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. 2000 Oct;20(19):7282-91.
doi: 10.1128/MCB.20.19.7282-7291.2000.

Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature

J Grunstein  1 , J J Masbad, R Hickey, F Giordano, R S Johnson
Affiliations

Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature

J Grunstein et al. Mol Cell Biol. 2000 Oct.

Abstract

Vascular endothelial growth factor (VEGF) is an essential regulator of vascularization. It is expressed as several splice variants; the major forms contain 120 amino acids, 164 amino acids, and 188 amino acids. We utilized transformed cells nullizygous for VEGF to specifically express each of these isoforms in isolation, in order to determine the role of each in tumorigenic neo-vascularization. We found that only the intermediate isoform, VEGF164, could fully rescue tumor growth; VEGF120 partially rescued tumor growth, and VEGF188 failed completely to rescue tumor expansion. Surprisingly, the vascular density of VEGF188 isoform-expressing tumors is significantly greater than that of wild-type VEGF cells and the other isoform-specific tumors. The failure of the hypervascular VEGF188-expressing tumors to grow may be due to inadequate perfusion of the massive number of microvessels in these tumors; three-dimensional imaging of the tumorigenic vasculature indicated little or no recruitment of the peripheral vasculature. This demonstrates that the VEGF isoforms perform unique functions which together enable tumorigenic vascularization.

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Figures

FIG. 1
FIG. 1
Expression levels in VEGF isoform-specific cell lines. (a) Northern blot analysis from retrovirally infected cell lines. Note the similar levels of isoform-specific RNA (5.0 to 5.2 kb). The cellular VEGF RNA (3.6 kb) is slightly smaller in the VEGF-null cell lines compared to that of wild type (WT) (3.7 kb) due to the absence of exon 2. The RNA filter was simultaneously probed with a fragment of GAPDH to control for gel loading differences. (b) Comparison of VEGF protein levels in conditioned medium from cell lines infected with isoform expressing adenoviruses. Cell lines were metabolically labeled and treated with heparin (100 μg/ml). (c) Comparison of cell growth rates of retrovirally infected, isoform-expressing cell lines and controls in tissue culture. (d) Northern blot analysis of neuropilin-1 (NP-1) in VEGF isoform-specific cell lines indicates similar expression levels of this receptor in each cell line used to generate tumors.
FIG. 2
FIG. 2
Tumorigenesis assay of VEGF isoform-specific cell lines. Shown is a comparison of the masses of fibrosarcomas generated from isoform-specific, stable cell lines and controls. A total of 107 cells in 100 μl of DMEM were injected subcutaneously, intracapularly into immunocompromised mice. Tumors were harvested and weighed 16 days postinjection (n = 9 animals per cell line). Error bars indicate 1 standard error.
FIG. 3
FIG. 3
Immunostaining of vascular endothelium from VEGF isoform-specific fibrosarcomas. (a) Representative anti-CD31 immunohistology of tumor cryosections from wild-type transformed fibroblasts. (b) The same cells following cre-mediated excision of the VEGF gene. (c) VEGF120-expressing tumors. (d) VEGF164-expressing tumors. (e) VEGF-188 expressing tumors. Magnification, ×100.
FIG. 4
FIG. 4
Microvessel density quantification by Chalkley analysis. For each tumor type, 10 representative fields from each of 3 individual tumors were scored for the maximum overlap of stained vessels with the random spot array on the Chalkley graticule. Error bars indicate 1 standard error.
FIG. 5
FIG. 5
VEGF immunostaining from paraffin-embedded tumor sections demonstrates relative local concentration of VEGF isoforms. (a) Wild-type control; (b) VEGF-deficient control; (c) VEGF120; (d) VEGF164; (e) VEGF188. Fluorescein isothiocyanate-conjugated secondary Ab recognizes anti-VEGF Ab-3 (JH121). Nuclear counterstain is Hoechst 33342 (blue). Magnification, ×400. Note the high degree of cell-associated VEGF staining in VEGF188-expressing tumors compared to the strictly vessel-associated staining found in VEGF120-expressing tumors.
FIG. 6
FIG. 6
Macroscopic view of tumor vasculature and surrounding stroma. Perfusion of tumor-bearing mice with liquid latex (Microfil) compound results in a three-dimensional cast of major vessels and microvessels. (a and b) Wild-type control tumor at magnifications of ×7 and ×20. (c and d) VEGF-null tumor at similar magnifications. (e and f) VEGF120. Note the extreme degree of host vasculature localized to the periphery of the tumor (arrows) and poor infiltration, coincident with the low local concentration of VEGF in the tumor. (g and h) VEGF164. Note the high degree of systemic vasculature directed to the tumor as well as significant microvasculature. (i and j) VEGF188. Note the presence of fewer systemic vessels directed to the tumor yet many microvessels within the tumor itself.
FIG. 7
FIG. 7
Tumor microenvironment selects for a gradient of VEGF expression. (a) Tumor masses resulting from subcutaneous injection of cell lines composed of every combination of isoform-specific cell lines. Note that there is a slight growth advantage to tumors which express all isoforms. (b) Southern blot analysis of isoform-specific VEGF levels in cell lines harvested from representative tumors in panel a. Input lanes represent the equal contribution from each cell line prior to tumor formation. Note the competitive advantage of VEGF164-expressing cells and the decrease in selective pressure in tumors expressing only VEGF120 and VEGF188, indicating that the tumor requires both soluble and cell-associated VEGF.
FIG. 8
FIG. 8
Proposed gradient model of tumorigenic VEGF signaling. VEGF120 produces a diffuse signal (light blue) which recruits peripheral vessels but does little to vascularize the tumor itself. VEGF164 can both recruit vessels with a partially diffusible signal and vascularize the tumor with a partially cell-associated signal. VEGF188 fails to adequately recruit the host vasculature, but vascular endothelium which is captured forms a hypervascular capillary network due to the high local concentration of VEGF.
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