Abstract
Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and 12 of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by Wnt3a application and ameliorated by the Wnt antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical Wnt signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor Wnt ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by Wnt3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day 12 and PA application correlated with Axin 2 induction, a universal reporter gene for canonical Wnt signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density.
Highlights
TEM8 is an integrin-like membrane protein that is highly expressed in endothelial cells lining blood vessels in colon tumors [3]
To determine whether TEM8 is expressed in chorioallantoic membrane (CAM) blood vessels, we used RT-PCR to evaluate the expression of TEM8 and Capillary Morphogenetic molecule 2 (CMG2)
We found that while TEM8 is expressed at low levels at early stages of CAM development and in the mature CAM, there is a transient increase in expression that peaks between days 10 and 12 (Fig. 1A)
Summary
TEM8 is an integrin-like membrane protein that is highly expressed in endothelial cells lining blood vessels in colon tumors [3]. Subsequent studies have shown that while this molecule is expressed at low levels in normal tissues, it is induced in the vascular compartment of multiple primary tumors and metastases, but not during normal vascularization occurring during wound healing or corpus luteum formation [4,5]. This distinct expression pattern poises TEM8 as a candidate molecule to selectively target tumor-associated vasculature. The physiological function of TEM8 in the vascular compartment remains unknown
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