Bioavailability of VEGF in tumor-shed vesicles depends on vesicle burst induced by acidic pH - PubMed (original) (raw)

Bioavailability of VEGF in tumor-shed vesicles depends on vesicle burst induced by acidic pH

Giulia Taraboletti et al. Neoplasia. 2006 Feb.

Abstract

Tumor angiogenesis is regulated by a dynamic cross-talk between tumor cells and the host microenvironment. Because membrane vesicles shed by tumor cells are known to mediate several tumor-host interactions, we determined whether vesicles might also stimulate angiogenesis. Vesicles shed by human ovarian carcinoma cell lines CABA I and A2780 stimulated the motility and invasiveness of endothelial cells in vitro. Enzyme-linked immunosorbent assay and Western blot analysis revealed relevant amounts of vascular endothelial growth factor (VEGF) and the two matrix metalloproteinases MMP-2 and MMP-9, but not fibroblast growth factor-2, contained in shed vesicles. An A2780 cell-derived clone transfected to overexpress VEGF shed the same amount of vesicles as did a control clone, but contained significantly more VEGF within the vesicles. Despite a greater amount of VEGF in vesicles of the overexpressing clone, vesicles of both clones stimulated endothelial cell motility to comparable levels, suggesting that VEGF was stored within the vesicle and was unavailable. Only following vesicle burst induced by acidic pH (a characteristic of the tumor microenvironment) was VEGF released, leading to significantly higher stimulation of cell motility. Thus, tumor-shed membrane vesicles carry VEGF and release it in a bioactive form in conditions typical of the tumor microenvironment.

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Figures

Figure 1

Figure 1

Scanning electron micrograph of CABA I (A) and A2780 (B) cell vesicle shedding.

Figure 2

Figure 2

Effect of tumor cell-shed vesicles on endothelial cell motility and invasiveness. HUVEC motility (A and C) was tested in the Boyden chamber using isolated vesicles shed by human ovarian carcinoma cells CABA I (A; triangles) and A2780 (C; triangles) used as attractants. VEGF (10 ng/ml; squares) was used as a reference stimulus. In the invasion assay (B and D), HUVECs were stimulated by vesicles (5 µg) shed by CABA I (B) or A2780 (D), or by VEGF (10 ng/ml) used as a reference stimulus. Data (mean and SD of triplicates) represent the number of cells that migrated in 10 high-power fields (representative of two to four experiments). *P ≤ .05.

Figure 3

Figure 3

Molecular characterization of vesicles shed by human ovarian carcinoma cell lines and transfected variants. (A) ELISA analysis of VEGF in vesicles. (B) Western blot analysis of vesicle-associated VEGF. (C) Zymographic analysis of vesicle-associated gelatinases. (D) Reverse zymographic analysis of vesicle-associated TIMP-1 and TIMP-2. Experiments were conducted as described in Materials and Methods.

Figure 4

Figure 4

Stimulation of endothelial cell motility by shed vesicles. (A) Vesicles shed from 1A9-VAS-3 (circles) and 1A9-VS-1 (triangles) were isolated and tested for their ability to stimulate HUVEC motility (see Figure 2). (B) Vesicles isolated from 1A9-VS-1 cells were resuspended in either PBS (open triangles) or water (filled triangles), and, after correction of molarity, tested for motogenic activity. Data (mean and SD of triplicates) represent the number of cells that migrated in 10 high-power fields.

Figure 5

Figure 5

Effect of pH on vesicle integrity and activity. Freshly isolated 1A9-VS-1-derived vesicle pellets were resuspended in buffers at the indicated pH and analyzed by TEM with negative staining for changes in morphology (A–C) and motogenic activity (D). (A) Pelleted vesicles resuspended in PBS (pH 7.4), showing intact rounded vesicle structures. (B) Pelleted vesicles resuspended in buffer at pH 6.0, showing both intact vesicles (short arrow) and small membrane portions derived from broken vesicles (arrows). (C) Pelleted vesicles resuspended in buffer at pH 5.6, revealing no intact vesicles and only membrane fragments (arrows), often aggregated in amorphous masses (inset). (D) Chemotactic activity for HUVECs by vesicles derived from 1A9-VS-1 cells and resuspended in buffers at the indicated pH (neutralized before the assay). Data (mean and SD of triplicates) are expressed as migration index (see Materials and Methods) (representative of three experiments). *P ≤ .05. Scale bars, 1 µm (A–C).

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