Monocyte attachment, adhesion, and infiltration into the vessel wall represent hallmarks of a wide range of vascular disorders including thrombosis, atherosclerosis, and inflammation. Endothelial cell surface expression of attachment molecules, such as E- and P-selectin, and adhesion molecules, such as ICAM-1 and VCAM-1, locally induced by inflammatory mediators, plays an important role in mediating monocyte attachment and adhesion by exposing binding sites for specific counter- receptors on the monocyte surface. The central premise of this proposal is that engagement of specific attachment receptors on monocytes is an important pathway of monocyte activation, and that attachment-induced activation triggers a unique intracellular signalling pathway that has particular relevance to atherogenesis. Preliminary data has been obtained demonstrating that when peripheral blood monocytes are co-cultured with cytokine-activated endothelial cells a specific phenotypic change is induced in the monocytes that includes surface expression of procoagulant tissue factor, increased surface expression of CD36 (a glycoprotein scavenger receptor and adhesion receptor), and secretion of the pro- inflammatory cytokine TNF. This phenotypic change is the result of increased transcription of specific genes and requires direct contact between the endothelial cells and the monocytes. Engagement of E-selectin receptors on monocytes has been shown to play a major role in inducing these phenotypic changes. Plans are outlined to define the surface, cytoplasmic and nuclear signalling pathways involved in monocyte activation by cellular attachment receptors. In particular the role of E- selectin ligands (ES-1 and CD15) will be explored using immuno-inhibition and anti-sense oligonucleotide approaches. Attachment of monocytes to E- selectin transfected cells will be used to explore specific intracytoplasmic signalling pathways and intranuclear gene regulation mechanisms induced by E-selectin engagement. These studies will involve close interactions with Dr. D. Hajjar and B. Hempstead who are experts in cell signalling pathways. Specific genes activated by this pathway will be characterized by northern analysis, PCR, and ELISA with particular attention to monocyte/macrophage effector functions relevant to vascular biology. This will involve close interactions with Dr. D. Hajjar and A. Marcus looking at scavenger receptors, cytokines, and eicosanoids; and Dr. K. Hajjar looking at regulators of coagulation and fibrinolysis. Novel genes activated by E-selectin engagement will be identified by PCR differential display and/or positive selection cloning techniques. It is expected that the proposed studies may provide novel mechanistic insight into cell signalling and ultimately may allow development of novel therapeutic strategies for vascular and inflammatory diseases.
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