9 of 10 Gas6−/− mice died within the first 12 hours of reperfusion, with 50% of the animals dying within 8 hours of reperfusion. In contrast, 90% of WT mice survived partial I/R (Fig. Roxadustat nmr 1C), and this was in line with our previous studies.23 The deaths of the Gas6−/− mice during hepatic I/R were most likely related to liver failure due to massive hepatocellular damage because serum aminotransferase levels 6 hours after reperfusion were dramatically elevated in GAS6-deficient mice versus WT mice (Fig. 2A). Moreover, this outcome mirrored histological findings revealing severe deterioration of the liver parenchyma after I/R exposure in GAS6-deficient mice with respect to WT mice (Fig. 2B). In addition, parallel liver sections from Gas6−/− mice undergoing I/R displayed extensive cell death detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, which
affected areas all over the hepatic parenchyma; this contrasted with the confined TUNEL-positive areas observed in WT mice (Fig. 2B). Finally, we examined whether GAS6 regulates PI3K inhibitor inflammatory mediators during I/R as previously proposed.20, 21 Interestingly, TNF and IL-1β mRNA levels were markedly elevated in the null mice versus the WT animals exposed to I/R (Fig. 2C,D) after 6 hours of reperfusion, and this suggests that GAS6 counterbalances the inflammatory response after hepatic ischemia. Thus, these findings clearly demonstrate overall that the lack of GAS6 sensitizes the liver to I/R and results in massive hepatic destruction incompatible with life. Although massive liver injury in GAS6-null mice was evident 3 to 6 hours after reperfusion, a significant increase in ALT levels was already detected as soon as 1 hour after ischemia in comparison with WT mice (1835 ± 893
U/mL for Gas6−/− mice versus 695 ± 248 U/mL for WT mice). Therefore, Carteolol HCl we next evaluated early changes in hepatic signaling responsible for the hepatic sensitization to I/R observed in Gas6−/− mice and focused particularly on protective/apoptotic pathways. Because c-Jun N-terminal kinase (JNK) activation has been shown to contribute to hepatic I/R injury, we analyzed the phosphorylation state of JNK 60 minutes after reperfusion. The robust phosphorylation of JNK p46 and p54 isoforms observed after reperfusion was comparable in the livers of both WT and GAS6-null mice (Fig. 3A); this particular pathway in the sensitization of GAS6-KO mice to hepatic I/R was discarded. Because AKT regulates cell survival, we examined the phosphorylation state of AKT during hepatic I/R. In contrast to JNK activation, hepatic AKT phosphorylation was clearly reduced in Gas6−/− mice versus I/R-exposed WT mice (Fig. 3B). In addition to AKT, nuclear factor kappa B (NF-κB) regulates hepatocellular susceptibility to I/R,28 and hence we compared the extent of activation of NF-κB in WT and GAS6-deficient mice.