Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis

Background: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of Apc(Min/+) mice and Apc(Min/+)/Vash2(-/-) mice. Methods: Apc(Min/+) mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. Apc(Min/+) mice were mated with Vash2(-/-) mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2(-/-) gene by PCR. Intestinal tumors from Apc(Min/+) mice and Apc(Min/+)/Vash2(-/-) mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. Results: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in Apc(Min/+) mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2(-/-) mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2(-/-) mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. Conclusion: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.