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Supplemental Information

Ephrin/ephrin receptor expression in ammonia-treated rat astrocytes

and in human cerebral cortex in hepatic encephalopathy

Karmela Sobczyk, Markus S Jördens, Ayse Karababa

Boris Görg and Dieter Häussinger

Clinic for Gastroenterology, Hepatology and Infectiology

Heinrich-Heine University, Düsseldorf, Germany

Supplemental Tables

Table 1: Patient demographics, cause of death and etiology of cirrhosis in cirrhotic patients with or without HE. 4 controls, 4 patients with cirrhosis without HE and 4 patients with cirrhosis with HE were obtained from the Australian Brain Donor Programs NSW Tissue Resource Centre.

Case / Patient Group / Age / Gender / Cause of Death Category / Etiology
1 / control / 63 / Male / coronary heart disease / -
2 / control / 63 / Male / coronary heart disease / -
3 / control / 49 / Female / suspected arrhytmogenic right ventricular dysplasia / -
4 / control / 52 / Male / undetermined / -
1 / cirrhosis without HE / 58 / Male / coronary heart disease / alcohol
2 / cirrhosis without HE / 52 / Male / alcohol intoxication / alcohol
3 / cirrhosis without HE / 37 / Male / alcohol intoxication / alcohol
4 / cirrhosis without HE / 57 / Male / coronary heart disease; acute bronchopneumonia / alcohol
1 / cirrhosis with HE / 73 / Male / bleeding ulcer, coronary heart disease / alcohol
2 / cirrhosis with HE / 62 / Female / drug toxicity / alcohol
3 / cirrhosis with HE / 60 / Male / drug toxicity / alcohol
4 / cirrhosis with HE / 47 / Female / lobar pneumonia / alcohol

Table 2: Liver and brain pathology and comorbidities of controls and cirrhotic patients with or without HE.4 controls, 4 patients with cirrhosis without HE and 4 patients with cirrhosis with HE as well as the clinical information and the corresponding pathology were obtained from the Australian Brain Donor Programs NSW Tissue Resource Centre.

Case / Patient Group / Liver and Brain Pathology, Comorbidities
1 / control / mild steatosis, normal brain, severe coronary artery atherosclerosis
2 / control / moderate macrovesicular steatosis, normal brain, coronary artery atherosclerosis
3 / control / normal liver, normal brain, arrhythmogenic right ventricular dysplasia
4 / control / mild steatosis, normal brain, ulcerative colitis
1 / cirrhosis without HE / cirrhosis
2 / cirrhosis without HE / cirrhosis, steatosis, normal brain
3 / cirrhosis without HE / mixed micro & macro nodular cirrhosis, fatty liver, severe macrovesicular steatosis
moderate cerebellar degeneration
4 / cirrhosis without HE / cirrhosis, normal brain, cholestasis, jaundice, bronchopneumonia
1 / cirrhosis with HE / cirrhosis, macrovesicular steatosis, normal brain, oesophageal varicies, gastric ulcers
2 / cirrhosis with HE / macrovesicular steatosis, and prominent perivascular fibrosis almost amounting to cirrhosis
3 / cirrhosis with HE / cirrhosis, steatosis
4 / cirrhosis with HE / evolving cirrhosis, fatty liver, steatohepatitis, hepatomegaly, cerebellar vermal atrophy

Table 3: PCR primer sequences used for realtime-PCR analysis. PCR primer sequences are given in 5´ to 3´ direction. f: forward, r: reverse.

target / accession no. / primer sequence
EphA1 (1)
NM_001107858.1 / f: TTGCCAACTTTGACCCTAGG;
r: CTTAAATCCTTGAATACTGCAG
EphA2 (2)
NM_001108977.1 / f: CCCGAGTGTCCATTCGGCTAC
r: TCACTTGGTCTTTGAGTCCCAG
EphA3 (1)
NM_031564.1 / f: GAGTTACGGGATTGTACTCTG
r: TGGCAATGGTGTCACAGGAGC
EphA4 (3)
NM001162411.1 / f: AGTTCCAGACCGAACACAGCTTG
r: GCCATGCATCTGCTGCATCTG
EphA5
NM_024367.1 / f: TCCACCTCACAGTTACACCC
r: GCGATTTTCCCCTTCTTCAC
EphA7 (3)
NM_134331.1 / f: CCATAAGCCCTCTTCTGGACCA
r: ACACTTGGATGCCGGTTCCGT
EphB1 (1)
NM_001104528.1 / f: AAGCCCCCTACCTCAAAGTTG
r: CACCATCCACTCTCCATCTCC
EphB2 (3)
NM_001127319.1 / f: CACTACTGGACCGCACGATAC
r: TCTACCGACTGGATCTGGTTCA
EphB3 (1)
NM_001105868.1 / f: GTAGGGTCAGGTGGGGATAAG
r: GACAGCACCAAGGGTAGGCAG
EphB6 (3)
NM 001107857.1 / f: GGACAGGCCTTCCCAGGCTCT
r: TGGCAGGTCTTCCAGGCTGA
ephrinA1 (1)
NM_053599.2 / f: ATCCCAAGTTCCGAGAGGAGG
r: CTCCTTGCCCAAGGTGAAAGGC
ephrinA2
NM_001168670.1 / f: ATACATGGTGAACGGCGAG
r: AGGGAAAAGGGGGTGAAGAG
ephrinA3 (1)
XM_001072657.2 / f:TCGCCTTCTTCCTCATGACG
r: CTGAGCACTGCCTTTATAGCC
ephrinA4
NM001107692.1 / f: GCTCCCAATACTCCGTCTTC
r: AAAGCTTCATCATGTCTGCC
ephrinA5(4)
NM_053903.1 / f: AACGGACCGCTGAAGTTCTCGG
r: TTTGTGCCGCGTTCTCTCCGCG
ephrinB1 (5)
NM_017089.2 / f: AGGCCTCTGGGCTCTGTGGG
r: GCAGGGCCAGGGGGCTATCT
ephrinB2 (6)
NM_001107328.2 / f: TGGAAGTACTGTTGGGGACT
r: TCACATCTTGGTCTGGTCTG
ephrinB3 (6)
NM_001100980.2 / f: : GGGACCGGCTAGATCTACTT
r: GGCTGTATTCCTGGAACTTG
HPRT
NM_012583.2 / f: TGCTCGAGATGTCATGAAGGA
r: CAGAGGGCCACAATGTGATG

1 Ivanov et al. (2005); 2 Buchert et al. (1999); 3 Biervert et al. (2001); 4 Lai et al. (1999); 5 Carmen Döbele (2011); 6 Zhuang et al. (2010).

Table 4: Effect of methionine sulfoximine, L-NAME and apocynin on EphR/ephrin and GLAST mRNA expression levels in cultured rat astrocytes. Astrocytes were treated withmethionine sulfoximine (MSO, 3mmol/l), L-NAME (1mmol/l) and apocynin (300µmol/l) or were left untreated for 72h before RNA was isolated and ephrin/ephrin receptor (EphR) and GLAST mRNA expression levels were analysed by realtime-PCR. mRNA expression levels in MSO-, L-NAME- or apocynin-treated astrocytes are given relative to untreated controls. *: statistically significantly different compared to untreated controls. n.s.: non statistically significantly different compared to untreated controls.

MSO (3mmol/l) / L-NAME (1mmol/l) / Apocynin (300µmol/l)
EphR A2 / 0.94±0.05 n.s. / 0.83±0.20 n.s. / 0.88±0.09 n.s.
EphR A3 / 0.90±0.15 n.s. / 0.67±0.19 n.s. / 0.85±0.17 n.s.
EphR A4 / 0.84±0.07 n.s. / 0.71±0.17 n.s. / 0.96±0.22 n.s.
EphR B3 / 0.85±0.17 n.s. / 0.88±0.09 n.s. / 1.05±0.09 n.s.
EphR B6 / 0.88±0.13 n.s. / 0.95±0.19 n.s. / 1.29±0.05 *
Ephrin A2 / 0.86±0.18 n.s. / 0.81±0.12 n.s. / 0.76±0.13 n.s.
Ephrin B3 / 0.99±0.29 n.s. / 1.08±0.30 n.s. / 1.09±0.41 n.s.
GLAST / 1.00±0.15 n.s. / 0.90±0.15 n.s. / 0.85±0.05 n.s.

Supplemental References

1. Ivanov AI, Steiner AA, Scheck AC, Romanovsky AA (2005) Expression of Eph receptors and their ligands, ephrins, during lipopolysaccharide fever in rats. Physiol Genomics21:152–60.

2. Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM (1999) The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. J Cell Biol144:361–71.

3. Biervert C, Horvath E, Fahrig T (2001) Semiquantitative expression analysis of ephrine-receptor tyrosine kinase mRNA’s in a rat model of traumatic brain injury. Neurosci Lett 315:25–8.

4. Lai KO, Ip FC, Ip NY (1999) Identification and characterization of splice variants of ephrin-A3 and ephrin-A5. FEBS Lett458:265–9.

5. Döbele C. Functional Characerisation of members of the microRNA-17-92 cluster in the vascular system (2001), Dissertation, Goethe Universität Frankfurt am Main

6. Zhuang Z, Yang B, Theus MH, Sick JT, Bethea JR, Sick TJ, Liebl DJ (2010)EphrinBs regulate D-serine synthesis and release in astrocytes.J Neurosci30:16015-24.

Supplemental Figure Legend

Supplemental Figure 1:Effect of hypoosmolarity on mRNA expression of ephrin receptor isoforms (A) and ephrins (B) in cultured rat astrocytes. Astrocytes were exposed to normoosmotic (320mosmol/l) or hypoosmotic (205mosmol/l) cell culture media for the indicated period of time before RNA was extracted and mRNA expression levels of various ephrin/ephrin receptor isoforms were analysed by realtime-PCR. mRNA expression levels in astrocytes treated with hypoosmotic cell culture media are given relative to the normoosmotic controls. *: statistically significantly different compared to untreated controls.

Supplemental Figure 2: Effect of NH4Cl on GLAST mRNA expression in cultured astrocytes. Astrocytes were either treated with NH4Cl (5mmol/l) or left untreated for the indicated time period. GLAST mRNA expression was analysed by realtime-PCR and expression level are given relative to the respective control. *: statistically significantly different compared to untreated controls.

Supplemental Figure 3:Effect of NH4Cl on mRNA expression of ephrin receptor isoforms (A) and ephrins (B) in cultured rat astrocytes. Astrocytes were either exposed to NH4Cl (5mmol/l) or left untreated for the indicated period of time before RNA was extracted and ephrin and ephrin receptor (Eph) mRNA expression levels were analysed by realtime-PCR. Ephrin receptor and ephrin mRNA expression levels in NH4Cl (5mmol/l)-treated astrocytes is given relative to the normoosmotic controls. *: statistically significantly different compared to untreated controls.

Supplemental Figure 4: Effect of NH4Cl on FGF receptor 1 expression in cultured astrocytes. Astrocytes were either left untreated or treated with NH4Cl (5mmol/l) for 72h before cells were fixed and FGF receptor 1 (FGFR1) was detected by immunofluorescence analysis. Nuclei were counterstained using Hoechst 34580.