Hepatic stellate cells (HSCs) and hepatic oval cells (OCs) are important cell types in liver repair. HSCs, the liver's principal fibrogenic cells, are activated by liver injury of any cause to move from a quiescent to an activated myofibroblastic phenotype. This myofibroblastic phenotype is proliferative, expresses α-smooth muscle actin, and synthesizes fibrogenic matrix proteins that accumulate during cirrhosis (Friedman, 2000). Activated myofibroblastic HSCs are also contractile and therefore may contribute to the pathogenesis of portal hypertension (Reynaert et al., 2002). Conversely, OCs are liver resident progenitor cells that help to regenerate the hepatic epithelial compartment. OC populations are activated when mature hepatocytes reach a critically low number, such as after severe liver injury, or when mature hepatocytes are prevented from dividing by hepatotoxic drugs (Evarts et al., 1996). Therefore, OCs promote the regeneration of damaged livers. Emerging evidence suggests that the sympathetic nervous system (SNS) effects the function of both HSCs and OCs and therefore directly regulates liver repair.
This chapter reviews recent evidence that the sympathetic nervous system (SNS) regulates liver repair by modulating the phenotypes of hepatic stellate cells (HSCs), the liver's principal fibrogenic cells, and hepatic epithelial progenitors, i.e., oval cells. SNS nerve fibers touch HSCs and these cells express adrenoceptors, suggesting that HSCs may be targets for SNS neurotransmitters. HSCs also contain catecholamine biosynthetic enzymes, release norepinephrine (NE), and are growth-inhibited by adrenoceptor antagonists. In addition, HSCs from mice with reduced levels of NE grow poorly in culture and exhibit inhibited activation during liver injury. Finally, growth and injury-related fibrogenic responses are rescued by adrenoceptor agonists. Thus, certain SNS inhibitors (SNSIs) protect experimental animals from cirrhosis. Conversely, SNSIs enhance the hepatic accumulation of oval cells (OCs) in injured livers. This response is associated with improved liver injury. Because SNSIs do not affect the expression of cytokines, growth factors, or growth factor receptors that are known to regulate OCs, and OCs express adrenoceptors, it is conceivable that catecholamines influence OCs by direct interaction with OC adrenoceptors. Given evidence that the SNS regulates the viability and activation of HSCs and OCs differentially, SNSIs may be novel therapies to improve the repair of damaged livers. © 2004 Wiley-Liss, Inc.
SNS REGULATION OF HSC: HISTORICAL EVIDENCE
HSCs Express Neuronal Markers
HSCs may function as hepatic neuroglia cells, receiving and integrating commands from the CNS, because SNS fibers abut HSCs (Moghimzadeh et al., 1983; Ohata, 1984; Bioulac-Sage et al., 1990), which possess functional α-adrenoceptors (Athari et al., 1994). That HSC may be neural crest-derived hepatic neuroglial cells is further supported by their expression of classical neuroglial proteins, such as glial acidic fibrillary protein (GFAP), nestin, neural cell adhesion molecule (NCAM), synatophysin, and neurotrophins, as well as by evidence that they contain synaptic vesicles (Buniatian et al., 1996; Cassiman et al., 1999, 2001; Niki et al., 1999).
SNS Activity Correlates With Liver Fibrosis
A growing body of indirect evidence supports the possibility that SNS overactivity may be involved in the aetiopathogenesis and progression of cirrhosis. First, the severity of carbon tetrachloride (CCL4)-induced fibrosis is greater in the spontaneously hypertensive rat, which has a hyperactive SNS compared with normotensive control Wistar-Kyoto rats (Hsu, 1992). Second, in livers pretreated ex vivo with D-galactosamine, the degree of injury is markedly enhanced by electrical stimulation of the attached sympathetic nerves. This effect is mimicked by infusions of low doses of norepinephrine (NE) that do not compromise perfusate flow to the liver (Iwai and Shimazu, 1996). Third, animals with low catecholamine levels and low SNS tone (e.g., leptin-deficient ob/ob mice) (Young and Landsberg, 1983; Liang and Cincotta, 2001) are resistant to liver fibrosis (Honda et al., 2002). Together, these findings suggest that activation of sympathetic nerves increases circulating cathecholamines that exacerbate liver injury. Conversely, chemical sympathectomy with 6-hydroxydopamine, or α1-adrenoceptor antagonism with prazosin, inhibits CCL4-induced liver fibrosis in rats (Dubuisson et al., 2002). Evidence that parasympathetic cholinergic agonists modulate local matrix production to regulate wound healing in other epithelial tissues (Heeschen et al., 2002; Jacobi et al., 2002) also supports the concept that the autonomic nervous system regulates liver injury and repair. Finally, activation of the SNS with increased levels of NE and its cotransmitter neuropeptide Y is well documented in patients with cirrhosis (Henriksen et al., 1984; Esler et al., 1992), particularly when the hepatorenal syndrome develops (Uriz et al., 2002). However, whether altered SNS activity is primary to the pathogenesis of cirrhosis or a secondary compensation for the circulatory disturbances that accompany cirrhosis is unknown.
SNS REGULATION OF LIVER FIBROSIS: NEW EVIDENCE FOR DIRECT SNS-HSC INTERACTIONS
Despite the cited indirect evidence suggesting that the SNS regulates the development of fibrosis, until recently there was no proof that neurotransmitters regulated liver fibrogenesis directly or what cell types might be targeted. Over the last 2 years, our group has been systematically investigating interactions between the SNS and HSCs (Oben et al., 2003d, e, f, 2004). Our overarching hypothesis is that HSCs are hepatic neuroglia that are regulated directly by the SNS and that provide a local source of cathecholamines. We evaluated our hypothesis by addressing the following questions (Oben et al., 2003f, 2004). Do HSCs contain NE synthesizing enzymes? Do HSCs release NE? And do HSC change function in response to exogenous or endogenous NE? The results of these studies are summarized subsequently.
HSCs Synthesize and Release NE and Other Neurotransmitters
The key enzymes in the synthesis of NE are tyrosine hydroxylase (which converts dihydroxyphenylalanine to dopamine) and dopamine-β-hydroxylase (which converts dopamine to NE). We evaluated the expression of these catecholamine biosynthetic enzymes in primary HSCs cultured from normal mice. Western blot analysis of HSC cell protein confirmed that HSCs express both dopamine-β-hydroxylase (Dbh) and tyrosine hydroxylase. Moreover, high-pressure liquid chromatography analysis of HSC-conditioned medium showed that normal HSCs also release NE. In contrast, NE was not detected in conditioned medium from Dbh−/− HSCs that are not able to synthesize NE because of a targeted deletion of the Dbh gene. In addition to NE, normal murine HSC lysates contain DA, serotonin (5-hydroxytryptamine; 5-HT), cathecholamine metabolites (dihydroxyphenylacetic acid; DOPAC), homovanillic acid (HVA), and the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), demonstrating that HSCs can function as neuroglia to provide catecholamines locally within injured livers (Oben et al., 2004).
HSCs Express Adrenoceptors
Although HSCs were known to express α1-adrenoceptors (Athari et al., 1994), which α-adrenoceptors subtypes are expressed, or if HSCs express β-adrenoceptors, was not known until our recent studies (Oben et al., 2004). Using RT-PCR analysis, we demonstrated that primary HSCs express α1B-, α1D-, β1-, and β2-adrenoceptor mRNAs (Fig. 1). A similar expression profile was noted with Western blot analysis.
NE Regulates HSC Growth in Culture
To determine if endogenous NE is important for HSC growth, we cultured normal HSCs in the presence and absence of an α1-adrenoceptor antagonist, prazosin (PRZ; 10 μM), or a β-adrenoceptor antagonist, propranolol (PRL; 10 μM), and assessed their growth (Mosmann, 1983; Isobe et al., 1999; Frank et al., 2000; Matsuoka et al., 2000; Saxena et al., 2002). PRZ and PRL each reduced HSC numbers by ∼ 20%, and the combination of PRZ + PRL decreased HSC growth by ∼ 50%, showing that the growth-inhibitory actions of the α- and β-adrenoceptor antagonists are additive. Studies of HSCs cultured from Dbh−/− (NE-deficient) and Dbh+/− (control) mice prove that NE is an autocrine growth factor for HSCs. HSCs from Dbh+/− mice proliferate to become nearly confluent by 4 days in culture, and their proliferative activity is inhibited significantly by PRZ. Proliferative activity is also significantly reduced in HSC from Dbh−/− mice (Fig. 2), while addition of exogenous NE rescues their growth. Together, these findings confirm the importance of NE for HSC growth in culture (Oben et al., 2004). Moreover, NE increases the growth of HSCs in a dose-dependent manner. Neuropeptide Y, an SNS cotransmitter neuropeptide that is released with NE from sympathetic nerve terminals, also promotes HSC growth (Oben et al., 2003f).
During culture, HSCs normally become activated and proliferate at a greater rate than they die. Therefore, increases in proliferative activity normally drive HSC growth in culture. To determine to what extent, if any, the NE-related differences in cell number might also reflect differences in apoptotic activity, HSCs were harvested, incubated with annexin V, and analyzed by flow cytometry. After 1 day in culture, slightly greater numbers of apoptotic HSCs were detected in cultures from Dbh−/− mice compared to Dbh+/− mice. Thus, the antiapoptotic effects of NE may help explain why endogenous NE is required for optimal HSC growth. In any case, our data show that cultured HSCs express NE synthesizing enzymes, release NE, and grow in response both to exogenous and endogenous NE.
SNS Regulates HSCs in Intact Mice
To study the effects of NE on HSC function in intact animals, NE was infused chronically into leptin-deficient ob/ob mice known to have low levels of NE (Knehans and Romsos, 1982; Young and Landsberg, 1983), but which are resistant to fibrosis, despite clear evidence of chronic liver injury (Ikejima et al., 2001; Honda et al., 2002; Leclercq et al., 2002; Oben et al., 2003c). Hepatic expression of GFAP was then analyzed as a readout of quiescent and activated HSCs (Cassiman et al., 2002a). We found that control ob/ob mice have significantly fewer GFAP+ HSCs than their lean littermates and NE replacement markedly stimulates the in vivo proliferation of HSCs, such that the numbers of GFAP+ HSCs in ob/ob mice approach that seen in lean controls (Oben et al., 2004).
Because leptin deficiency might have confounded the effects of NE in ob/ob mice, we next studied Dbh−/− mice, which also have reduced NE but are not leptin-deficient (Thomas and Palmiter, 1997a, 1997b). Dbh−/− mice and Dbh+/− mice were fed an antioxidant-depleted hepatotoxic and fibrogenic diet (Leclercq et al., 2002). After 4 weeks of treatment, control Dbh+/− mice exhibit a striking accumulation of HSCs that express ASMA, an accepted marker of HSC activation (Fig. 3a and c). In contrast, ASMA+ HSCs could not be demonstrated in Dbh−/− mice (Fig. 3b and c), while blood vessel walls in these mice clearly express ASMA+, arguing against this effect being a staining artifact (Fig. 3a). Moreover, compared to Dbh+/− controls, Dbh−/− mice also exhibit significantly less hepatic expression of collagen and TGF-β1, two other indicators of HSC activation (Fig. 4). Ribonuclease protection analysis also demonstrates a small (∼ 30–40%) but statistically significant (P < 0.05) reduction in the induction of tissue inhibitor of metalloproteinase (TIMP)-2 transcripts in the Dbh−/− group.
To verify that it was reduced adrenergic activity that prevented HSC activation, we implanted osmotic minipumps containing vehicle or isoprenaline (ISO), a β-adrenoceptor agonist, into Dbh−/− mice and repeated the feeding experiment. Infusion of ISO rescues HSC activation in Dbh−/− mice and returned numbers of ASMA+ HSCs to levels exhibited by Dbh+/− mice also fed the hepatotoxic diet (Fig. 3c). ISO infusion similarly normalizes induction of TGF-β in Dbh−/− mice (Fig. 4b). In parallel experiments, we examined the effect of NE, an α-adrenoceptor agonist, on HSC activation in ob/ob mice. Compared to control ob/ob mice, ob/ob mice treated chronically with NE minipumps have significantly increased liver expression of TGF-β1, collagen mRNA (Oben et al., 2004), and histological evidence of fibrosis (Oben et al., 2003c). Therefore, both α-predominant NE and β-predominant ISO adrenoceptor agonists affect HSC activation in vivo. Despite this increase in HSC activation, alanine aminotransferase (ALT) values in NE-treated ob/ob mice were lower than their littermate controls. As such, NE-related increases in fibrogenesis are not easily attributed to NE exacerbation of liver injury (Oben et al., 2003c).
These studies then confirm that HSCs express key enzymes for cathecholamine biosynthesis, actually produce NE and other cathecholamines, and that HSCs are directly regulated by the SNS both in vitro and in whole animals. HSCs appear to use cathecholamines to autoregulate their growth, because increases in HSC number are significantly attenuated by culturing normal HSCs with α- or β-adrenoceptor antagonists. HSCs from NE-deficient animals also grow poorly in culture. In vivo activation of HSCs, as judged by the expression of ASMA, TGF-β and collagen along with histological evidence of fibrosis, is markedly reduced in NE-deficient animals.
SNS REGULATION OF LIVER FIBROSIS: NE INTERACTIONS WITH OTHER PEPTIDES
That NE may subserve functions other than its classically assigned role of neurotransmission is established in other organs. For example, cardiac remodeling in heart failure involves mitogenic and fibrogenic actions of NE that are mediated via adrenoceptors (Fisher and Absher, 1995; Xiao et al., 2001; Akiyama-Uchida et al., 2002). The in vivo sources that might provide NE for HSC regulation include HSCs themselves, SNS nerve terminals that abut HSCs, and the adrenal medulla that releases NE and adrenaline into the circulation under stressful conditions, such as liver injury. The relative importance of these three sources in the regulation of HSC function in vivo is as yet unclear. It is also likely that NE interacts with other peptides to modulate HSC function. Indeed, our studies identify at least three other factors, acetylcholine (Ach), neuropeptide Y (NPY), and leptin, that may modulate SNS-HSC interactions to influence liver fibrosis.
Like NE, the parasympathetic neurotransmitter Ach appears to function extraneuronally (Heeschen et al., 2001, 2002; Jacobi et al., 2002). In keeping with this concept, we demonstrated that Ach promotes proliferation and induces collagen gene expression in activated HSCs (Oben et al., 2003d). Whether HSCs also synthesize and release Ach is unknown. More studies are also needed to elucidate the role of the parasympathetic nervous system in HSC biology.
The sympathetic neurotransmitter NPY is coreleased with NE from SNS terminals. We demonstrated that NPY promotes the proliferation of cultured HSCs. Indeed, NPY has much greater mitogenic potency than NE, because peak HSC proliferation was observed at 1 μM NE, but occurred at as little as 0.1 nM NPY (Oben et al., 2003f). As has been shown in studies of other mesenchymal cell types (Kanevskij et al., 2002), we noted that these two cotransmitters interact to influence HSC proliferation. Increasing concentrations of NE attenuate NPY-induced proliferation of HSCs (Oben et al., 2003f). NE and NPY also appear to have divergent effects on collagen gene expression by HSCs. While NE increases the expression of collagen1-α2, neither low nor high concentrations of NPY alter collagen1-α2 mRNA levels. Thus, although both NE and NPY increase HSC proliferation, only NE induces collagen gene expression (Oben et al., 2003f). The clinical significance of NE-NPY interactions for regulating HSC biology remains untested.
In bone and fat, leptin modulates tissue remodeling by regulating NE production. Our recent studies of Dbh−/− mice and ob/ob mice suggest that a similar process applies in the liver, at least during fibrogenesis. Dbh−/− mice (which are leptin-replete but lack NE) do not activate their HSCs in response to injury. Conversely, supplemental NE normalizes HSC numbers and activation in leptin-deficient ob/ob mice. Whether or not leptin induces production of NE by HSCs, as it does in adipocytes (Commins et al., 1999), is not yet known. It is also conceivable that leptin promotes HSC activation by inducing the expression or function of adrenoreceptors or components of the postreceptor signaling pathways that mediate NE effects. More work is required to delineate the exact nature of leptin-NE interactions.
SNS EFFECTS ON HEPATIC OVAL CELLS
SNS Inhibition Promotes Liver Regeneration: Historical Evidence
Oval cells are another major cell type that is involved in liver repair. Hepatic oval cells (HOCs) are facultative stem cells that are activated when mature hepatocytes reach a critically low level, as after subtotal necrosis, or when mature hepatocytes are prevented from dividing. The latter occurs in animals and humans with various chronic liver diseases (Roskams et al., 2003). To determine whether the SNS effects HOC-mediated liver regeneration, we investigated the effects of SNS inhibition on the HOC response to chronic liver injury. Although earlier work from other groups had shown that SNS inhibition enhanced hepatic regeneration after a partial hepatectomy (Kato and Shimazu, 1983; Kiba et al., 1994), it was not known if (or how) the SNS influenced HOCs themselves or if SNS-HOC interactions played any role in the recovery from chronic liver damage.
SNS Inhibition Promotes Accumulation of HOCs and Reduces Liver Injury: New Evidence
We used an established model of OC activation (methionine-choline-deficient diets plus ethionine) to injure the liver, inhibit mature hepatocyte replication, and induce HOC accumulation (Akhurst et al., 2001). We hypothesized that SNS inhibition would promote further HOC accumulation and reduce liver damage. Compared to control mice that were fed only the antioxidant-depleted diets, mice fed the same diets with prazosin (an α1-adrenoceptor antagonist) or 6-hydroxydopamine (6-OHDA, an agent that induces chemical sympathectomy) had significantly increased numbers of HOCs (Fig. 5). Increased HOC accumulation was accompanied by less hepatic necrosis (Fig. 6a) and steatosis (Oben et al., 2003a, b), lower serum aminotransferases (Fig. 6b), and greater liver and whole body weights (Oben et al., 2003a, 2003b).
Neither PRZ nor 6-OHDA affected the hepatic expression of granulocyte, granulocyte/macrophage, or macrophage colony stimulating factor (G-CSF, GM-CSF, M-CSF), interleukin (IL)-6, IL-7, IL-11, leukemia inhibitory factor, stem cell factor, hepatocyte growth factor, or vascular endothelial growth factor (VEFG) and its receptors VEGFR1 and -3 cytokines, growth factors, or growth factor receptors that are known to regulate progenitor cells (Oben et al., 2003a, 2003b). Hence, the SNS may exert its inhibitory effects on HOCs directly, by interacting with HOC adrenoceptors (Fig. 7) (Oben et al., 2003a, 2003b). More work is required to evaluate this possibility. Nevertheless, it is likely that autonomic control of hepatic progenitors is important given recent evidence that the parasympathetic nervous system, acting via the vagus nerve, also regulates the accumulation of HOCs (Cassiman et al., 2002b).
SNS REGULATION OF HSC AND HOC: THERAPEUTIC IMPLICATIONS
Our studies of the SNS and HSCs extend understanding of the mechanisms by which cathecholamines regulate the repair of injured livers. The aggregate data support the notion that SNS activation during liver disease (Henriksen et al., 1984; Esler et al., 1992) promotes liver fibrosis (by activating HSCs) and may simultaneously inhibit liver regeneration (by reducing HOC accumulation). Our goal is to exploit this knowledge to develop novel treatments for liver disease. Because NE promotes HSC activation, targeted interruption of catecholamine signaling in HSCs may be a useful therapeutic approach to constrain liver fibrosis. Similarly, targeted inhibition of SNS actions on HOCs may enhance liver regeneration by negating NE's inhibitory actions on liver repair by HOCs (Fig. 8). The recent delineation of the adrenoceptor subtypes that are expressed by HSCs and OCs is a step toward this goal. Remaining challenges include the development of adrenoceptor antagonists that are devoid of vascular effects that would otherwise limit their utility in patients with liver disease.