GB appears as a nodular structure with a centrally placed lipid-containing cell (lip) and comprised of several different cell types: E, endothelial cells; P, pericytes; lip, lipid-containing cell with a cytoplasmic lipid body, LB; MO, monocyte; F, fibroblasts. also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid body severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF164 expression declined, glomeruloid body devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid body and indicate that VPF/VEGF164 is sufficient for their induction and necessary for their maintenance. Glomeruloid body (GBs) are structures that form in a number of different types of tumors and malformations and are so named because of their resemblance to renal glomeruli. 1 They are of two general types depending on whether they are primarily epithelial or vascular in nature. 1,2 Vascular GBs, the subject of this statement, are one of the defining histological characteristics Col13a1 of glioblastoma multiforme brain tumors 2,3 and are found, although less generally, in gastrointestinal carcinomas 4 and thymomas; 5 they have also been explained in cutaneous vascular tumors and malformations. 6,7 Vascular GBs have not been well characterized and their pathogenesis is usually primarily unknown. To elucidate the actions and mechanisms of pathological and physiological angiogenesis, we recently designed adenoviral vectors to express angiogenic cytokines and have used these as vehicles for expressing these cytokines in mice and rats. 1 Because of its prominent role in both angiogenesis and vasculogenesis, we selected vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) as the cytokine for initial study 1,8,9 In the course of these studies, we noted that common GB created in romantic association with infected cells that expressed VPF/VEGF. 1 Therefore, this system provided an excellent animal model for investigating the pathogenesis of GB formation. The present study was undertaken to elucidate the origins, composition, and fate of VPF/VEGF164-induced vascular GBs. Materials and Methods Animals and Adenoviral Vectors Four- to 6-week-old female athymic nude mice on two backgrounds were utilized for these studies with equivalent results: BALB/c ByJ hfhHybridization Tissues were fixed in RNase-free 4% paraformaldehyde in PBS, pH 7.4, for 4 hours at 4C and were transferred to 30% sucrose in PBS overnight at 4C before embedding in OCT compound. Cryostat sections were hybridized with antisense and sense Mitochonic acid 5 (control), single-stranded, 35S-labeled RNA probes to murine VPF/VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, Tie-1, and Tie-2 as previously explained. 32,33 Double Staining and Confocal Imaging One hundred-m cryostat sections were fixed in 100% acetone at 4C for 20 moments and rehydrated in PBS made up of 0.2% Tween 40. Sections were blocked with normal goat serum made up of 2% 3-omega fatty acid for 1 hour at room temperature. Sections were then incubated for 2 hours with rabbit anti-mouse NG2, rinsed, and incubated with a second main antibody, biotinylated rat anti-mouse CD31, for 2 hours. After rinsing, sections were incubated with avidin-coupled fluorescein isothiocyanate (FITC) for 2 hours. Sections were then Mitochonic acid 5 washed 5 in distilled water and mounted with Vectashield (Vector Laboratories). Perfusion Studies To determine whether the GBs that experienced created in mouse ears were perfused with blood, mice were injected intravenously with 4 mg of TRITC-dextran (MW, 70,000) and 4 mg of FITC-dextran (MW, 2,000,000) in 0.9% NaCl. Fifteen minutes later mice were sacrificed, ears were fixed in a 7:3 (vol:vol) mixture of complete ethanol and 10% formalin for 4 hours at room temperature, and were then processed for paraffin embedding. 12 Forty-micron optical sections were evaluated in a Bio-Rad MRC-1024 confocal microscope equipped with an argon/krypton laser. Sections were digitized, filtered with edge definition and median filters, and viewed as compiled images. Alternatively, anesthetized mice were perfused through the left ventricle with heparin-saline and then with 20 ml of a 1:4 dilution of Sumi black Mitochonic acid 5 ink (Yasutomo and Co.,.
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