Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature.

Endothelial cells are highly sensitive to ionizing radiation, and exposure leads to multiple adaptive changes. Remarkably, part of this response is the translocation of normally intracellular proteins to the cell surface. It is unclear whether this ectopic expression has a protective or deleterious function, but, regardless, these surface-exposed proteins may provide unique discriminatory targets for radiation-guided drug delivery to vascular malformations or tumor vasculature. We investigated the ability of an antibody-thrombin conjugate targeting mitochondrial PDCE2 (E2 subunit of pyruvate dehydrogenase) to induce precision thrombosis on irradiated endothelial cells in a parallel-plate flow system. Click-chemistry was used to create antibody-thrombin conjugates targeting PDCE2 as the vascular targeting agent (VTA). VTAs were injected into the parallel-plate flow system with whole human blood circulating over irradiated cells. The efficacy and specificity of fibrin-thrombus formation was assessed relative to non-irradiated controls. The PDCE2-targeting VTA dose-dependently increased thrombus formation: minimal thrombosis was induced in response to 5 Gy radiation; doses of 15 and 25 Gy induced significant thrombosis with equivalent efficacy. Negligible VTA binding or thrombosis was demonstrated in the absence of radiation or with non-targeted thrombin. PDCE2 represents a unique discriminatory target for radiation-guided drug delivery and precision thrombosis in pathological vasculature.

The Evolution of Safe and Effective Coaguligands for Vascular Targeting and Precision Thrombosis of Solid Tumors and Vascular Malformations.

In cardiovascular and cerebrovascular biology, control of thrombosis and the coagulation cascade in ischemic stroke, myocardial infarction, and other coagulopathies is the focus of significant research around the world. Ischemic stroke remains one of the largest causes of death and disability in developed countries. Preventing thrombosis and protecting vessel patency is the primary goal. However, utilization of the body's natural coagulation cascades as an approach for targeted destruction of abnormal, disease-associated vessels and tissues has been increasing over the last 30 years. This vascular targeting approach, often termed "vascular infarction", describes the deliberate, targeted delivery of a thrombogenic effector to diseased blood vessels with the aim to induce localized activation of the coagulation cascade and stable thrombus formation, leading to vessel occlusion and ablation. As systemic delivery of pro-thrombotic agents may cause consternation amongst traditional stroke researchers, proponents of the approach must suitably establish both efficacy and safety to take this field forward. In this review, we describe the evolution of this field and, with a focus on thrombogenic effectors, summarize the current literature with respect to emerging trends in "coaguligand" development, in targeted tumor vessel destruction, and in expansion of the approach to the treatment of brain vascular malformations.

Targeting of externalized αB-crystallin on irradiated endothelial cells with pro-thrombotic vascular targeting agents: Potential applications for brain arteriovenous malformations.

BACKGROUND: Vascular targeting uses molecular markers on the surface of diseased vasculature for ligand-directed drug delivery to induce vessel occlusion or destruction. In the absence of discriminatory markers, such as in brain arteriovenous malformations (AVMs), stereotactic radiosurgery may be used to prime molecular changes on the endothelial surface. This study explored αB-crystallin (CRYAB) as a radiation induced target and pre-tested the specificity and efficacy of a CRYAB-targeting coaguligand for in vitro thrombus induction. METHODS: A parallel-plate flow system was established to circulate human whole blood over a layer of human brain endothelial cells. A conjugate of anti-CRYAB antibody and thrombin was injected into the circuit to compare binding and thrombus formation on cells with or without prior radiation treatment (0-25 Gy). RESULTS: Radiation increased CRYAB expression and surface exposure in human brain endothelial cells. In the parallel-plate flow system, the targeted anti-CRYAB-thrombin conjugate increased thrombus formation on the surface of irradiated cells relative to non-irradiated cells and to a non-targeting IgG-thrombin conjugate. Fibrin deposition and accumulation of fibrinogen degradation products increased significantly at radiation doses at or above 15 Gy with conjugate concentrations of 1.25 and 2.5 µg/mL. CONCLUSIONS: CRYAB exposure can be detected at the surface of human brain endothelial cells in response to irradiation. Pro-thrombotic CRYAB-targeting conjugates can bind under high flow conditions and in the presence of whole blood induce stable thrombus formation with high specificity and efficacy on irradiated surfaces. CRYAB provides a novel radiation marker for potential vascular targeting in irradiated brain AVMs.