The objective of the work in this proposal is to determine modes of regulation involved in the tissue-specific and modulated expression of von Willebrand factor and certain other endothelial cell functions. The long term objective is to define the regulatory circuitry coordinating the expression of hemostatic factors expressed in endothelium. Vascular endothelial cells differentially express many functions important in hemostasis and thrombosis, including von Willebrand factor. The hypothesis that the relevant genes are in a more accessible conformation or are chemically modified in differentiated cells will be tested. Von Willebrand factor DNA from endothelial cells will be compared to the equivalent sequences from relatively undifferentiated cells in terms of DNAse hypersensitivity and in terms of methylation to determine whether either of these two mechanisms may be involved in the differentiated expression of von Willebrand factor. The relative amounts of endothelial cell products in the blood are modulated in response to physiological conditions. For instance, von Willebrand factor levels are increased during pregnancy and chronic inflammation. The hypothesis that production of this factor may be significantly controlled at transcriptional levels will be tested using cultured cells under conditions which have been shown to affect the levels of von Willebrand factor expressed. Transcript levels, messenger RNA stability, and rate of transcription will be determined using cloned DNA probes and the continuous human endothelial cell line derived in this laboratory. The differentiated functions of endothelial cells include not only thrombogenic agents like von Willebrand factor, but also antithrombogenic agents like antithrombin III, and fibrinolytic agents like tissue plasminogen activator. Chromatin properties associated with differentiated expression and modes of transcriptional regulation will be compared for these three examples of opposing endothelial cell functions.
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