Table 1.
RT-PCR primers used to amplify adrenoceptors transcripts.
Table 2.
RT-PCR primers used to amplify neuropeptide Y receptors (NPY) transcripts.
Table 3.
List of antibodies, and relative dilutions, utilized for immunoblotting.
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 auto-fluorescence, 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, fully-activated) with basal proliferative activity less at day 30 compared to day 15. *p<0.05, **p<0.0001, n = 5, compared to control response.
Figure 2.
Primary hHSC express adrenoceptors and NPY receptors, upregulated in NAFLD cirrhosis.
a, b) Semi-quantitative Rt-PCR and Western blot analyses showing that cultured activated hHSC express α1A, β1, β2 and β3 adrenoceptorsm but not absent α1B or α1D adrenoceptors as confirmed by protein level expression. PCR reactions were carried out for 35 cycles. c) Immunohistochemistry for hHSC, showed significant upregulation of α1A, β1, β2 and β3 in livers of patients with NASH cirrhosis patients compared to near normal controls. *p<0.05.
Figure 3.
Expression of NPY receptors in hHSC.
a, b) hHSC also express NPY receptors with abundant Y1, Y4, and Y6 NPY receptors at mRNA and protein levels with little or absent expression of Y2 and Y5.
Figure 4.
Primary hHSC expressed NPY receptors are upregulated in NAFLD.
Semi-quantitative RT-PCR analysis, each lane normalised against its corresponding GAPDH, of liver RNA from patients with NASH fibrosis scored as F0 (n = 3 patients) or F4 (cirrhosis, n = 4 patients) showing human livers with F0 fibrosis having little expression of Y1, Y4 or Y6 NPY but exuberant expression of Y1, Y4 and Y6 NPY in F4 fibrosis (cirrhosis); *p<0.001 compared to F0 control for each receptor subtype, O.D. = optical density.
Figure 5.
Primary hHSC express the cathecholamine synthesising enzyme dopamine-β-hydroxylase and synthesize/release NE plus EPI to regulate hHSC basal growth in culture.
a) We first established by immunohistochemistry that hHSC expressed dopamine-β-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 μM) or PRL (10 μM) 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 ±SEM, n = 3.
Figure 6.
Exogenous NE stimulates proliferation of hHSC.
a) NE induced a dose-dependent, biphasic, enhancement of proliferation of hHSC, with a maximal effect at 10 nM. Each bar represents mean ± SEM of triplicate responses in one typical experiment, expressed as a percentage of the response in serum free control wells. Similar results have been obtained on at least three other occasions; SF, serum free; * p<0.001, ** p<0.0001. b) Phase contrast micrographs are shown as visual confirmation of actual cell number increase in the presence of increasing concentrations of NE.
Figure 7.
Exogenous EPI stimulate proliferation of hHSC.
a) EPI, as with NE, similarly induced proliferation of cultured activated hHSC, at EPI concentrations of 1 nM–1 µM with a maximal effect at 10 nM. b) Prazosin (10 µM) significantly inhibited NE induced hHSC proliferation. PRL (10 µM) also inhibited EPI induced hHSC proliferation. Results are expressed as a percentage of each agonist control (100%).
Figure 8.
Exogenous NPY stimulate proliferation of hHSC.
NPY induced proliferation of cultured activated hHSC, at concentrations as low as 10 pM. Results are expressed as a percentage of each agonist control (100%).
Figure 9.
Exogenous NE and EPI stimulate proliferation of hHSC through p38 MAP, PI3K and MEK.
a) Pre-treatment for 2 hours with PT (G-protein inhibitor, 100 nM) and SB203580 (p38MAP inhibitor, 10 µM) significantly reduced NE induced hHSC proliferation, with WT wortmannin (PI3K inhibitor, 100 nM), and PD98059 (MEK inhibitor, 100 nM) also slightly but non-significantly reducing NE-induced proliferation. b) The proliferative effects of EPI were also inhibited by PT and furthermore by WT, PD and RO (PKC inhibitor, 1 µM).
Figure 10.
Exogenous NPY stimulate proliferation of hHSC through p38 MAP, PI3K and MEK.
NPY proliferative effects on hHSC were inhibited by PT and WT. Results (mean ±SEM of triplicate responses in one typical experiment) are expressed as a percentage of the response in serum free control wells. Similar results have been obtained on at least three other occasions. SF, serum free; *p<0.05, **p<0.01, ***p<0.001.
Figure 11.
Lack of Endogenous NE increases apoptosis of hHSC.
Absence of serum induced marked apoptosis (a), reduced by presence of serum (b), PDGF (c) or NE (d). PRZ markedly induced hHSC apoptosis more than serum deprivation (e), the effect of PRZ was abrogated by NE (f). Results shown are from 1 typical experiment. Similar results have been obtained on at least 2 other occasions. PRZ significantly induced apoptosis compared to serum alone or compared to NE alone (Serum alone 3±0.6% vs PRZ 11±3%, p<0.05,n = 3; NE 4±0.2% vs PRZ 11±3%, p<0.05,n = 3).
Figure 12.
Exogenous NE induced collagen expression through TGF-β.
a) NE upwards of 10nM significantly increased hHSC collagen-1a2 gene expression, with absence of serum and TGF-β as negative and positive controls. b) NE significantly induced expression TGF-β. Results (mean ±SEM of triplicate responses in one typical experiment) are expressed as a percentage of the response in serum free control wells, vs SF, *p<0.05. Similar results have been obtained on at least three other occasions.