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note
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164 was used. This line of K14-VEGF mice was phenotypically similar to those generated by Detmar et al. (3). In a previous study we examined single-allele K14-Ang1 mice (2), whereas in this study we used double-allele K14-Ang1 mice. As previously reported, both transgenic strains of mice were fertile and overtly healthy (2, 3). The ear skin of both K14-VEGF and K14-Ang1 mice was redder than that of wild-type FVB/N mice. Otherwise the skin of K14-Ang1 mice appeared normal, and histological sections revealed that the ear skin was similar to that of wild-type mice in terms of both thickness and cellular components; the only obvious difference in the K14-Ang1 skin was an increased number of large vessels. In contrast, the ear skin of K14-VEGF mice appeared thicker than normal (3), and by the time the mice were 10 to 12 weeks of age focal red skin lesions began to develop in their ears. We focused on the ear skin of mice because (i) the vasculature is not obscured by dense hair, (ii) the ear vasculature can easily be examined in whole mounts, (iii) the ears can easily be examined while the mice are alive, and (iv) the ear skin seemed to resemble the remainder of the skin. Different amounts of Ang1 or VEGF expression in different regions of skin, perhaps as a result of different density of hair follicles, could result in quantitative differences in vessel length and area density.
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The phenotype of the ear microvessels was determined by examining the venule-specific endothelial cell markers P-selectin and von Willebrand factor (vWF) (23) and the pericyte marker desmin. In K14-Ang1 mice we found strong immunoreactivity for P-selectin and vWF in venules and in vessels in the anatomic position of capillaries, but in wild-type mice we found strong immunoreactivity for these markers only in venules. Further, the pericytes of these vessels in K14-Ang1 mice had the highly branched morphology of venules instead of the elongated morphology of capillaries.
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0342855129
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2), whereas the average capillary diameter was normal (8.7 ± 0.1 μm) due to averaging unusually large and small vessels. Supplementary material is available at www.sciencemag.org/feature/data/1043334.shl
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0343290106
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An inflammatory agent, mustard oil or serotonin, known to cause plasma leakage in mouse skin (9, 24), was applied to the ear 1 min after injection of Evans blue dye. Mustard oil (Sigma) was diluted to 5% in mineral oil and applied to the dorsal and ventral surfaces of the ear with a cotton tip; it was reapplied 15 min later. Nothing was done to the other ear of mice treated with mustard oil (baseline control). Serotonin (Sigma) was dissolved at 0.22 mg/ml in sterile 0.9% NaCl plus 0.005 N acetic acid (vehicle). About 10 μl was injected (28G needle) intradermally into the dorsal ear skin, and a similar volume of vehicle was injected into the other ear (control). Thirty minutes after the stimulus, the vasculature was perfusion-fixed (1% paraformaldehyde in 50 mM citrate buffer, pH 3.5) for 2 min. Ears were removed, blotted dry, and weighed. Evans blue was extracted from the ears with 1 ml of formamide overnight at 55°C and measured spectrophotometrically at 610 nm.
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Injection of PAF (80 μg/ml) in the ear skin of wild-type mice increased Evans blue content to 41.2 ± 10.0 ng/mg, whereas in K14-Ang1 mice the amount was 23.2 ± 4.1 ng/mg (n = 3).
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0342855124
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Because ricin binds more avidly to components of the basement membrane (25) than to the endothelial cell surface, it can be used to identify the number and location of sites of exposed basement membrane (26). Perfusion of ricin after fixation helps to eliminate the role of blood flow and pressure.
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We found fewer ricin-labeled leaky vessels in double transgenic K14-Ang1/VEGF mice than in K14-VEGF mice after treatment with mustard oil. This difference corresponded to the difference in the amount of Evans blue leakage. The few leaky sites in inflamed ears of K14-Ang1/VEGF mice were located in small venules and in capillary-like vessels, similar to the location of leaky vessels in K14-VEGF mice.
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0343290097
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Supported in part by NIH grants HL-59157 and HL-24136. We thank P. Baluk and J. W. McLean for helpful discussions; M. Simmons, T. Murphy, and N. Glazer for technical assistance; and C. Murphy and E. Burrows for help with graphics.
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