Extracellular matrix protein components of vascular basement membranes profoundly affect endothelial cell morphology and growth behavior. Our central hypothesis is that FAK (ppl25FAK, focal adhesion kinase) is essential to the organization of the cytoskeleton and the facilitation of migration in vascular endothelial cells. The proposed studies will test the role of FAK signal transduction during the formation and remodeling of focal adhesions. The first specific aim (A) will focus on the effects of altered FAK expression on endothelial cell adhesion and migration. Modulation of FAK interactions with potential substrates will be accomplished by microinjection of FAK fragments or anti-FAK antibodies, and by cDNA transfection or antisense oligonucleotides. FAK interactions with other focal adhesion proteins, and the activation state and phosphotyrosine content of FAK will be studied in these cells. The second specific aim (B) is to identify the relationship of FAK to the initiation of cytosolic free calcium oscillations during integrin-mediated endothelial cell adhesion. Digitized video microscopy, fluorescent calcium indicators, and caged mediators of intracellular calcium flux will be used to study endothelial cells with normal and altered FAK expression. The third specific aim (C) is to identify the domains of fibronectin required for endothelial cell signalling through FAK during focal adhesion assembly. This will be done by studying FAK activation and tyrosine phosphorylation in parallel with focal adhesion morphology. Endothelial cells will be imaged during the process of adhesion to various fibronectin domains with immunofluorescence, interference reflection, and laser scanning confocal microscopy. Vascular endothelial dysfunction is central to the pathogenesis of vital organ failure due to trauma, inflammation, and sepsis. Understanding the ways in which the vascular endothelium supports microvascular integrity may facilitate the treatment of critically ill children. These studies may provide insights into signalling mechanisms that occur during endothelial cell adhesion to extracellular matrix during vascular growth, development, and responses to trauma. Therapeutic strategies that modify these mechanisms may have far reaching implications for disorders as diverse as congenital heart disease, circulatory failure during septicemia, pulmonary vascular disease in acute respiratory failure, and cerebral ischemia after trauma. Long range goals include the development of therapeutic interventions targeting adhesion protein expression and signal transduction pathways.
