This Project will further develop the theme of sickle disease as an example of reperfusion injury physiology and focus on the endothelial activation endpoint of abnormal expression of tissue factor by pulmonary vein ndothelium. This focus is justified because: [a] it will extend the understanding of TF biology; [b] it comprises a robust endpoint indicator of endothelial cell activation; and [c] it is of great potential importance n sickle disease, in which affected individuals have a coagulopathy and thromboses. We will examine 5 Specific Aims. [1] Identify what is different about sickle mice, that leads them - but not normal mice - to develop endothelial TF expression. Our Key Hypotheses are that inflammation plays the primary role, and that this is mediated by peripheral blood monocytes, and modulated by NO from endothelial eNOS. [2]deleted. [3] Assess the location, functionality, and relevance of endothelial TF expression. Our Key Hypotheses are: that endothelial TF is functional and expressed on the lumen side of the endothelial cell; and that inhibition of TF expression (in mouse and human) will be paralleled by diminution in coagulation activation state. [4] Identify, at molecular and whole mouse levels, the mechanism underlying endothelial TF expression and inhibition. Our Key Hypotheses is that while NFKB is permissive for endothelial TF expression in sickle mouse model, Egr-1 is essential. [5] Examine why there is endothelial heterogeneity in TF expression. Endothelial TF expression is confined to the lungs, and within this organ to the veins, and only some veins. Understanding the reason for this very interesting example of endothelial phenotype heterogeneity is not only of general biological interest, but also of some medical importance. Here, our Key Hypotheses is that the TF positive veins exhibit this phenotype because of greater prior hypoxic stress than the TF negative veins. LAY SUMMARY: We will study sickle cell mice to determine why they are abnormally susceptible to hypoxia and express tissue factor, the trigger of blood clotting. The importance of these studies is that they well help us develop a novel therapy for sickle disease that will prevent the large stroke problem in affected kids.
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