A selective tumor microvasculature thrombogen that targets a novel receptor complex in the tumor angiogenic microenvironment.

We have previously shown that part of the heparin-binding domain of the vascular endothelial growth factor (VEGF), designated HBDt, localizes very selectively to surfaces of the endothelial cells of i.t blood vessels. Here, we have coupled the HBDt to the extracellular domain of tissue factor (TFt), to locally initiate the thrombogenic cascade. In tumor-bearing mice, infusion of this HBDt.TFt results in rapid occlusive thrombosis selective only for tumor microvasculature with resultant infarctive destruction of tumors. We now show that infusion of an optimal combination of this HBDt.TFt and its requisite cofactor (factor VIIa) in tumor models results in significant tumor eradication. Binding studies and confocal microscopy indicate that the target for the HBDt.TFt seems to be a trimolecular complex of chondroitin C sulfate proteoglycan, neuropilin-1, and VEGF receptor-2, overexpressed together only in highly angiogenic sites of the tumor microenvironment. The HBDt.TFt was also colocalized with the trimolecular receptor complex in endothelial sprouts from tumor tissues, and its binding inhibited the growth of such sprouts. In vitro, we show that the HBDt structure has its highest affinity for chondroitin 6 sulfate. We show the potential of this HBDt.TFt as a candidate therapeutic and elucidate its target in vivo.

Regulation of tissue factor--mediated initiation of the coagulation cascade by cell surface grp78.

OBJECTIVE: To test the hypothesis that Grp78 negatively regulates cell surface tissue factor (TF) procoagulant activity and whether this is mediated by physical interaction. METHODS AND RESULTS: Biopanning with phage-displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78-kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF-mediated procoagulant activity on murine brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface. CONCLUSIONS: The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

A novel vascular endothelial growth factor heparin-binding domain substructure binds to glycosaminoglycans in vivo and localizes to tumor microvascular endothelium.

Vascular endothelial growth factor has an exon 7-encoded heparin-binding domain. To explore the expression of complementary ligands on endothelial surfaces in vivo and to assess potential for localization within the vascular tree, we introduced a truncated version of this domain (HBDt) into a modified M13 phage. Despite the small size and trace-level expression, this HBDt endowed the phage with affinity for heparin to which it bound in vitro. It also preferentially and selectively localized the phage in vivo to vascular endothelial surfaces, especially of tumors. Competition assays demonstrated that accumulation and localization of this phage was attributable to expression of the HBDt on the phage surface and sequence comparison suggests its novelty. We propose to use this novel HBDt structure to explore the expression of the ligand glycosaminoglycans within the vascular tree. This structure may facilitate directed delivery of therapeutic molecules.

Prostate-specific membrane antigen directed selective thrombotic infarction of tumors.

Prostate-specific membrane antigen (PSMA), a glutamyl preferring carboxypeptidase, is found in prostate and other carcinomas present on both tumor cells and associated microvascular lining cells. We find that the channel structures delineated by PSMA-expressing cells in human and rat prostate tumors are in functional continuity with the vasculature and thus form part of tumor microvasculature. The PSMA-positive cell-outlined channels are CD31 negative and mutually exclusive of CD31-positive cell-lined channels elsewhere in the tumor consistent with tumor cells adapted to a pseudoendothelial phenotype in vasculogenic mimicry. To assess the functional potential of such PSMA-lined microvasculature to selectively direct infarctive tumor therapy, we coupled the soluble extracellular domain of tissue factor to a PSMA catalytic site inhibitor to create a PSMA-directed selective tumor vascular thrombogen (STVT). This protein induced selective local in vivo infarctive necrosis of the rat Mat Lu prostate tumor when administered i.v. The combined administration of this STVT with low-dose doxorubicin produced a profound tumoricidal effect, resulting in complete eradication of some tumors. This is consistent with the therapeutic potential for a PSMA-directed STVT and expands the potential for selective infarctive ablation of tumors.