Correspondence to:
Professor A M Diehl
Division of Gastroenterology, Johns Hopkins University School of Medicine, 912 Ross Research Building, 720 Rutland Ave, Baltimore, MD 21205, USA; ude.imhj@lheidma

Accepted 2003 Sep 22.

Abstract

Background and aims: Hepatic stellate cells (HSC) are activated by liver injury to become proliferative fibrogenic 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 expressed adrenoceptors, catecholamine biosynthetic enzymes, released norepinephrine (NE), and were growth inhibited by α- and β-adrenoceptor antagonists. HSC from dopamine β-hydroxylase deficient (Dbh^−/−) mice, which cannot make NE, grew poorly in culture and were rescued by NE. Inhibitor studies demonstrated that this effect was mediated via G protein coupled adrenoceptors, mitogen activated kinases, and phosphatidylinositol 3-kinase. Injury related fibrogenic responses were inhibited in Dbh^−/− mice, as evidenced by reduced hepatic accumulation of α-smooth muscle actin^{HSC and decreased induction of transforming growth factor β1 (TGF-β1) and collagen. Treatment with isoprenaline rescued HSC activation. HSC were 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 induced HSC proliferation, upregulated hepatic TGF-β1 and collagen, and increased liver fibrosis.}

Conclusions: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitters to promote hepatic fibrosis.

Keywords: sympathetic nervous system, neurotransmitters, liver, fibrogenesis, hepatic stellate cells

Abstract

Liver injury activates hepatic stellate cells (HSC) to move from a quiescent phenotype to a proliferative, fibrogenic, myofibroblastic phenotype.¹ These activated myofibroblastic cells are responsible for the progressive accumulation of collagen that occurs as injured livers become cirrhotic. HSC are also contractile and may therefore contribute to portal hypertension.² The mechanisms that initiate and perpetuate the fibrogenic response in injured livers are not understood.

Sympathetic nervous system (SNS) inhibitors markedly reduce experimentally induced liver fibrosis,³ and the spontaneously hypertensive rat, which has an overactive SNS, develops unusually severe liver fibrosis when given hepatotoxins.^4,⁵ Although these observations suggest that SNS activity promotes hepatic fibrosis, the cellular target of the SNS in mediating these effects, and the mechanisms involved, are not known.

HSC may be targets for SNS regulation because we showed that cultured HSC proliferate and express collagen mRNA in response to SNS neurotransmitters.^6,⁷ Given that HSC also express stereotypical neuroglial proteins,^2,^8–¹¹ possess synaptic vesicles,¹⁰ and are innervated by autonomic fibres,^12,¹³ we hypothesise that HSC produce SNS neurotransmitters and function as an effector arm of the SNS, automodulating their own activation via SNS neurotransmitters.

Acknowledgments

This work was supported by NIAAA RO1-10154 (AMD), NIAAA RO1-12059 (AMD), and FWO Vlaanderen G.0139.00N (TR).

Acknowledgments

Abbreviations

ASMA, alpha smooth muscle actin
Dbh, dopamine β-hydroxylase
DOPAC, 3,4-dihydroxyphenylacetic acid
GFAP, glial acidic fibrillary protein
HPLC, high pressure liquid chromatography
HSC, hepatic stellate cells
HVA, homovallinic acid
MCD, methionine restricted choline deficient
MMP, matrix metalloproteinase
NE, norepinephrine
5-HT, 5-hydroxytryptamine (serotonin)
RT-PCR, reverse transcription-polymerase chain reaction
SNS, sympathetic nervous system
TGF-β1, transforming growth factor β1
TH, tyrosine hydroxylase
TIMP, tissue inhibitor of metalloproteinase
5-HIAA, 5-hydroxyindoleacetic acid

Abbreviations

REFERENCES

References

1. Friedman SLMolecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000;275:2247–50. [[PubMed][Google Scholar]
2. Reynaert H, Thompson MG, Thomas T, et al. Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension. Gut 2002;50:571–81.
3. Dubuisson L, Desmouliere A, Decourt B, et al. Inhibition of rat liver fibrogenesis through noradrenergic antagonism. Hepatology 2002;35:325–31. [[PubMed]
4. Hsu CTThe role of the sympathetic nervous system in promoting liver cirrhosis induced by carbon tetrachloride, using the essential hypertensive animal (SHR). J Auton Nerv Syst 1992;37:163–73. [[PubMed][Google Scholar]
5. Hsu CTThe role of the autonomic nervous system in chemically-induced liver damage and repair-using the essential hypertensive animal model (SHR). J Auton Nerv Syst 1995;51:135–42. [[PubMed][Google Scholar]
6. Oben JA, Yang S, Lin H, et al. Norepinephrine and neuropeptide Y promote proliferation and collagen gene expression of hepatic myofibroblastic stellate cells. Biochem Biophys Res Commun 2003;302:685–90. [[PubMed]
7. Oben JA, Yang S, Lin H, et al. Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells. Biochem Biophys Res Commun 2003;300:172–7. [[PubMed]
8. Buniatian G, Hamprecht B, Gebhardt RGlial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast-like phenotypes. Biol Cell 1996;87:65–73. [[PubMed][Google Scholar]
9. Niki T, Pekny M, Hellemans K, et al. Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells. Hepatology 1999;29:520–7. [[PubMed]
10. Cassiman D, van Pelt J, De Vos R, et al. Synaptophysin: A novel marker for human and rat hepatic stellate cells. Am J Pathol 1999;155:1831–9.
11. Cassiman D, Denef C, Desmet VJ, et al. Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors. Hepatology 2001;33:148–58. [[PubMed]
12. Bioulac-Sage P, Lafon ME, Saric J, et al. Nerves and perisinusoidal cells in human liver. J Hepatol 1990;10:105–12. [[PubMed]
13. Ueno T, Sata M, Sakata R, et al. Hepatic stellate cells and intralobular innervation in human liver cirrhosis. Hum Pathol 1997;28:953–9. [[PubMed]
14. Thomas SA, Palmiter RDImpaired maternal behavior in mice lacking norepinephrine and epinephrine. Cell 1997;91:583–92. [[PubMed][Google Scholar]
15. Akhurst B, Croager EJ, Farley-Roche CA, et al. A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver. Hepatology 2001;34:519–22. [[PubMed]
16. Leclercq IA, Farrell GC, Schriemer R, et al. Leptin is essential for the hepatic fibrogenic response to chronic liver injury. J Hepatol 2002;37:206–13. [[PubMed]
17. Mackintosh CA, Feith DJ, Shantz LM, et al. Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy. Biochem J 2000;350:645–53.
18. Vecchione C, Fratta L, Rizzoni D, et al. Cardiovascular influences of (alpha)1b-adrenergic receptor defect in mice. Circulation 2002;105:1700–7. [[PubMed]
19. Saxena NK, Yang Y, Floyd J, et al. Leptin is mitogenic, anti-apoptotic and increases fibrogenic response genes in rat hepatic stellate cells. Hepatology 2002;36:316A. [PubMed]
20. Hakuno D, Fukuda K, Makino S, et al. Bone marrow-derived regenerated cardiomyocytes (CMG cells) express functional adrenergic and muscarinic receptors. Circulation 2002;105:380–6. [[PubMed]
21. Zou Y, Komuro I, Yamazaki T, et al. Both Gs and Gi proteins are critically involved in isoproterenol-induced cardiomyocyte hypertrophy. J Biol Chem 1999;274:9760–70. [[PubMed]
22. Tangkijvanich P, Santiskulvong C, Melton AC, et al. p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts. J Cell Physiol 2002;191:351–61. [[PubMed]
23. Ajizian SJ, English BK, Meals EASpecific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. J Infect Dis 1999;179:939–44. [[PubMed][Google Scholar]
24. Xiao L, Pimental DR, Amin JK, et al. MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes. J Mol Cell Cardiol 2001;33:779–87. [[PubMed]
25. Hansel DE, Eipper BA, Ronnett GVNeuropeptide Y functions as a neuroproliferative factor. Nature 2001;410:940–4. [[PubMed][Google Scholar]
26. Wu H-L, Albrightson C, Nambi PSelective inhibition of rat mesangial cell proliferation by a synthetic peptide derived from the sequence of the C2 region of PKC[beta]. Peptides 1999;20:675–8. [[PubMed][Google Scholar]
27. Frank S, Stallmeyer B, Kampfer H, et al. Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair. J Clin Invest 2000;106:501–9.
28. Mosmann TRapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. [[PubMed][Google Scholar]
29. Isobe I, Michikawa M, Yanagisawa KEnhancement of MTT, a tetrazolium salt, exocytosis by amyloid beta-protein and chloroquine in cultured rat astrocytes. Neurosci Lett 1999;266:129–32. [[PubMed][Google Scholar]
30. Matsuoka M, Wispriyono B, Igisu HIncreased cytotoxicity of cadmium in fibroblasts lacking c-fos. Biochem Pharmacol 2000;59:1573–6. [[PubMed][Google Scholar]
31. Hutchinson DS, Evans BA, Summers RJ(beta)1-Adrenoceptors compensate for (beta)3-adrenoceptors in ileum from (beta)3-adrenoceptor knock-out mice. Br J Pharmacol 2001;132:433–42. [Google Scholar]
32. Ladenheim B, Krasnova IN, Deng X, et al. Methamphetamine-induced neurotoxicity is attenuated in transgenic mice with a null mutation for interleukin-6. Mol Pharmacol 2000;58:1247–56. [[PubMed]
33. Koteish A, Yang S, Lin H, et al. Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation. J Biol Chem 2002;277:13037–44. [[PubMed]
34. Chomczynski P, Sacchi NSingle-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9. [[PubMed][Google Scholar]
35. Elenkov IJ, Chrousos GP, Wilder RL. Neuroendocrine regulation of IL-12 and TNF-alpha/IL-10 balance. Clinical implications. Ann NY Acad Sci U S A 2000;917:94–105. [[PubMed]
36. Athari A, Hanecke K, Jungermann KProstaglandin F2 alpha and D2 release from primary Ito cell cultures after stimulation with noradrenaline and ATP but not adenosine. Hepatology 1994;20:142–8. [[PubMed][Google Scholar]
37. Knehans AW, Romsos DRReduced norepinephrine turnover in brown adipose tissue of ob/ob mice. Am J Physiol 1982;242:E253–61. [[PubMed][Google Scholar]
38. Young JB, Landsberg LDiminished sympathetic nervous system activity in genetically obese (ob/ob) mouse. Am J Physiol 1983;245:E148–54. [[PubMed][Google Scholar]
39. Ikejima K, Honda H, Yoshikawa M, et al. Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals. Hepatology 2001;34:288–97. [[PubMed]
40. Honda H, Ikejima K, Hirose M, et al. Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver. Hepatology 2002;36:12–21. [[PubMed]
41. Cassiman D, Libbrecht L, Desmet V, et al. Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers. J Hepatol 2002;36:200–9. [[PubMed]
42. Ikejima K, Takei Y, Honda H, et al. Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat. Gastroenterology 2002;122:1399–410. [[PubMed]
43. Titus MA, Saxena NK, Floyd JJ, et al. Leptin signal transduction in hepatic stellate cells involves activation of the mitogen activated protein kinase (MAPK) p44/42 or ERK, as well as the signal transducer and activator of transcription 3 (Stat 3). Gastroenterology 2003;124:A634. [PubMed]
44. Ikejima K, Lang T, Yoshikawa M, et al. Leptin enhances PDGF-dependent cell growth in hepatic stellate cells: involvement of the PI3K-AKT pathway. Gastroenterology 2003;254:A254. [PubMed]
45. Commins SP, Marsh DJ, Thomas SA, et al. Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue. Endocrinology 1999;140:4772–8. [[PubMed]
46. Thomas SA, Palmiter RDThermoregulatory and metabolic phenotypes of mice lacking noradrenaline and adrenaline. Nature 1997;387:94–7. [[PubMed][Google Scholar]
47. Akiyama-Uchida Y, Ashizawa N, Ohtsuru A, et al. Norepinephrine enhances fibrosis mediated by TGF-beta in cardiac fibroblasts. Hypertension 2002;40:148–54. [[PubMed]
48. Fisher SA, Absher MNorepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat cardiac fibroblasts in vitro. Am J Physiol 1995;268:C910–17. [[PubMed][Google Scholar]