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Endogenous cannabinoids are ubiquitous lipid signaling molecules that are able to partially mimic the actions produced by Δ9-tetrahydrocannabinol, the compound responsible for most of the psychological effects of marijuana. Endocannabinoids are involved in an important number of central and peripheral physiological effects and are derived from arachidonic acid. They include anandamide (arachidonylethanolamide [AEA]), 2-arachydonyl glycerol (2-AG), N-arachydonoyldopamine, noladin ether, and virodhamine,1 the former two being the most extensively investigated. The interaction of these substances with two different types of receptors results in most of their biological effects. By coupling to Gi/o proteins the cannabinoid CB1 receptor may inhibit adenylate cyclase activity and voltage-activated Ca2+ channels or else stimulate protein kinase activity and inwardly rectify K+ channels. The cannabinoid CB2 receptor transduces similar biological responses, with the exception of those acting through voltage-activated Ca2+ and inwardly rectifying K+ channels.2, 3 Moreover, AEA also can interact with the transient receptor potential vanilloid type 1 protein (TRPV1), which is also known as the VR1 receptor and belongs to the large family of TRP ion channels.4, 5 The cannabinoid CB1 receptor is densely distributed in areas of the brain related to motor control, cognition, emotional response, motivated behavior, and homeostasis. In addition, the CB1 receptor is also peripherally located in vessels, in endothelial6 and smooth muscle cells,7, 8 and in the terminal endings of perivascular nerves, which also express TRPV1 receptors.9 By contrast, CB2 receptors are mainly restricted to immune tissues and cells.

In addition to displaying psychoactive, analgesic, hypothermic, antiemetic, and locomotor inhibitory properties, endocannabinoids also exhibit vasorelaxing and anti-inflammatory activities and stimulate food intake. Several investigations have also suggested that these substances may be antitumoral agents, since they induce the regression of skin tumors and gliomas, a phenomenon thought to be due to their antiproliferative and apoptotic properties.10, 11

Endogenous cannabinoids are released upon demand from lipid precursors in a receptor-dependent manner. AEA is produced from the enzymatic cleavage of N-arachydonyl phosphatidylethanolamine by a Ca2+-dependent N-acetyltransferase in several cell types, including neurons, human umbilical vein endothelial cells, neuroblastoma cells, leukocytes, monocytes, and macrophages.12 Endocannabinoids are transported into cells by a specific uptake system and degraded by two well-characterized enzymes, fatty acid amidehydrolase and monoacylglycerol lipase.13, 14 Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and antagonists, anandamide uptake blockers, and selective inhibitors of endocannabinoid degradation. These pharmacological tools have also enabled investigation of the role of endocannabinoids in physiological and pathological conditions.15

Awareness of the pathophysiological importance of the endogenous cannabinoid system in advanced liver disease has grown during the past few years. Initially, investigations focusing on the vasorelaxing properties of endocannabinoids demonstrated that these substances are involved in the pathogenesis of the cardiovascular dysfunction occurring in advanced liver disease.16, 17 Systemic administration of a selective CB1 receptor antagonist, SR141716A, increased arterial pressure and total peripheral resistance and decreased mesenteric blood flow and portal pressure in rats with cirrhosis and ascites, but not in control rats. These effects were peripherally mediated because injection of the antagonist in the fourth cerebral ventricle of both control rats and rats with cirrhosis failed to alter blood pressure. Furthermore, a significant reduction in mean arterial pressure was observed in recipient animals in response to circulating blood cells of rats with cirrhosis, a phenomenon not observed when cells were collected from control rats. A remarkably similar pattern of cardiovascular behavior was observed in recipient animals in response to isolated circulating monocytes of rats with cirrhosis and ascites.17 This hypotensive effect induced by isolated monocytes of rats with cirrhosis was prevented by the previous administration of the CB1-specific receptor antagonist. Since it has been shown that AEA can elicit arterial hypotension through CB1 receptors, it is likely that this endocannabinoid mediates, at least in part, the hypotension induced by monocytes of rats with cirrhosis. Increased circulating levels of AEA have also been described in patients with cirrhosis.18 More recently, experiments in isolated third-order mesenteric arteries of rats with cirrhosis19 and ascites have shown that these vessels display an altered and differential response to anandamide. This seems to occur via two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in the perivascular sensory nerve terminals of rats with cirrhosis (Fig. 1). Experimental evidence also suggests that increased local production of endocannabinoids, acting through a CB1-mediated signaling pathway, could be involved in the pathogenesis of the cirrhotic cardiomyopathy observed in bile duct–ligated rats with cirrhosis.20 Overall, these results point to the endocannabinoid system as a potentially significant pathogenic mediator of the splanchnic arteriolar vasodilation found in advanced liver disease.

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Figure 1. Immunodetection of CB1 receptors in whole mounted third-order mesenteric arteries of control and CCl4-induced rats with cirrhosis and ascites. (Courtesy of Dra. Ana Maria Briones, Universidad Autonoma, Madrid, Spain). AC, adventitial cells; PN, perivascular nerves.

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An increasing body of evidence indicates that in addition to playing a role in the systemic circulation in advanced cirrhosis, the endogenous cannabinoid system could also be of relevance in the pathogenesis of liver fibrosis and portal hypertension. In fact, in a paper on this subject Siegmund et al.21 demonstrated that AEA is a selective killer of hepatic activated stellate cells (HSCs) in vitro, suggesting that AEA could be used as a potentially therapeutic antifibrogenic mediator. The data presented indicate that AEA dose-dependently induces necrosis in HSCs at μmol/L concentrations, a phenomenon not dependent on CB1, CB2, or TRPV1 receptor activation. Rather, the authors suggest that AEA induces cellular necrosis through its interaction with the membrane cholesterol, resulting in reactive oxygen species formation, intracellular Ca2+ release and cell death.

As to whether AEA-induced necrosis is a specific effect for HSCs but not for hepatocytes is a matter of controversy. Biswas et al.22 previously reported that HepG2 cells and primary hepatocytes undergo apoptosis when these cells are challenged with AEA. Nevertheless, most experiments in this study were performed in HepG2 cells, which may present a different sensitivity to AEA-mediated cell death compared with primary hepatocytes. These differences may explain the discrepancies between the results of Siegmund et al.21 and those of Biswas et al.22 Whatever the case, the important conceptual hypothesis raised by Siegmund et al.21 is that AEA may induce antifibrogenic effects by inhibiting HSCs at low concentrations and by inducing HSC death at higher concentrations. Therefore, AEA could be therapeutically useful to prevent or reverse the progression of liver fibrosis. The hypothesis that endocannabinoids may behave as antifibrogenic agents has been strengthened by the results obtained by Julien et al.23 These authors recently showed that CB2 receptors are strongly induced during human liver cirrhosis and are expressed in nonparenchymal cells and biliary cells located within and at the edges of fibrotic septa. CB2 receptor activation resulted in growth inhibition and apoptosis. These effects were observed at different cannabinoid concentrations because submicromolar doses inhibited growth without affecting cell viability, whereas μmol/L concentrations induced apoptosis. Moreover, they also showed that mice treated with CCl4 lacking CB2 receptors develop more fibrosis than wildtype CCl4-treated mice.

However, substantial issues dealing with the specifity of the AEA effects still remain to be solved. For instance, it has been shown that by interacting with CB1 or CB2 receptors, AEA may mediate other important biological functions in human HSCs, including intracellular Ca2+ mobilization, cell contraction, and proinflammatory cytokine secretion.24 Moreover, it is important to elucidate whether AEA-induced cell death may also affect other cell types throughout the organism, since the biological responses to cannabinoids critically depend on drug concentration and cellular context. In fact, previous studies have shown that AEA treatment at μmol/L concentrations may cause death in extra hepatic cells. This, in turn, raises important concerns regarding the systemic effects of AEA treatment in cirrhosis. Therefore, in vitro experiments should be complemented with investigations in experimental models of liver fibrosis in which the therapeutic utility of the pharmacological treatment with AEA could be tested.

In summary, regardless of whether the experiments are performed in vivo or in vitro, or in activated stellate cells or human liver biopsies, the endocannabinoid system emerges as an important modulator of cell growth and viability in nonparenchymal hepatic cells. These results, therefore, open new avenues for the therapeutic regulation of fibrosis and portal hypertension in advanced liver disease.

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