Microvascular endothelial cell heterogeneity in the central nervous system

描述（由申请人提供）：微血管内皮异质性问题极大地影响健康和疾病中的血管功能，并可能使使用基于组织培养的模型研究内皮行为严重复杂化。然而，尽管这一主题已经在外周血管床中进行了相当深入的探索，但令人惊讶的是，很少有人关注中枢神经系统（CNS）的微血管轴。尽管如此，描述特定基因的无关报道已经有力地表明，内皮基因表达的多样性可能存在于节段水平（即，在小动脉、毛细血管和小静脉之间）以及CNS的区域水平，并且接近或超过在外周中发现的水平。考虑到微血管网络在形成血脑屏障和血脊髓屏障中的优势，以及CNS的炎症、感染、变性和创伤性疾病中的显著微血管参与，对这一临床重要问题进行系统和详细的评价至关重要。更重要的是，这种分析应在原位微血管组织上进行，”因为内皮细胞基因表达非常容易受到环境调节。因此，我们提出以下具体目的：1）验证和优化脑微血管内皮细胞的激光捕获显微切割（LCM）与DMA微阵列分析的全局基因表达谱的耦合方法。这将包括通过最大化仅在毛细血管中检测到的转录物的百分比（P调用率）来证明可行性，并通过鉴定微阵列数据中的变异的技术和生物来源来建立再现性;以及2）使用LCM/微阵列方法来比较来自毛细血管、小静脉和小动脉的内皮细胞的全局基因表达谱。选择的R21格式是专门为探索性项目设计的，可能涉及相当大的风险，但会导致开发可能对研究领域产生重大影响的新技术。在这方面，这里获得的结果将为评估神经血管单位的区域基因表达（即，内皮细胞和密切相关的神经细胞）沿着CNS微血管树。反过来，这些研究将使制定更精确的内皮模型，研究脑微血管的生理和病理生理过程的分子基础。

DESCRIPTION (provided by applicant): 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.