High-LET radiation-induced response of microvessels in the Hippocampus.

The hippocampus is critical for learning and memory, and injury to this structure is associated with cognitive deficits. The response of the hippocampal microvessels after a relatively low dose of high-LET radiation remains unclear. In this study, endothelial population changes in hippocampal microvessels exposed to (56)Fe ions at doses of 0, 0.5, 2 and 4 Gy were quantified using unbiased stereological techniques. Twelve months after exposure, mice that received 0.5 Gy or 2 Gy of iron ions showed a 34% or 29% loss of endothelial cells, respectively, in the hippocampal cornu ammonis region 1 (CA1) compared to age-matched controls or mice that received 4 Gy (P < 0.05). We suggest that this "U-shaped" dose response indicates a repopulation from a sensitive subset of endothelial cells that occurred after 4 Gy that was stimulated by an initial rapid loss of endothelial cells. In contrast to the CA1, in the dentate gyrus (DG), there was no significant difference in microvessel cell and length density between irradiated groups and age-matched controls. Vascular topology differences between CA1 and DG may account for the variation in dose response. The correlation between radiation-induced alterations in the hippocampal microvessels and their functional consequences must be investigated in further studies.

Uniform overexpression and rapid accessibility of alpha5beta1 integrin on blood vessels in tumors.

Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung.

Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. 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, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes.