Karyotypes of HMVECs exposed to hypoxia.

<p>Karyotypes of HMVECs after exposure to normoxia, hypoxia (1% O2), and hypoxia-reoxygenation were analyzed by Q band analysis. A karyotype of one cell in each condition is shown. (A) HMVECs in normoxia were essentially diploid. (B, C) HMVECs exposed to hypoxia and hypoxia-reoxygenation had complex abnormal karyotypes and were aneuploid. (D) The chromosome number of each condition was counted and shown.</p

Hypoxia induces aneuploidy in HMVECs.

<p>(A, B) After HMVECs were cultured in each condition for 7 days, FISH analysis using a chromosome-7 probe revealed that approximately 22.8% ECs in the hypoxic condition (1% O2) and 19.8% ECs in the hypoxia-reoxygenation condition were aneuploid, whereas 6.4% ECs in the normoxic condition were aneuploid. (C, D) FISH analysis using a chromosome-8 probe revealed that approximately 19.1% ECs in the hypoxic condition and 23.3% of ECs in the hypoxia-reoxygenation condition were aneuploid, whereas 8.5% ECs in the normoxic condition were aneuploid.</p

Tumor endothelial cell (TEC) characteristics.

<p>(A) Cultured NECs and TECs are positive for CD31, CD105, CD144, VEGFR1, and VEGFR2 and negative for CD11b and CD45. Human HB-EGF expression is shown in CD31 negative cells and human tumor cells (Melanoma and Oral carcinoma: Oral-Ca) using a human primer, but not in TECs. Abbreviation: NCT = Negative Control Template. (B) TECs showed a higher proliferation rate compared with NECs. (C) Expression levels of the angiogenesis-related gene VEGF in ECs by real-time PCR. Expression levels of VEGF were higher in TECs than in NECs. (D) Cultured ECs were stained with FITC-conjugated BS1-B4 lectin, and FISH was performed using a Cy3-mouse chromosome-17 locus-specific BAC probe. Red, chromosome 17; green, BS1-B4. FISH revealed aneuploidy in 42% melanoma ECs, 40.2% oral carcinoma ECs, and 7.9% NECs. Freshly isolated and noncultured TECs were cytospun onto a glass slide and immunostained for CD31 (green) and subjected to FISH. Aneuploidy was observed in 35.6% melanoma ECs, 35.0% oral carcinoma ECs, and 8.7% NECs. Scale bar, 10 μm. Experiments were performed in triplicate. *<i>p</i> < 0.01.</p

Hypoxia elevates ROS production and VEGF signaling increases aneuploidy in ECs exposed to hypoxia.

<div><p>(A)Cellular ROS in HMVECs were analyzed after exposure to normoxia, hypoxia (1% O2), and hypoxia-reoxygenation for 7 days. The experiment was repeated three times. Representative data is shown. (B) The ROS inducer pyocyanin increased EC aneuploidy in a concentration-dependent manner. (C) Inhibition of RO.</p> <p>(B)S by the ROS scavenger N-acetyl-cysteine (NAC) decreased hypoxia-induced aneuploidy in ECs. (D) Involvement of VEGF signaling in ROS production. Hypoxia-stimulated HMVECs reduced ROS production in the presence of the VEGFR inhibitor Ki8757. The experiment was repeated three times. Representative data is shown. (E) Aneuploidy induced by the hypoxic condition in HMVECs was significantly suppressed by Ki8751. *<i>p</i> < 0.05.</p></div

Association between cell cycle and EC aneuploidy in HMVECs exposed to hypoxia.

<p>(A) Cell number and the cell cycle in HMVECs exposed to hypoxia were analyzed by flow cytometry after staining fixed cells with propidium iodide. In each phase, cultured ECs proliferated for 7 days (normoxia, 2.5-fold; hypoxia (1% O₂), 1.6-fold; hypoxia-reoxygenation, 2.4-fold versus Day 0 cell numbers). (B) We analyzed the distribution of ECs throughout the cell cycle by flow cytometry. There was no significant difference in cell-cycle distribution between conditions of normoxia and hypoxia. The experiment was repeated three times, and representative data is shown.</p

Hypoxia induces HIF-1α and VEGF-A expression in ECs.

<p>(A) The hypoxic area in human tumor xenografts in nude mice was analyzed using the hypoxia marker pimonidazole and CA IX. Tumor tissues were double-stained with anti-CD31 (red) and anti-pimonidazole antibodies or anti-CA IX (green) to visualize hypoxic areas. Pimonidazole staining revealed that tumor vessels were exposed to hypoxia to some extent. Scale bar, 100 μm. (B) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. (C) HMVECs were cultured and treated for 8 h under normoxia or hypoxia. HIF-1α protein was upregulated 8 h after hypoxia, as revealed by western blotting. Densitometry analysis revealed that HIF-1α was induced by hypoxia. The experiment was repeated three times. Representative data is shown. (D) mRNA levels of VEGF-A were significantly increased by hypoxia in HMVECs. Experiments were performed in triplicate. *p < 0.01.</p

Assessment of tumor tissue hypoxia and analysis of ploidy in TECs by FISH Hypoxic areas in supermetastatic tumor cryosections were detected using hypoxyprobe.

<p>To analyze the hypoxic area in TECs in in-vivo tumors, frozen sections of tumors were immunostained with anti-CD31 (red) and anti-pimonidazole (green) followed by FISH using a spectrum red-conjugated mouse chromosome-17 locus-specific probe (red spot). Representative CD31-positive ECs in tumor tissue. (A, D, G) The blood vessels in tumor tissue were pimonidazole-positive. (B, E, H) Nuclei are stained with DAPI (blue). Aneuploidy was also observed in ECs of tumor blood vessels exposed to hypoxia. (C, F, I) Pimonidazole-nagative ECs in tumor tissue did not exhibit aneuploidy. (J, K, L) ECs in normal tissue did not exhibit aneuploidy. (M, N, O) Scale bar, 20 μm.</p

LY294002 inhibited TMV-promoted random motility and tube formation in NEC.

<p>(A) When treated by the PI3K inhibitor LY294002, cell motility was not stimulated by TMV (50 µg/ml) anymore. The results are presented as mean velocities ± SE (DMSO; n = 15, DMSO+TMV; n = 15, LY294002; n = 15, LY294002+TMV; n = 15). *<i>P</i><0.01. (B) In addition, LY294002 suppressed increase of tube formation in NEC treated by TMV (50 µg/ml). The results are presented as mean tube length per field ± SE. (DMSO; n = 10, DMSO+TMV; n = 10, LY294002; n = 10, LY294002+TMV; n = 10). *<i>P</i><0.01. These results indicate that TMV-induced proangiogenic phenotype is mediated by PI3K/Akt pathway, at least in part.</p

Characterization of isolated normal endothelial cells (NEC) from mice.

<p>(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.</p

TMV promote random motility and tube formation in NEC.

<p>Time-lapse observation revealed that TMV promote random motility in NEC. When treated by the endocytosis inhibitor dynasore, cell motility was not stimulated anymore. (A) Migrated trajectories of TMV-treated NEC (with/without 50 µM dynasore) were plotted and velocity was calculated using ImageJ. (B) The results are presented as mean velocities ± SE (DMSO; n = 15, DMSO+TMV; n = 15, dynasore; n = 15, dynasore+TMV; n = 15). *<i>P</i><0.01. TMV also promote tube formation in NEC. Dynasore canceled increase of tube formation even in NEC cultured with TMV. (C) Phase-contrast images of control and TMV-treated NEC (with/without 50 µM dynasore) cultured on a matrigel. Bar = 100 µm. Capillary-like structure enhanced with TMV. Total length of capillary-like tubes was analyzed using ImageJ. (D) The results are presented as mean tube length per field ± SE. (DMSO; n = 10, TMV; n = 10, dynasore; n = 10, dynasore+TMV; n = 10). *<i>P</i><0.01.</p