Full Length ArticleStable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations
Introduction
Brain arteriovenous malformations (AVMs) result from abnormal vascular development resulting in direct arterial-to-venous connections without intervening capillaries, forming tangled collections of vessels called the ‘nidus’. This causes high pressure arterial blood to flow directly to veins increasing the risk of vessel rupture [1]. Hemorrhage can lead to significant neurological morbidity or mortality [2,3]. Current treatment approaches include surgical resection [4], endovascular embolization [5] and radiosurgery [6] with the goal to achieve complete AVM vessel removal or occlusion, and prevent hemorrhage. Depending on size and location of the lesion, over 90% of large AVMs are not treated safely using current treatments [7]. Hence there is a need to develop a new treatment approach to achieve complete obliteration. This could be made possible using a vascular-targeting approach by inducing rapid thrombosis precisely on targeted AVM vessels without affecting surrounding normal vessels.
In vascular targeting, a target molecule is recognized on the surface of the endothelium and a ligand or antibody to the target is bound to a drug or effector of interest [8]. The concept was initially developed for the treatment of cancer, where the inherent difference between normal and tumor vessels is utilized to direct therapeutic agents selectively to the abnormal tumor endothelium [9]. To date, no valid targets have been found on the AVM endothelial surface that makes it significantly different from normal vessels. Hence, we aim to use radiosurgery to prime the AVM endothelium to induce potential targets. Our previous studies demonstrated that radiation can induce up-regulation of surface biomarkers on the AVM endothelium thus discriminating it from normal vessels [[10], [11], [12], [13], [14]]. These proteins may serve as potential targets for ligand-directed pro-thrombotic conjugates, enhancing the thrombotic response when induced with radiation. Our overall aim is to develop pro-thrombotic conjugates that target these radiation-stimulated proteins. However, it is not economical to test multiple combinations of target molecules with thrombotic agents using an animal model. Hence we are developing an in vitro parallel-plate flow chamber model to simulate in vivo fluid shear stresses which rapidly tests the potency of conjugate-driven thrombus formation on irradiated endothelial cells when exposed to dynamic fluid flow [15]. Parallel-plate flow perfusion devices are widely used to evaluate platelet function, endothelial cell function, and thrombus formation [[16], [17], [18], [19]].
In this study, we aimed to investigate the pro-thrombotic efficacy of a phosphatidylserine (PS)-targeting annexin V-thrombin conjugate in irradiated human microvascular endothelial cells using the parallel-plate flow system. PS is a negatively charged phospholipid present in the membrane of all cells. It normally localizes to the inner membrane leaflet that faces the cytosol, but under certain conditions it translocates to the outer leaflet of the plasma membrane where it plays a role in various cellular processes [20,21]. PS plays a vital role during apoptosis where it is exposed on the surface of apoptotic cells to trigger phagocytosis [22]. PS externalization has been shown to occur on tumor endothelium, allowing it to be exploited for imaging and vascular targeting using PS-directed antibodies and immunoconjugates [[23], [24], [25]]. In addition, radiation therapy has been reported to enhance PS exposure on tumor endothelium, serving as a potential radiation-stimulated target in future clinical applications [25,26]. Our recent studies also demonstrated significant externalization of endothelial PS upon radiation both in vitro, in brain microvascular endothelial cells, and in vivo, using a rat model of AVM [11,12]. This suggests PS could be used as a vascular target in irradiated AVMs.
In the current study, we investigated the efficacy of a PS-targeting annexin V-thrombin conjugate at inducing thrombus formation under parallel-plate flow on irradiated human brain microvascular endothelial cells. Using whole human blood perfusion, we investigated the effect of conjugate dose, radiation dose and methods of thrombus analysis, in this system.
Section snippets
Annexin V-thrombin conjugate preparation
Thrombin (Jomar Life Research) was conjugated with annexin V (Jomar Life Research) using a Lys-Lys protein-protein conjugation kit (Click Chemistry Tools Bioconjugate Technology Company) according to the manufacturer's instructions. Briefly, 300 μL thrombin (2 mg/mL) in BupH buffer (pH 7.5) was labelled using 12-fold molar excess tetrazine (Tz) reagent, and 300 μL annexin (1 mg/mL) in BupH buffer was labelled using 12-fold molar excess trans-cyclooctene (TCO) reagent. Annexin-TCO and
Average flow rate and shear stress
Flow conditions were optimized to ensure that cells remained adherent to the surface until the end of the experiment. The stroke rate was optimized at 38 strokes/min. The flow volume and flow time were found to be optimal at 2.4 mL/min and 15 min respectively (Supplementary Fig. S2A, B, C). The average wall shear stress using these parameters was calculated based on bead velocity over time and measured as 3.1 dynes/cm2. Increasing any of these parameters led to decreased cell confluency. The
Discussion
In this study, we investigated the efficacy of an annexin V-thrombin conjugate to bind selectively to irradiated endothelial cells and induce thrombosis under flow using an in vitro parallel-plate flow chamber. This demonstrated the successful development and use of an in vitro flow device to test the effect of a pro-thrombotic conjugate in stimulating platelet aggregation and fibrin formation. Here, we successfully used fresh human whole blood with irradiated endothelial cells to demonstrate
Conclusions
Our findings demonstrate the first evidence of stable thrombus formation on irradiated endothelial cells in a parallel-flow device by designing and testing a pro-thrombotic conjugate targeting PS. Using this system, we established dose combinations of conjugate and radiation that could effectively induce highly stable thrombus formation in the presence of whole blood that can now be applied in a pre-clinical animal study. Further, development of this system also enables us to rapidly screen
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2020, Thrombosis ResearchCitation Excerpt :Overexpression of activated RAS in vascular endothelial cells could be used to examine this in vitro, and further biodistribution studies in pre-clinical models are required to assess any potential for off-target binding in other vascular beds, however collectively, our data suggests strong potential for radiation specificity at this stage. We previously used the in vitro parallel-plate flow chamber to successfully examine PS as a radiation target with an annexin V-thrombin conjugate as the targeting coaguligand [27]. As observed for the CRYAB conjugate, the annexin V-thrombin conjugate demonstrated dose-dependent and synergistic thrombus induction in response to increasing radiation doses, with the higher combined dose of radiation (25 Gy) and conjugate (2.5 μg/mL) causing significantly greater thrombus formation compared to the lower doses with both conjugates.
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