Sympathetic Nervous System Catecholamines and Neuropeptide Y Neurotransmitters Are Upregulated in Human NAFLD and Modulate the Fibrogenic Function of Hepatic Stellate Cells

Background Sympathetic nervous system (SNS) signalling regulates murine hepatic fibrogenesis through effects on hepatic stellate cells (HSC), and obesity-related hypertension with SNS activation accelerates progression of non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease. NAFLD may lead to cirrhosis. The effects of the SNS neurotransmitters norepinephrine (NE), epinephrine (EPI) and neuropeptide Y (NPY) on human primary HSC (hHSC) function and in NAFLD pathogenesis are poorly understood. Aims to determine the mechanistic effects of NE/EPI/NPY on phenotypic changes in cultured hHSC, and to study SNS signalling in human NAFLD livers. Methods Freshly isolated hHSC were assessed for expression of cathecholamine/neuropeptide Y receptors and for the synthesis of NE/EPI. The effects of NE/EPI/NPY and adrenoceptor antagonists prazosin (PRZ)/propranolol (PRL) on hHSC fibrogenic functions and the involved kinases and interleukin pathways were examined. Human livers with proven NAFLD were then assessed for upregulation of SNS signalling components. Results Activated hHSC express functional α/β-adrenoceptors and NPY receptors, which are upregulated in the livers of patients with cirrhotic NAFLD. hHSC in culture synthesize and release NE/EPI, required for their optimal basal growth and survival. Exogenous NE/EPI and NPY dose-dependently induced hHSC proliferation, mediated via p38 MAP, PI3K and MEK signalling. NE and EPI but not NPY increased expression of collagen-1α2 via TGF-β without involvement of the pro-fibrogenic cytokines leptin, IL-4 and IL-13 or the anti-fibrotic cytokine IL-10. Conclusions hHSC synthesize and require cathecholamines for optimal survival and fibrogenic functionality. Activated hHSC express directly fibrogenic α/β-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.


Introduction
The sympathetic nervous system (SNS), a major branch of the autonomic nervous system, is responsible for up-and downregulating many homeostatic mechanisms in living organisms. Preand post-ganglionic neurons are involved in the transmission of signals through the sympathetic system. At the synapses within the ganglia, preganglionic neurons release acetylcholine, a neurotransmitter that activates nicotinic acetylcholine receptors on postganglionic neurons. In response to this stimulus postganglionic neurons release catecholamines such as norepinephrine, which activate adrenergic receptors on the target tissues. The activation of target tissue receptors causes the effects associated with the sympathetic system such as vasoconstriction and hypertension.
Hepatic stellate cells (HSC), also known as peri-sinusoidal cells or Ito cells, are pericytes found in the peri-sinusoidal space between the sinusoids and hepatocytes in the liver. HSC are the principal hepatic fibrogenic cells generating scar tissue in response to persisting liver injury [1]. Emerging evidence suggests that HSC express functional adrenoceptors [2,3], and SNS neurotransmitters induce in vitro HSC characteristic pro-fibrotic phenotypic changes [4,5]. Additionally, mice with genetic deletion of dopamine-b-hydroxylase, lacking cathecholamines, are poorly fibrogenic [2]. Neuropeptide Y (NPY), co-released with cathecholamines at SNS nerve terminals modulates in turn cathecholamine release [6,7] and induces murine HSC proliferation but not collagen expression [5]. The applicability of these findings to human liver fibrosis is unknown. Nearly 4 decades ago it was envisaged that SNS activation is an adaptive response to overeating helping to stabilise body weight [8] with underfed rodents suppressing their SNS activity. Similar effects observed in humans provide support for a key role for the SNS in obesity. Overfeeding concurrently stimulates cardiac sympathetic nerves, which are proposed to be a reflex response to circulatory overload [9,10]. Hypertension observed with SNS activation is a component of the metabolic syndrome and obesity [9][10][11]. In 90% of cases obesity is associated with non-alcoholic fatty liver disease (NAFLD), an accumulation of intra-hepatic triglycerides that is often considered the hepatic manifestation of insulin resistance. NAFLD is the most common cause of chronic liver disease in Western countries with up to one third of the US population affected [12]. NAFLD is a spectrum of hepatic disorders that encompass varying degrees of liver damage ranging from steatosis to non-alcoholic steatohepatitis (NASH) characterized by hepatocellular injury and fibrosis which may progress to cirrhosis and hepatocellular carcinoma [13][14][15]. There is currently no pharmacological treatment for NAFLD, other than loss weight and physical exercise [16]. The aim of this study was to determine if human HSC (hHSC) synthesize and respond to cathecholamines or NPY, and explore the signalling pathways therein involved. Moreover, we have studied changes in SNS signalling components in liver specimens from NAFLD patients.

Materials and Methods
Isolation and culture of hHSCs Human HSCs were isolated as described [17] from resected normal liver tissue, in patients undergoing clinically indicated resection of liver metastases. These liver specimens were obtained with appropriate local Ethics Committee approval (UCL -Royal Free Hospital, London, UK). Written informed consent from the donors was obtained for use of samples in research. Experiments were performed with hHSCs in culture at 7-30 days. Proliferation experiments were performed at first hHSC activation [5,18] and not on multiply passaged cells. A subset of experiments was performed with freshly isolated, quiescent HSCs.

Near normal and cirrhotic non-alcoholic steatohepatitis liver specimens
With appropriate ethical approval and consent for research, liver specimens were obtained from adult patients undergoing clinically indicated investigations for NAFLD or liver transplantation in our units. A total of 10 human liver specimens were used in the current study: 5 were from liver biopsies in patients in whom the final diagnosis was near normal liver -Brunt-Kleiner [19] NAFLD liver fibrosis score = F0; and 5 were from peri-transplant resected specimens in whom the final diagnosis was cirrhotic NAFLD, BK NAFLD fibrosis score = F4. The histopathological evaluations were performed by an expert liver histopathologist.  Immunocytochemistry and confirmation of hHSC identity hHSC were prepared and stained for alpha smooth muscle actin (ASMA) and glial fibrillary acidic protein (GFAP) with controls as described [5,18]. Images were captured with a Zeiss Axiophot microscope and a Leica DMRE fluorescence microscope fitted with an SP confocal head.

High performance liquid chromatography (HPLC) analysis
Cathecholamines were extracted from HSC conditioned medium as described [2] and an aliquot was injected into a reverse phase ion pair HPLC system with electrochemical detection using ESA Coulochem5100 A.

Apoptosis assay
hHSC (5610 5 / ml) were plated into 6 mm petri dishes with test agents. Cells without serum served as positive control of apoptosis and cells cultured with PDGF served as a negative control of apoptosis. At harvest, 48 hours later, apoptotic activity was assessed with the Vybrant (annexin V) apoptosis assay kit 2 (Molecular Probes, Invitrogen). FACS analysis was performed using a Becton-Coultor flow cytometer.

RT-PCR
RT-PCR was performed as previously described [20]. RNA was isolated from activated hHSCs using TRizol (Invitrogen) and cDNA synthesized using QuantiTect Rev. Transcription kit (Invitrogen). Quantitative RtPCR was performed using a Rotorgene RG-3000 instrument (Corbett Research), SYBR GreenER and a 2 Step qRT-PCR kit (Invitrogen). Target gene levels in treated samples are presented as a ratio to levels detected in corresponding control samples, according to the DDCt method. For semi-quantitative PCR, Superscript III one-step RT-PCR with platinum Taq kit (Invitrogen) and classic II 18s internal standard kit (Ambion) was used. The cycle number and 18 S primer/ competimer pair ratio for each primer set were determined by the manufacture's instruction. PCR products were separated by electrophoresis and quantified by densitometry using Figure 1. Characteristics of cultured hHSC: hHSC express GFAP plus ASMA and basal proliferative activity of hHSC is maximal at after 15 days in culture and reduces thereafter. a) Freshly isolated cells hHSC in culture were confirmed as HSC by autofluorescence, and expression of ASMA and GFAP verified at day 4, by immunocytochemistry. b) Basal proliferative activity of hHSC was maximal at day 15 (non-passaged, not fully activated), compared to day 7 (non-passaged, not fully-activated) or day 30 (passaged, fullyactivated) with basal proliferative activity less at day 30 compared to day 15. *p,0.05, **p,0.0001, n = 5, compared to control response. doi:10.1371/journal.pone.0072928.g001

Immunohistochemistry
Immunohistochemistry staining was performed as previously described [22,23]. Briefly, four-mm-thick frozen sections of human liver were cut, dried overnight at room temperature, fixed in acetone and washed in PBS. All sections were incubated, 30 minutes, room temperature with primary antibodies. Primary antibodies (Table 3) were from Santa Cruz Biotech, Heidelberg, Germany. For DBH, b1-b2 and b3-AR samples were incubated with an anti-rabbit peroxidase-conjugated EnVision antibody (Dako) 30 minutes/room temperature. For a1A-AR, a1B-AR, a1D-AR and b3-AR, we used a three-step immunoperoxidase procedure: peroxidase labeled rabbit-anti goat IgG (Dako, Denmark) followed by peroxidase labeled swine anti-rabbit IgG (Dako). The secondary and tertiary antibodies were diluted (1650 and 16100 respectively) in PBS, pH 7.2 containing 10% normal human serum. For negative controls the primary antibody was omitted. Number of hHSC positive for protein expression of a1A, b1, b2 and b3 adrenoceptors in livers of patients with NASH compared to near normal controls were assessed as previously described [24].
Induction of leptin, IL-4, IL-10, IL-13 gene expression and induction of leptin or IL-13 protein expression by NE hHSC were cultured under conditions identical to those used above to detect NE effects on hHSC proliferation. After 48 hours, RNA was extracted from the cells and analysed by Q-RT-PCR for expression of leptin, IL4, 10 and 13. Additionally, NE conditioned hHSC media was assayed by ELISA for leptin and IL-13 with a methodology as supplied -R&D systems, Abingdon, UK.

Drugs
All from Sigma, unless stated otherwise.

Statistical analysis
Data expressed as mean (6SEM). Statistical analyses performed using the Mann-Whitney test, significance accepted as p,0.05.

Characteristics of cultured hHSC
Upon isolation, primary hHSC cell identity and activation was confirmed by their expression of established indicators, ASMA and GFAP, verified at day 4 by immunocytochemistry ( Figure 1a). We confirmed with quantitative RT-PCR that after 10 days in culture these cells had increased expression of ASMA by about 600%, decreased GFAP expression by about 50% and increased collagen gene expression by about 400% compared to freshly isolated cells at day 0 (results not shown). We then studied the basal proliferative activity of hHSC after 7, 15 or 30 days in culture (Figure 1b). Basal hHSC proliferation was maximal at day 15, compared to day 7 or 30 ( Figure 1b). Therefore, subsequent proliferation experiments were performed with hHSCs at day 15.

Primary hHSC express adrenoceptors and NPY receptors which are upregulated in NAFLD Cirrhosis
We previously showed that murine HSC express mediocre amounts of a1A, but exuberant a1B, a1D, b1 and b2 adrenoceptor subtypes [2]. The presence of some adrenoceptor subtypes was recently shown in both activated hHSC and human whole liver [3]. Here, we found by semi-quantitative RT-PCR that culture activated hHSC express a1A, but had no detectable expression of a1B or a1D adrenoceptors (Figure 2a). We also observed expression of b1 and b2 adrenoceptors (Figure 2a). To confirm protein level expression of these receptors, and assay their possible functional significance in vivo, we first performed Western blot analysis on hHSC lysate, and then immunohistochemistry for hHSC on clinical liver specimens, either with a nearly normal histology -fibrosis score, F0, or having NAFLD cirrhosis -fibrosis score, F4. Western blot analysis confirmed protein level expression of the b-adrenoceptors (Figure 2b) and immunohistochemistry similarly showed a significantly upregulated number of hHSC positive for protein expression of a1A, b1, b2 and b3 adrenoceptors in livers of patients with NAFLD cirrhosis compared to near normal controls (Figure 2c).
We have previously shown that NPY, a SNS neurotransmitter co-released with NE [7], induces murine HSC proliferation. The expression of NPY receptors by hHSC is not known. Here, we found that hHSC robustly express Y1, Y4, and Y6 NPY receptors with little or no expression of Y2 and Y5 (Figure 3a, b). To confirm the functional relevance of these receptors, we now analysed liver RNA extracted from liver biopsies in patients with near-normal livers, fibrosis score F0 (n = 3 patients) or cirrhotic NAFLD with fibrosis score = F4 (n = 4 patients). As shown in Figure 4a and b, patients with F0 fibrosis had little expression of Y1, Y4 or Y6 NPY receptors whereas patients with F4 fibrosis (cirrhosis) had exuberant expression of Y1, Y4 and Y6 NPY receptors indicating that some NPY receptors as with adrenoceptors may be implicated in the pathogenesis of NAFLD fibrosis since these receptors are upregulated with fibrosis stage.
Primary hHSC express the cathecholamine synthesising enzyme dopamine-b-hydroxylase and synthesize/release NE plus EPI to regulate hHSC basal growth Although murine HSC synthesize and release NE in culture and use this endogenously released NE for optimal basal growth [2], the existence of a similar pathway in hHSC is not known. We therefore examined by immunohistochemistry the expression of DBH, the rate limiting enzyme in the biosynthesis of cathecholamines. DBH was expressed by near normal livers and markedly upregulated in livers with NASH cirrhosis compared to near normal controls (mean counts of DBH positive cells 1761 vs. 262, n = 5, p,0.001). HPLC analysis of hHSC conditioned medium showed that hHSC release NE and EPI, with NE released at about 8369 pg/ml, more abundantly produced than EPI (Figure 5a). To investigate the importance of endogenous NE and EPI and the functionality of hHSC-expressed adrenoceptors, we cultured hHSC with PRZ, a predominant a1-adrenoreceptor antagonist, and PRL a predominant b-adrenoceptor antagonist. PRZ (10 mM) and PRL (10 mM) singly or combined significantly reduced hHSC basal growth (Figure 5b). Therefore, hHSC endogenously produced cathecholamines are important for hHSC basal growth via hHSC expressed adrenoceptors.

Exogenous NE, EPI and NPY stimulate proliferation of hHSC through p38 MAP, PI3K and MEK
We then investigated whether exogenous NE and EPI as with murine HSC [2] induced hHSC proliferation. Activated hHSC were cultured for 48 hours in serum free medium, as negative controls, or in medium with 10% FBS as controls or with 10% FBS plus varying concentrations of NE (1 nM-1 mM). NE induced a dose-dependent, biphasic, enhancement of proliferation of hHSC with maximal effect at 10 nM (Figure 6a, b). EPI similarly induced hHSC proliferation at 1 nM -1 mM with maximal effect at 10 nM (Figure 7a). The effect of EPI on hHSC proliferation was mostly mediated by b-adrenoceptors, since PRL significantly inhibited it (Figure 7b, top panel) whilst the effect of NE on hHSC proliferation was mostly mediated by a-adrenoceptors, since PRZ significantly inhibited NE induced proliferation (Figure 7b, bottom panel). NPY as with NE and EPI also induced Figure 5. Primary hHSC express the cathecholamine synthesising enzyme dopamine-b-hydroxylase and synthesize/release NE plus EPI to regulate hHSC basal growth in culture. a) We first established by immunohistochemistry that hHSC expressed dopamine-b-hydroxylase (DBH) (data not shown). HPLC analysis of hHSC conditioned medium showed hHSC release of NE and EPI with NE more abundantly produced than EPI. b) Activated hHSC cultured basally for 48hours with PRZ (10 mM) or PRL (10 mM) singly or in combination significantly had reduced basal proliferation. Proliferation under each treatment condition was normalised to the corresponding serum free control in each experiment; *p,0.05, **p,0.0001, each bar represents mean 6SEM, n = 3. doi:10.1371/journal.pone.0072928.g005 biphasic hHSC proliferation with concentrations as low as 10 pM inducing significant increases in hHSC proliferation (Figure 8).
To investigate the role of the G-protein, PI3K, MEK, p38 MAPK and PKC intra-cellular signalling pathways on NE and EPI-induced proliferation of hHSC, we now examined the effect of their respective inhibitors at the concentrations shown: pertussis toxin (PT, 100 ng/ml); wortmannin (WT, 100 nM); PD98059 (PD, 20nM); SB202190 (SB, 10 mM) and RO-320432 (RO, 1 mM) as previously detailed [2,17]. Pertussis toxin pre-treatment significantly reduced NE induced hHSC proliferation, while pre-treatment with wortmannin, PD98059 and RO also slightly but non-significantly reduced NE-induced proliferation. However, pre-treatment with SB202190 markedly and significantly inhibited NE-induced hHSC proliferation (Figure 9a). Therefore, the proliferative effects of the predominant a1-adrenoceptor agonist NE, on hHSC, are mediated by G-protein coupled adrenoceptors with downstream effects mostly involving p38 MAPK. Conversely, the proliferative effects of the predominant b-agonist EPI involves G-protein coupled adrenoceptors mediated downstream by PI-3K and MEK with minor contributions from p38 MAP and PKC, since EPI effects were inhibited by PT and furthermore by WT and PD (Figure 9b). The hHSC NPY receptors, as with the hHSC adrenoceptors, appear also to be coupled to G-proteins with downstream pathways involving PI-3K since PT and WT inhibited NPY induced hHSC proliferation ( Figure 10).

Lack of Endogenous NE increases apoptosis of hHSC
In the absence of serum (serum free, SF) ( Figure 11a) as expected, marked apoptosis was observed, which was reduced by the presence of serum (Figure 11b), platelet derived growth factor, PDGF (Figure 11c) or NE (Figure 11d). PRZ markedly induced hHSC apoptosis to an even a greater extent than serum deprivation (Figure 11e). The effect of PRZ was abrogated by NE ( Figure 11f). Therefore, hHSC endogenously produced NE and exogenously applied NE appears to be a survival factor for hHSC.

Exogenous NE induced collagen expression through TGF-b
We now investigated whether NE applied to hHSC upregulated collagen gene expression in tandem with increased proliferation. NE significantly increased collagen-1a2 gene expression, at concentrations upwards of 10 nM (Figure 12a). Additionally, NE induced expression of TGF-b (Figure 12b), indicating that at least some of the increased collagen expression could be mediated through TGF-b. Pro-fibrotic effects of NE on hHSC are not mediated through leptin, IL-4 or IL-13 and NE does not induce production of IL-10 Besides TGF-b, leptin, IL-4 and IL-13 [25,26] are also involved in hepatic fibrogenesis consequent to various aetiologies and IL-10 may be anti-fibrotic [25][26][27][28]. To determine if leptin, IL-4 or IL-13 are upregulated by NE in order to explain the pro-fibrotic effect of NE, and whether there is simultaneous induction of IL-10 by NE, we initially assayed by quantitative RT-PCR the expression of these factors by hHSC cultured under conditions identical to those used above to detect NE effects on hHSC proliferation. We found no appreciable evidence of induction by NE of leptin, IL-4, IL-10   or IL-13 gene expression (results not shown). We also confirmed by ELISA that there was no appreciable induction of leptin or IL-13 protein (results not shown). Therefore, the pro-fibrotic effect of NE on hHSC does not involve appear to involve leptin, IL-4 or IL-13. These results are in keeping with our previous findings that in HSC from mice NE induced pro-fibrogenic without a requirement for leptin [4].

Discussion
We have shown here that hHSC express adrenoceptors, which are markedly upregulated in human livers with NAFLD cirrhosis. Activated hHSC in culture express a1A, b1, b2 and b3 adrenoceptors, while no convincing expression of a1B or a1D adrenoceptors was observed. This is to our knowledge the first comprehensive report of the adrenoceptor profile of primary hHSC including their expression of b3-adrenoceptors. hHSC also robustly express Y1, Y4, and Y6 NPY receptors with little expression of Y2 and Y5. These receptors are functional and are possibly implicated in NAFLD fibrosis, since patients with F0 fibrosis had reduced expression of a1A, b1, b2 and b3 adrenoceptors or Y1, Y4 and Y6 NPY receptors, whereas patients with NAFLD F4 fibrosis (cirrhosis) had exuberant expression of a1A, b1, b2 and b3 adrenoceptors plus Y1, Y4 and Y6 NPY receptors. These results suggest also that hHSC are potential targets for the pathogenic effect of catecholamines in the liver, consistent with recent data indicating that catecholamines contribute to tissue repair and inflammation, in addition to regulating vascular homeostasis [29]. Furthermore, hHSC express functional dopamine-b-hydroxylase and synthesize/release the cathecholamines NE plus EPI to regulate hHSC basal growth since PRZ (a1-adrenoreceptor antagonist) and PRL (b-adrenoceptor antagonist) reduced hHSC basal growth. Endogenous NE is also necessary for hHSC survival since there is markedly increased hHSC apoptosis in the presence of PRZ. DBH expression was markedly upregulated in livers with NAFLD cirrhosis.
NE is known to stimulate key intracellular kinases and signalling pathways implicated in the hepatic fibrogenesis [add Ref 2,[3].
Here we found that exogenous NE, EPI and NPY also regulate hHSC fibrogenic functions since NE, EPI and NPY dosedependently induced hHSC proliferation through G-protein coupled receptors. The downstream effects of NE on hHSC are shown here to be mediated by p38 MAP and that of EPI mediated most likely by PI3K and MEK, with perhaps some minor contribution from p38 MAP and PKC. The involvement of these latter 2 pathways in hHSC proliferation needs clarifying in future experiments. The intra-cellular pathways involved in the actions of NE and EPI on hHSC as shown here, are similar to those reported for their actions in murine HSC and other cell types [2,30]. Importantly, for our finding that the intra-cellular action of EPI on hHSC probably involves MEK; the MEK inhibitor PD98059 has recently been shown to reduce injury and fibrosis in a murine model of lung fibrosis [31]. Additionally, the proliferative effects of NPY on hHSC as shown here are similar to previous findings of the effect of NPY on murine HSC [5], but the current findings are to our knowledge the first comprehensive description of NPY receptors on HSC and the signalling pathways therein.
NE and EPI induced hHSC proliferation were maximal at 10nM. NE induced hHSC proliferation is an a-adrenoceptor mediated (PRZ inhibited) whilst EPI induced hHSC proliferation is b-adrenoceptor mediated (PRL inhibited). NPY as with NE and EPI dose-dependently induced hHSC proliferation with concentrations as low as 10 pM inducing significant increases in hHSC proliferation. Exogenously administered NE and EPI, moreover, induced collagen expression through TGF-b because NE and EPI applied to hHSC upregulated collagen gene expression in tandem with increased proliferation. Therefore, at least some of the increased collagen expression induced by NE and EPI in hHSC is mediated through TGF-b, in line with our previous study in mice [2]. It has been shown previously that HSC secrete proinflammatory chemokines such as interleukins [32,33] and inflammatory mediators derived from lymphocytes can stimulate HSC fibrogenic potential [33], such that chemokine secretion by NE might enhance inflammation in chronic liver disease. In our study however, we did not find appreciable evidence of induction by NE of leptin, IL-4, IL-10 or IL-13. In support of our findings here, a study using a Mdr2(2/2) mouse model, displaying blockade of b-adrenoceptors has been shown to be a promising therapeutic strategy in the treatment of human primary sclerosing cholangitis [34]. Moreover, b-adrenoceptors mediate liver NE modulation of pro-inflammatory responses, cardiac dysfunction and portal hypertension in cirrhotic patients and rodent models of the disease [35][36][37][38][39]. In summary, our study clearly shows that hHSC express adrenergic and NPY receptors which are upregulated in vivo with fibrosis. Additionally, hHSC synthesise and require the cathecholamines NE and EPI for optimal survival and NE, EPI and NPY stimulate the proliferation of hHSC with NE also shown to induce collagen gene expression in cultured hHSC. These findings suggest therefore that adrenergic and NPY antagonists may be novel anti-fibrotic agents in NAFLD.