Diannexin, a novel annexin V homodimer, provides prolonged protection against hepatic ischemia-reperfusion injury in mice.

BACKGROUND AND AIMS: Ischemia-reperfusion injury (IRI) remains an important cause of liver failure after hepatic surgery or transplantation. The mechanism seems to originate within the hepatic sinusoid, with damage to endothelial cells, an early, reproducible finding. Sinusoidal endothelial cells (SECs), damaged during reperfusion, activate and recruit inflammatory cells and platelets. We hypothesized that a recombinant human annexin V homodimer, Diannexin, would protect SECs from reperfusion injury. METHODS: We tested this proposal in a well-characterized in vivo murine partial hepatic IRI model. RESULTS: Whether administered 5 minutes or 24 hours before or 1 hour after ischemia-reperfusion, Diannexin (100-1000 microg/kg) almost completely protected against liver injury. The protective efficacy conferred by Diannexin was highly visible at the microcirculatory level. Thus, although IR in this model is associated with early swelling and gap formation in SECs, Diannexin ameliorated these effects as shown by >80% reduction in alanine aminotransferase values during the early phase of reperfusion injury (2 hours) and near normalization of liver necrosis and inflammation in the late phase of inflammatory recruitment (24 hours). Consistent with the proposed role of SEC injury in hepatic IRI, Diannexin also decreased hepatic expression of proinflammatory molecules (MIP-2, ICAM-1, VCAM), abolished leukocyte and platelet adherence to damaged SECs, and, by in vivo microscopy, Diannexin preserved microcirculatory blood flow and hepatocyte integrity during reperfusion. CONCLUSIONS: Diannexin is an apparently safe therapeutic protein that provides prolonged protection against hepatic IRI via cytoprotection of SECs, thereby interrupting secondary microcirculatory inflammation and coagulation.

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform.

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D(3), the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D(3) was greater in COS1 and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D(3)-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D(3) and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D(3) than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand.