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Local Expression of Hemostatic Genes

D. Loskutoff, G. Belogrudov, G. Deng, K. Fujisawa, L. Hervio, G. Hu, H. Ihara, Y. Okumura, F. Samad, A. Stiko, K. Yamamoto, Q. Zhang

Vascular hemostasis occurs because of the exquisitely regulated interaction and balance between the coagulation and fibrinolytic systems, and imbalances in either system promote vascular disease. However, little is known about the cells that express hemostatic genes during normal physiologic conditions or how expression of these genes changes in various pathologic states. One hypothesis of our work is that expression of hemostatic genes is highly regulated and not restricted to the liver and that changes in the expression of these genes in tissues alter local hemostatic balance. A second hypothesis is that expression of these genes may regulate local cell adhesion and migration. We have been using quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis of tissues from murine models of thrombotic disease (e.g., obesity) to test the first hypothesis and cell adhesion assays to test the second.

REGULATION OF HEMOSTATIC GENES IN OBESITY

Obesity is an independent risk factor for the development of atherosclerosis and cardiovascular disease and is associated with related metabolic disorders such as hypertriglyceridemia, hyperinsulinemia, and non--insulin-dependent diabetes mellitus. Interestingly, in clinical studies, levels of plasminogen activator inhibitor-1 (PAI-1) (another risk factor for cardiovascular disease) are consistently elevated in obese subjects. Despite this finding, little is known about the origins of PAI-1 in obesity or the events that regulate this inhibitor. To address these questions, we are using genetically obese mice (ob/ob and db/db) and their lean counterparts.

We observed that PAI-1 antigen and mRNA levels were elevated 5-fold in the plasma and adipose tissues, respectively, of obese mice, and that in the adipose tissue, PAI-1 mRNA was increased in mature adipocytes, vascular smooth muscle cells, and a few endothelial cells. The elevated levels of PAI-1 in the plasma appear to be due to transforming growth factor-ß (TGF-ß), insulin, and TNF-, all of which are elevated in obesity and induce PAI-1 in adipocytes. The dramatic increase in PAI-1 most likely disrupts normal hemostatic balance and creates a severe hypofibrinolytic state.

In separate studies, we found that expression of the gene for tissue factor (TF) also was significantly higher in the epididymal and subcutaneous fat pads in ob/ob mice than in lean mice and that the level of expression of the gene in the obese mice increases with age and the degree of obesity. Cell fractionation and in situ hybridization analysis of adipose tissues indicated that TF mRNA is increased in adipocytes, possibly through TGF-ß, because administration of this growth factor increased TF mRNA expression in adipocytes in vivo and in vitro. These observations suggest that TF and TGF-ß also may contribute to the increased cardiovascular disease that accompanies obesity and related non--insulin-dependent diabetes mellitus and that the adipocyte itself plays a key role in this process. The recent demonstration that TF also influences angiogenesis, cell adhesion, and signaling suggests that the exact role of TF in both normal physiologic and pathologic conditions in adipose tissue may be complex.

ADHESION OF CELLS TO VITRONECTIN

PAI-1 binds to the somatomedin B domain of vitronectin and, in U937 cells, directly competes with the receptor for u-plasminogen activator (uPAR) for binding to this same site. Thus, PAI-1 appears to play a dynamic regulatory role in uPAR-mediated adhesion of U937 cells to vitronectin. Many cells, however, use a different class of receptors, the integrins, to adhere to the single arginine--glycine--aspartic acid (RGD) sequence in vitronectin. The proximity of this RGD site to the somatomedin B domain suggests that PAI-1 may also block integrin binding, and a number of recent observations support this hypothesis. We did a series of cell adhesion experiments to further test this idea and to more completely define the relative contributions of u-PAR and integrins to adhesion.

In one set of experiments, we showed that MCF7 cells adhere to vitronectin and that preincubating the vitronectin-coated wells of the tissue culture plate with PAI-1 blocked this adhesion. The adhesive behavior of MCF7 cells is similar to that of U937 cells, because this blocking effect occurs in a PAI-1 dose-dependent manner and can be reversed by urokinase. However, unlike U937 cells, once attached to vitronectin, MCF7 cells rapidly become resistant to dissociation by PAI-1. Moreover, RGD-containing peptides that have no effect on the binding of U937 cells to vitronectin release MCF7 cells from a vitronectin substratum. Thus, adhesion of U937 cells is mediated primarily by uPAR, whereas that of MCF7 cells is mediated primarily by integrins.

In a second set of experiments, we examined the adhesion of HT-1080 cells to vitronectin. In this case, adhesion was strongly inhibited by PAI-1, and the RGD peptide had little effect (although spreading was inhibited). Unexpectedly, in cell-detachment experiments, neither PAI-1 nor the RGD peptide alone was particularly effective in detaching HT-1080 cells from vitronectin. However, the addition of both PAI-1 and the RGD peptide led to the release of the cells. These observations indicate that the adhesion of cells to vitronectin occurs in a cell-specific manner, one that may be mediated by uPAR, integrins, or both. PAI-1 potentially can regulate each of these adhesive steps.

PUBLICATIONS

Estellés, A., Gilabert, J., Grancha, S., Yamamoto, K., Thinnes, T., Espana, F., Aznar, J., Loskutoff, D.J. Abnormal expression of type 1 plasminogen activator inhibitor and tissue factor in severe preeclampsia. Thromb. Haemost. 79:500, 1998.

Fearns, C., Loskutoff, D.J. Role of tumor necrosis factor alpha in induction of murine CD14 gene expression by lipopolysaccharide. Infect. Immun. 65:4822, 1997.

Loskutoff, D.J., Samad F. The adipocyte and hemostatic balance in obesity: Studies of plasminogen activator inhibitor 1. Arterioscler. Thromb. Vasc. Biol. 18:1, 1998.

Samad, F., Loskutoff, D. The fat mouse: A powerful genetic model to study elevated plasminogen activator inhibitor 1 in obesity/NIDDM. Thromb. Haemost. 78:652, 1997.

Samad, F., Pandey, M., Loskutoff, D.J. Tissue factor gene expression in the adipose tissues of obese mice. Proc. Natl. Acad. Sci. U.S.A., in press.

Samad, F., Schneiderman, J., Loskutoff, D. Expression of fibrinolytic genes in tissues from human atherosclerotic aneurysms and from obese mice. Ann. N.Y. Acad. Sci. 811:350, 1997.

Schneiderman, J., Bordin, G.M., Adar, R., Smolinsky, A., Seiffert, D., Engelberg, I., Dilley, R.B., Thinnes, T., Loskutoff, D.J. Patterns of expression of fibrinolytic genes and matrix metalloproteinase-9 in dissecting aortic aneurysms. Am. J. Pathol. 152:703, 1998.

van Aken, B.E., Seiffert, D., Thinnes, T., Loskutoff, D.J. Localization of vitronectin in the normal and atherosclerotic human vessel wall. Histochem. Cell Biol. 107:313, 1997.

Yamamoto, K., de Waard, V., Fearns, C., Loskutoff, D.J. Tissue distribution and regulation of murine von Willebrand gene expression in vivo. Blood, in press.

Yamamoto, K., Loskutoff, D.J. Extrahepatic expression and regulation of protein C in the mouse. Am. J. Pathol., in press.

Yamamoto, K., Loskutoff, D.J. The kidneys of mice with autoimmune disease acquire a hypofibrinolytic/procoagulant state that correlates with the development of glomerulonephritis and tissue microthrombosis. Am. J. Pathol. 151:725, 1997.