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Department of Anesthesiology and Critical Care Medicine, University Hospital, Freiburg, Germany
Address correspondence to B. H. J. Pannen, MD, Anaesthesiologische Universitaetsklinik, Hugstetterstrasse 55, D-79106 Freiburg, Germany. Address e-mail to pannen{at}nz.ukl.uni-freiburg.de Reprints will not be available from the author.
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Introduction |
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 Top Introduction References |
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There is ample evidence that the sequence of hepatic I/R may cause liver injury. In addition to postoperative bleeding, the development of hepatic failure is the main cause of death in the hospital after warm I/R of the liver (2,3). Moreover, in patients undergoing hepatectomy, the degree of the resulting impairment of hepatic perfusion and oxygenation determines the extent of liver injury, as well as the incidence of liver failure and mortality (4,5). Similar results have also been reported in patients after cold I/R associated with liver transplantation. For example, Klar et al. (6) observed an inverse correlation between the intraoperatively measured hepatic microvascular blood flow rate and the maximum postoperative enzyme release from the liver. In addition, Nakatani et al. (7) reported that the degree of impairment of the hepatic mitochondrial redox state determines the survival rate of patients after hemorrhagic shock and resuscitation.
The extent of liver injury caused by I/R depends primarily on the presence of preexisting liver diseases, such as cirrhosis (3,8), and on the duration of ischemia (9). If the injury is severe enough to cause liver dysfunction, this is associated with a profound deterioration of the prognosis (10). Because the liver plays a central role in the metabolic and immunologic response to stress (11), this increased mortality is most likely due to the subsequent progression to multiple organ failure (12,13). Although the development of bioartificial liver devices is an area of extensive research, none of these systems is readily available for routine clinical use (14). Therefore, identification of the underlying pathophysiological mechanisms is of major importance, because this may form the basis for the development of new strategies aimed at limiting IR-induced liver injury.
Mechanisms of Liver Injury During I/R
In addition to the direct ischemic insult, hepatic injury is in large part the result of processes that occur during reperfusion. During the first hours of reperfusion, the local macrophages residing within the liver, i.e., the Kupffer cells, get stimulated by complement factors such as C5a (15). Kupffer cells induce inflammation by producing and releasing proinflammatory cytokines such as tumor necrosis factor-
or interleukin-1ß. In addition, these cells seem to be the major source of reactive oxygen species generated during early reperfusion (<6 h), leading to a primarily intravascular oxidative stress. The sum of these processes may either directly or indirectly damage nonparenchymal cells of the liver, such as sinusoidal endothelial cells and parenchymal cells. As a result, circulating neutrophils accumulate in the postischemic liver. During this later phase of reperfusion (>6 h), the recruited neutrophils may also induce hepatocellular injury by releasing oxidants and proteases. The contribution of this inflammatory response and the resulting oxidative stress to hepatic reperfusion injury has long been recognized and is reviewed in detail elsewhere (16–18).
Accumulating evidence suggests that alterations of hepatic perfusion may also play a major role in the pathogenesis of I/R-mediated liver injury. For example, it has been reported that hepatosplanchnic blood flow was still reduced in humans at 60 min of reperfusion after 55 min of hypovolemia, even though arterial blood pressure, cardiac output, and blood flow to the muscles, skin, and kidneys were already fully restored (19). In addition, perturbations of the hepatic microcirculation have been observed in different animal models after I/R, including decreases in sinusoidal diameters and flow, increases in the heterogeneity of hepatic microvascular perfusion, and even complete cessation of blood flow within individual sinusoids (20).
The impaired restoration of hepatic microvascular flow correlates with the extent of liver injury after hemorrhagic shock and resuscitation (21) and during reperfusion after portal triad cross-clamping (22). Physical prevention of microvascular shutdown by use of a flow-controlled reperfusion mode largely prevents parenchymal cell necrosis after I/R of the liver, suggesting that the degree of microvascular failure determines the extent of lethal hepatocyte injury (23). Similar relationships between liver blood flow and injury could be observed in humans (5,24). Over the past decade, our knowledge about the pathogenesis of hepatic perfusion failure after I/R increased substantially. This review summarizes the most important findings that led to these new insights. If not specified otherwise, the results described were obtained in animal studies.
Levels of Regulation of Hepatic Blood Flow
Hepatic blood flow is regulated at different levels. Changes at each of these levels can ultimately cause alterations in nutritive blood flow through the hepatic sinusoids and may thus contribute to the subsequent development of hepatocellular injury described previously. The first level of regulation is at the systemic circulation. Decreases in cardiac output, reductions in the arterial inflow resistance to other vascular beds, and increases in right atrial pressure above a level of approximately 3–4 mm Hg (i.e., above the portal venous critical closing pressure) may all reduce blood flow through the liver (25,26).
The second level of regulation is at the regional macrocirculation. Blood is supplied to the liver via the hepatic artery and the portal vein. The amount of blood flowing through the hepatic artery is determined by its vascular resistance. Because the hepatic artery has only a small autoregulatory capacity, the ability to maintain the hepatic arterial flow rate constant in the presence of a decrease in arterial perfusion pressure is limited (27,28). Hepatic arterial resistance may change so as to buffer the effect of portal flow alterations on total hepatic blood flow, thus tending to maintain total hepatic flow at a constant level. This mechanism has been called the "hepatic arterial buffer response" (29). Ezzat and Lautt (27) have provided evidence that both the hepatic arterial pressure-flow autoregulation and buffer response are mediated through changes in the washout of locally produced adenosine. The total resistance to flow from the portal vein to the inferior vena cava is low, and the relative contribution of individual anatomical sites, such as the hepatic veins, may vary significantly depending on the conditions and the species studied (30–33). The portal vein passively drains most of the venous blood from the splanchnic compartment. Therefore, portal venous flow is primarily determined by the arterial inflow resistance to these organs. However, splanchnic resistance may increase linearly with increases in portal venous pressure (34). Consequently, in the presence of such a "portal-splanchnic response," alterations in portal flow may well be accomplished by changes in portal venous resistance.
The third level of flow regulation is at the hepatic microcirculation. Changes in hepatic microvascular flow have long been exclusively interpreted as the result of systemic and regional macrohemodynamic changes and of obstructions of the sinusoidal lumen by swollen perisinusoidal cells, trapped blood cells, or thrombotic material. However, the findings described below show that sinusoidal flow can also be actively regulated and redistributed at the level of the microcirculation.
Local Control of Vascular Tone
Changes in vascular resistance have been traditionally ascribed to alterations in autonomic nervous activity and to the influence of circulating hormones. However, the discovery that endothelial cells can actively control various functions of the vasculature, including local control of vascular tone, had a profound effect on our understanding of how organ blood flow is regulated. In 1980, Furchgott and Zawadzki (35) reported that the vascular relaxation by acetylcholine depends on the presence of endothelial cells. In 1987, Palmer et al. (36) suggested that the gas nitric oxide (NO) accounts for the biological activity of the "endothelium-derived relaxing factor." However, Hickey et al. (37) reported in 1985 about the existence of an "endothelium-derived contracting factor." Shortly thereafter, Yanagisawa et al. (38) identified a peptide responsible for this vasoconstrictive action, named "endothelin" (ET). Meanwhile, there are indications that the endogenous generation of another gaseous monoxide, i.e., carbon monoxide (CO), may also exert local vasodilatory effects (39). During the past decade, evidence accumulated that these "endothelial" mediators may control hepatic blood flow under physiologic and, in particular, under pathologic conditions, such as I/R of the liver. Although all these mediators have also been shown to exert numerous other effects that could affect the extent of liver injury, this review focuses on their vasoregulatory properties. The major findings described within the following paragraphs are summarized in Figure 1.
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Hemodynamic Effects of ETs in the Normal Liver
Binding sites for ET-1 in liver tissue are almost exclusively confined to nonparenchymal perivascular and vascular cells of the portal vein, the hepatic sinusoids, and the central vein (41). Similarly, Housset et al. (42) reported that ET receptor messenger RNA expression was greatest on hepatic stellate cells (HSCs) located in the perisinusoidal space of Dissé, followed by sinusoidal endothelial cells and Kupffer cells. Exogenous ET elicits a sustained increase in portal venous vascular resistance (43). This seems to be mediated not only via extrasinusoidal sites such as terminal portal and hepatic venules (44), but also via sinusoidal sites. This conclusion is based on the observation that intraportal infusion of ET-1 decreases the mean sinusoid diameter and increases the heterogeneity of sinusoidal perfusion (45). Both of these changes caused by exogenous ETs in the normal liver are hallmarks of I/R-induced alterations of hepatic blood flow.
This raises the question of how hepatic blood flow is regulated at the level of the hepatic microcirculation in the absence of vascular smooth muscle cells within this compartment of the hepatic vasculature. In situ microscopic analyses revealed that the ET-1-induced decrease in sinusoid diameter is most pronounced at sites that colocalize with the body of HSCs that are situated in the perisinusoidal space with long stellate appendages that wrap around the sinusoids (46). Further support for the premise that these liver-specific pericytes may serve as a contractile target for ETs came from studies showing that isolated stellate cells in vitro are capable of constriction in a reversible and graded manner in response to these agents (47). Although the contribution of endogenously generated ETs to total portal resistance is rather limited in the normal liver, this seems to be different under pathological conditions (see below).
ET Production and Receptor Expression After I/R
Reactive oxygen intermediates, bacterial lipopolysaccharides, proinflammatory cytokines, hypoxia, and increases in vascular shear stress can induce ET gene expression (40,48). Because most of these factors play an essential role during I/R of the liver, increased generation of ETs has been observed after hemorrhagic hypotension (49) and after lobar or complete ischemia of the liver (50). Increases of the plasma concentration of ETs have also been reported in patients after cardiopulmonary bypass (51), in response to blood loss during major abdominal surgery (52), and during hepatic resection (53). Potential sources for ET in the liver are (sinusoidal) endothelial cells, Kupffer cells, and stellate cells. Evidence suggests that the transcription factors activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and nuclear factor-1 (NF-1) may play a role in the transcriptional control of ET genes, in particular in response to hypoxia (54). Besides the I/R-induced increase in the generation of ETs, changes in the pattern of ET receptor expression have also been reported. In I/R livers, ETB receptor expression is increased, whereas expression of the ETA receptor gene is reduced (55).
Hepatic Hemodynamic Effects of ETs After I/R
The portohepatic contractile response to ET-1 is enhanced in models of hepatic stress (56,57), including hemorrhagic shock and hepatic lobar I/R (58,59). Together with the increased generation of ETs, this may contribute to the impairment of hepatic perfusion and integrity under these conditions. After hemorrhagic shock, blockade of the ETA receptor increases cardiac output and portal venous flow (49). In addition, the administration of inactivating anti-ET-1 antibodies or ET receptor antagonists after hepatic ischemia in vitroor in vivo attenuates hepatic perfusion failure (50,60). Moreover, this improvement in hepatic blood flow is associated with reduced liver dysfunction or injury (50,60). However, ETs not only induce vasoconstriction under these pathological conditions, but may also exert vasodilatory effects. For example, pharmacological studies with different ET receptor antagonists have provided indirect evidence that simultaneous ETB1 receptor stimulation can enhance blood flow through the liver and may attenuate the vasoconstrictive effects mediated via ETA and ETB2 receptors (49,60,61). Evidence suggests that in the absence of these ET-mediated vasodilatory effects, the extent of IR-induced liver injury would be even larger (60).
The Role of NO
NO is a gaseous molecule generated from the amino acid L-arginine by NO synthases (NOS). Three different isoforms have been identified—i.e., neuronal constitutive NOS (nNOS; Type I NOS), inducible NOS (iNOS; Type II NOS), and endothelial "constitutive" NOS (eNOS; Type III NOS). The enzymatic activity of nNOS and eNOS is Ca2+ and calmodulin dependent. In contrast, once it has been expressed, iNOS produces much larger amounts of NO in a Ca2+-independent fashion. The vasodilatory effect of NO depends on its reaction with the ferrous iron in the heme prosthetic group of the soluble guanylate cyclase that increases the concentration of cyclic guanosine monophosphate (cGMP) within the respective target cell, thus mediating its relaxation (62).
Hemodynamic Effects of NO in the Normal Liver
NO can exert vasodilatory effects within the normal liver. The addition of either L-arginine or of NO donors increases portal flow in the isolated perfused liver (63). Moreover, exogenous NO may not only antagonize the macrohemodynamic effects of ET-1, but also inhibit its microcirculatory actions. For example, gene transfer of the nNOS to sinusoidal endothelial cells, HSCs, and hepatocytes in vivo inhibits the contraction of stellate cells in response to ET-1 (64).
The administration of NOS inhibitors revealed a substantial basal NO-mediated vasodilatory effect within the hepatic arterial vascular bed, whereas this effect was only small in the portal vein (65,66). This is consistent with the concept that NO exerts a greater vasodilatory effect in arteries than in veins under physiologic conditions. The primary source for NO in the normal liver is most likely the eNOS, which is expressed not only by endothelial cells of the large resistance vessels but also by sinusoidal endothelial cells (67). Its activity increases in response to increases of vascular shear stress, thus counteracting the hemodynamic effects of vasoconstrictive drugs in both vascular beds of the liver (68).
Generation of NO After I/R
The generation of NO requires the presence of nicotinamide adenine dinucleotide phosphate and molecular oxygen (62). Although the availability of both molecules is limited during the ischemic period, the intracellular calcium concentration increases rapidly upon reperfusion, giving rise to an increase in eNOS activity (69). Besides this allosteric upregulation of eNOS, hypoxia may induce an increase in gene transcription of this "constitutive" NOS isoform in endothelial cells (70). Consequently, an increased generation of NO has been observed during early reperfusion after hemorrhagic shock and after low-flow I/R of the liver (49,60). Discrepancies between studies regarding the amount of NO detected during early reperfusion most likely reflect differences in the level of vascular shear stress, a key determinant of eNOS activity.
Bacterial endotoxins and proinflammatory cytokines can induce iNOS expression in parenchymal and nonparenchymal cells of the liver within 4 to 6 h (71), presumably via signal transduction pathways that involve the activation of the nuclear transcription factor-
B (72), a central mediator of the immune response (73), of AP-1, and of HIF-1 (74,75). Once induced, iNOS produces large quantities of NO until the protein is degraded. In patients undergoing partial hepatectomy with Pringle’s maneuver, an increased iNOS expression can be detected in the liver after reperfusion, associated with an increased production of NO (76). Thus, the amount of NO generated during later reperfusion depends primarily on the magnitude of the accompanying inflammatory response (77).
Hepatic Hemodynamic Effects of NO After I/R
There is evidence that the increased endogenous generation of NO maintains hepatic blood flow during early reperfusion. Under these conditions, NOS inhibitors reduce hepatic arterial and portal venous flow and increase the degree of sinusoidal perfusion failure (49,60). This is associated with impaired hepatic oxygenation and an increase in lethal hepatocyte injury (60), which could be explained by the antagonizing effect of NO on ET-1-induced increases in total hepatic and sinusoidal resistance described previously.
The small increase in the amount of NO generated and the time course of de novo iNOS expression would suggest that eNOS is the primary source for NO during early reperfusion. This assumption is supported by a study that used mice genetically deficient in either eNOS or iNOS. Whereas liver injury was significantly enhanced in eNOS-deficient animals subjected to I/R compared with postischemic wild-type mice, this was not the case for iNOS-deficient animals (78). Even at later time points of reperfusion, several studies failed to demonstrate a role of NO generated by iNOS in the regulation of hepatic perfusion and the subsequent development of liver injury (79,80). However, if the magnitude of the inflammatory response that accompanies I/R is large enough, iNOS-derived NO may well contribute to the changes in vascular tone and reactivity that act to maintain liver blood flow during the later reperfusion period (77,81).
The Role of CO
During the past decade, evidence has accumulated suggesting that another endogenously generated gaseous monoxide, i.e., CO, may function as a second messenger gas (82). The catabolism of the heme molecule by the microsomal enzyme heme oxygenase (HO) yields equimolar amounts of biliverdin, iron, and CO. So far, three different isozymes have been identified. Whereas the HO-1 gene is highly inducible by a variety of stimuli, HO-2 is constitutively expressed. In contrast, HO-3 is a poor heme catalyst and may be involved in heme binding. Like NO, CO may induce vasorelaxation as the result of an increased generation of cGMP via activation of the soluble guanylate cyclase by binding to the prosthetic heme moiety of this enzyme (39). However, recent evidence suggests that CO may also modulate vascular resistance by a cGMP-independent mechanism involving the inhibition of cytochrome P450 (83).
Hemodynamic Effects of CO in the Normal Liver
Inhibition of HO activity in isolated perfused livers decreases the amount of CO released and increases portal resistance (84). The administration of either exogenous CO or of cGMP analogs prevents this increase in resistance. These vasorelaxing properties of CO seem to involve HSCs, because the major sites of the constriction associated with HO blockade correspond to sinusoidal segments colocalized with the body of these cells (84). Evidence suggests that the vasodilatory effect of CO represents a physiologic regulatory mechanism that is also found in the intact organism in vivo to keep portal resistance and pressure low, whereas no such intrinsic CO-mediated vasodilation can be detected in the hepatic artery (65).
Within the normal liver, HO-2 is primarily expressed by hepatocytes, whereas HO-1 can be detected in Kupffer cells (85,86). Although both could provide a source for CO, the CO responsible for the vasodilatory effects in the portohepatic circulation under physiologic conditions seems to be primarily derived from HO-2 expressed by parenchymal cells of the liver (86).
Generation of CO After I/R
Several factors that play a key role during I/R of the liver are inducers of HO-1 (32-kd heat shock protein). These include the increased generation of reactive oxygen intermediates, impaired tissue oxygenation, the release of heme from necrotic cells, and increases in shear stress, as well as bacterial lipopolysaccharides and proinflammatory cytokines (87–89). Consequently, increases in total hepatic HO-1 expression have been observed after lobar ischemia of the liver (90) and after hemorrhagic shock (85,91), which is primarily the result of HO-1 gene expression in sinusoidal lining cells and pericentral hepatocytes (85). Recent evidence suggests that this HO-1 induction results primarily from an activation of the transcription factor AP-1 by reactive oxygen intermediates released from Kupffer cells and that it is associated with an increase in the enzymatic activity of HO (92,93). However, HO-1 induction may also depend on other transcription factors such as HIF-1, signal transducers and activators of transcription (STAT1, STAT3, and STAT5), or nuclear factor-erythroid 2 related factor (NF-2) (88,94,95).
Hepatic Hemodynamic Effects of CO After I/R
Chemically induced overexpression of HO-1 in normal rat livers increases hepatic venous CO flux, reduces basal portal resistance, and attenuates the vasoconstrictive effects of exogenous ET-1 (96). This suggests that this protective effect may not be confined to chemical overexpression of HO-1 but could also be a result of the induction of HO-1 after I/R of the liver. This is supported by the following findings. The increase in portal resistance after HO blockade is much more pronounced after hemorrhagic shock as compared with in normal livers and results in a profound decrease in portal flow (91). Under these conditions, endogenously generated CO has been shown to preserve hepatic sinusoidal perfusion via a relaxing mechanism involving HSCs (79). Moreover, this serves to limit I/R-induced perturbations of hepatic mitochondrial redox state, secretory function, and integrity (79,97).
Interactions Between the Different Mediator Systems
All three mediator systems can interact on different levels. NO and CO may exert synergistic vasodilatory effects, because both are able to stimulate soluble guanylate cyclase. Although the relative potency of CO to activate this enzyme is lower, NO can enhance the stimulatory effect of CO. Because NO may interact with superoxide anions that are generated in increased amounts upon reperfusion, the resulting formation of peroxynitrite may limit its availability and in turn further increase the relative importance of CO under these conditions, which cannot react with these compounds. Because the NOS moieties are also hemoproteins, an increased hepatic HO activity after I/R could accelerate the degradation and turnover of NOS. Moreover, because CO is generated in increased amounts under these conditions, it could bind to the prosthetic heme moiety of existing NOS, which will inactivate this enzyme (82). However, NO may either attenuate or enhance hepatic HO-1 gene expression, depending on the amount of NO available (92,98). Although the vasodilatory effects of ETs are at least in part mediated by NO (see previously), both gaseous monoxides can functionally antagonize the vasoconstrictive effects of ETs in the liver (99) (see previously). In addition, smooth muscle cell-derived CO can inhibit the expression of ET-1 by endothelial cells in response to hypoxia (100). Similarly, NO has been shown to limit the release of ET-1 from endothelial cells by a cGMP-independent mechanism (101). Moreover, NO may terminate ET signaling by displacement of bound ET-1 from its receptor and by interference with its postreceptoral signal transduction pathway that involves an attenuation of Ca2+ mobilization (102) (Fig. 1).
Potential Implications for the Development of New Clinical Strategies
Most of the results summarized previously have been obtained in animal studies. Although the mediator systems of interest have been highly preserved during evolution and some of the findings derived from animal studies have been confirmed in humans, extreme caution is necessary if they are to be used as a basis for the development of new clinical strategies aimed at limiting I/R-induced impairments of hepatic perfusion and integrity. This implies that all concepts derived from these data will require prior thorough evaluation in future animal and human studies to decide whether they are suitable to improve not only nutritive hepatic blood flow, but also the function of the liver. Moreover, I/R injury of the liver may lead to remote organ failure of the lungs, the heart, and the systemic circulation (17,103). Consequently, it will be of major importance to determine whether these interventions may also improve the outcome after I/R of the liver.
The Concept of a "Critical Balance"
Under physiologic conditions, the influence of vasoconstrictors and vasodilators on hepatic blood flow is only small, and their effects are balanced. After I/R of the liver, there is an upregulation of constrictor and dilator influences. However, because this upregulation is frequently not matched in time and space, it may result in an imbalance of these antagonistic influences at all levels of blood flow regulation that can cause severe perturbations of hepatic hemodynamics (Fig. 2). For example, the net increase in portal resistance after endotoxemic shock is, at least in part, due to a profound increase in ET-mediated sinusoidal and presinusoidal vasoconstrictive tone that is only transiently and partially counterbalanced by increases in NO-mediated vasodilator influences (66). Moreover, the profound alterations of nutritive sinusoidal flow described previously and the subsequent functional impairments may occur, even in the absence of overt macrohemodynamic changes, as a result of an I/R-induced heterogeneity of hepatic microvascular flow patterns (104,105). This concept has several important implications for the development of new therapeutic strategies. First, it must be the goal of potential interventions to reestablish a new balance. Thus, complete blockade of a single "noxious" mediator is unlikely to be successful. Second, although this concept has been primarily developed on the basis of the interactions between ETs and gaseous monoxides, it can and must be applied to other vasoactive mediators involved in the pathogenesis of disorders of hepatic perfusion after I/R. Finally, two principal ways may lead to a reestablishment of a new balance after I/R, i.e., either an adapted attenuation of constrictive influences or a graded enhancement of dilator influences. Different methodological approaches could be applied to meet these goals. These include the use of receptor antagonists, of inhibitors of enzymatic activity, of pharmacological drugs that provide an exogenous source of mediators, and of chemical inducers of target genes, as well as transgenic approaches aimed at modulating the expression of genes of interest.
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On the basis of the results of these studies, it is likely that the new insights into the regulation of hepatic blood flow after I/R may ultimately lead to the development of new therapeutic strategies aimed at limiting the subsequent development of liver injury.
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Acknowledgments |
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The author would like to thank Hans-Joachim Priebe, MD, and Albert Benzing, MD, for critically reviewing the manuscript.
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