WO2005020969A2

WO2005020969A2 - A method of treating fibrosis

Info

Publication number: WO2005020969A2
Application number: PCT/GB2004/001324
Authority: WO
Inventors: Jude A. Oben; Anna Mae Diehl
Original assignee: Oben Jude A; Anna Mae Diehl
Priority date: 2003-03-28
Filing date: 2004-03-29
Publication date: 2005-03-10

Abstract

A method of treating fibrosis comprising administering to a subject at risk of or suffering from a fibrotic disease, particularly of the liver, an amount of a neurotransmitter antagonist, for example prazosin and/or propanolol, effective to reduce the activity of hepatic stellate cells.

Description

A METHOD OF TREATING FIBROSIS

Recovery from liver damage might be enhanced by encouraging repopulation of the liver by endogenous hepatic progenitor cells. Bone marrow-derived progenitors may differentiate into oval cells - resident hepatic stem cells that promote liver regeneration and repair. Little is known about the mediators that regulate the homing or accumulation of these cells in the liver. The sympathetic nervous system (SNS) innervates bone marrow, and adrenergic inhibition mobilizes hematopoeitic precursors into the circulation. Thus, we hypothesized that SNS inhibition would promote hepatic accumulation of progenitor cells and reduce liver damage in mice fed anti-oxidant depleted diets to induce liver injury. Our results confirm this hypothesis. Compared to control mice that were fed I "' only the' anti-oxidant depleted diets, mice fed the same diets with Prazosin (PRZ, an alpha- 1 . adrenoceptor antagonist) or 6-Hydroxydopamme (6-OHDA, an agent that induces chemical sympathectomy), had significantly increased numbers both of oval cells and putative bone marrow- / derived hepatic progenitors. Increased hepatic progenitor cell accumulation was accompanied by less hepatic necrosis and steatosis, lower serum aminotransferases, and greater liver andwhole body weights. Neither PRZ nor 6-OHDA affected the expression of cytokines, growth factors or growth factor receptors that are known to regulate progenitor cells. In conclusion, stress-related sympathetic activity modulates progenitor cell accumulation in damaged livers and SNS blockade with alpha-adrenoceptor antagonists enhances hepatic progenitor cell accumulation and improves recovery from liver damage.

Abbreviations: /

^■ Sympathetic Nervous System (SNS), Prazosin (PRZ), 6-Hydroxydopamine (6-OHDA), Hepatic Progenitor Cell (HPC), Autonomic Nervous System (ANS), Norepinephrine (NE), Natural Killer T (NK-T) cells, Half Methionine-Choline Deficient plus Ethionine (HMCDE), Control Methionine Choline Diet (CMCD), Stem Cell Factor (SCF), Interleukin (IL), Leukaemia Inhibitory Factor (LIF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Granulocyte Colony Stimulating Factor (G -CSF), Vascular Endothelial Growth Factor (NEGF), Hepatocyte Growth Factor (HGF). The liver's progenitor cell compartment is activated if the resident mature hepatocytes reach a critically low number, such as after severe hepatic injury, or if the mature hepatocytes are prevented from dividing by hepatotoxic drugs (1). One hepatic progenitor cell (HPC) compartment, the oval cells, are resident within the liver's canals of Herring - the terminal branches of the biliary tree. The source of oval cells themselves is debated but there is some evidence that they may be derived from pluripotent progenitors that reside in the bone marrow (2). The factors involved in expanding hepatic progenitor cell populations within the liver are not well understood. The identification of such factors is an important therapeutic goal because they may be useful to support patients with acute liver failure until a suitable organ is found for transplant. Indeed, if successful, targeted expansion of endogenous HPC may even obviate the need for orthotopic liver transplantation. Emerging evidence suggests that the autonomic nervous system (ANS) may regulate the accumulation of HPC in the liver. The parasympathetic nervous system appears to promote this ^•process because vagotomy reduces the expansion of HPC numbers in rats with drug induced • hepatitis. Similarly, after transplantation (which surgically denervates the liver), human livers that develop hepatitis have fewer HPC than native, fully innervated livers, with similar degrees of liver injury (3). The decreased accumulation of HPC in transplanted livers may alter their regenerative response to injury because the rate of fibrosis is often accelerated in liver transplant recipients with chronic hepatitis (4). Although the sympathetic nervous system (SNS) is known to modulate both liver regeneration (5) and hepatic fibrogenesis (6,7) it is not known if these effects reflect the ability of the SNS to influence HPC accumulation in injured livers. Thus, the aim of the present study was to test the hypothesis that the SNS affects the expansion of HPC. We used established models of HPC accumulation involving administration of anti-oxidant depleted diets plus ethionine to cause liver injury and inhibit mature hepatocyte replication (8). We then manipulated the SNS by adrenoceptor antagonism with prazosin (PRZ) or chemical sympathectomy with 6-hydroxydopamine (6-OHDA), in order to reduce the activity or production of the SNS neurotransmitter, norepinephrine (NE). HPC numbers in control and SNS-inhibited livers were analysed by both flow cytometry and immunohistochemistry (9,3) . Because the SNS is known to promote the hepatic accumulation of natural killer T (NK-T) cells (10), liver NK-T cells were evaluated concurrently to monitor the physiological efficacy of SNS inhibition. Our results demonstrate that SNS inhibition significantly enhances the accumulation of HPC and reduces liver injury. This suggests that adrenoreceptor blockade might be used therapeutically to expand HPC and promote liver regeneration in circumstances that prevent the replication of mature hepatocytes.

Materials and Methods Animals C57BL-6 mice, 10-18 weeks old, were from Jackson Laboratory (Bar Harbor, ME).

Diets and Drugs The diet was a modification of the half-choline deficient diet (ICN, Aurora, OH) that haξ, been shown to cause hepatic accumulation of HPC within 2 weeks (8). In addition to choline deficiency the diet used here was also 50% deficient in methionine to enhance oxidative injury to the, liver. This diet was administered with ethionine (0.15%) in drinking water (8) and the combination treatment is referred to as half methionine choline deficient diet plus ethionine (HMCDE) hereafter. The control methionine choline diet (CMCD) was also from ICN. Prazosin (PRZ) and DL-Ethionine (E) were from Sigma, St Louis, MO).

Chemical Sympathectomy Chemical sympathectomy was achieved by intra-peritoneal (IP) injection of 6- Hydroxy dopamine (6-OHDA) lOOmg/kg for 5 consecutive days as described (11). Thereafter, 6- OHDA was administered at lOOmg/kg i.p., three times per week to ensure continued sympathectomy (7). The dose and dosing regimen for 6-OHDA has been previously shown to virtually deplete norepinephrine in rodent tissues (7,11).

Experimental Design

Mice were divided into 4 groups, with each group containing 10 to 12 animals. Group 1 - Control diet; Group 2 - HMCDE plus saline i.p.; Group 3 - HMCDE plus prazosin in drinking water; Group 4 - HMCDE plus 6-OHDA i.p. Experiments were performed on 2 separate occasions. Therefore, final results are derived from -100 mice (10-12 mice/group/experiment x 2 experiments). All mice were weighed at the beginning of the feeding period and weekly thereafter until killed. At the time of sacrifice, sera were collected from all the animals in each group and liver tissue from half the animals in each group. Collected liver tissues were either fixed in buffered formalin, preserved in OCT compound (Sakura, Torrance, CA) and processed for histology or snap frozen in liquid nitrogen and stored at -80 °C until RNA was isolated. The livers from the remaining animals in each group were prepared for flow cytometry as described below. All experiments satisfied the Guidelines of our institutions Animal Care Committees and the National Institutes of.

Health, USA. >

Histology Wedges of liver from each of the mice were prepared for histology and immunochemistry as we have described previously (3,12). For histology, tissues were formalin fixed, paraffrn embedded and 5-micron sections were stained with hematoxylin and eosin (H&E). Coded samples were examined by an experienced liver pathologist who was blinded to treatment groups. Hepatocellular fat accumulation was scored as, no fat = 0, focal fat accumulation in < 1% of the hepatocytes = F, fat in 1-30% of the hepatocytes = 1+, fat in 31-60% of the hepatocytes = 2+, and fat in 61-100% of the hepatocytes = 3+. To evaluate the amount of hepatocyte necrosis, the number of necrotic hepatocytes was counted in 10 randomly selected fields with a 20X lens.

Immunohistochemistry

Immunohistochemical analysis of HPC was performed with a mouse monoclonal OV6-type antibody (a kind gift from Dr Stuart Sell, Albany Medical College, Albany, NY) reacting with cytokeratins 14 and 19; a rabbit polyclonal antibody against 56 and 64 kD human callus cytokeratins (Dako, Denmark) and a rat monoclonal antibody to cytokeratin 19 as described

(3,13,14). Details of the staining procedures are as we have detailed previously (3,14). Briefly, 4 um thick paraffin sections were deparaffimzed and rehydrated, followed by heating in a microwave oven for 10 minutes at 750 Watt in citrate buffer, pH 6.0. Incubation with the primary antibodies was performed at room temperature for 30 minutes. Mouse monoclonal OV6 antibody and rat anti- cytokeratin 19 were detected using the DAKO Animal Research Kit, peroxidase (Dako, Denmark).

The rabbit polyclonal antibody against 56 and 64 kD human callus cytokeratins was detected by anti-rabbit Envision (Dako, Denmark) as described previously (14). . . ^• HPC were defined as small cells with an oval nucleus and little cytoplasm. These cells occur either singularly or Organized in arborizing, ductular structures. They have strong reactivity for liver type cytokeratins, OV-6 and bile duct type cytokeratin 19 (3,13,14). To evaluate the effect of treatments on the HPC compartment, coded samples were examined by an experienced liver pathologist blinded to treatment groups. For each liver section, the number of HPC in 5, randomly selected, non-overlapping, high power (x40 objective) fields was counted. Interlobular bile ducts, were defined as bile ducts with a lumen, associated with a branch of the hepatic artery. Interlobular bile ducts were not considered progenitor cells and, thus, were not counted as such. The presence of alpha- 1 adrenergic receptors on HPC was detected on frozen sections using a rabbit polyclonal anti-alpha 1 adrenergic receptor antibody (sc 10721, Santa Cruz Biotech, Santa Cruz, CA, dilution 1/20), followed by undiluted anti-rabbit Envision (Dako, Denmark). For immunofluorescence studies, the anti-alpha- 1 adrenergic receptor antibody was combined with a polyclonal antibody against 56 and 64 kDa human callus cytokeratins (Dako, Denmark; dilution 1:100). The primary antibodies were applied sequentially and subsequently detected with swine- antirabbit FITC or TRITC conjugates. In controls sections primary antibodies were omitted. All stainings were performed on 4 representative sections.

Serum Markers of Liver Injury

Sera from all the animals were analysed for alanine aminotransferase (ALT) activity by the Clinical Chemistry Laboratory of the Johns Hopkins Hospital.

RNA isolation and Ribonuclease Protection Assay

Total RNA was isolated from frozen liver samples according to the method of Chomczynski I ^■ . , . and Sacchi (15) as we have described (16). RNA concentration was determined by optical density and quality was assessed by agarose gel electrophoresis and ethidium bromide staining. Commercial ribonuclease protection assay (RPA) kits with probes for murine cytokines (PharMingen, San Diego, CA) were used to evaluate factors that might be involved in the recruitment and expansion of HPC after Hver injury. The factors studied were Stem Cell Factor (SCF), Hepatocyte Growth Factor (HGF), Interleukin-7 (IL-7), IL-11, Leukaemia Inhibitory Factor (LIF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Granulocyte Colony Stimulating Factor (G-CSF), Vascular Endothelial Growth Factor (VEGF), and its receptors, VEGFR1 and NEGFR3. Details of the RPA are as we have described previously (14).

Flow Cytometry

The hepatic non-parenchymal cell fraction, which contains the oval cell population and the ΝK-T cell populations, were isolated by previously described techniques (9,16). Briefly, livers were carefully removed and homogenized in Stomacher80 (Seawood, England). The homogenate was then passed through a 100-micron wire mesh and liver cells were collected by centrifugation at 450g. Mononuclear cells were purified from this fraction by centrifugation at 900g over 35%

Percoll gradients (Amersham Pharmacia Biotech) and incubated with normal mouse serum (Sigma,

St Louis, MO) and Fc-receptor block (anti-CD 16/CD32) to prevent non-specific binding, plus APC- conjugated anti-mouse Thy-1.2 (the C57BL-6 form of the Thy-1 antibody) and antibodies directed against hematopoeitic lineage markers (LIN, a mix of anti-mouse CD4, CD8, CD3, CD45, CD19,

Mac-1, Gr-1, Terl l9). For NK-T cell labeling, the mononuclear cells were incubated with FITC- conjugated anti-mouse NK-1.1 and PE-conjugated anti-mouse CD3. All antibodies were from

Pharmingen except anti-mouse Terll9, which was from Cedarline lab, Canada. After incubation, pellets were washed to remove unbound antibodies, fixed with 2% formaldehyde and evaluated by

FACS (Becton Dickenson). As described (2, 9) LIN^"ve/Thy-l^+ve cells, were classified as putativebone marrow-derived, hepatic progenitor cells. Data was analyzed by Cell Quest software (Becton ! Dickenson) to determine changes in these cell populations in different treatment groups.

Statistical Analysis

All values are expressed as mean ± SEM. The group means were compared by unpaired t-test or ANOVA using Graphpad Prism 3.03 (San Diego, CA).

Results

To determine the gross effects of the diets on our experimental animals, the weights of the animals at the start and end of the experiments were compared. Mice fed the control diet gained a mean of 3g (12% of starting body weight) during the course of the study (Fig. 1). In contrast, mice fed the HMCDE diet lost a mean of 3 g (12% of starting body weight). Mice fed the HMCDE diet in the presence of PRZ or 6-OHDA, however, only lost a mean of 2 g (7% and 8% of starting body weight). Therefore, SNS inhibition slightly, but significantly, attenuates the weight loss that occurs during consumption of antioxidant-depleted diets. The treatments also influenced liver mass (Fig. 2a, b). In mice with an intact SNS, as well as in those treated with SNS inhibitors, the HMCDE diet caused an increase in liver mass (Fig 2a), I ^{' ■} . - ,.. as well as liver/body mass ratio (Fig. 2b) above that of the control diet. Increases in both parameters tended to be greater in mice that were treated with SNS inhibitors, but the differences in liver mass achieved statistical significance only for the HMCDE + PRZ treated' group. Thus, although SNS inhibition reduced diet-related loss of body mass, it tended to enhance diet-induced hepatomegaly. Liver histology confirms that, as expected, HMCDE diets caused hepatic steatosis and necrosis (Fig. 3a-c). Histologic evidence of liver injury was accompanied by significant increases in serum ALT values (Fig.3d). Treatment with 6-OHDA, but not PRZ, significantly reduced the fat score (Fig. 3b). However, both SNS inhibitors significantly reduced hepatic necrosis (Fig. 3c) and serum ALT values (Fig. 3d). These findings demonstrate that PRZ and 6-OHDA-related increases in liver mass occured despite improvements in hepatic steatosis (6-OHDA) and/or necrosis (PRZ and 6-OHDA) and suggest that SNS inhibition might improve liver regeneration. Diet induced liver injury itself elicits a compensatory regenerative response, as evidenced by the accumulation of HPC in control mice that were fed the HMCDE diet. The increased HPC were demonstrated immunohistochemically by an increase in the numbers of bile duct type cytokeratin - positive oval cells (Fig. 4a, b) and by flow cytometry quantification of bone marrow lineage marker negative (LIN 8-) cells that expressed Thy 1.2 (Fig. 4c). SNS inhibition with either PRZ or 6- OHDA significantly augments diet-induced HPC expansion by both assays (Fig. 4a-c). The hepatic accumulation of HPC is a fairly specific consequence of SNS inhibition because, as expected (10), the numbers of NK-T cells in the livers of HMCDE-treated mice (8 + 1% liver mononuclear cells) decrease significantly after treatment with either PRZ (3.5 + 0.5%, P < 0.05) or 6-OHDA (3.6 + 0.6%, P < 0.05). Given that SNS inhibition also reduces HMCDE-induced liver injury (Fig. 3) and stabilizes body weight (Fig. 1), it seems unlikely that SNS inhibition generates a greater requirement for hepatic HPC accumulation. Rather, these findings suggest to us that HPC expansion might contribute to the hepatoprotective effects of SNS inhibition. Other groups have shown that the hepatocyte mitogen, hepatocyte growth factor (HGF), ) induces oval cell proliferation, promotes liver regeneration and protects the liver from hepatotoxicity (17). Given the similarities between the effects of SNS inhibition and HGF, it was important to determine if SNS inhibition increased hepatic HGF expression. Consistent with other reports that liver injury induces compensatory expression of HGF and other factors that promote regeneration. (18), we found that treatment with HMCDE increased the hepatic expression of HGF more than 2 fold above control (P < 0.04 versus CMCD) - data not shown. However, SNS inhibition with PRZ or 6-OHDA did not augment this response. Therefore, the hepatoprotective effects of SNS inhibition are not easily explained by HGF induction, although our studies do not exclude the possibility that SNS inhibition sensitizes the liver to HGF actions. Oval cells and bone marrow-derived hepatic progenitors express c-kit, the receptor for stem cell factor (SCF) (9). Other cytokines, such as interleukin (IL)-7 and LU, may also promote progenitor cell accumulation in injured tissues because after cardiac injury, these factors help to recruit bone marrow-derived stem cells to the injured heart (19). IL-6 is expressed by bone marrow derived cells in regenerating livers (20) and this cytokine has an important hepatoprotective effect because mice that are genetically deficient in IL-6 exhibit inhibited liver regeneration after partial hepatectomy (21). Other cytokines, such as G-CSF, that signal through gp-130 receptors may be able to compensate for IL-6 deficiency and promote regeneration when the latter cytokine is deficient (22). Vascular endothelial growth factor (VEGF) may also play some role in the expansion of HPC because it is a growth factor for hematopoietic stem cells, which express VEGF receptors (22). To begin to clarify the mechanisms, by which SNS inhibition enhances HPC accumulation in injured livers, we evaluated the effects of SNS inhibition on the hepatic expression of G-CSF, GM- CSF, IL-6, IL-7, IL-11, LIF, SCF, VEGF and its receptors VEGFR1 and 3. RPA of whole liver RNA was used to compare the expression of these factors in control (CMCD) mice and mice treated with HMCDE pluέ or minus SNS inhibitors. No appreciable GM-CSF, IL-6, IL-7,- IL- 1-1, SCF or • • .. ' LIF expression could be demonstrated by this assay (data not shown). HMCDE-treatment, however, I increased G-CSF expression about 2 fold, regardless of SNS inhibition (P < 0.05 all HMCDE groups versus CMCD). VEGF and its receptors were expressed in both control and all HMCDE- treated mice, but SNS inhibition did not alter the expression of these factors (data not shown). Thus, although these experiments do not exclude the possibility that the expression of one or more of these factors may have changed in some small population of liver cells after SNS inhibition, these progenitor cell trophic factors do not appear to be the major targets for SNS regulation. To determine if the effects of SNS inhibition on the HPC compartment might be mediated via direct interaction between NE and adrenoceptors on HPC, we used immunohistochemistry to determine if HPC express alpha- 1 adrenoceptors. Our results show that bile duct type cytokeratin- positive oval cells do express apha-1 adrenoceptors (Fig. 5a,b). Therefore, direct regulation of this HPC compartment by NE is plausible. Discussion Critical shortages of donor livers for orthotopic liver transplantation have become a major limiting factor in efforts to reduce mortality of patients with end-stage liver disease (24). Therefore, alternative strategies to replace severely damaged livers must be developed. Studies in mice with massive toxin-induced liver injury have demonstrated that liver cell transplantation can effectively regenerate the liver (reviewed in (25). Hence, many groups are working to optimize cell transplantation strategies. An alternative, but complementary, approach that might be used to enhance regeneration of injured livers involves treatment to encourage repopulation of the liver by endogenous hepatic progenitors. The general feasibility of this strategy is supported by recent evidence that the administration of cytokine mixtures to mobilize native, bone marrow-derived progenitor cells heals experimentally-induςed myocardial infarcts in mice (19). However, although certain bone marrow cells can differentiate into oval cells (2) and mature hepatocytes (26), the • relative importance of bone marrow-derived progenitors, as opposed to resident hepatic progenitors (i.e. oval cells) and mature hepatocytes for liver regeneration remains uncertain (25). Moreover, even if certain progenitor cell populations do contribute to recovery from liver injury, little is known about the mediators that regulate their accumulation within the liver. Therefore, the identification of these factors is an important first step in the development of treatments that seek to expand hepatic progenitor cell populations. Presumably, endogenously produced factors that induce the hepatic accumulation of liver progenitor cells are increased, to some extent, during liver damage because regenerative responses are evident in most injured livers (18). However, other factors that increase during injury might inhibit progenitor cell expansion and this would compromise reconstruction of a healthy organ, if the progenitors play a role in liver regeneration. Thus, one way to enhance recovery from liver injury might be to neutralize the actions of endogenous factors that limit the expansion of native HPC populations. To explore the validity of this concept, we studied mice that were treated with half strength, methionine/choline deficient diets supplemented with ethionine (HMCDE), because this murine model of liver injury is known to increase hepatic oval cells (8). Our results show that stress-related SNS activity is one of the endogenous factors that modulate HPC accumulation in damaged livers. However, the mechanisms for this remain uncertain because we found no effect of SNS inhibition on several factors that are thought to promote progenitor cell accumulations. On the other hand, at least one mechanism that regulates the accumulation of oval cells in the livers of choline deficient mice has been reported. Fausto and colleagues showed that TNF-α increases in mice that are fed choline-deficient diets and demonstrated that proliferating hepatic oval cells produce this cytokine (27). Moreover, they found that TNF-α is required for oval cell expansion because this response is abrogated by genetic disruption of TNFRl. Their observations - are particularly intriguing because TNF-α and TNFRl are necessary for liver regeneration after partial hepatic resection and other types of liver injury (28,29). Although we did not evaluate potential interactions between TNF-α and the SNS in our model, work in many other systems, demonstrates clear evidence for cross talk between signaling mechanisms that are .activated by TNF-α and those that are modulated by sympathetic neurotransmitters, such as NE (reviewed in (30). At the very least, these interactions may explain our observation that PRZ and 6-OHDA reduced HMCDE-induced liver injury, because NE inhibits cytokine inducible nitric oxide (NO) production in hepatocytes and NO protects hepatocytes from TNF-toxicity (31). Thus, NE promotes TNF-α-mediated hepatotoxicity and agents that block NE generally inhibit this (32). Whether or not NE-TNFα interactions influence HPC expansion has not been investigated, but merits evaluation because NE regulates TNF production and vice versa (30, 33-36). Thus, given that cytokine-neurotransmitter interactions influence liver injury and SNS-regulated cytokines modulate both oval cell expansion and liver regeneration, SNS inhibition may promote HPC accumulation and recovery from liver injury indirectly, by effecting cytokine activity. Theoretically, neurotransmitters may also promote HPC expansion by directly interacting with their receptors on oval cells or their precursors. Such direct effects have been demonstrated for at least one SNS neurotransmitter, NPY, which interacts with its receptors on neuronal progenitors to induce their proliferation (37). Although we have shown here that oval cells express apha-1 adrenoceptors, it remains to be seen if their putative bone marrow-derived progenitors also express such receptors. It is tempting to speculate, however, that SNS manipulation might have exerted a direct effect on one or more of the HPC populations, because the bone marrow receives SNS innervation (38,39) and adrenoceptors have been demonstrated on certain types of bone marrow derived progenitor cells (38,40). Moreover, treatment of mice with PRZ or 6-OHDA mobilizes bone marrow-derived hematopoietic progenitors into the circulation (38,40), suggesting that i ' ■

-*• injury/inflammation-related increases in NE might normally limit accumulation of HPC. If so, then ) ^•' SNS inhibition would be_*, expected to dis-inhibit this process, permitting expansion of HPC populations within damaged livers. The observation that treatment with PRZ or 6-OHDA increased hepatic accumulation of Thy-1 expressing cells that lack appreciable surface markers for the ^'l r hematopoietic lineage is consistent with this hypothesis because Petersen et al have demonstrated that such cells can be isolated from the bone marrow of adult rats and induced to differentiate into hepatic oval cells (2). Despite these uncertainties about the mechanism(s) through which SNS inhibition promotes HPC expansion, the observation that this process can be induced by PRZ, a widely available, relatively safe, oral agent, has potential therapeutic implications. In our study, PRZ treatment was well tolerated - none of the PRZ-treated mice died and most developed less cachexia, as well as less liver necrosis and more liver regeneration, than the liver disease controls. These findings complement those of an earlier study which demonstrated that PRZ prevents the development of cirrhosis in carbon tetrachloride-treated rats (7). Taken together, these results suggest that alpha adrenoceptor blockade might be an effective strategy to arrest liver disease progression. References

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35. Spengler RN, Chensue SW, Giacherio DA, Blenk N, Kunkel SL. Endogenous norepinephrine regulates tumor necrosis factor-alpha production from macrophages in vitro. J. Immunol. 1994; 152: 3024-3031 36. De Lugi, A, Terreni L, Sironi, M, De Simoni MG. The sympathetic nervous system tonically inhibits peripheral interleukin-lbeta and interleukin-6 induction by central lipopolysaccharide. Neuroscience 1998;83: 1245-1250.

37. Hansel DE, Elpper BA, Ronnett GV. Neuropeptide Y functions as a neuroproliferative factor. Nature 2001; 410: 940-944

38. Marino F, Cosentino M, Bombelli R, Ferrari M, Maestroni GJ, Conti A, Lecchini S, et al. Measurement of catecholamines in mouse bone marrow by means of HPLC with electrochemical detection. Haematologica 1997; 82: 392-394

39., Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, Armstrong D, et al. Leptin regulates bone formation via the sympathetic nervous system. Cell 2002; 111: 305-317 40. Maestroni GJM, Conti A. Modulation of hematopoiesis via alpha 1 -adrenergic receptors on bone marrow cells. Exp. Hematology 1994; 22: 313-320.

Figure Legends

Figure 1. Effect of control and antioxidant-depleted diets on body weight.

Mean + SEM body weights of mice before and after 4 weeks of feeding. Only mice fed the control diet (CMCD) gained weight (* P< 0.04 vs baseline); all groups that were fed half methionine choline deficient diets (HMCDE) lost weight (*P<0.001 for post- versus pre-HMCDE, P< 0.008 for post- versus pre-HMCDE + PRZ, P< 0.03 for post- versus pre- HMCDE + 6OHDA). However, HMCDE +PRZ and HMCDE + 6 OHDA groups lost less weight than the HMCDE group (*P < 0.05).

-Figure 2. Effect of SNS inhibition on liver mass in mice with diet-induced liver \ damage. "^■

a) Compared to mice fed control diets (CMCD), absolute liver mass was greater in all groups fed HMCDE diets (*P < 0.01). Absolute liver mass in the HMCDE + PRZ group was greater than the group fed HMCDE alone ( P < 0.04). b) Liver/body weight ratios also increased on HMCDE diets (*P < 0.02 for all groups versus CMCD) and tended to be greater in HMCDE-treated mice that received SNS inhibitors, although the difference between these groups and those fed HMCDE diets alone did not achieve statistical significance.

Figure 3. Effect of SNS inhibition on diet-induced liver injury.

a) Liver Histology. Images were captured with a 25X lens. Hematoxylin and eosin stained sections of representative mice that were fed control diet (CMCD) (top left) showed no fat accumulation or necrosis. A section from a representative HMCDE fed animal showed 2+ fat accumulation and areas of hepatocyte death - arrowed (top right), while one from a HMCDE + PRZ fed mouse showed 1+ fat accumulation and reduced liver cell death (bottom left). The liver section from a representative HMCDE + 60HDA fed animal showed focal (F+) fat accumulation and minimal necrosis (bottom right). b) Fat Score. Compared to mice fed control diets (CMCD), the HMCDE and HMCDE + PRZ groups had more fat (*P < 0.0004). The HMCDE +6OHDA treated group had significantly less fat than the HMCDE alone group (#p<0.0001). c) Necrosis score. Compared to controls (CMCD), all HMCDE-fed groups had more necrotic hepatocytes (*P < 0.01), but compared to mice that were fed the HMCDE diet alone, the numbers of necrotic hepatocytes were reduced in HMCDE + PRZ (*P < 0.05) or HMCDE + 6OHDA

., d. Serum alanine aminotransf erase (ALT). Serum levels of ALT, a marker of liver injury, were ' increased in all HMCDE-fed groups compared to CMCD controls (*P< 0.01). Compared to ^'HMCDE-fed mice, mice treated with HMCDE +PRZ or HMCDE +6OHDA had lower ALT levels (ⁿP< 0.03). /

Figure 4. Effect of SNS inhibition on the numbers of hepatic progenitors in livers with diet-induced damage. a) Immunohistochemistry for oval cells, in representative mice that were fed control diet (CMCD) (top left), HMCDE (top right), HMCDE diet + PRZ (bottom left) or HMCDE + 6OHDA (bottom right). Oval cells are stained brown. b) The numbers of oval cells were increased in all HMCDE-fed groups compared to CMCD controls (*P< 0.0001). Both groups treated with SNS inhibitors had more oval cells than mice that were fed HMCDE diets alone (^#i°<0.001 ). c) When putative bone marrow-derived hepatic progenitors (i.e., LIN^"v7Thy-l^+ve) are quantified by flow cytometry, livers from groups treated with HMCDE + PRZ or HMCDE + 6OHDA contain more of these cells than CMCD controls (*P<0.01), although HMCDE feeding alone did not expand this compartment. Compared to mice fed HMCDE diets alone, mice fed HMCDE +PRZ or HMCDE + 6OHDA had more LIN-^v7Thy-l^+ve cells (^# P< 0.03 and < 0.05, respectively).

Figure 5

Oval cells express Alpha-1 adrenoceptors a) Immunohistochemistry for alpha-1 adrenoceptors on bile duct type cytokeratin-positive oval cells in a liver section from a representative mice fed HMCDE. Oval cells expressing alpha-1 adrenoceptors are stained brown, b) Immunofluorescence smdies confirms the co-localisation of i ^{' •} . . - -- . alpha-1 adrenoceptors on bile duct type cytokeratin-positive oval cells. Without the primary antibodies, binding of the secondary antibodies was negligible (not shown). Alpha-1 adrenoceptors - red, cytokeratins -green, co-localization - yellow.

Figure 1

CMCD HMCDE HMCDE HMCDE +P Z +60HDA

Figure 2a

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA

Figure 2b

CMCD HMCDE HMCDE+PRZ rWCDE+fcOHDA

Figure 3a

CMCD HMCDE

Figure 3b

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA

Figure 3c

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA

Figure 3d

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA Figure 4 a

Figure 4b

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA

Figure 4c

CMCD HMCDE HMCDE+PRZ HMCDE+6-OHDA

Figure 5

a) Oval cells express alpha-1 adrenoceptors.

b) Immunofluorescence confirmation of Oval cells expression of alpha-1

adrenoceptors.

Effect of Genotype on Hepatic Progenitor Cell Number

o Genotype

All publications, patents and patent applications (including US provisional applications 60/458,644 filed 3/28/03 and 60/458,450 filed 3/28/03), and disclosed herein are incorporated into this application by reference in their entirety.

The sympathetic nervous system (SNS) nerve terminals contain both Norepinephrine (NE) plus NPY and other molecules. Prazosin blocks only the alpha-1 adrenoceptor mediated effects of NE.

6-OHDA (6-hydroxy dopamine) however depletes the SNS nerve terminals of NPY and NE. Therefore, that a larger number of oval cells and bone marrow derived progenitor cells are seen with 6-OHDA treatment suggests that NPY is inhibitory and that removing NPY removes this inhibition and leads to larger numbers of liver stem cells.

Sympathetic nervous system activation is thought to promote hepatic fibrosis, but the mechanisms involved are obscure. Hepatic stellate cells (HSC), the liver's major collagen producers, express stereotypical neuronal proteins. Thus, HSC may function as hepatic neuroglia, producing neurotransmitters to affect fibrogenesis locally. To evaluate this hypothesis, we assessed whether HSC synthesize and release norepinephrine (NE), and determined if NE is an autocrine regulator of HSC. We found that HSC express tyrosine hydroxylase, the rate- limiting enzyme in cathecholamine biosynthesis, and release NE in culture. HSC from dopamine beta hydroxylase (DβH) -/- mice, which cannot make NE, grow poorly in culture. Conversely, exogenous NE promotes HSC proliferation, and prazosin, an alpha- 1 adrenoceptor antagonist, reduces HSC growth. When treated with hepatotoxic diets, intact DβH -I- mice exhibit inhibited induction of TGF-βl, do not accumulate alpha smooth muscle actin (+) HSC, and express significantly less collagen mRNA. Histologic liver injury and serum alanine aminotransferase levels are also reduced. These results support our hypothesis that HSC function as neuroglia in the liver and demonstrate that NE is a critical regulator of hepatic fibrogenesis.

Abbreviations: Tyrosine hydroxylase (TH), Norepinephrine (NE), Dopamine beta hydroxlase (DBH), Alpha smooth muscle actin (ASMA), Glial acidic fibrillary protein (GFAP), Methionine restricted, choline deficient (MCD), Autonomic nervous system (ANS), Sympathetic nervous system (SNS), Hepatic stellate cells (HSC), ethionine (E), Prazosin (PRZ), Norepinephrine (NE), Dopamine (DA), 3,4-dihydroxyphenylacctic acid (DOPAC), Serotonin (5-hydroxytrptamine, 5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and Homovallinic

acid (HVA). The mechanisms that initiate and perpetuate the fϊbrogenic response in the injured liver are not well understood. It is clear, however, that after liver injury hepatic stellate cells (HSC) are activated, moving from a quiescent phenotype to become proliferative and fibrogenic myofibroblasts (1). These activated myofibroblastic cells constitute the major matrix-producing cells in the injured liver. These cells are also contractile and may, therefore, contribute to portal hypertension, a major complication of hepatic fibrosis (2). There is accumulating experimental evidence linking the autonomic nervous system (ANS), in particular, the sympathetic nervous system (SNS), with the pathogenesis of cirrhosis and its complications (3- 11). For example, the spontaneously hypertensive rat, which has an over-active SNS, develops more severe liver fibrosis after carbon tetrachloride-induced liver injury than does its wild type control (4,5). Although these early studies did not identify the cellular targets of the ANS, recent work from our group shows that HSC themselves respond to ANS neurotransmitters (9,10). Therefore, HSC may be an effector arm of the ANS, mediating ANS regulation of liver fibrosis after injury. This possibility is supported by evidence that the ANS modulates wound healing in other epithelial tissues (12,13). The embryonic derivation of HSC is uncertain. It has been suggested that HSC may be derived from the neural crest because they express glial fibrillary acidic protein (GFAP), nestin, neural cell adhesion molecule (NCAM), synaptophysin, and neurotrophins (2,14-17), have synaptic vesicles (16), and receive innervation by autonomic fibres (18,19). Therefore, HSC may function as resident neuroglial cells in the liver (17). Given evidence suggesting involvement of the SNS in hepatic fibrosis and the putative neuroglial role of HSC, mice with a targeted deletion of dopamine β-hydroxylase (DβH -/-), were used to test the hypothesis that sympathetic neurotransmitters modulate hepatic fibrosis. Dopamine _/^-hydroxylase converts dopamine to norepinephrine (NE) in the cathecholamine biosynthetic pathway. DβH null mice, therefore, are unable to synthesis NE, the principal SNS neurotransmitter, or its product, epinephrine. Using HSC from our DβH-/- mice, as well as from normal mice and rats, we also tested the hypothesis that HSC synthesize and release the SNS neurotransmitter, norepinephrine (NE), and that this neurotransmitter functions as an autocrine regulator of HSCs. We applied the neurotransmitter identification criteria of Sir Henry Dale (as reviewed in 20) to assess whether or not HSC have the enzymatic machinery to synthesize cathecholamines, actually synthesis and release these factors, and use catecholamines to modulate cellular physiology. Our results confirm both hypotheses: HSC synthesize and release NE as an autocrine regulatory factor and this sympathetic neurotransmitter is necessary for hepatic fibrosis.

Methods

Animal Experiments

Male C57BL-6 mice, 10-18 weeks old were from Jackson Laboratory (Bar Harbor, ME) and Sprague- Dawley rats 250-300g, 10-16 weeks old, were from Charles River Laboratories (Wilmington, MA). DβH knockout mice were generated and maintained as previously described (21) and used at 30-40 weeks old. Animals were allowed access to diets and water ad libitum. For some studies, DβH -I- mice (n=12) and DβH +/- littermates (n=12) were fed methionine restricted, choline-deficient (MCD) diets (ICN, Aurora, OH) for 4 weeks, with DL-efhionine (E) added to the drinking water (final concentration 0.15%) to induce hepatic oxidative stress and steatohepatitis (22). These mice were weighed at the beginning of the feeding period and weekly thereafter until sacrifice. At the end of the feeding periods the mice were sacrificed to obtain liver tissu and sera. Liver tissues were fixed in buffered formalin or preserved in OCT compound (Sakura, Torrance, CA and processed for histology; alternatively they were snap frozen in liquid nitrogen and stored at -80°C for further analysis. All experiments satisfied the Guidelines of our Institutions Animal Care Committee and the National Institutes of Health.

Stellate Cell Isolation and Culture Using pronase and collagenase liver digestion, HSC were isolated from DβH+l-, DβH-l-mke or rats. I each experiment, HSC were pooled from 6 mice of each genotype. A single rat provided sufficient HSC for a experiment. All experiments were replicated at least twice. Cell identity was confirmed by autofluorescence

and expression of alpha smooth muscle actin (ASMA) and Glial Fibrillary Acidic Protein (GFAP) were verifie both by immunoblots and immunocytochemistry (9). Using a hemocytometer, the numbers of autofluorescen cells were also counted in an aliquot of each preparation and demonstrated that HSC preparation purity wa always > 95%. HSC were cultured in 10% serum-supplemented RPMI 1640 medium. For determination o cellular cathecholamines or HSC released cathecholamines, HSC and their conditioned media were harveste after 4 days in culture. The cells were washed twice with ice-cold PBS, and rapidly analysed for cathecholamine content by HPLC. Conditioned media was stored at -70°C until analysed.

Apoptosis Assays

To evaluate the effect of the alpha-i adrenoceptor antagonist, prazosin (PRZ) or DβH genotype on cell viability, equal numbers of HSC were plated on 6mm petri dishes and grown in the presence and absence of lOμM PRZ. Fresh PRZ was added every 2 days, when the medium was changed. At various time points, cells were harvested and apoptotic activity was assessed with the Vybrant (Annexin V) apoptosis assay kit (Molecular Probes, Eugene, OR).

Cell Proliferation Assay

Freshly isolated HSC were seeded into culture flasks and grown to subconfluence. At this time point (~ day 7-10), cultures were harvested. HSC were resuspended in serum-free medium, and then re-plated on 96- well plates at 5,000 cells/well. Twenty-four hours later, when the cells had become quiescent, NE (lOμM) ± PRZ (lOμM) were added to some wells, with 0.1% serum as described (23). After 44 hours, cell numbers were assessed by a further 4 h incubation with WST-8 tetrazolium reagent ( Dojindo Molecular Technologies, Gaithersburg, MD) as described (9,23). In viable cells, the tetrazolium salt is metabolized to a colorimetric dye and cell number is proportional to the signal intensity, at 450nm (9).

Immunoblot HSC were harvested after various times in culture, cell homogenates were prepared and protein content was quantified by BSA assay (Pierce, Rockford, IL) using bovine serum albumin standards. Proteins (lOμg/lane) were then resolved by polyacrylamide gel electrophoresis and transferred to nylon membranes. After membranes were incubated with primary antibodies (mouse monoclonal anti-alpha smooth muscle actin (ASMA), Sigma, St. Louis, MO, 1:3000 dilution; mouse monoclonal anti-tyrosine hydroxylase (TH), Novus Biologies, Littleton, CO, 1:100 dilution), peroxidase-conjugated anti-mouse antibodies were added, and antigens were demonstrated by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

HPLC Analysis Catecholamines were extracted from HSC pellets and HSC-conditioned medium with perchloric acid. Norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5- hydroxy tφtamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) and homovallinic acid (HVA) were analyzed in these extracts by HPLC as described (24), using dihydroxybenzilamine as the internal standard. Peak heights andsample concentrations of NE, DOPAC, DA, 5-HIAA, HVA and 5-HT were calculated with a Hewlett Packard integrator.

Histology Pieces of liver were formalin fixed, embedded in paraffin and 5-micron sections were stained with hematoxylin and eosin (H&E). Coded samples were examined by an experienced liver pathologist blinded to treatment groups. Hepatocellular fat accumulation was scored as follows: 0 = no fat; F = focal fat accumulation in < 1% hepatocytes; 1+ = fat in 1-30% hepatocytes; 2+ = fat in 31-60% hepatocytes, and 3+ = fat in 61-100% hepatocytes. To evaluate the amount of hepatocyte necrosis, the number of necrotic hepatocytes was counted in 10 randomly selected fields with a 20X lens (25, 26).

Immunohistochemistry In liver tissues, HSC expression of alpha smooth muscle actin (ASMA), was used as a marker of HSC activation (1). Details of the staining procedure are essentially as wc have detailed previously (11). Briefly, 4 um thick paraffin liver sections were deparaffinized and rehydrated, followed by heating in a microwave oven for 10 minutes at 750 Watt in citrate buffer, pH 6.0. Incubation with the primary antibody, mouse monoclonal anti-ASMA antibody (1/40 dilution, DAKO, Denmark), was performed at room temperature for 30 minutes. Primary antibody binding was revealed with the DAKO Animal Research Kit, peroxidase (Dako, Denmark).

Serum Markers of Liver Injury Sera were analyzed for alanine aminotransferase (ALT) activity by the Clinical Chemistry Laboratory of the Johns Hopkins Hospital.

RNA isolation and Ribonuclease Protection Assay Total RNA was isolated from liver samples according to the method of Chomczynski and Sacchi (27) as we have described (28). Collagen- 1-αl and TGF-βl gene expression were evaluated by commercial ribonuclease protection assay (RPA) kits with probes for collagen- l-rx2 and TGF-βl (PharMingen, San Diego, CA) as we described previously (29).

Statistics Statistical analysis by unpaired t-test or the Mann- Whitney test was performed with Graphpad Prism software (San Diego, CA). Significance was accepted as *p< 0.05.

Results To determine if HSC from rats and mice are capable of producing neurotransmitters, we performed immunoblot blot analysis for TH, the rate-limiting enzyme in the catecholamine biosynthetic pathway (Figure 1). HSC from both species express TH (Figure 2). TH expression is also apparent in HSC cultured from DβH -/- mice, which cannot produce NE because they lack dopamine β-hydroxylase (DβH), the enzyme that converts dopamine to NE (Figure 1). Next, we asked if cultured HSC actually synthesis and release cathecholamines. HPLC analysis of HSC lysates and HSC-conditioned medium demonstrate that the cells contain NE and release NE into their milieu (Table 1, Figure 3). HSC also contain dopamine (DA), serotonin (5-Hydroxytryptamine, 5-HT) and the cathecholamine metabolites Dihydroxyphenylacetic acid, (DOPAC), 5-Hydroxyindoleacetic acid (5-HIAA) and, hoπiovanillic acid (HVA). As a negative control conditioned medium from cultured DβH-/- HSC was also analysed for cathecholmines. As expected this did not contain NE (results not shown).

We then investigated the physiological relevance of HSC catecholamine synthesis. In the final steps of catecholamine biosynthesis, NE is produced from DA in a reaction that is catalyzed by DβH (Figure 1). Because DβH-/- mice, in which the DβH gene has been experimentally deleted, cannot produce NE (21), their HSC might be abnormal, if NE normally regulates HSC. To evaluate this possibility, we harvested HSC from DβH -I- and DβH +/- mice, and plated identical numbers of cells from each group. As an experimental control, some HSC from DβH+/- mice (which can produce NE (Table 1, Fig 3) were incubated with prazosin, an alpha- 1 adrenoceptor antagonist. During culture, striking differences in the numbers of HSC became apparent when NE was inhibited. For example, after 4 days in culture, there were significantly more untreated HSC from DβH+/- controls (Fig 4a) than DβH+A HSC treated with prazosin (Fig 4b) or untreated HSC from DβH-/- mice (Fig 4c). These differences in cell number paralleled differences in culture protein concentrations (data not shown). To determine if the NE-related differences in cell number might be explained by differences in apoptotic activity, HSC were harvested, incubated with Annexin V and analyzed by flow cytometry. There was a greater proportion of apoptotic cells in HSC from DβH-/- mice compared to DβH+/- mice after 1 day in culture (data not shown). Therefore, NE appears to promote the viability of HSC, at least in vitro. We then determined if NE also promoted the proliferation of HSC in culture. We have previously shown that NE induced the proliferation of murine HSC in culture (9) but to assure that these effects were not species-specific, NE with and without prazosin was also added to HSC cultured from healthy adult rats. Incubation of rat HSC with NE for 48 hours significantly increased HSC numbers. Prazosin blocked these trophic effects of NE (Figure 5). Although the aforementioned results suggest that autocrine production of NE regulates the phenotype of cultured HSC, we wanted to determine if NE had any impact on HSC function in intact animals. Therefore, we fed DβH-/- mice and DβH+/- control mice antioxidant-depleted diets and ethionine to induce steatohepatitis (22) and provoke stellate cell activation, the primary event in the liver's fibrogenic response (1). After 4 weeks of treatment, control DβH+/- mice exhibited a striking accumulation of HSC that express ASMA, an accepted marker of HSC activation (Figure 6a,b). In contrast, ASMA ^ve HSC could not be demonstrated in DβH-/- ice (Figure 6a,c). This finding is unlikely to be a staining artifact, because ASMA ^e vessel walls are easily

identified in the DβH -/- group (Figure 6c). Consistent with evidence that HSC activation is inhibited in NE-

deficient mice, DβH-/- mice also express significantly less collagenl-αl mRNA than DβH+/- mice, which accumulate activated, ASMA^+V6 HSC during hepatic oxidant stress (Figure 7a, b). To begin to delineate the mechanism for reduced HSC activation and collagen gene expression in NE-deficient mice, we compared hepatic expression of TGF- βl, a pro-fibrogenic cytokine, in DβH-/- and DβH+/- mice. NE-deficient mice express significantly less hepatic TGF-βl than DβH+/- controls (Figure 7a,c). Further analysis of the livers from DβH-/- mice demonstrated that the NE-deficiency is also hepatoprotective. Compared to DβH+/- controls that were exposed to hepatotoxic diets, identically treated DβH-/- mice exhibit significantly less histologic steatosis (Figure 8a) and necrosis (Figure 8b) and lower serum ALT values (Figure 8c). Discussion The results presented here confirm a role for sympathetic neurotransmitters in hepatic fibrogenesis. They also support our hypothesis that HSC, the liver's principal collagen producing cells, function as liver resident neuroglial cells, producing neurotransmitters to regulate, in an autocrine manner, their own function. We have shown that HSC not only contain tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of cathecholamines, but actually make and release NE and other cathecholamines into their milieu. Moreover, cultured HSC proliferate in response to NE. NE also appears to be necessary for activation of HSC in vivo, because induction of ASMA was absent in NE-deficient mice. Hepatic gene expression of collagen and TGF- βl, a key proiϊbrogenic cytokine, are also markedly reduced when NE is deficient, suggesting that NE may promote HSC collagen gene expression by increasing TGF-βl. Prior to our study, evidence suggested a role for the SNS in hepatic fibrosis (3-11). Several studies demonstrated positive correlations between SNS activity and hepatic fibrosis in intact animals, but none of these identified the cellular targets of SNS regulation (4-7). Recent, in vitro studies of cultured murine HSC by our group suggested that HSC were likely to mediate the profibrogenic actions of the SNS (9,10). However, the physiological relevance of our earlier findings remained questionable because we studied HSC in the absence of other cells that might modify their phenotype. The approach adopted here uses a combination of in vitro and in vivo models to overcome the limitations of the previous studies. Moreover, studies of cultured HSC and intact mice that are genetically incapable of producing NE provide definitive proof that this neurotransmitter plays a key role in hepatic fibrogenesis. Sympathetic neurons and the adrenal medulla are the major sources of NE in vivo. HSC receive adrenergic autonomic fibers (18, 19, 30) and Athari and colleagues demonstrated that HSC have adrenoceptors (31). Here and elsewhere (10), we show that prazosin reverses the proliferative effect of exogenous NE on cultured HSC. Therefore, neuronal NE is likely to regulate HSC in intact animals. In addition, we present novel evidence that HSC, themselves, produce NE. HSC-derived NE appears necessary for optimal growth of HSC in culture, because simply adding prazosin to cultures of DβH +/- HSC reduces their proliferation. Moreover, HSC from DβΑ -I- mice grow poorly in vitro. However, the relative importance of NE produced by HSC, as opposed to neuron-derived NE, in regulating HSC biology in living animals remains uncertain. It is tempting to speculate that autocrine regulation of HSC is important under at least some circumstances, because transplanted livers, which remain denervated (32), clearly can develop cirrhosis (33). That NE may sub-serve functions other than its classically assigned role of neurotransmission is becoming well established in other systems. For example, cardiac remodeling in heart failure involves mitogenic and fϊbrogenic actions of NE that are mediated via adrenoceptors (34-36). Emerging evidence also demonstrates that there are multiple alpha adrenoceptor subtypes, some of which selectively mediate mitogenic effects of NE, and others which selectively regulate NE vasopressor effects (37,38). In the present study we used a non-specific alpha antagonist - prazosin, that binds to alpha-1 adrenoceptors in general, and demonstrated a physiological effect, confirming that HSC express this receptor family. However, the subtype identities of HSC adrenoceptors remain to be defined. Indeed delineation of the specific signals that are activated by NE-adrenoceptor interaction will clarify if NE regulates TGF-β expression in HSC, as it does in vascular smooth muscle cells and cardiac fibroblasts (36,39). This possibility is suggested by our observation that induction of TGF-βl mRNA is inhibited in the livers of ΩβΑ -I- mice. HSC are an important source of TGF-βl (40) and TGF-βl is necessary for optimal activation of HSC to myofibroblastic, collagen-producing cells during liver injury (41). Consistent with the possibility that NE promotes HSC activation by inducing TGF-βl, our T>βΑ -I- mice failed to accumulated activated, ASMA(+) HSC in their livers and exhibited significantly less induction of hepatic collagen gene expression during treatment with hepatotoxic diets. Together, these findings suggest that NE is upstream of TGF- βl in the hepatic fibrogenic cascade and that liver TGF-β expression is regulated by NE. The latter may also explain why pharmacologic inhibition of NE was hepatoprotective for the wild-type mice in our studies, because decreasing NE activity reduces local exposure to TGF-βl, an acknowledged hepatocyte apoptotic factor (42). However, given evidence that multiple types of liver cells, including hepatocytes, Kupffer cells and resident lymphocytes express adrenoceptors and respond to NE (43-45), more complicated mechanisms may also be involved. In any case, our findings suggest a novel paracrine mechanism, i.e., local release of NE by HSC, that may regulate multiple aspects of liver injury and repair. This, in turn, identifies novel therapeutic targets to prevent liver fibrosis and its sequalae.

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Hepatology 2000; 31 : 907-915 Table 1. HSC Contain and Release NE into their Culture Medium

Figure Legends

Figure 1. Catecholamine biosynthetic pathway.

Figure 2. Expression of tyrosine hydroxylase (TED by cultured HSC. HSC were cultured from D H-/- mice that cannot produce NE, heterozygous DβH +/- littermates that produce NE, and healthy adult rats. After 4 days in culture, cell lysates were evaluated for tyrosine hydroxylase expression using immunoblot analysis (10 μg protein/lane). Results from representative immunoblots are shown.

Figure 3,Table 1. Norepinephrine (NE) production by cultured HSC. HSC were cultured from normal mice and the production of NE and other catecholamines were evaluated by HPLC analysis of cell lysates and conditioned media after 4 days in culture (b). The retention times of the HSC products were compared to those of a concurrently analyzed standard (a).

Figure 4. Effect of NE on HSC growth in culture. HSC were isolated from NE-producing Oβϋ +/- mice and cultured in the absence (a) or presence (b) of prazosin (PRZ, 10 μM), an alpha-adrenoceptor antagonist, for 4 days. Results were compared to 4 day-old HSC cultures from D/33 -/- mice (c) which cannot produce NE. Photomicrographs of representative cultures are shown. Compared to vehicle-treated controls, PRZ-treated cultures and HSC cultures from DySH -/- mice have -40-50% less protein.

Figure 5. Effect of NE on the growth of rat HSC. HSC were isolated from healthy rats and cultured with vehicle (control), NE (10 μM) or NE + prazosin (PRZ, 10 μM) for 2 days. At the end of the treatment period, the numbers of cells in culture were evaluated by a colorimetric assay that measures the reduction of the tetrazolium salt, WST-8, by viable HSC. Figure 6. HSC activation in DβH-l- and DβH+l- mice. Mice were fed methionine choline deficient diets with ethionine in the drinking water to cause steatohepatitis and stellate cell activation. After 4 weeks of treatment, liver samples were obtained, fixed in formalin and paraffin-embedded. Alpha smooth muscle actin (ASMA) expression was demonstrated by immunohistochemistry. ASMA(+) sinsusoidal cells were counted in 5 randomly selected fields/section from 4 mice/group. Mean (SEM) results of one experiment are graphed (a). Virtually identical results were obtained in a second experiment that studied an additional 4 mice/group. Photomicrographs from representative DβH+l- mice (b) and DβH -I- mice (c) are shown. Arrows indicate typical ASMA(+) HSC.

Figure 7. Hepatic expression of collagen 1-αl and TGFβ-1 in DβH-l- and DβH+l- mice. Total liver RNA was obtained from the mice described in the legend to figure 6. Gene expression was evaluated by ribonuclease protection analysis using commercially available reagents that included probes for GAPDH, which was used to control for quantitative differences in input RNA. a) A representative phosphoimage of gene expression in two DβH+l- mice and two DβH-l- mice, b) Mean (SEM) collagen 1 α-land c) TGFβ-1 gene expression in duplicate experiments (4 mice/group/experiment). **P<0.03 for TGF-βl gene expression in DβH +/- versus mice.

Figure 8. Liver injury in DβH-l- and DBH +/- mice. Hepatic steatosis (a) and necrosis (b) were graded on hematoxylin and eosin-stained liver sections from the mice described in the legend to figure 6. Serum alanine aminotransferase (ALT) values were also measured in these mice (c). The steatosis scoring system ranged from 0 (no fat) to 3+ fat (fat accumulation in >61% hepatocytes). Numbers of necrotic hepatocytes were counted in 10 fields/section. Results are the mean (SEM) from 4 mice/group in one experiment. These results were reproduced in a second study that involved 4 other mice/group. Figure 1.

Tyrosine tyrosine hydroxylase

Dihydroxyphenylalanine (DOPA) dopa decarboxylase

Dopamine dopamine- β-hydroxylase

Norepinephrine

PNMT

Epinephrine

PNMT — Phenylethanolamine N-methyltransferase

Figure 2.

Mice Rat DβH+/- DβH-/-

66kD *WS^{: ■} βM'^{: β} d-άH

Figure 3. a) Standard

b) Day 4 HSC lysate

Figure 4 a) DβH +/- Control b) DβH +/- plus PRZ

c) DβH -/- Control

_i--A

Figure 5

Ccrtrrj bEtfW. Treafrrert

Figure 6 a)

DBH +/- DBH-/- D B H G enotype

Figure 7 Reduced Expression of Collagen and ΥGF-βi in DβH-/- mice

b)

DBH +/- DBH-/- Genotype c) Effect of Genotype on TGF-B1 Expression

DBH +/- DBH-/- Genotype

Figure 8 a)

DBH +/- DBH -/- Genotype

b)

DBH+/- DBH-/- DBH Genotype

c)

DBH +/- DBH -/- Genotype

Hepatic stellate cells (HSC) are activated by liver injury to become proliferative, fibrogemc myofibroblasts. This process may be regulated by the sympathetic nervous system (SNS), but the mechanisms involved are unclear. Methods: We studied cultured HSC, and intact mice with liver injury, to test the hypothesis that HSC respond to and produce SNS neurotransmitters to promote fibrogenesis. Results: HSC express adrenoceptors, catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine β-hydroxylase (Dbh)), release norepinephrine-NE, and are growth inhibited by α- and β-adrenoceptor antagonists. HSC from DbK^f', which cannot make NE, grow poorly in culture and are rescued by NE. Inhibitor studies demonstrate that this effect is mediated via G-protein-coupled adrenoceptors, mitogen activated kinases, andphosphatidylinositol 3-kmase. Injury-related fibrogemc responses are inhibited in Z M ^" mice, as evidenced by reduced hepatic accumulation of α-smooth muscle actin^+vc HSC and decreased induction of TGF-βl and collagen. Treatment with isoprenaline, a β-adrenoceptόr agonist, rescues HSC activation. HSC are also reduced in leptin-deficient ob/ob mice, which have reduced NE levels and are resistant to hepatic fibrosis. Treating ob/ob mice with NE, an α-adrenoceptor agonist, induces HSC proliferation, up-regulates hepatic TGF-βl and coEagen, and increases liver fibrosis. Conclusions: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitteis. The SNS directly regulates liver fibrogenesis by acting on HSC.

Abbreviations: Alanine aminotransferase (ALT), Alpha smooth muscle actin (ASMA), Autonomic nervous system (ANS), Dopamine beta-hydroxylase (DBH), Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), Ethionine (E), Glial acidic fibrillary protein (GFAP), Hepatic stellate cells (HSC), Homovallinic acid (HVA), Methionine restricted, choline deficient (MCD), Neural cell adhesion molecule (NCAM), Norepinephrine (NE), Prazosin (PRZ), Serotonin (5-hyάYoxytryptarnine, 5-HT), Sympathetic nervous system (SNS), Tyrosine hydroxylase (TH), 5-hydroxyindoϊeacetic acid (5-HIAA). Liver injury activates hepatic stellate ceils (HSC) to move from a quiescent phenotype to a proliferative fibrogenic, myofibroblastic phenotype [1]. These activated myofibroblastic cells constitute the major matrix producing cells in the injured liver. As such, they are responsible for the progressive accumulation of collage that occurs as injured livers become cirrhotic. HSC are also contractile and may, therefore, contribute to porta hypertension, a major complication of cirrhosis [2]. The mechanisms that initiate and perpetuate the fibrogeni response in injured livers are not understood. It has been reported that sympathetic nervous system (SNS) inhibitors markedly reduce experimentally induced liver fibrosis [3], and that the spontaneously hypertensive rat, which has an over-active SNS, develops unusually severe liver fibrosis after carbon tetrachloride-induced liver injury [4, 5]. These observations suggest that SNS activity promotes hepatic fibrosis. However, the cellular target of the SNS in mediating these effects, and the mechanisms involved, are not known. Identification of such targets and mechanisms is an important goal because such information will facilitate development of new therapies to prevent cirrhosis. The HSC themselves may be targets for SNS regulation of liver fibrosis. Our earlier studies show that cultured HSC proliferate and express collagen rnRNA in response to SNS neurotransmitters [6, 7]. Therefore, HSC may be an effector arm of the SNS, mediating its regulation of liver fibrosis. Further support for this liver neuroglial role of HSC is provided by evidence that HSC express stereotypical neuroglial proteins [2, 8-11], possess synaptic vesicles [10], and are innervated by autonomic fibers [12, 13]. Therefore, we hypothesized that the SNS promotes liver fibrosis in vivo via direct actions on HSC, that the in vivo fibrogenic response requires SNS neurotransmitters, and that HSC themselves are an autocrine source of NE, the principal SNS neurotransmitter. Methods

Drugs All chugs were from Sigma except pertussis toxin, wortmannin, SB202190, PD98059 and RO-32-0432 which were purchased from Calbiochem, San Diego, CA.

Animal Experiments Male, lean C57BL/6 mice, and ob/ob mice, 10-18 weeks old were from ackson Laboratory (Bar

Harbor, ME). Sprague-Dawley rats 250-300g, 10-16 weeks old, were from Charles River Laboratories (Wilmington, MA). Dbh ^~'^~ mice were generated and maintained as previously described [14], and used at 30-40 weeks of age. Animals were allowed access to diets and water ad libitum.

For some studies, Dbh^'1' mice (n=12) and Dbh^+/' littermates (n=12) were fed methionine restricted, choline-deficient (MCD) diets (ICN, Aurora, OH) for 4 weeks, to induce hepatic oxidative , stress and steatohepatitis [15]. A similar diet has previously been shown to cause steatohepatitis and fibrosis in mice [16]. Some Dbh^'1' mice were also implanted with subcutaneous osmotic minipumps (Alzet, Cupertino, CA) and infused with isoprenaline (ISO) 20mg kg/day. The dose of isoprenaline is known to induce significant SNS activation and cardiac hypertrophy when infused chronically into normal mice [17]. In other studies, ob/ob (n=5) mice were infused with NE (2.5mg/kg/day) for 4 weeks. This dose of NE is known to induce significant SNS activation with a parallel elevation of blood pressure when infused chronically into normal mice [18]. All mice were weighed at the beginning of the feeding period and weekly thereafter until sacrifice. At sacrifice, liver tissues were fixed in buffered formalin or optimal cutting temperature (OCT) fixative (Sakura, Torrance, CA) and processed for histology; alternatively, tissues were snap frozen in liquid nitrogen and stored at -80°C for further analysis. All experiments satisfied the Guidelines of our Institutions Animal Care Committee and the National Institutes of Health. Stellate Cell Isolation and Culture Using pronase and collagenase liver digestion, HSC were isolated from wild type mice, Dbh^+/~, Dbh^'1' mice or healthy rats. In each experiment, HSC were pooled from 6 mice of each genotype. A single rat provided sufficient HSC for an experiment. All experiments were replicated at least twice. Cell identity was confirmed by aufofluorescence, and expression of alpha smooth muscle actin (ASMA) and glial fibrillary acidic protein (GFAP) by immunoblots and immunocytochemistry [7]. HSC preparation purity was always > 95% [7]. HSC were routinely cultured in 10% serum-supplemented RPMI 1640 medium. For determination of cellular cathecholamines or HSC released cathecholamines, HSC and their conditioned media were harvested after 4 days in culture. The cells were washed twice with ice-cold PBS, and rapidly analyzed for cathecholamine content by HPLC. Conditioned media was stored at -70°C until analyzed.

Apoptosis Assays To evaluate the effect of the alpha-j adrenoceptor antagonist, prazosin (PRZ) or Dbh genotype on cell viability, equal numbers of HSC were plated on 6mm petri dishes and grown in the presence and absence of lOμM PRZ. Fresh PRZ was added every 2 days, when the medium was changed. At various time points, cells were harvested and apoptotic activity was assessed with the Vybrant (Annexin V) apoptosis assay kit (Molecular Probes, Eugene, OR).

Cell Proliferation Assay Freshly isolated HSC were seeded into culture flasks and grown to subconfluence. At this time point

(~ day 7-10), cultures were harvested. HSC were resuspended in serum-free medium, and then re-plated on 96- well plates at 5,000 cells/well. Twenty-four hours later, when the cells had become quiescent, NE (lOμM) ± various inhibitors, e.g., PRZ (lOμM), pertussis toxin (lOOng/ml), wortmannin (lOOnM ), SB202190 (lOμM ), PD98059 (20μM ), or RO-32-0432 (lμM) were added to some wells, as described [6, 19-26]. After 44 hours, cell numbers were assessed by a further 4 h incubation with WST-8 tetrazolium reagent (Dojindo Molecular Technologies, Gaithersburg, MD) as described [7, 19, 27]. In viable cells, the tetrazolium salt is metabolized to a colorimetric dye and cell number is proportional to the signal intensity at 450nm [7]. Therefore, we [7] and others [19] have used this assay to evaluate HSC proliferation.

RT-PCR RNA was extracted from HSC using RNeasy kits (Qiagen, Valencia, CA). Concentration and purity were assessed by absorbance at 260/280 nm. One-step RT-PCR was performed with Superscript one-step RT- PCR with platinum Taq kits (Invitrogen, Carlsbad, CA) with Ambion's QuantumRNA Classic II 18S internal standard (Ambion, Austin, TX). Products were separated by electrophoresis on a 1.5% agarose gel. Primer sequences and conditions were as reported [20, 28].

HPLC Analysis Catecholamines were extracted from HSC pellets and HSC-conditioned medium with perchloric acid.

Norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5- hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) and homovallinic acid (HVA) were analyzed in these extracts by HPLC as described [29], using dihydroxybenzilamine as the internal standard. Peak heights and sample concentrations of NE, DOPAC, DA, 5-HIAA, HVA and 5-HT were calculated with a Hewlett Packard integrator.

Immunoblot HSC were harvested after various times in culture, cell homogenates were prepared and protein content was quantified by BSA assay (Pierce, Rockford, IL) using bovine serum albumin standards. Proteins (lOμg lane) were then resolved by polyacrylamide gel electrophoresis and transferred to nylon membranes. After membranes were incubated with primary antibodies to ASMA (1:3000, Sigma, St. Louis, MO), TH (1:100, Novus Biologies, Littleton, CO), DBH (1:250, Research Diagnostics, Flanders, NJ), α-l_A, C -IB, and α- 1_D adrenoceptors (1:100, Santa Cruz Biotech, Santa Cruz, CA), and βl, β2, and β3 adrenoceptors (1:200, Santa Cruz Biotech), peroxidase-conjugated secondary .antibodies were added, and antigens were demonstrated b enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) as we have described previously [30].

Immunohistochemistry In liver tissues, HSC expression of alpha smooth muscle actin (ASMA), was used as a marker of HS activation [1], whilst GFAP expression was used to identify both activated and quiescent HSC. Details of th staining procedure are essentially as we have detailed previously [11]. Briefly, 4 urn thick paraffin liver sectio were deparaffimzed and rehydrated, followed by heating in a microwave oven for 10 minutes at 750 Watt i citrate buffer, pH 6.0. Incubation with the primary antibodies, mouse monoclonal anti-ASMA antibody (1/4 dilution, Dako, Denmark), or rabbit polyclonal anti-GFAP (1/300 dilution, Dako, Denmark), was performed room temperature for 30 minutes. Primary antibody binding was revealed with the peroxidase animal researc Kit (Dako).

Histology Pieces of liver were formalin fixed, embedded in paraffin and 5-micron sections were stained Masso trichrome. Coded samples were examined by an experienced liver pathologist (MT) who was blinded t treatment groups. The degree of fibrosis was scored as 0 = no fibrosis; 1 = mild fibrosis; 2 = moderate fibrosis 3 = severe fibrosis.

RNA isolation and Ribonuclease Protection Assay Total RNA was isolated from liver samples according to the method of Chomczynski and Sacchi [31] as we have described [32]. Collagen- 1-αl and TGF-βl gene expression were evaluated by commercial ribonuclease protection assay (RPA) kits with probes for collagen- 1-αl and TGF-βl (PharMingen, San Diego, CA) as we described previously [32]. ^Jt^*ιιti sties _* Statistical analysis by unpaired t-test or the Mann-Whitney test was performed with Graphpad Pris software (San Diego, CA). Significance was accepted as *p< 0.05.

Results

HSC express catecholamine biosynthetic enzymes We began to evaluate our hypothesis that HSC are resident liver neuroglial cells that produce an respond to SNS neurotransmitters by determining if HSC express TH and Dbh, key enzymes in th catecholamine biosynthetic pathway. Immunoblot analysis of lysates from primary cultures of normal mic HSC demonstrates that these cells express both TH and Dbh (Figure 1), suggesting that HSC may be capable o producing NE.

Primary HSC synthesize and release NE in culture To confirm actual synthesis and release of NE, HPLC analysis was performed on HSC lysates and conditioned media. Analysis of conditioned medium from wild-type cells demonstrates NE (69 ± 6 ng/ml). However, no NE was detected in conditioned medium from Dbh^'1" HSC or in unconditioned medium that had not been exposed to HSC. Lysates of wild type HSC were similarly analyzed. NE, as well as DA, serotonin (5- Hydroxytryptamine, 5-HT) and the cathecholamine metabolites dihydroxyphenylacetic acid, (DOPAC) and homovanillic acid (HVA) plus the 5-HT metabolite 5-Hydroxyindoleacetic acid (5-HIAA) were demonstrated (Figure 2).

HSC express multiple adrenoceptor subtypes We next investigated the physiological relevance of HSC catecholamine synthesis. If NE directly regulates HSC, then these cells should express adrenoceptors. In fact, HSC have been shown to express α adrenoceptors [33]. However, it is not known which particular α-adrenoceptors subtypes are expressed. Nor is it known if HSC express β-adrenoceptors subtypes. To address these questions, we performed RT-PCR analysis and western blot analysis of primary HSC cultures. HSC express O-IA α-l_B, α-l_D, βl and β2 adrenoceptors (Figure 3). No β3 expression was detectable. A similar expression profile was seen at the protein level wit Western blot analysis (not shown).

Adrenoceptor antagonists inhibit the growth of primary HSC cultures To assess the importance of endogenous NE for HSC growth in culture, we plated wild type HSC in th presence and absence of the αl-adrenoceptor PRZ (lOμM) and the β-adrenoceptor antagonist propranolol (PRL lOμM). PRZ and PRL significantly inhibited HSC proliferation. Moreover, the growth-inhibitory actions o the α-adrenocεptor and β-adrenoceptor antagonists were additive because PRZ and PRL each reduced HS numbers by -20%, but the combination of PRZ + PRL decreased HSC growth by ~50% (Figure 4). Thes findings demonstrate that endogenous production of NE is required for the optimal growth of HSC in culture and suggests that NE functions as an autocrine growth factor for HSC.

HSC that are genetically incapable of producing NE grow poorly in culture and exogenous NE rescues proliferative activity To further evaluate the possibility that NE is an autocrine growth factor for HSC, we studied the proliferation of HSC cultured from mice that are genetically deficient in Dbh and their heterozygous littermates. In the final steps of catecholamine biosynthesis, NE is produced from DA in a reaction that is catalyzed by Dbh. Because Dbh^'1' mice, in which the Dbh gene has been disrupted through targeting, cannot produce NE [14], their HSC might not grow well in culture if endogenous NE normally promotes HSC proliferation. HSC were harvested from Dbh^"'^' and Dbh*^1' mice. The same numbers of cells from each group were plated. In addition, to assure that the culture conditions permitted NE activity, HSC from Dbh^+I~ mice, which can produce NE, were incubated with PRZ (lOμM) as an experimental control. During 4 days in culture, HSC from Dbh*^1' control mice proliferated to become nearly confluent (Figure 5a). This proliferative activity was inhibited significantly by PRZ, an α-adrenoceptor antagonist (Figure 5b). Proliferative activity was also significantly inhibited in HSC from Dbh^'1' mice (Figure 5c), which cannot produce NE. To be certain that the reduced growth of HS from Dbh^''^' mice was not an artifact that might have resulted from plating fewer Dbh^''^' HSC at the start of th experiment, we repeated these studies and asked if exogenous NE could rescue the proliferative activity of HS from Dbh^''^' mice. HSC from Dbh^"'^' mice were cultured in the presence and absence of NE (lOμM). Exogenou NE rescued the growth of HSC from Dbh^''^' ice (Figure 5d), confirming the importance of endogenous NE fo HSC growth in culture.

NE is an autocrine growth factor for HSC Both apoptosis and proliferation control the number of cells that accumulate during culture. Norm HSC become spontaneously activated during culture and proliferate at a greater rate than they die, such that th plated HSC population gradually expands. Therefore, increases in proliferative activity normally drive HS growth in culture. To determine to what extent, if any, the NE-related differences in cell number might als reflect differences in apoptotic activity, HSC were harvested, incubated with Annexin V and analyzed by flo cytometry. After one day in culture, a slightly greater proportion of apoptotic HSC were detected in culture from Dbh^''^' mice (12.4±0.26%) compared to Dbh*^1' mice (9.96±0.15%) (p=0.007). Therefore, endogenous N appears to promote the viability of HSC, at least in vitro. This anti-apoptotic effect may help to explain wh endogenous NE is required for optimal HSC growth in culture.

NE activates adrenoceptor G protein-coupled mechanisms that induce mitogenic and survival pathways Given that HSC produce NE and that endogenous NE is required for optimal HSC growth in vitro, we wanted to verify our earlier observation that wild type HSC also respond to exogenous NE. Previously, we showed that adding NE to cultures of murine HSC caused a dose-related increase in proliferative activity that was blocked by co-administration of the α-adrenoceptor antagonist, PRZ [6]. To be certain that the proliferative response to exogenous NE was not an unusual property of murine HSC, we cultured rat HSC with NE in the presence and absence of PRZ. Incubation with NE for 48 hours significantly increased rat HSC numbers and PRZ blocked these trophic effects (Figure 6a), demonstrating that the proliferative effects of NE extend across at least two rodent species. Because NE is a generally important HSC growth factor, we next addressed potential mechanisms for this effect. Adrenoceptors are known to be coupled to G-proteins in other mesenchymal cells, in which they induce proliferation by activating inositol triphosphate and mitogen activated protein kinases [20, 21, 24]. Therefore, we investigated the possibility that similar signaling pathways may mediate NE-induced proliferation in cultured HSC. Primary HSC from normal mice were cultured with NE in the presence and absence of pertussis toxin (PT), a specific G-protein inhibitor; wortmannin (WT), a specific IP3-kinase inhibitor; SB202190 (SB), a specific inhibitor of p38 MAP kinase; PD98059, a specific MEK inhibitor; and RO-32-0432, a specific Protein kinase C inhibitor. As shown in Figure 6b, PT, WT and PD significantly reduced NE induced proliferation while SB and RO had no effect. These results suggest that the proliferative effects of NE on HSC are mediated by G-protein coupled receptors and the downstream effectors involve IP-3 and the Erk family of mitogen activated protein kinases.

NE regulates HSC activation in intact mice

Mice with decreased NE levels have fewer HSC and treatment with adrenergic agonists restores HSC numbers Although the aforementioned results show that autocrine production of NE regulates the phenotype of cultured HSC and that NE stimulates the proliferation of HSC in vitro, we wanted to determine if NE had any impact on HSC function in intact animals. Therefore, NE was infused chronically into obese, ob/ob mice which have low levels of NE [34, 35], and are resistant to fibrosis despite having chronic steatohepatitis [16, 36, 37]. Immunohistochemistry was used to demonstrate GFAP (+) cells in the livers because this marker provides a reliable estimate of both quiescent and activated HSC [38]. Control ob/ob mice have significantly fewer HSC than their lean littermates. Continuous infusion of NE, an α-adrenoceptor agonist, via subcutaneous minipumps for 4 weeks, markedly stimulated HSC proliferation in ob/ob mice, such that the numbers of GFAP(+) HSC in NE-treated ob/ob mice doubled, leading to HSC numbers that approached those of their control littermate (Figure 7).

Injury-related activation of HSC and fibrogenesis is inhibited in mice with decreased NE levels an treatment with adrenergic agonists restores HSC activation and improves the fibrogenic response ob/ob C57Bl/6mice, however, are genetically incapable of producing leptin, a factor that is known t activate HSC proliferation by direct interaction with HSC receptors [19, 39], Therefore, it is conceivable tha the effects of NE might have been confounded by leptin deficiency. To evaluate this possibility, we studie another strain of C57B1-6 mice, Dbh^'1' mice and their Dbh* ^' littermates, that is not leptin deficient but that i genetically incapable of producing NE due to experimental disruption of the Dbh gene. To further evaluate th role of the SNS in hepatic fibrosis, we fed Dbh^'1' mice and Dbh*^' control mice antioxidant-depleted diets t induce steatohepatitis [15] and provoke stellate cell activation, the primary event in the liver's fibrogeni response [1]. Normal mice develop steatohepatitis and fibrosis after eating these diets for 4-8 weeks [16] Consistent with this literature, after 4 weeks of treatment, control Dbh^*'^' mice exhibit a striking accumulation o HSC that express ASMA, an accepted marker of HSC activation (Figure 8a,b). In contrast, ASMA^+ve HS cannot be demonstrated in Dbh^''^' mice (Figure 8a,c). This finding is unlikely to be a staining artifact, becaus ASMA ^ve vessel walls are easily identified in the Dbh^''^'. group (Figure 8c). Moreover, compared to Dbh*¹ controls, Dbh^'1' mice exhibit significantly less hepatic expression of TGF-βl and collagen, two other widely accepted markers of HSC activation (Figure 9a,b). Nevertheless, to verify that it was the absence o adrenoceptor agonists that prevented HSC activation in Dbh^'1" mice, we implanted osmotic minipumps that contained either vehicle or isoprenaline (ISO), a β-adrenoceptor agonist, into Dbh^''^' mice and repeated the feeding experiment. Infusion of ISO rescued HSC activation in Dbh^''^' mice and returned numbers of ASMA (+) HSC to levels exhibited by Dbh*^1' mice that were also fed the hepatotoxic diet (Fig 8a). ISO infusion similarly returned to normal the reduced induction of TGF-β in untreated Dbh^"'^' mice (Figure 9b). Finally, to complete our evaluation of the role of adrenoceptor activation in hepatic fibrogenesis, w examined the effect of NE, an α-adrenoceptor agonist, on HSC activation in ob/ob mice. As mentioned earlie these mice have obesity-related steatohepatitis, but have reduced levels of NE and are unusually resistant to th development of cirrhosis. Compared to control ob/ob mice that received vehicle-containing minipumps, ob/o mice that were treated with NE minipumps for 4 weeks had significantly increased liver expression of TGF- β and collagen mRNA (Figure 10a,b). Indeed, in 2 out of 5 ob/ob mice, the NE treatment increased hepati fibrosis sufficiently for scarring to be detected simply by staining liver sections with Mason trichrome reage (Figure 10c). No such fibrosis was detected in twice as many control ob/ob liver sections that were processe similarly. Therefore, both α-predominant (NE, Figure 7) and β-predominant (ISO, Figure 8a, 9b adrenoceptor agonists affect HSC activation in vivo. Despite this increase in HSC activation, ALT values in N treated ob/ob mice were lower than their littermate controls (Figure lOd). Therefore, NE-related increases i fibrogenesis are not easily attributed to NE exacerbation of liver injury in the leptin-deficient mice.

Discussion The in vitro and in vivo results presented here confirm a role for sympathetic neurotransmitters i hepatic fibrogenesis. They also support our hypothesis that HSC, the liver's principal collagen producing cells, function as resident hepatic neuroglial cells, producing neurotransmitters to regulate, in an autocrine manner, their own function. We have shown that HSC not only contain tyrosine hydroxylase and dopamine β- hydroxylase, key enzymes in the biosynthesis of cathecholamines, but actually make and release NE and othe cathecholamines into their milieu. These findings complement and extend our previous studies which showed that cultured HSC proliferate and up-regulate collagen gene expression in response to NE [6], NE is also necessary for activation of HSC in vivo, because injury-related induction of ASMA is absent in mice with acquired or genetic deficiency of NE. Expression of collagen and TGF-βl, a key profibrogenic cytokine, are also markedly reduced in injured livers when NE is deficient, suggesting that NE may promote HSC collagen gene expression by increasing TGF-βl. Prior to this study, evidence suggested a role for the SNS in hepatic fibrosis. Several studies demonstrated positive correlations between SNS activity and hepatic fibrosis in intact animals, but none of these identified the cellular targets of SNS regulation [3-5, 40]. Recently, in vitro studies of cultured murine HSC by our group suggested that HSC were likely to mediate the profibrogenic actions of the SNS [6, 7]. However, the physiological relevance of our earlier findings remained questionable because we studied HSC in the absence of other cells that might modify their phenotype. The approach adopted here uses a combination of in vitro and in vivo models to overcome the limitations of the previous studies. Moreover, evidence that treatment with NE rescues activation of cultured HSC from mice that are genetically incapable of producing NE, and restores injury-related HSC activation in intact mice that are NE-deficient, provides definitive proof that interactions between HSC and this neurotransmitter play a key role in hepatic fibrogenesis. Sympathetic neurons and the adrenal medulla are the major sources of NE in vivo. HSC receive adrenergic autonomic fibers [12, 13, 41, 42]. Athari and colleagues demonstrated that HSC have a._\- adrenoceptors [33]. The present studies identify _tthe α-adrenoceptor subtypes and also show that these cell express β-adrenoceptors. Moreover, we found that NE induces HSC proliferation by activating G protein coupled mechanisms that result in the induction of mitogenic kinases and cellular survival pathways. This resul is supported by the recent findings that leptin induces similar pathways in HSC [43]. Here and elsewhere [6] w report that prazosin reverses the proliferative effect of exogenous NE on cultured HSC. Therefore, neuronal N is likely to regulate HSC in intact animals. In addition, we present novel evidence that HSC, themselves produce NE. HSC-derived NE appears necessary for optimal growth of HSC in culture, because simply addin either an α-adrenoceptor antagonist or a β-adrenoceptor antagonist to cultures of normal HSC reduces thei proliferation. Combinations of α-and β-adrenoceptor antagonists are maximally growth inhibitory, suggestin that the growth-stimulatory effects of the two major adrenoceptor subtypes are additive. Finally, HSC fro Dbh^"1' mice that cannot make NE grow poorly in vitro and adding NE restores growth. Hence, NE functions a an autocrine growth factor for HSC. However, the relative importance of NE produced by HSC, as opposed to adrenal medulla or neuron-derived NE, in regulating HSC biology in living animals remains uncertain. It is tempting to speculate that autocrine regulation of HSC is important under at least some circumstances, because transplanted livers, which remain denervated [44] clearly can develop cirrhosis [45], although circulating epinephrine- an α and β-adrenoceptor agonist - may also contribute to this process. That NE may sub-serve functions other than its classically assigned role of neurotransmission is becoming well established in other systems. For example, cardiac remodeling in heart failure involves mitogenic and fibrogenic actions of NE that are mediated via adrenoceptors [24, 46, 47], There are multiple alpha- adrenoceptor subtypes, some of which selectively mediate mitogenic effects of NE, and others which selectively regulate NE vasopressor effects [48, 49]. In the present study we used NE, which activates α- adrenoceptors predominately, and prazosin, a non-specific alpha antagonist that binds to α-1 adrenoceptors in general, and demonstrated physiological effects, thus validating and extending our RT-PCR and Western blot evidence that HSC express this receptor family. HSC also express β-adrenoceptors and this class of adrenoceptors is also functional in intact animajs because isoprenaline, a selective β-adrenoceptor agonist restores HSC activation in Dbh^'1' mice that lack both NE and its metabolite, epinephrine. More work is needed to characterize the intracellular pathways that transduce signals initiated b specific adrenoceptor subtypes. The delineation of the specific signals that are activated by NE-adrenocepto interaction will clarify if NE regulates TGF-β expression in HSC, as it does in vascular smooth muscle cells an cardiac fibroblasts [47, 50]. This possibility is suggested by our observation that induction of TGF-βl mRN is reduced in the livers ϊDbh^"1' mice and ob/ob mice, both of which have reduced levels of NE. HSC are a important source of TGF-βl [51]and TGF-βl is necessary for optimal activation of HSC to myofibroblastic collagen-producing cells during liver injury [52]. Consistent with the possibility that NE promotes HS activation by inducing TGF-βl, both Dbh^"1' mice and ob/ob mice failed to accumulate activated, ASMA ^v

HSC in their livers and exhibited significantly less induction of hepatic collagen gene expression during live injury. Together, these findings suggest that NE is upstream of TGF-βl in the hepatic fibrogenic cascade an that liver TGF-β expression is regulated by NE. However, given evidence that multiple types of liver cells, including hepatocytes, Kupffer cells and resident lymphocytes express adrenoceptors and respond to NE [53- 55], more complicated mechanisms may also be involved. In any case, our findings suggest a novel paracrine mechanism, i.e., local release of NE by HSC, that may regulate multiple aspects of liver injury and repair. This, in turn, identifies novel therapeutic targets to prevent liver fibrosis and its sequelae.

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Figure Legends

Figure 1. Expression of tyrosine hydroxylase and dopamine β-hydroxylase by cultured HSC. HSC were cultured from Dbh^'1' mice that cannot produce NE, heterozygous Dbh* ^' littermates that produce NE, and health adult rats. After 4 days in culture, cell lysates were evaluated for tyrosine hydroxylase (TH) and dopamine β- hydroxylase (Dbh) expression using immunoblot analysis (10 μg protein/lane). Results from representative wild type mice immunoblots are shown.

Figure 2. Primary HSC synthesize and release NE in culture.

HSC were cultured from normal mice and the production of NE and other catecholamines were evaluated by HPLC analysis of cell lysates and conditioned media after 4 days in culture. The retention times of the HSC products were compared to those of a concurrently analyzed standard.

Figure 3. HSC express multiple adrenoceptor subtypes.

HSC were cultured from normal mice and extracted RNA analyzed by RT-PCR for the expression of adrenoceptors. Results from a representative RT-PCR analysis of HSC RNA pooled from 4 normal mice are shown. The first lane shows the DNA ladder (500-200bp, arrowed). Each subsequent pair of lanes is a replicate analysis of adrenoceptor genes, the 18S band (324bp) in each lane. is shown as a control. HSC express low levels of α-l_A and βl adrenoceptor mRNA, and much higher levels of mRNAs for α-l_B, α-lo, and β2 adrenoceptors. There was no detectable expression of β3 adrenoceptor.

Figure 4. Adrenoceptor antagonists inhibit the growth of primary HSC cultures

Freshly isolated HSC from normal wild type mice were cultured with the αi -adrenoceptor antagonist prazosin

(PRZ, lOμM) and the β-adrenoceptor antagonist propranolol (PRL, 10 μM). After 48 hours, the numbers of cells in culture were evaluated by a validated colorimetric assay that measures the reduction of the tetrazolium salt, WST-8, by viable HSC. Results are the mean, ±SD of 2 or more separate determinations. * p< 0.05 for PR or PRL-treated HSC vs. control, ** p < 0.01 for PRZ + PRL (PP)-treated HSC vs. control.

Figure 5. HSC that are genetically incapable of producing NE grow poorly in culture and exogenous N rescues proliferative activity.

HSC were isolated from NE-producing Dbh*^1' mice and cultured in the absence (a) or presence (b) of PRZ (10 μM), an alpha-adrenoceptor antagonist, for 4 days. Results were compared to 4 day-old HSC cultures fro control Dbh^"'^' mice (c) which cannot produce NE. Photomicrographs of representative cultures are shown. Compared to vehicle-treated controls, PRZ-treated cultures and HSC cultures from control Dbh^'!' mice have ~40-50% less protein.

(d) Dbh ^''^' HSC were plated in medium containing no added NE (control) or NE (10 μM) for 4 days and cell numbers were quantified by the WST-8 assay. NE increased Dbh^'1' growth significantly, *p < 0.05.

Figure 6. NE activates adrenoceptor G protein-coupled mechanisms that induce mitogenic and survival pathways in rat HSC.

HSC were isolated from healthy rats and mice, (a) Rat HSC were cultured with vehicle (control), NE (10 μM) or NE + PRZ (10 μM) to determine if the trophic effects of NE extend across species. After 2 days, HSC numbers were evaluated in triplicate wells by the WST-8 assay. Mean (+SEM) results of duplicate experiments are graphed, (b) Experiments were repeated with murine HSC and inhibitors of mitogen and/or survival pathways, were added to some wells to determine if any of these inhibited NE effects on HSC growth. PT = pertussis toxin, WT = wortmannin, SB = SB2021 0, PD = PD98059, RO = RO-32-0432, * p < 0.05 for treated groups versus NE alone.

Figure 7. NE increases HSC activation in NE-deficient ob/ob mice.

NE or control vehicle were infused for 4 weeks into obese, NE-deficient, ob/ob mice. At the end of the treatment period, liver samples were obtained and fixed in OCT. GFAP expression, as a marker of both quiescent and activated HSC, was assessed by immunohistochemistry. HSC were counted in 5 randomly selected high power fields/section from each of 5 animals per treatment group. Mean + SD data are graphed. * p < 0.05 for ob/ob control vs. lean control; # p < 0.05 for ob/ob + NE vs ob/ob control.

Figure 8. Reduced activation of HSC in NE-deficient Dbh ^"^mice. Dbh^'1' and their control Z?M^{+ "}littermates were fed methionine choline deficient (MCD) diets to cause steatohepatitis and stellate cell activation. A subgroup of the Dbh^''^' mice were infused with isoprenaline (ISO), a β-adrenoceptor agonist. After 4 weeks, liver samples were obtained, fixed in formalin and paraffin-embedded, (a) Alpha smooth muscle actin (ASMA) expression was demonstrated by immunohistochemistry. ASMA

sinusoidal cells were counted in 5 randomly selected fields/section from 4 mice/group. Mean ±SD results of one experiment are graphed. Virtually identical results were obtained in a second experiment that studied an additional 4 mice/group, (b) Photomicrograph from representative Dbh ^' mice, (c) Photomicrograph fro typical Dbh^'1' mice. Arrows indicate typical ASMA HSC.

Figure 9. NE regulates hepatic expression of collagen and TGF-β Liver RNA was isolated from NE producing Dbh*'^" mice (n = 3) and NE-deficient Dbh^'1' mice (n = 3 after feeding a hepatotoxic MCD diet for 4 weeks. Another group of the Dbh^''^' mice were infused wit isoprenaline (ISO), a β-adrenoceptor agonist (n = 3). Hepatic expression of collagenl-α-1 (a) and TGF-β (b genes were evaluated by ribonuclease protection assay (20μgRNA assay). Normalized mean (SD) collagen an TGF-βl gene expression in duplicate experiments (3 mice/group/experiment) is graphed. *p<0.05 Dbh*^1' versu Dbh^'1' mice. #p<0.05 Dbh^''^' versus Dbh^"'^' + ISO.

Figure 10. NE increases hepatic expression of collagen and TGF-β without increasing liver injury in NE deficient ob/ob mice.

Liver RNA was isolated from control ob/ob mice or NE-treated ob/ob mice. Hepatic expression of collagen 1-α- 1 (a) and TGF-β (b) genes were evaluated by ribonuclease protection assay (20μgRNA/assay). Representative phosphoimages from 2 animals per group are shown. Normalized mean (SD) collagen and TGF-βl gene expression in duplicate experiments (4 mice/group/experiment) is graphed. *p<0.05 control ob/ob versus ob/ob+NE. (c) Representative Mason-trichrome stained sections from a vehicle-treated control ob/ob mice and from an NE-treated ob/ob mouse with pericellular and sinusoidal fibrosis are shown, (d) ALT values in control ob/ob mice and NE treated ob/ob mice. *p< 0.05 for NE-treated ob/ob group vs. ob/ob controls.

Claims

1- A method of treating fibrosis comprising administering to a subject at risk of or suffering from a fibrotic disease an amount of a neurotransmitter antagonist e ective to reduce the activity of hepatic stellate cells.

2. A method accordin to claim 1, wherein the fibrotic disease is of the liver.

3. A method according to either of the preceding claims , therein the neurotransmitter antagonist comprises an α-adrenoceptor antagonist or a β-adrenoceptor antagonist.

4. A method according to claim 3, wherein the neurotransmitter antagonist comprises prazosin.

5. A method according to claim 3, wherein the neurotransmitter antagonist comprises propanolol.

6. A method according to claim 4 or claim 5, wherein the neurotransmitter antagonist comprises both prazosin and propanolol .