set of experiments, we studied a structurally unrelated Rho-kinase inhibitor, Y-27632 (10 mg/kg), against PBS in endotoxin-treated mice. Six hours prior to surgery and intravital observation, mice were pretreated i.p. with a combination of LPS (10 µg/mouse, Sigma Chemical Co.) and D-galactosamine (Gal; 18 mg/mouse, Sigma Chemical Co.). Mice, which were not exposed to LPS/Gal, served as controls.
Intravital fluorescence microscopy
A transverse, subcostal incision was performed, and the ligamentous attachments from the liver to the diaphragm and the abdominal wall were released gently. The animals were positioned on their left side, and the left liver lobe was exteriorized carefully onto an adjustable stage for analysis of hepatic microcirculation by use of intravital fluorescence microscopy as described previously [6
]. An equilibration period of 5 min was allowed before starting microscopical observation. For observations of the liver microcirculation, we used a modified Olympus microscope (BX50WI, Olympus Optical Co. GmbH, Hamburg, Germany) equipped with different water immersion lenses [x40 numerical aperture (NA) 0.75/x63 NA 0.9]. The image was televised using a charge-coupled device video camera (FK 6990 Cohu, Pieper GmbH, Schwerte, Germany) and recorded on CD-ROM for subsequent off-line evaluation. Blood perfusion within individual microvessels was studied after contrast enhancement by fluorescein isothiocyanate-dextran (0.1 ml, 2 µmol/kg, Sigma Chemical Co.). In vivo labeling of leukocytes with rhodamine-6G (0.1 ml, 0.05 mg/ml, Sigma Chemical Co.) enabled quantitative analysis of leukocyte flow behavior in sinusoids and postsinusoidal venules. Five postsinusoidal venules with connecting sinusoids were evaluated in each animal. Microcirculatory analysis included determination of sinusoidal perfusion by measuring the number of nonperfused sinusoids given as a percentage of the total number of sinusoids observed. Within postsinusoidal venules, leukocyte rolling was measured by counting the number of cells rolling in the venule during 30 s and is expressed as cells/min. Leukocyte adhesion was measured by counting the number of cells that adhered along the venular endothelium and remained stationary during the observation period of 30 s and is expressed as cells/mm2 venular endothelial surface. Hepatocyte apoptosis was determined morphologically by topical application of the fluorochrome Hoechst 33342 (0.02 ml, 0.2 µg/ml, Molecular Probes, Leiden, the Netherlands) onto the liver surface for staining of hepatocyte DNA. Hoechst 33342 is a fluorescent dye, which has been widely used for analysis of nuclear morphology, e.g., nuclear condensation and fragmentation in cultured hepatocytes and endothelial cells [28
]. Six microscopical fields (using a x63 lens) were recorded for off-line quantification of hepatocyte nuclei showing signs of apoptosis (chromatin condensation and fragmentation). Hepatocyte apoptosis is given as the percentage of the number of hepatocyte nuclei showing apoptotic features from the total number of hepatocyte nuclei observed. After intravital observations, animals were killed, and blood was drawn from the inferior vena cava for analysis of liver enzymes, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), using standard spectrophotometric procedures.
Myeloperoxidase (MPO)
Liver tissue was collected, weighed, and homogenized in 10 ml 0.5% hexadecyltrimethylammonium bromide. Next, the sample was freeze-thawed, after which the MPO activity of the supernatant was assessed. The MPO activity was determined spectrophotometrically as the MPO-catalyzed change in absorbance occurring in the redox reaction of H2O2 (460 nm, 25°C). Values are expressed as MPO units/g tissue.
Caspase-3 protease activity
Caspase-3 protease activity in the liver tissue was measured using a caspase-3 colorimetric assay kit (R&D Systems, Oxon, UK) according to the manufacturer’s instructions. Briefly, after homogenization of whole liver tissue in cell lysis buffer, homogenates were centrifuged for 1 min at 10,000 g, and the supernatant was incubated with Asp-Glu-Val-Asp-p-nitroanilide (pNA) and reaction buffer for 90 min at 37°C. Levels of the chromophore pNA released by caspase-3 activity were quantified spectrophotometrically. The data were normalized to liver weight and are given as fold-increases in caspase-3 activity of test livers relative to PBS-treated control livers.
Enzyme-linked immunosorbent assay (ELISA)
The right liver lobe was weighed, washed, and homogenized in PBS containing 1% penicillin and streptomycin and fungizone (100 U/ml) and then kept cool in cold, serum-free Dulbecco’s modified Eagle’s medium. After centrifugation, supernatants were collected and stored in –20°C until analysis of TNF-, KC, and MIP-2 by using double antibody Quantikine ELISA kits (R&D Systems) using recombinant murine TNF-, KC, and MIP-2 as standards. The minimal detectable protein concentrations were less than 0.5 pg/ml.
Histology
Samples were taken from the left lobe of liver and fixed in 4% formaldehyde phosphate buffer overnight. Dehydrated, paraffin-embedded, 6 µm sections were stained with hematoxylin and eosin (H&E) and analyzed under light microscopy. The number of extravascular leukocytes was quantified randomly in 40 high-power fields and expressed as number of cells/mm2.
Statistical analyses
Data are presented as mean values ± SEM. Statistical evaluations were performed using Kruskal-Wallis one-way ANOVA on ranks followed by multiple comparisons versus control group (Dunn’s method). P < 0.05 was considered significant, and n represents the number of animals.
RESULTS
Hepatocellular damage and apoptosis
Endotoxin challenge caused substantial hepatocellular injury, manifested by a more than 26-fold increase in liver injury enzymes (Fig. 1A
and 1B
; P<0.05 vs. control, n=5–13). Pretreatment with fasudil significantly reduced ALT and AST from 25.9 ± 8.7 µKat/L and 22.2 ± 6.2 µKat/L to 2.5 ± 1.4 µKat/L and 2.5 ± 1.0 µKat/L, respectively, in endotoxemic mice (Fig. 1 A and B
; P<0.05 vs. PBS+LPS/Gal, n=5–13). Thus, inhibition of Rho-kinase signaling decreased liver enzyme release by more than 89%. Apoptosis constitutes a hallmark in endotoxin-induced liver damage. In the present study, we determined apoptosis morphologically and biochemically. By use of the DNA-binding fluorescent dye Hoechst 33342, which stains the nuclei of hepatocytes, we quantified the percentage of cells with nuclear condensation and fragmentation [28
]. It was observed that the percentage of apoptotic hepatocytes was 1.8 ± 0.4% in control mice, which was enhanced markedly by almost eightfold up to 13.3 ± 1.2% in endotoxemic animals (Fig. 2A
; P<0.05 vs. control, n=5–10). It was found that pretreatment with fasudil reduced LPS-induced apoptosis down to 4.6 ± 0.8%, corresponding to a 75% reduction (Fig. 2A
; P<0.05 vs. PBS+LPS/Gal, n=5–10). Moreover, it was observed that the hepatic caspase-3 activity was elevated almost eightfold in mice challenged with endotoxin (Fig. 2B
; P<0.05 vs. control, n=5–9). It is notable that this LPS-induced increase in caspase-3 activity was reduced by 79% in fasudil-treated mice (Fig. 2B
; P<0.05 vs. PBS+LPS/Gal, n=5–9). Treatment with fasudil alone had no effect on ALT, AST, or apoptosis compared with PBS in nonendotoxemic mice (not shown). Microscopic analysis of H&E-stained liver sections revealed normal hepatic architecture in PBS-treated controls (Fig. 3A
), whereas severe destruction of the liver architecture characterized by massive panlobular hemorrhage and necrosis as well as infiltration of neutrophils was observed in endotoxemic animals (Fig. 3B)
. Administration of fasudil markedly attenuated LPS-induced morphologic liver injury (Fig. 3C)
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and CXC chemokines from 22.9 ± 11.0 pg/g tissue in controls to 157.1 ± 49.0 pg/g (Fig. 6A
; P<0.05 vs. control, n=5–9). Pretreatment with fasudil decreased LPS-induced TNF- expression to 37.4 ± 9.8 pg/g (Fig. 6A
; P<0.05 vs. PBS+LPS/Gal, n=5–9). Leukocyte extravasation in septic liver damage is mediated by secreted CXC chemokines [11
]. Hepatic levels of CXC chemokines in negative controls were low but detectable (Fig. 6B
and 6C)
. Formation of MIP-2 and KC in livers of endotoxin-treated mice increased markedly, i.e. from 12 ± 7 pg/g and 175 ± 37 pg/g in controls to 7047 ± 2110 pg/g and 4673 ± 2144 pg/g tissue, respectively (Fig. 6 B and C
; P<0.05 vs. control, n=5–9). It is interesting that pretreatment with fasudil significantly decreased LPS-induced expression of MIP-2 and KC to 1025 ± 251 pg/g and 750 ± 136 pg/g liver tissue, respectively (Fig. 6 B and C
; P<0.05 vs. PBS+LPS/Gal, n=5–9). Thus, we observed that inhibition of Rho-kinase signaling attenuated hepatic expression of CXC chemokines by more than 69% in endotoxemic animals.
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Our data provide evidence suggesting a fundamental role of Rho-kinase signaling in endotoxin-induced liver injury. Thus, this study demonstrates that treatment with fasudil markedly reduced hepatic expression of TNF- and CXC chemokines in endotoxemic mice. It is notable that it was found that fasudil significantly decreased endotoxin-induced hepatocellular apoptosis and damage. Of interest, fasudil reduced leukocyte adhesion in postsinusoidal venules and abolished extravascular accumulation of leukocytes in the liver of endotoxemic mice. Taken together, our novel data indicate that Rho-kinase plays a key role in endotoxin-provoked generation of proinflammatory mediators, leukocyte recruitment, and apoptosis and that targeting the Rho-kinase signaling pathway may be an attractive way to ameliorate septic liver injury.
Rho-kinase-mediated signaling has mainly been considered to regulate cytoskeletal dynamics, such as cell contraction and vesicular trafficking, but an accumulating body of evidence suggests that Rho-kinase is also involved in the regulation of several aspects of innate immunity, including leukocyte chemotaxis, phagocytosis, and ROS formation [14
, 15
]. Herein, we found that pretreatment with fasudil, a specific Rho-kinase inhibitor, abolished endotoxemic liver injury; i.e., the LPS-induced increase in ALT and AST was reduced by more than 89%. Our results are the first data in the literature to indicate that inhibition of the Rho-kinase signaling pathway protects against LPS-induced hepatic damage. This notion is in line with a recent study showing that inhibition of Rho-kinase with Y-27632 protects against endotoxin-provoked lung injury [29
]. Thus, the present findings add septic liver injury to the growing list of pathological conditions, which may be ameliorated by interference with Rho-kinase signaling, including ischemia-reperfusion [16
, 17
], vascular diseases [19
, 20
], and tissue fibrosis [18
]. Convincing data have shown that TNF- plays an important role in the pathogenesis of endotoxin-provoked hepatotoxicity [30
, 31
]. It is interesting that we observed that blocking Rho-kinase function decreased hepatic levels of TNF- by nearly 90% in septic liver injury, supporting the idea that inhibition of Rho-kinase activity indeed may exert a protective effect on septic liver damage.
It is widely held that a fundamental function of proinflammatory cytokines in sepsis is to activate endothelial cells and support mobilization of circulating leukocytes to the extravascular space. Tissue accumulation of leukocytes is an essential component in the inflammatory response following tissue injury and microbial infection [8 ], but under certain situations, such as ischemia-reperfusion, graft rejection, and endotoxemia, activation and infiltration of leukocytes may cause organ damage [32 ]. In fact, numerous studies have documented that leukocyte recruitment constitutes a rate-limiting step in septic liver injury by demonstrating that LPS-induced hepatotoxicity is markedly attenuated in adhesion molecule (P-selectin and LFA-1)-targeted and in neutrophil-depleted animals [9 , 10 , 33 ]. In the present study, it was found that fasudil markedly reduced the endotoxin-induced increase of MPO activity, indicating an inhibitory effect of fasudil on leukocyte accumulation in the liver. By use of intravital microscopy, we observed that fasudil inhibited leukocyte adhesion but not leukocyte rolling in postsinusoidal venules in endotoxemic animals. Moreover, endotoxin-provoked accumulation of leukocytes in the extravascular space in the liver was markedly reduced by fasudil. Knowing that leukocyte recruitment is a multistep process [7 , 8 ], it is conceivable that the decreased, firm adhesion helps explain the inhibitory effect of fasudil on leukocyte accumulation in the extravascular space in the liver of endotoxemic mice. These findings collectively suggest that leukocyte adhesion and extravasation are regulated by the Rho-kinase signaling pathway upon exposure to endotoxin. Indeed, our data may be of special importance, knowing that transmigration of leukocytes out into the extravascular space is required for provoking hepatocellular injury [34 ].
Considering that chemokines are key regulators of leukocyte adhesion and chemotaxis, it is interesting to note that we found, herein, that fasudil reduced endotoxin-provoked expression of CXC chemokines by more than 84%. This decrease in CXC chemokine production may account for the inhibitory effect of fasudil on leukocyte extravasation in the liver. In fact, that notion is supported by a recent study demonstrating that CXC chemokines play a critical role in endotoxin-induced hepatic injury by inducing leukocyte extravasation [11
]. On top of that, our findings indicate that CXC chemokine production is regulated by Rho-kinase signaling in endotoxemic liver injury, and it may be suggested that the protective effect of fasudil is related to an inhibition of CXC production and leukocyte extravasation into the liver parenchyma. In this context, it is important to note that a previous study has shown that Rho-kinase can regulate chemoattractant-induced leukocyte migration in vitro [19
]. Thus, our data do not exclude that such a direct, inhibitory effect of fasudil on the motile functions of leukocytes may also contribute to the abolished leukocyte accumulation in the extravascular space, knowing that cytoskeletal functions coordinate chemotaxis [14
]. Moreover, it is worth noting that these findings do not necessarily exclude a potential role of other protein kinases, such as stress-activated mitogen-activated protein kinases (MAPKs) in endotoxin-induced hepatotoxicity, which have been shown to integrate LPS signaling and regulate TNF- production in monocytes [32
]. For instance, it has recently been observed that p38 MAPK plays a key function in septic liver injury [30
]. Nonetheless, this is the first study indicating that the Rho-kinase signaling pathway may be involved in regulating endotoxin-induced expression of CXC chemokines and leukocyte recruitment in the liver.
Hepatocyte apoptosis is considered to be a central feature in septic liver injury [35
]. Herein, we analyzed apoptosis morphologically by Hoechst 33324 staining and biochemically by quantifying caspase-3 activity in the liver. It was found that endotoxin challenge induced clear-cut apoptosis and that administration of fasudil decreased the percentage of apoptotic hepatocytes by 75%, which corresponded well to the 79% decrease in hepatic levels of caspase-3, suggesting that Rho-kinase is involved in the regulation of apoptosis in septic liver injury. However, it should be noted that the exact role of apoptosis in endotoxemic liver injury is a matter of discussion. Some investigators advocate an active role of apoptosis in the initial phase of hepatic damage by suggesting that endotoxin challenge provokes apoptosis prior to actual recruitment of leukocytes [36
, 37
]. In contrast, others have reported that direct inhibition of leukocyte recruitment, by interfering with specific adhesion molecules, such as P-selectin and LFA-1 or chemokines, protects against apoptosis in endotoxemia [9
10
11
]. Moreover, considering that TNF- may induce hepatocellular apoptosis directly or indirectly (i.e., through stimulating hepatic infiltration of leukocytes), it has been suggested that there may be an escalating pathophysiological mechanism in the liver, involving chemokine-induced leukocyte recruitment at inflammatory foci, which causes apoptosis on one hand, and apoptosis-induced secretion of chemokines, which causes leukocyte infiltration on the other [11
]. Our present findings, which show that fasudil decreases TNF- production and leukocyte infiltration, may help to explain the potent, antiapoptotic impact exerted by fasudil in endotoxin-induced liver damage.
Taken together, our novel results demonstrate that inhibition of Rho-kinase protects against septic liver damage. Indeed, administration of fasudil attenuated endotoxin-induced synthesis of TNF- and CXC chemokines, leukocyte recruitment, as well as hepatocellular apoptosis in the liver. Thus, these findings not only indicate important signaling mechanisms in septic liver injury but also suggest that targeting Rho-kinase activity may be an effective strategy to treat pathological inflammation in the liver.
ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Medical Research Council (2002-955, 2002-8012, 2003-4661), Crafoordska stiftelsen, Blanceflors stiftelse, Einar och Inga Nilssons stiftelse, Harald och Greta Jaenssons stiftelse, Greta och Johan Kocks stiftelser, Fröken Agnes Nilssons stiftelse, Franke och Margareta Bergqvists stiftelse för främjande av cancerforskning, Magnus Bergvalls stiftelse, Mossfelts stiftelse, Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Svenska Läkaresällskapet (2001-907), Teggers stiftelse, Allmäna sjukhusets i Malmö stiftelse för bekämpande av cancer, MAS fonder, Malmö University Hospital, and Lund University. K. T. and J. E. S. contributed equally to this work.
Received July 22, 2005; revised November 12, 2005; accepted November 19, 2005.
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