Title

Endothelial heterogeneity of blood-brain barrier gene expression along the brain microvasculature

Date of Completion

January 2009

Keywords

Biology, Molecular|Biology, Neuroscience|Biology, Cell

Degree

Ph.D.

Abstract

The blood-brain barrier (BBB) refers to the network of microvessels that selectively restricts the passage of substances between the circulation and central nervous system (CNS). This microvascular network is comprised of arterioles, capillaries and venules, yet the respective contribution of each of these to the BBB awaits clarification. In this regard, it has been postulated that brain microvascular endothelial cells (BMEC) from these different tributaries might exhibit considerable heterogeneity in form and function, with such diversity underlying unique roles in physiological and pathophysiological processes. Means to begin exploring such endothelial differences within the CNS in situ, free from caveats associated with cell isolation and culturing procedures, are crucial to comprehending the nature and treatment of CNS diseases with vascular involvement. Here, a unique approach was used to analyze BBB gene expression patterns of endothelial cells retrieved in situ from the distinct microvascular segments: immuno-laser capture microdissection (immuno-LCM) coupled to quantitative real-time PCR (qRT-PCR). After validating the reproducibility of this combined approach, an initial assessment of endothelial heterogeneity was obtained by contrasting expression patterns of a handful of BBB genes between arterioles, capillaries and venules. Due to the close proximity of smooth muscle cells to arteriolar BMEC and given the resolution of the PixCell Ile LCM machine, great difficulty was encountered in ensuring endothelial purity for this branch. For this reason, efforts focused on contrasting LCM-procured capillary versus venular BMEC for the full panorama of BBB genes. From profiling 87 genes encoding proteins shown to play a role in BE3B function or enriched in BMEC compared to parenchyma but with unknown contribution to BBB function, results imply that most BBB properties reside in both segments, but that capillaries preferentially express some genes related to solute transport, while venules tend toward higher expression of genes involved in inflammatory-related tasks. Moreover, preliminary studies support employing this approach to detect age-related differences in BBB gene expression. Performed under physiological conditions, these studies call attention to the breadth of endothelial diversity and its functional consequences with respect to the BBB and set the stage for the role played by endothelial heterogeneity under CNS disease. ^