To better define the biological role of VEGF in intestinal neoplasia, we generated transgenic mouse lines with augmented VEGF production from intestinal epithelium. We found that VEGF has potent effects in normal mucosa, inducing a thickened, hypercellular mucosa with recruitment of endothelial cells and myofibroblasts and augmented proliferation of the epithelium. Moreover, neoplasia of all stages was stimulated. The increase in microscopic neoplasia, well below the size of the typical angiogenic switch, suggests relatively direct effects of VEGF. For example, VEGF may foster proliferation in part through paracrine-stimulated supportive interactions between VEGF-secreting epithelium and recruited endothelial cells and fibroblasts. Proof of principle that proximity to endothelial cells per se can stimulate human colonocyte proliferation was obtained from organotypic cultures.
The results of our transgenic studies argue for a tumor-promoting role for the increased VEGF levels observed in sporadic human and mouse colon tumors (2
). Transgenic VEGF overexpression in skin and breast has also accelerated tumor development (13
). Conversely, targeted deletion of the VEGF gene in lung reduces lung epithelial cell proliferation during development (26
). Histopathological features overlapping those observed in vilVEGF mice are seen in some constitutional proliferative diseases of the gut. Polycystic kidney disease cysts afflict kidney and liver. The epithelium of cyst walls is proliferative and associated prominently with vessels and VEGF expression (27
), suggesting that VEGF may play a role. We did not observe kidney cysts in vilVEGF mice, but VEGF expression in this tissue may not have been high enough to yield a phenotype or may not be sufficient, at any level. Both vilVEGF-Min mice and humans with Juvenile Polyposis (JP) display intestinal tumors with cysts and prominent stroma. Deregulated BMP1-SMAD signaling underlies at least some JP (28
). Evidence has been obtained that the BMP pathway may directly regulate VEGF transcription (32
), suggesting that increased VEGF expression may occur in JP and experimental settings of compromised BMP signaling, contributing to their pathology. Mutation of smooth muscle actin causes a cystic expansion of intestinal epithelium in zebra fish (33
), underscoring the potential role for mesenchymal cells in such phenotypes.
VEGF receptors (e.g. Flk-1/KDR) are preferentially expressed on endothelial cells, where they act through paracrine and autocrine routes (34
). VEGF receptors have also been identified on some malignant cells (35
). In these cases, it has generally been assumed that an autocrine loop developed through genetic and/or epigenetic instability. We have not ruled out autocrine stimulation of intestinal epithelium by VEGF. However, the recruitment of endothelial cells and fibroblasts by VEGF in vivo and the ability of these cell types to stimulate proliferation of colonocytes in vitro are consistent with paracrine effects. Although emerging evidence indicates that truncating mutations in APC confer some genetic instability in early neoplastic and pre-neoplastic cells (37
), the proliferative stimulus mediated by VEGF in early neoplastic lesions and non-neoplastic tissue suggests function through normal pathways. Direct supportive interactions between gut epithelial cells and endothelial cells have been described in developing liver and pancreas and adult liver (39
Stimulation of intestinal neoplasia by VEGF suggests an oncogenic role in this tissue and validates VEGF as a target of tumor suppression by p16 (11
). Both the vilVEGF1-Min and p16-null-Min phenotypes show an increase in colon tumors and tumors with advanced features. A difference is the stronger stimulation of early neoplasia in the vilVEGF1-Min mice. A stronger increase in VEGF in early neoplasia of vilVEGF mice and/or differences in genetic background could contribute. Augmented VEGF was not sufficient to increase vascularity of vilVEGF1-Min tumors yet was still oncogenic.
It is unclear why only some crypts form cysts. Different expression levels of VEGF might be one factor. The cysts generally show greater investment with mesenchymal cells than non-cystic epithelia. These mesenchymal cells may alter epithelial cell fate through paracrine signaling and/or exert mechanical effects that inhibit epithelial cell migration to the lumen. Whereas the mesenchymal cells surrounding cysts express endothelial markers CD31 and CD34, fewer stained for vWF than those lining vessels. Contact with blood might shape differentiation of the vascular endothelial cells, while reciprocal interactions with epithelial cells may modify the differentiation program. Some endothelial cells may be locally derived, others recruited from the bone marrow (42
Cells of the cyst interior appear to be derived in part by delamination of cells lining the wall opposite the crypt. This pattern may reflect the normal developmental program of intestinal epithelial cells, as many are ultimately shed from the surface into the intestinal lumen. Alternatively, some cysts appear to be open on the end opposite the crypt, with invasion of mesenchymal cells. Cyst epithelial cells of the far wall show evidence for loss of epithelial differentiation, in the form of reduced expression of E-cadherin and claudin-7. Preliminary data showing a lack of distinct staining for vimentin (data not shown) do not support a mesenchymal transition (43
). The loss of differentiation in cysts may reflect local environmental influences, such as mechanical forces. On the other hand, similar changes were observed in some tumors of vilVEGF1-Min mice and may reflect an additional oncogenic effect of VEGF.
Our results have implications for VEGF-targeted therapy by directly demonstrating that VEGF drives growth of intestinal neoplasia. Moreover, VEGF's effects are evidently not confined to large tumors that require angiogenesis, because neoplasia of all sizes was stimulated. Given that normal intestine is highly vascular, it seems unlikely that oxygenation is limiting for formation and growth of microadenomas, although a role for enhanced delivery of oxygen, cells, and/or serum-borne factors from the submucosa is not excluded. Our results dovetail well with evidence that treatment of Min mice with VEGF receptor antagonists can reduce tumor burden (45
) and recent results from conditional deletion of the VEGF gene and administration of anti-VEGF antibodies (19
). These latter interventions blunted growth of tumors 1 mm or larger in diameter. Korsisaari et al. inferred that the angiogenic switch occurs earlier than generally thought in intestinal tumors. Our findings extend these studies by showing that VEGF stimulates growth of microadenomas. Our interpretation is also different, in that we suspect that angiogenesis is not wholly responsible. Similarly, VEGF may augment growth of micrometastases in humans. These findings underscore that VEGF is a potent mediator of intestinal tissue growth, through pathways inherent to the involved cell types. The limited efficacy thus far of single agent anti-VEGF therapy may reflect technical obstacles to complete inactivation of the pathway and/or redundant signaling in tumors. Our results imply a potential role for anti-VEGF therapy against different stages of intestinal neoplasia, including microscopic lesions.