Hypertension and cigarette smoking are two of the most important ischemic stroke risk factors. Endothelin, a vasoconstrictive peptide has been found to be increased in the plasma of hypertensive patients and induces hypertension in animals, and cigarette smoking is an important risk stroke risk factor. Furthermore, patients with infection or other inflammatory processes may be subjected to increased stroke risk. The goal of this study is therefore to determine how nicotine and endothelin, plasma factors associated with stroke risk factors, affect specific functions of endothelial cells, and whether inflammatory cytokines, i.e., tumor necrosis factor (TNF), IL-1 or interferon gamma (IFN-gamma), modulate this activity. To achieve this goal we will analyze the effects of nicotine and endothelin on endothelial cells derived from the central nervous system (CNS). CNS-derived endothelial cultures have been established in this laboratory and have been shown to be greater than 98% pure, with gamma-glutamine transpeptidase activity. Endothelial cells will be examined for the production of the coagulation factors (e.g. plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), thrombomodulin, prostacyclin, tissue factor (TF)), vasoregulatory factors (e.g. endothelin and nitric oxide), and cytokine production (e.g. IL-1 IL-6, IL-8). The effects of endothelin and nicotine will be analyzed in the absence and presence of inflammatory cytokines (e.g. TNF, IL-1, IFN-gamma) and negative-regulatory cytokines (e.g. IL-4, transforming growth factor Beta (TGFBeta)). Coagulation factors will be identified using ELISA and functional assays. Vasoregulatory factors will be measured by immunocytochemical techniques, and the enzymatic assay to identify nitric oxide synthase activity. Cytokine synthesis and secretion will be determined using in situ hybridization, Northern blot analysis, immunostaining and ELISA techniques. TNF receptor regulation will be monitored using flow cytometry; membrane signalling will be examined by measuring phosphokinase C, phospholipase C and phospholipase C and phospholipase A enzymatic activity. The data obtained from these studies will determine how hemostatic functions are altered by nicotine and endothelin and how inflammatory cytokines in the local cerebral microenvironment affect CNS-derived endothelial cell activity. This information will provide new insights into the brain regulation of hemostasis and lead to specific therapeutic measures capable of altering brain hemostasis function to reduce stroke risk.
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