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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