Regulation of Inducible Gene Expression in Monocytes

Univ Of North Carolina Chapel Hill, Chapel Hill NC

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Gram-negative sepsis is a major cause of death in intensive care units in the United States. Lipopolysaccharide (LPS [endotoxin]) from Gram-negative bacteria induces monocytes and endothelial cells to express the procoagulant protein tissue factor (TF), which initiates disseminated intravascular coagulation (DIG) and microvascular thrombosis. Monocytes and endothelial cells (ECs) also express a variety of inflammatory cytokines that contribute to morbidity and mortality. Recent studies indicate that administration of anticoagulants or anti-inflammatory agents reduces mortality in animal models of endotoxemia and sepsis. The long-term objectives of this proposal are: i) to develop anti-TF therapies that target different cell types, and ii) to understand the role of the phosphatidylinositol-3 kinase (PISK)-Akt pathway in suppressing LPS- induced coagulation and inflammation. We hypothesize that monocytes, ECs and platelets express TF during endotoxemia. Selective inhibition of TF expressed by these cell types may reduce DIG without compromising hemostasis. Our second hypothesis is that the anti-inflammatory activity of a variety of drugs is mediated by activation of the PI3K-Akt pathway in monocytes and endothelial cells. To test these hypotheses, we will employ both genetic and pharmacologic approaches. We will use the Cre-loxP system to determine the effect of selectively deleting the TF gene in myeloid cells or endothelial cells on LPS-induced coagulation. We will also evaluate the role of platelet TF in microvascular thrombosis by depleting platelets in wild type mice and replacing them with platelets from either low TF or wild type mice. Finally, we will use a combination of a species-specific anti-TF antibody, bone marrow transplantation and humanized TF mice to determine the effect of selectively inhibiting TF expression in hematopoietic cells on endotoxemia. We anticipate that this strategy will protect the mice without inducing hemorrhage. In parallel studies, we will use mice with either increased or decreased PI3K activity and drugs that activate the PI3K-Akt pathway. This proposal contains two Specific Aims. Specific Aim 1 will analyze LPS regulation of TF activity in monocytes, ECs and platelets, and determine the contribution of TF expression by these different cell types to LPS- induced coagulation and microvascular thrombosis. Specific Aim 2 will determine the role of the PI3K-Akt pathway in the inhibition of LPS-induced gene expression by various anti-inflammatory drugs. These studies will provide new insight into the role of TF and the PI3K-Akt pathway in endotoxemia. This information can be used to develop improved therapeutic strategies for the treatment of patients with severe sepsis. [unreadable] [unreadable] [unreadable]