Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal (neovascularizationyvessel regressionyhemorrhageytetracycline-inducible expression)

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously ‘‘on,’’ tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis. It is well established that a rate-limiting step in solid tumor growth is the ability to recruit blood vessels from the host tissue (for a review, see ref. 1). Angiogenesis has become a major target for antitumor therapy on the premise that limiting angiogenesis will retard tumor growth and will inhibit metastatic spread of tumor cells. The tumor ‘‘angiogenic switch’’ is determined by the net balance of angiogenic stimulators and natural inhibitors of (2, 3). VEGF is a potent angiogenic factor mediating developmental, physiological, and pathological neovascularization. In the context of tumor angiogenesis, VEGF expression is upregulated as a consequence of oncogene activation or a loss of tumor suppressor (4–8). In addition to a genetic angiogenic switch, VEGF expression is induced in tumors by hypoxia andyor hypoglycemia generated whenever the angiogenic response is insufficient and vascular growth is lagging behind tumor growth (9–11). Augmented VEGF expression is correlated with increased tumor growth and vascularity (12, 13) and the inhibition of VEGF production or function leads to inhibition of tumor growth (14–17). Thus, it appears that VEGF plays a key role in the promotion of tumor angiogenesis. In the present study we examined whether the level of VEGF produced by tumor cells may determine the size and shapes of tumor vessels. In particular, we examined the thesis that excessive production of VEGF may lead to formation of hyperfused vessels, such as those found in hemangioblastomas. Growth factors, in general, may also function as survival factors for the respective target cell (18, 19). We have recently shown that VEGF is required for the maintenance of newly formed blood vessels in a natural developmental setting of retina neovascularization (20). Importantly, dependence on VEGF for survival is transient and, upon their maturation, vessels switch to a VEGF-independent state. Tumor expansion is associated with a continuous formation of new vessels and remodeling of existing vessels. We reasoned that these vessels may be sensitive to loss of VEGF resulting in regression of the existing tumor vasculature. To test this hypothesis, we constructed an inducible VEGF expression system in a xenografted C6 glioma tumor. We show that regression of preformed tumor vessels can be induced by VEGF withdrawal. The finding that VEGF is required for the maintenance of immatureyremodeling tumor vessels may be exploited to increase the efficiency of anti-angiogenesis tumor therapy.