Cellular senescence is associated with aging and is considered a potential contributor to age-associated neurodegenerative disease. Exposure to ionizing radiation increases the risk of developing premature neurovascular degeneration and dementia but also induces premature senescence. As cells of the cerebrovascular endothelium are particularly susceptible to radiation and play an important role in brain homeostasis, we investigated radiation-induced senescence in brain microvascular endothelial cells (EC). Using biotinylation to label surface proteins, streptavidin enrichment and proteomic analysis, we analyzed the surface proteome of stress-induced senescent EC in culture. An array of both recognized and novel senescence-associated proteins were identified. Most notably, we identified and validated the novel radiation-stimulated down-regulation of the protease, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 is an important modulator of amyloid beta protein production, accumulation of which is central to the pathologies of Alzheimer's disease and cerebral amyloid angiopathy. Concurrently, we identified and validated increased surface expression of ADAM10 proteolytic targets with roles in neural proliferation and survival, inflammation and immune activation (L1CAM, NEO1, NEST, TLR2, DDX58). ADAM10 may be a key molecule linking radiation, senescence and endothelial dysfunction with increased risk of premature neurodegenerative diseases normally associated with aging.

Stereotactic radiosurgery (SRS) is an established treatment for brain arteriovenous malformations (AVMs) that drives blood vessel closure through cellular proliferation, thrombosis and fibrosis, but is limited by a delay to occlusion of 2-3 years and a maximum treatable size of 3 cm. In this current study we used SRS as a priming tool to elicit novel protein expression on the endothelium of irradiated AVM vessels, and these proteins were then targeted with prothrombotic conjugates to induce rapid thrombosis and vessel closure. SRS-induced protein changes on the endothelium in an animal model of AVM were examined using in vivo biotin labeling of surface-accessible proteins and comparative proteomics. LC-MS/MS using SWATH acquisition label-free mass spectrometry identified 280 proteins in biotin-enriched fractions. The abundance of 56 proteins increased after irradiation of the rat arteriovenous fistula (20 Gy, ≥1.5-fold). A large proportion of intracellular proteins were present in this subset: 29 mitochondrial and 9 cytoskeletal. Three of these proteins were chosen for further validation based on previously published evidence for surface localization and a role in autoimmune stimulation: cardiac troponin I (TNNI3); manganese superoxide dismutase (SOD2); and the E2 subunit of the pyruvate dehydrogenase complex (PDCE2). Immunostaining of AVM vessels confirmed an increase in abundance of PDCE2 across the vessel wall, but not a measurable increase in TNNI3 or SOD2. All three proteins co-localized with the endothelium after irradiation, however, more detailed subcellular distribution could not be accurately established. In vitro, radiation-stimulated surface translocation of all three proteins was confirmed in nonpermeabilized brain endothelial cells using immunocytochemistry. Total protein abundance increased modestly after irradiation for PDCE2 and SOD2 but decreased for TNNI3, suggesting that radiation primarily affects subcellular distribution rather than protein levels. The novel identification of these proteins as surface exposed in response to radiation raises important questions about their potential role in radiation-induced inflammation, fibrosis and autoimmunity, but may also provide unique candidates for vascular targeting in brain AVMs and other vascular tissues.

The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 μg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies.

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To achieve accurate dose calculations in radiation therapy the electron density of patient tissues must be known. This information is ordinarily gained from a computed tomography (CT) image that has been calibrated to allow relative electron density (RED) to be determined from CT number. When high density objects such as metallic prostheses are involved, direct use of the CT data can become problematic due to the artefacts introduced by high attenuation of the beam. This requires manual correction of the density values, however the properties of the implanted prosthetic are not always known. A method is introduced where the RED of such an object can be determined using the treatment beam of a linear accelerator with an electronic portal imaging device. The technique was tested using a metallic hip replacement that was placed within a container of water. Compared to the theoretical RED of 6.8 for cobalt-chromium alloy, these measurements calculated a value of 6.4 ± 0.7. This would allow the distinction of an implant as Co-Cr or steel, which have similar RED, or titanium, which is much less dense with an RED of 3.7.

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A computed tomography number to relative electron density (CT-RED) calibration is performed when commissioning a radiotherapy CT scanner by imaging a calibration phantom with inserts of specified RED and recording the CT number displayed. In this work, CT-RED calibrations were generated using several commercially available phantoms to observe the effect of phantom geometry on conversion to electron density and, ultimately, the dose calculation in a treatment planning system. Using an anthropomorphic phantom as a gold standard, the CT number of a material was found to depend strongly on the amount and type of scattering material surrounding the volume of interest, with the largest variation observed for the highest density material tested, cortical bone. Cortical bone gave a maximum CT number difference of 1,110 when a cylindrical insert of diameter 28 mm scanned free in air was compared to that in the form of a 30 × 30 cm(2) slab. The effect of using each CT-RED calibration on planned dose to a patient was quantified using a commercially available treatment planning system. When all calibrations were compared to the anthropomorphic calibration, the largest percentage dose difference was 4.2 % which occurred when the CT-RED calibration curve was acquired with heterogeneity inserts removed from the phantom and scanned free in air. The maximum dose difference observed between two dedicated CT-RED phantoms was ±2.1 %. A phantom that is to be used for CT-RED calibrations must have sufficient water equivalent scattering material surrounding the heterogeneous objects that are to be used for calibration.