Tissue factor (TF) expression is associated with life-threatening thrombosis in a variety of human diseases, including sepsis, cancer, and atherosclerosis. Recently, it was shown that inactivation of the murine TF (mTF) gene results in embryonic lethality. To date, despite extensive studies on the regulation of the TF promoter in vitro, no studies have examined the cis-acting regulatory elements that control TF gene expression in vivo. Here we report that a human TF (hTF) minigene containing the human TF promoter and human TF cDNA directed a low level (approximately 1% relative to mouse TF) of both constitutive and LPS-inducible human TF expression in transgenic mice. Importantly, the human TF minigene rescued the embryonic lethality of murine TF null embryos, suggesting that human TF substituted for murine TF during embryogenesis. Rescued mice (mTF-/-, hTF+), which expressed low levels (approximately 1%) of TF activity, developed normally with no signs of a bleeding diathesis, suggesting that low TF expression can maintain hemostasis compatible with normal survival. These studies establish a novel mouse model system that can be used to examine the regulation of the human TF gene in vivo and the impact of low TF levels on the hemostatic balance in various thrombotic diseases.

Recognition of bacterial endotoxin (LPS) elicits multiple host responses, including activation of cells of the innate immune system. LPS exposure occurs repeatedly during septicemia, making strict regulation of gene expression necessary. Such regulation might prevent, for example, the continuous production of proinflammatory cytokines such as tumor necrosis factor (TNF), which could lead to severe vascular collapse. Tolerance to LPS is characterized by a diminished production of TNF during prolonged exposure to LPS, and is therefore likely to represent an essential control mechanism during sepsis. In the present study, which uses mice with genetic deletions of the proteins of NF-B complex, we provide data demonstrating that increased expression of the p50 subunit of NF-B directly results in the downregulation of LPS-induced TNF production. This contention is supported by the following observations: (1)

Functional inhibition of tissue factor (TF) has beenshown to improve coronary blood flow after myocardial ischemia/reperfusion (I/R) injury. TF initiates the coagulation protease cascade, resulting in the generation of the serine protease thrombin and fibrin deposition. Thrombin can also contribute to an inflammatory response by activating various cell types, including vascular endothelial cells. We used a rabbit coronary ligation model to investigate the role of TF in acute myocardial I/R injury. At-risk areas of myocardium showed increased TF expression in the sarcolemma of cardiomyocytes, which was associated with a low level of extravascular fibrin deposition. Functional inhibition of TF activity with an anti-rabbit TF monoclonal antibody administered either 15 minutes before or 30 minutes after coronary ligation reduced infarct size by 61% (P ‫؍‬ 0.004) and 44% (P ‫؍‬ 0.014), respectively. Similarly, we found that inhibition of thrombin with hirudin reduced infarct size by 59% (P ‫؍‬ 0.014). In contrast, defibrinogenating the rabbits with ancrod had no effect on infarct size, suggesting that fibrin deposition does not significantly contribute to infarct size. Functional inhibition of thrombin reduced chemokine expression and inhibition of either TF or thrombin reduced leukocyte infiltration. We propose that cardiomyocyte TF initiates extravascular thrombin generation, which enhances inflammation and injury during myocardial I/R. (Am J Pathol 2000, 157:1849 -1862)

Exposure of blood to tissue factor (TF) activates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this study, we found that mice expressing low levels of human TF (Ϸ1% of wild-type levels) in an mTF ؊/؊ background had significantly shorter lifespans than wild-type mice, in part, because of spontaneous fatal hemorrhages. All low-TF mice exhibited a selective heart defect that consisted of hemosiderin deposition and fibrosis. Direct intracardiac measurement demonstrated a 30% reduction (P < 0.001) in left ventricular function in 8-month-old low-TF mice compared with age-matched wild-type mice. Mice expressing low levels of murine FVII (Ϸ1% of wild-type levels) exhibited a similar pattern of hemosiderin deposition and fibrosis in their hearts. In contrast, FIX ؊/؊ mice, a model of hemophilia B, had normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired hemostasis. We propose that TF expression by cardiac myocytes provides a secondary hemostatic barrier to protect the heart from hemorrhage. E xpression of tissue factor (TF) by adventitial fibroblasts and vascular smooth muscle cells surrounding blood vessels provides a hemostatic barrier that activates coagulation when vascular integrity is disrupted (1). TF is also expressed by cardiac muscle but not by skeletal muscle (1). TF functions as the high-affinity cellular receptor for FVII͞VIIa (2). The coagulation protease cascades are comprised of the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways, which together maintain hemostasis (3).Many murine models of coagulation have been generated that provide new insights into the role of the various procoagulant and anticoagulant proteins in hemostasis (4). For instance, FV Leiden/Leiden mice, which express an FV variant that is resistant to inactivation by activated protein C, and TM Pro/Pro mice, which express a mutated version of thrombomodulin (TM) with reduced thrombin binding, both exhibit prothrombotic phenotypes with increased fibrin deposition in select tissues (5-7). Mice with prohemorrhage phenotypes include models of hemophilia A (FVIII Ϫ/Ϫ ) and B (FIX Ϫ/Ϫ ), as well as fibrinogen-deficient mice (Fbg Ϫ/Ϫ ) and thrombocytopenic mice (NF-E2 Ϫ/Ϫ ) (8-12). Mice with complete deficiencies in TF, FVII, FX, FV, and prothrombin die in utero or shortly after birth (4). We and others have generated mice expressing low levels (Ͻ0.1-1% of wild-type levels) of human TF, murine FVII, and murine FV (13-15). We have shown that low-TF mice have impaired uterine hemostasis (16). A similar phenotype is observed with low-FVII mice.In this study, we performed a detailed characterization of low-TF mice. These mice exhibited shorter lifespans than wildtype mice. Histological analysis of various tissues of low-TF mice revealed hemosiderin deposition and fibrosis selectively in their hearts. Our data suggest that cardiac fibrosis in low-TF mice is caused by hemorrhage from cardiac vessels due to impaired hemostasis. M...