A major advance has been the understanding of the role of both endothelin-I (ET-]) and NF-KappaB in the pathogenesis of chagasic vasculopathy. We propose to focus on molecular, cellular, and tissue level studies of how the parasite causes vascular pathology. Two pathogenic pathways will be investigated, vasoconstriction/ischemia and inflammation. T. cruzi infection of the cells of the vascular endothelium modulates vessel tone by increasing the synthesis of the vasoactive peptide, ET- I causing vascular spasm and myocardial ischemia. The NIAPK cascades regulate AP-1 activation resulting in ET-1 expression. We will determine which kinase cascade (Raf-ERK or MEKK-JNK) is dominant in regulating the activation of AP-1 in endothelial cells. The methods to be used include small G-protein guanidine nucleotide assays and NIAPK activity assays. We will co-transfect dominant negative genes of the kinase cascades with AP-I and ET-I reporter genes to delineate the activation of pathways. Since the MAPK cascade also contributes to smooth muscle proliferation and vascular remodeling of damaged vessels we plan to examine the consequences of T. cruzi infection on NIAPKs in these cells by exposure to supernatants of infected endothelial cells. In addition, we will examine the effect of T. cruzi infection on the activation of the MAPKS, AP-I and ET-I expression in the vasculature and myocardium of mice. The role of ET-I in the pathogenesis of chagasic heart disease will be investigated by infecting mice in which ET-l has been deleted in vascular endothelial cells and cardiac myocytes. The pathophysiological consequences of infection on myocardial structure and function will be evaluated by invasive and non-invasive methods. Infection of endothelial cells with T. cruzi induces expression of cytokines, adhesion molecules, leading to inflammation and pathology. We hypothesize that an important mechanism responsible for myocardial inflammation is activation of the vasculature NF-KappaB pathway. Accordingly, we will examine the mechanism by which T. cruzi activates the NF-KappaB pathway in endothelial and vascular smooth muscle cells by investigating the role of the generation of reactive oxygen species and cytoplasmic phospholipase A2 activity. We will also employ co-transfection of dominant negative genes of the kinase cascade with NF-KappaB reporter to define the pathway. Finally, we plan to examine the consequences of T. cruzi infection on the activation of NF-KappaB and the outcome of ROS inhibitors in vivo. These studies will delineate the mechanisms by which T. cruzi causes vasculopathy leading to cardiomyopathy.
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