α_v Integrin is expressed on intestinal ECs after allo-HCT and can be targeted by cilengitide. Allo-HCT was performed as described in the “Methods” section. (A) Intestinal CD34⁺ ECs of mice euthanized on day 14 after allo-HCT express high levels of α_v integrin as shown for 1 representative fluorescence-activated cell sorter histogram (left panel). The experiment was repeated 3 times with at least 3 animals resulting in a total of 11 analyzed mice. (B) PET imaging of mice on day 22 after allo-HCT with previous cilengitide or PBS treatment. The signal intensity of [⁶⁸Ga]NODAGA-c(RGDfK) was significantly reduced in animals pretreated with cilengitide (P = .001) as shown for 1 representative mouse (left panel) and when quantified (right panel). The experiment was performed once with 3 animals per group. (C) Small and large bowels were isolated on day 14 after allo-HCT from the indicated groups and analyzed for the number of CD34⁺ ECs by histology. Cilengitide treatment significantly reduced the number of CD34⁺ ECs in the small (P = .0001) and large intestines (P = .001) as compared with PBS-treated animals. The experiment was performed 3 times with 3 mice in each group (animals were pooled: each data point represents an individual animal). (D) The complete ITs of transplanted mice were digested and analyzed on day 14 after allo-HCT for the number of CD34⁺ ECs using flow cytometry. Cilengitide treatment reduced the number of CD34⁺ ECs in the total IT. Three independent experiments (at least 3 animals per group/experiment) were pooled; each data point represents an individual animal. (E) Small bowel was isolated on day 14 after allo-HCT from the indicated groups and analyzed for the number of CD31⁺ ECs by immunofluorescence staining. Ten sections per sample were analyzed, and vessel density was assessed by quantification of CD31⁺ area/total area ± SD (n = 3 per group). One representative picture from each group is shown. (F) Transplanted BALB/c recipients were treated with pimonidazole HCl on day 7 after allo-HCT for 1 hour. Small and large bowels were isolated and analyzed for the abundance of hypoxia by peroxidase staining. Three sections per sample were analyzed, and hypoxia was assessed by quantification of pimonidazole adducts⁺ cells/per high power field ± SD (n = 7 per group). One representative picture from each group is shown. Two independent experiments (at least 3 animals per group per experiment) were pooled.