Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF

Science

Volumn 284, Issue 5422, 1999, Pages 1994-1998

  Holash, J ^a   Maisonpierre, P C ^a   Compton, D ^a   Boland, P ^a   Alexander, C R ^a   Zagzag, D ^b   Yancopoulos, G D ^a   Wiegand, S J ^a  

^a REGENERON PHARMACEUTICALS INC   (United States)

^b NEW YORK UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

VASCULOTROPIN;

ANGIOGENESIS; ARTICLE; METASTASIS; NEOVASCULARIZATION (PATHOLOGY); PRIORITY JOURNAL; PROTEIN EXPRESSION;

ADENOCARCINOMA; ANGIOPOIETIN-1; ANGIOPOIETIN-2; ANIMALS; APOPTOSIS; BLOOD VESSELS; ENDOTHELIAL GROWTH FACTORS; ENDOTHELIUM, VASCULAR; GLIOBLASTOMA; GLIOMA; IN SITU HYBRIDIZATION; LYMPHOKINES; MALE; MEMBRANE GLYCOPROTEINS; MICE; MICE, INBRED C57BL; MUSCLE, SMOOTH, VASCULAR; NEOPLASM TRANSPLANTATION; NEOPLASMS, EXPERIMENTAL; NEOVASCULARIZATION, PATHOLOGIC; PROTEINS; RATS; RATS, SPRAGUE-DAWLEY; UP-REGULATION; VASCULAR ENDOTHELIAL GROWTH FACTOR A; VASCULAR ENDOTHELIAL GROWTH FACTORS;

EID: 0033580889     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5422.1994     Document Type: Article

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47. Animals were anesthetized and either decapitated or perfused with 4% paraformaldehyde. Brains for thick sliding microtome sections (40 μm) were post-fixed in 4% paraformaldehyde overnight and then equilibrated in 35% sucrose. Fixed brains for thin sections (10 μm) were equilibrated in 17% sucrose. Fixed and fresh brains were frozen in methylbutane, chilled in dry ice, and sectioned on a cryostat For TUNEL labeling (25), brains were immersion-fixed in 10% neutral buffered formalin and embedded in paraffin. Fixed sections were immunostained with a monoclonal antibody to rat endothelial cell antigen (RECA1; 1:250; Serotec) and a biotinylated horse anti-mouse secondary antibody (1:1500; Vector) or with a monoclonal antibody to PECAM (CD31; 1:100; Pharmingen) and a biotinylated rabbit anti-rat secondary antibody (1:150; Vector) as previously described (26). A similar protocol was used for double labeling. Sections were initially labeled with a monoclonal antibody to alpha smooth muscle actin (SMA; 1:500; DAKO) and a biotinylated goat antimouse immunoglobulin C IIa secondary antibody (1:1250; Amersham). SMA staining was visualized with a Vectastain Elite kit (Vector), and a black reaction product was generated by nickel sulfate enhancement After SMA labeling, sections were then reblocked and labeled with antibody to RECA (1:100). A brown reaction product was used.
48. 4 cells were plated in 96-well microwells and grown for 24 hours in basal medium plus 0.5% fetal bovine serum. Cells were re-fed with the same medium plus purified factors and grown for 20 hours, with 1 mCi tritiated thymidine (80 Ci/mmol; Amersham) being present for the last 3 hours of incubation. Cells were rinsed and fixed with trichloroacetic acid, and thymidine incorporation was measured by standard liquid scintillation techniques. Ang-1* (ANG1*) was a modified form of human Ang-1, described previously (9); VEGF was murine VEGF-164, produced and purified from baculovirus-infected insect cells; bFGF was human basic fibroblast growth factor (R&D Systems). For assessing resistance to apoptosis, plates of ∼80% confluent HUVECs were rinsed twice with basal medium and grown for 18 to 20 hours in basal medium and bovine serum albumin (0.5 mg/ml). plus or minus purified factors. Both adherent and non-adherent cells were harvested, pooled, and fixed in 70% ethanol at -20°C overnight. Cells were washed in PBS, incubated for 30 min with ribonuclease A (5 kunitz units/ml; Sigma) and propidium iodide (50 (μg/ml: Sigma). Cellular DNA content, as judged by propidium iodide fluorescence, was measured by flow cytometry (MoFlo, Cytomation, Fort Collins, CO).
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50. 35S-labeled cRNAs (27). Probes for VEGF and Ang-1 and Ang-2 have been described (9). For Tie1, a 1.3-kb fragment of rat Tie1 spanning the last 309 codons and 375 base pairs of the 3′ untranslated sequence was used, and for Tie2 a 460-base pair fragment spanning codons 771 through 924 within the kinase domain was used. This probe does not cross-hybridize to Tiel mRNA in Northern blots.
51. We thank B. Luan, J. Zheng, P. Burfeind, S. Zabski, and F. Martin for excellent technical assistance; E. Burrows and C, Murphy for graphics work; A. Hooper and D. Friedlander for data on human gliomas; and M. Grumet for intellectual discussions. Supported in part by a grant from the Children's Brain Tumor Foundation to D.Z. and by Procter & Gamble Pharmaceuticals, Inc. All animal studies were done in accordance with institutional guidelines.