The issue of microvascular endothelial heterogeneity greatly impacts vascular function in both health and disease, and potentially severely complicates the use of tissue culture-based models to study endothelial behavior. Yet, despite this topic having been explored in considerable depth in peripheral vascular beds, surprisingly little attention has yet been focused on the microvascular axis of central nervous system (CNS). Unrelated reports describing particular genes have, nonetheless, provocatively suggested that a diversity in endothelial gene expression may exist at the segmental level (i.e., between arterioles, capillaries and venules) as well as the regional level of the CNS, and approximate or surpass that found in the periphery. Given the preeminence of the microvascular network in forming both the blood-brain barrier and blood- spinal cord barrier, and the significant microvascular involvement in inflammatory, infectious, degenerative and traumatic conditions of the CNS, it is critical that there be a systematic and detailed evaluation of this clinically important issue. It is of further importance that such analysis be performed on microvascular tissue in situ,"""""""" as endothelial cell gene expression is exquisitely prone to environmental modulation. Accordingly, we propose the following Specific Aims: 1) To validate and optimize the approach of coupling laser capture microdissection (LCM) of brain microvascular endothelial cells, with global gene expression profiling by DMA microarray analysis. This will include demonstrating feasibility, by maximizing the percentage of transcripts detected (P call rate) in capillaries only, and establishing reproducibility, by identifying both technical and biological sources of variance in microarray data; and 2) To use the LCM/microarray approach to compare the global gene expression profiles of endothelial cells from capillaries, venules and arterioles. The R21 format chosen is specifically designed for projects that are exploratory and may involve considerable risk, but lead to the development of novel techniques that could have major impact on a field of research. In this regard, results obtained here will set the stage for evaluating regional gene expression by the neurovascular unit (i.e., endothelial cells and intimately associated neural cells) along the CNS microvascular tree in both health and disease. In turn, these studies will enable formulation of more precise endothelial models to study the molecular basis of physiological and pathophysiological processes of the cerebral microvasculature. ? ? ?
