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1 Department of Cardiovascular Research Institute, Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA, USA
* To whom correspondence should be addressed. E-mail: dmcd{at}itsa.ucsf.edu.
Profiling gene expression in endothelial cells provides a strategy for developing a molecular understanding of normal vascular physiology and disease processes involving angiogenesis and vascular remodeling. However, the purification of endothelial cells for this purpose has been challenging because of the difficulty of isolating the cells and their low abundance in many organs and tumors. Here we describe an approach that exploits the propensity for certain types of endothelial cells to take up fluorescently labeled cationic liposomes from the bloodstream and use this property to isolate the cells from lung capillaries by FACS rapidly and with high purity. Out of some 39,000 genes and expressed sequence tags (ESTs) evaluated on custom oligonucleotide arrays, the expression of 234 genes and 321 ESTs was enriched in the endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGFR-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin 37, CD31, and CD34. Also enriched were genes in the semaphorin/neuropilin (Sema3c, Nrp1), ephrin/Eph (ephrin A1, B1, B2, EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, Siah1b), and Wingless (Frizzled-4, Tle1) signaling pathways that are expressed in vascular development and angiogenesis. The expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube1, serpinh1, SNF related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can also be used to profile genes expressed in angiogenic blood vessels in tumors, chronic inflammation, and other sites where endothelial cells avidly take up cationic liposomes.
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