1. Volume 136, Issue 7, Pages 2334-2344.e1 (June 2009)
Angiotensin II Activates IκB Kinase Phosphorylation of RelA at Ser536 to Promote Myofibroblast Survival and Liver Fibrosis 
Fiona Oakley, Victoria Teoh, Gemma Ching–A–Sue, Ramon Bataller, Jordi Colmenero, Julie R. Jonsson, Aristides G. Eliopoulos, Martha R. Watson, Derek Manas, Derek A. Mann 
Gastroenterology 
Volume 136, Issue 7, Pages e1 (June 2009)
DOI: /j.gastro
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2. Figure 1
P-Ser536-RelA is a marker of activated hepatic myofibroblasts. (A) Nuclear localization of P-Ser536-RelA in human myofibroblasts (red arrows) and cytoplasmic localization (yellow arrows) was determined by indirect immunofluorescence. Fluorescent images using FITC (P-Ser536-RelA) and DAPI (nuclei) filters were taken using laser scanning confocal microscopy (63 × 1.9 zoom magnification). Photomicrographs represent 2 independent cell isolations. (B) Whole cell extracts were isolated from quiescent (day 0) and activated (day 10) rat hepatic stellate cells, and levels of RelA and P-Ser536-RelA were assessed by Western blotting (n = 3). (C) Photomicrographs (original magnification, 40×) show liver sections from near normal human liver or patients with primary billiary cirrhosis (PBC) and alcoholic liver disease (ALD) immunohistochemically stained for RelA-phospho-Ser536. Arrows indicate high levels of P-Ser536-RelA in myofibroblastic cells, and cell counts show an average of 52.6% ± 4.6% and 50.4% ± 2.9% P-Ser536-RelA positive cells in fibrotic bands in PBC and ALD, respectively.
Gastroenterology  , e1DOI: ( /j.gastro )
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3. Figure 2
P-Ser536-RelA regulates nuclear translocation and myofibroblast survival. (A) Representative fluorescent confocal scanned images (original magnification, 63×) of RelA−/− mouse embryonic fibroblasts (MEFS) transfected with RelA-EGFP or RelAS536A-EGFP (S536A-EGFP) plasmids. Nuclear positive cells were counted in RelA−/− MEFS transfected with expression vectors: EGFP, RelA-EGFP, and RelAS536A-EGFP. Ser536 to Ala mutation statistically significantly retarded RelA nuclear localization, **P < .01. An average of 150 cells/plasmid/experiment were counted, and data are expressed as mean ± SEM, n = 4. (B) RelA−/− MEFS were transfected with RelA-EGFP overnight, serum starved for 24 hours, and then treated with cell-permeable peptides RelA-P6 (Ser529/536) or RelA-P1 (Ser276) for 30 minutes prior to serum stimulation. Representative fluorescent confocal scanned images (original magnification, 63×) of RelA-EGFP (green) and nuclei (blue) at 0 hours or 4 hours serum stimulation with competing peptides. (C) RelA nuclear positive cells were counted at time 0 (24 hours serum free); 30 minutes; and 1, 2, and 4 hours postserum stimulation ± 30-minute pretreatment with 50 μmol/L competing peptides. Results were expressed as average percentage RelA nuclear positive cells/10× field ± SEM, n = 6, vehicle (♢), RelA-P1 (■), and RelA-P6 (●). (D) Rat hepatic myofibroblasts were transiently transfected with IκBα-Luc or TIMP1-Luc promoter reporter plasmid for 16 hours, then treated with cell-permeable RelA-P6 (Ser529/536) or controls for a further 6 hours. (E) Apoptosis of rat myofibroblasts incubated with 50 μmol/L cell-permeable RelA phosphorylation competing peptides for 24 hours was determined using acridine orange staining. Respective P values for ser529/536, ala529/536, ser529/ala536, and ala529/ser536 were ***P < .001, ns, ns, and ***P < Results were expressed as percentage apoptotic cells in 4 independent experiments ± SEM, and P values were determined using 1-way ANOVA.
Gastroenterology  , e1DOI: ( /j.gastro )
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4. Figure 3
Constitutive IKK activity regulates P-Ser536-RelA and myofibroblast survival. (A) IKK activity assay. Total IKK complex was immunoprecipitated from freshly isolated rat hepatic stellate cells and culture activated rat myofibroblasts using anti-IKKγ and incubated with kinase buffer, γ32P-ATP, and recombinant GST-IκBα, then quenched with Laemelli buffer. Reactions were resolved by SDS-PAGE, and GST-P-IκBα was visualized upon exposure to x-ray film. Total levels of IKKα/β were assessed in whole-cell extracts (middle) or input control (bottom) isolated from quiescent and activated rat myofibroblasts by Western blotting. (B) Whole-cell extracts isolated from human myofibroblasts treated ± 10-8 mol/L angiotensin II with or without 50 μmol/L Calbiochem IKK inhibitor VI, relative levels of total RelA, P-Ser536-RelA, and β-actin were assessed by Western blotting. (C) Relative mRNA levels of BCl-2, Gadd45β, or β-actin were assessed by RT-PCR in rat myofibroblasts treated ± 50 μmol/L Calbiochem IKK inhibitor VI for up to 4 hours. (D) Rat myofibroblast apoptosis was induced after 16-hour incubation with 10 nmol/L–50 μmol/L IKK inhibitor VI and quantified by acridine orange staining with respective P values of *P < .05, **P < .01, ***P < .001 for 1–50 μmol/L. Results were expressed as percentage apoptotic cells for at least 4 independent experiments ± SEM, and P values were determined using a 1-way ANOVA. Gels are representative of at least 2 or 3 independent experiments.
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5. Figure 4
IKK and P-Ser536-RelA are under the autocrine control of angiotensin II. Whole-cell extracts isolated from human myofibroblasts treated with (A) 10-8 mol/L angiotensin II ± 10 μmol/L ZD7155 and (B) relative levels of total and P-IKKα/β or total RelA or P-Ser536-RelA and β-actin were assessed by Western blotting. (C) Human myofibroblasts were transiently transfected with IκBα-Luc promoter reporter plasmid for 16 hours, serum starved for 24 hours, and treated ± angiotensin II (10-8 mol/L) or ZD7155 (10 μmol/L) for a further 6 hours. Reporter gene activity was expressed as a percentage relative to unstimulated control ± SEM (n = 4); P values, *P < .05. (D) Relative levels of angiotensinogen (AGT) or β-actin were determined via RT-PCR in human myofibroblasts treated with either 30 μg/mL TAT-IκBα-SR (SR) or control protein TAT-GST. Gels are representative of 2 independent cell preparations.
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6. Figure 5
Angiotensin II prevents JNK-dependent myofibroblast apoptosis. (A) Human myofibroblast apoptosis was determined via acridine orange staining after 16-hour exposure to enalapril (10–500 μmol/L); respective P values, *P < .05, **P < .01, and ***P < (B) Percentage apoptosis was determined in human myofibroblasts treated with ZD7155 (0.01–10 μmol/L); respective P values, *P < .05, ***P < (C) Relative mRNA levels of BCl-2, Gadd45-β, and β-actin were determined by RT-PCR after 0–4-hour treatment with 10 μmol/L ZD7155. (D) Quantification of myofibroblast apoptosis after treatment with 1 and 10 μmol/L ZD7155 ± 50 μmol/L JNK inhibitor SP or vehicle, DMSO; respective P values, *P < .05. Statistical significance was calculated using a 1-way ANOVA.
Gastroenterology  , e1DOI: ( /j.gastro )
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7. Figure 6
Myofibroblast P-Ser536-RelA is angiotensin dependent and profibrogenic in acute liver injury. (A) Mice were injured once with CCl4, then treated with ZD7155 (10 mg/kg) at 20 or 22 hours postinjury. Hepatic myofibroblasts were isolated 24 hours after CCl4 injury and stained for P-Ser536-RelA using indirect immunofluorescence. Fluorescent images using FITC (P-Ser536-RelA) and DAPI (nuclei) filters were taken using laser scanning confocal microscopy (original magnification, 63×). Positively stained cells were expressed as a percentage of total cells counted in 15 fields ± SEM. Statistical significance was *P < .05 calculated using a 1-way ANOVA. (B) Mice given a single dose of CCl4, then treated with cell-permeable P-Ser536-RelA competing peptides (P6) or control 30 minutes prior to and 6 and 20 hours postinjury. Livers were harvested; RNA was isolated 24 hours after CCl4 administration; and mRNA levels of procollagen I, TIMP1, and α-SMA were assessed using Taqman qRT-PCR. Relative level of transcriptional difference between olive oil and treatment groups was calculated and expressed as an average ± SEM, from 4 or 5 independent experiments.
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8. Figure 7
Blockade of IKK activity or angiotensin II signaling promotes regression of established fibrosis in rodents. (A) Rats were treated with captopril (100 mg/kg/day) commencing 2 weeks after BDL and livers harvested after a further 2 weeks injury. Photomicrographs (original magnification, 40×) show rat liver sections without (top) or with captopril treatment (bottom), immunostained with anti-P-Ser536-RelA antibodies. Positive myofibroblastic cells in fibrotic tissue (red arrows) were reduced upon captopril treatment (yellow arrows). Cell counts show the average number of P-Ser536-RelA-positive cells in fibrotic bands, expressed as mean ± SEM, n = 8, and ***P < (B) Photomicrographs (original magnification, 20×) of liver sections from 4 weeks CCl4 injured rats ± 1 IP administered dose (100 mg/kg) of sulphasalazine, immunostained for P-Ser536-RelA. Graphical representation of cell counts showing the average number of P-Ser536-RelA-positive cells in fibrotic bands from CCl4 and CCl4/sulphasalazine-treated rats. Cell counts are the mean of up to n = 5 ± SEM. A statistically significant reduction in P-Ser536-RelA-positive cells was observed in the treatment group, ***P < All P values were determined using an unpaired 2-tailed Student t test.
Gastroenterology  , e1DOI: ( /j.gastro )
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9. Figure 8
Regression of human fibrosis in response to losartan is associated with loss of P-Ser536-RelA-positive cells. (A) Representative photomicrographs (original magnification, 100×) of human liver biopsy samples taken from hepatitis C virus-infected fibrotic patients pre- and post-18-month treatment with losartan. Sections were immunostained with antibodies recognizing P-Ser536-RelA. Positively stained cells prelosartan treatment in nonresponders and responders are denoted by yellow and red arrows, respectively, whereas black arrows show cells postlosartan treatment. Images are subdivided into 2 groups: patients who responded to losartan treatment (right) and nonresponders (left). (B) Graphical representations of average percentage area of tissue stained positively for P-Ser536-RelA in fibrotic bands of human liver biopsy specimens. Densitometric analysis was used to calculate mean percentage area of up to 5 patients/group ± SEM. A statistically significant reduction in immunoreactivity was observed in the responder group, P = .025 (2-tailed paired Student t test).
Gastroenterology  , e1DOI: ( /j.gastro )
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