MS# G99-306/RRR

Expression of Angiopoietin-1 in Human Glioblastomas Regulates Tumor-Induced Angiogenesis: In Vivo and In Vitro Studies.

Enrica Audero1,2, Ilaria Cascone1,2, Ilaria Zanon1,2, Stefano C. Previtali3, Roberto Piva4, Davide Schiffer4 and Federico Bussolino1,2

MATERIAL TO BE PUBLISHED ON LINE

METHODS

Quantitative Analysis

To quantify microvessels or EC clusters (more than 3 cells) expressing Ang1, Ang2 or CD31, the most highly vascularized areas of each specimen were identified by scanning the CD31-stained sections at low power (10x). Individual microvessels expressing CD31 were counted in three high-power fields (40x). Ang1 or Ang2 expression was quantified by counting positive microvessels or EC clusters in the same regions where CD31 expression was determined. The number of non-EC (CD31 negative) expressing Ang1 and Ang2 was evaluated in eight high-power fields (40x). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test (Statistic Software, Bio-Soft).

RNA extraction and RT-PCR

Total cellular RNA from cell cultures was isolated using the RNAzol B Reagent (Tel-Test, Inc., Friendswood, Texas). Reverse transcription (RT) was performed on 10 ml reaction mixture containing 1 mg of total RNA, isolated using RNAzol B Reagent (Tel-Test, Inc., Friendswood, Texas), and 2.5 mM oligodeoxythymidine (Perkin Elmer, Branchburg, New Jersey) at 65°C for 10 minutes. After cooling, 20 units of ribonuclease inhibitor (Perkin Elmer) and 50 units of Moloney’s murine leukemia virus reverse transcriptase (Perkin Elmer) were added in a final 20 ml reaction mixture containing 1 mM each dNTP (Amersham Pharmacia Biotech AB, Uppsala, Sweden), incubated for 15 minutes at 42°C, heated 5 minutes at 95°C and diluted to 50 ml. For the polymerase chain reaction (PCR), a 50 ml reaction containing 5 ml of cDNA-RNA hybrids, 200 mM each dNTP, 0.2 mM each oligonucleotide primer, and 1.25 units of Ampli Taq Gold polymerase (Perkin Elmer) was subjected to the following cycles: 30 s of denaturing at 95°C, 30 seconds of annealing at 60°C (b-actin), 55°C (VEGF-A) or 52°C (Ang1 and Ang2), and 1 minute of extension at 72°C. After 40 cycles, an extension step was carried out for 7 minutes. The primers used for amplification were specified in the legend to Figure III.

Immunoprecipitation and Immunoblotting

To test recombinant Ang1 activity, confluent HUVECs were made quiescent by 20 hours starvation in M199 0.5% FCS and then stimulated for 10 minutes at 37 °C with 200 ng/ml Ang1 alone or pre-incubated overnight at 4°C with the polyclonal anti-Ang1 antibody (1:50). Cells lysates (1.5 mg proteins) were incubated with protein-A sepharose (Sigma) and a rabbit polyclonal antibody against the C-terminus peptide of human Tie2 (Santa Cruz) for 2 hours at 4°C. Solubilized proteins were separated by SDS-PAGE, and immunoblotted with anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY) or anti-Tie2 antibodies.


LEGEND FOR ON-LINE FIGURE

Figure I

Immunohistochemical staining of low-grade astrocytomas (A-D,F-H) and normal brain (E) for CD31 (A,F), Ang1 (B,D,E) and Ang2 (G). In F black arrows indicate EC of blood vessels stained with CD31. In G arrows indicate blood vessels (black) and astrocytes (red) stained with Ang2. In the insert a positive astrocyte is shown at higher magnification. C and H are negative control showing respectively lack of staining of Ang1 and Ang2 antibodies pre-absorbed with the specific immunizing peptides. Panels A,B,C and F,G,H show 5 mm adjacent sections. Scale bar: 20 mm. Magnification: 40x for A-C, F-H; 100x for D,E and insert in G.

Figure II

Immunohistochemical staining of anaplastic astrocytomas 5 mm adiacent sections for CD31 (A,C), Ang1 (B) and Ang2 (D). In D arrows indicate tumor cells (red) and blood vessels (black) stained with Ang2. Scale bar: 20 mm. Magnification: 40x.

Figure III

RT-PCR demonstrating expression of Ang1, Ang2 and VEGF-A mRNA in endothelial (HUVEC, HMEC) and glioblastoma (ADF, DF, LI, U87, U373) cell lines. Amplification of b-actin demonstrates comparable RNA amount and quality among samples. Sizes of predicted PCR products are indicated by arrows. L, DNA ladder, bp, base pair. For amplification the following primers were used: Ang1 GCCTCCTCTCTCAGACTGCAG (pos. 706-727) and CATACTGTGAATAGCCTCGG ( pos. 1457-1477) (PCR product of 771 base pair); Ang2 CTGAAGAAAGA ATGTGGCAG (pos. 339-358) and TGGTCTGGTCCAAAATCTGT (pos. 865-884) (545 base pair); VEGF-A CATGAACTTTCTGCTGTCTTGG (pos. 56-77) and TCACC GCCTCGGCTTGTCACAT (pos. 611-632) (PCR products of 452, 584, 656 base pair corresponding to the VEGF-A isoforms 121, 165, 189); b-actin TGACGGGGT CACCCACACTGTGCCCATCTA (pos. 1038-1067) and CTAGAAGCATTTGCGGTGG ACGATGGAGGG (pos. 1876-1905) (661 base pair).

Figure IV

Activation of Tie2 receptor by recombinant Ang1 in EC. Tie2 was immunoprecipitated from lysate of stimulated and unstimulated HUVECs and proteins were separated by SDS-PAGE. Blot was stained with anti-phosphotyrosine mAb or with Tie2 polyclonal antibody and bands were visualized by chemioluminescence technique (NEN Life Science Products, Inc.). The pre-incubation of Ang1 by non-immune rabbit serum did not neutralize the observed effect on Tie-2 phosphorylation. This experiment is representative of three experiments performed with three different preparation of recombinant Ang1.