News and Publications

Novel Roles of Tissue Factor in Inflammation and Angiogenesis

N. Mackman, G. Parry, J.E. Erlich, M. O'Connell, R. Santucci

We investigated the lipopolysaccharide signaling pathway in human monocytic cells. We showed that lipopolysaccharide activates both IB kinases IKK1 and IKK2 in human monocytes and THP-1 monocytic cells. Dominant-negative IKK2 inhibited lipopolysaccharide induction of B-mediated transcription in THP-1 cells in a dose-dependent manner, whereas dominant-negative IKK1 had no effect. These studies indicate that lipopolysaccharide activates the IKK2 kinase in the signalsome complex. Subsequent phosphorylation and proteolytic degradation of both IB and IBß leads to nuclear translocation of the transcription factor NF-B and induction of gene expression.

We examined the regulation of tissue factor (TF) in the heart after experimental ischemia-reperfusion in rabbits in collaboration with E. Boyle, T. Pohlman, and E. Verrier at the University of Washington, Seattle. The left circumflex coronary artery in New Zealand White rabbits was ligated for 45 minutes and then reperfused for 2 hours. Cardiomyocytes in the area "at risk" had a dramatic increase in the expression of TF mRNA. Animals pretreated with an inhibitory monoclonal antibody to rabbit TF had a 62% reduction in infarct size. These findings suggest that TF contributes to ischemia-reperfusion injury in the heart.

The role of TF in angiogenesis was investigated in a rabbit corneal model in collaboration with M. Friedlander and E. Aguilar, Department of Cell Biology. TF was expressed by cells of the developing neovasculature. Inhibition of TF activity with a monoclonal antibody to rabbit TF reduced angiogenesis by 58%. These findings suggest that TF plays a role in this model of angiogenesis induced by basic fibroblast growth factor.

High levels of TF in the placenta are consistent with a hemostatic role for TF during pregnancy. We have generated mice that express low levels (1% of the level of wild-type mice) of TF. We investigated the affect of low levels of TF during pregnancy by breeding low-TF females with either low-TF or wild-type males. Fatal postpartum hemorrhage occurred in both groups (Table 1). Surprisingly, only the low-TF females that had low-TF embryos had fatal hemorrhage at embryonic day 11--13. Autopsies of low-TF females that died at embryonic day 11--13 revealed massive uterine hemorrhage, which appeared to be due to rupture of maternal vessels in the placenta. These studies uncovered an unexpected role of TF in the structural integrity of placental blood vessels.

Inactivation of the gene for TF results in embryonic death in 80--90% of murine TF (mTF) null embryos at embryonic day 10.5. We have successfully "rescued" mTF null embryos by using a transgene that expresses full-length human (hTF). To determine if the cytoplasmic domain of TF is required for embryonic development, we did similar rescue experiments with a transgene (hTFmutID) that expresses human TF without the cytoplasmic domain. We determined the genotype of 90 P1 pups from a breeding of mTF^+/-, hTFmutID x mTF^+/- mice (Table 2). Thirteen of the pups were mTF^-/-, hTFmutID, and 1 was mTF^-/-. These findings strongly suggest that expression of the truncated human TF protein rescued mTF^-/- pups. These results indicate that the extracellular domain of TF functions in embryogenesis independently of the cytoplasmic domain.

PUBLICATIONS

Brand, K., Eisele, T., Kreusel, U., Page, M., Page, S., Haas, M., Gerling, A., Kaltschmidt, C., Mackman, N., Neumann, F.-J., Baeuerle, P.A., Walli, A.K. Neumeier, D. Dysregulation of monocytic nuclear factor-B by oxidized low density lipoprotein. Artherioscler. Thromb. Vasc. Biol. 17:1901, 1997.

Krikun, G., Schatz, F., Mackman, N., Guller, S., Lockwood, C.J. Transcriptional regulation of the tissue factor gene by progestins in human endometrial stromal cells. J. Clin. Endocrinol. Metab. 83:926, 1998.

Oeth, P., Yao, J., Tan, S.-T., Mackman, N. Retinoic acid selectively inhibits lipopolysaccharide induction of tissue factor gene expression in human monocytes. Blood 91:2857, 1998.

Parry, G.C.N., Erlich, J.H., Luther, T., Mackman, N. Low levels of tissue factor are compatible with development and hemostasis in mice. J. Clin. Invest. 101:560, 1998.

Parry, G.C.N., Mackman, N. NF-B-mediated transcription in human monocytic and endothelial cells. Trends Cardiovasc. Med. 8:138, 1998.

Parry, G.C.N., Mackman, N. Role of CREB-binding protein in cyclic AMP inhibition of NF-B-mediated transcription. J. Immunol. 159:5450, 1997.