Radiosurgery Alters the Endothelial Surface Proteome: Externalized Intracellular Molecules as Potential Vascular Targets in Irradiated Brain Arteriovenous Malformations.

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.

Angiographic, hemodynamic, and histological changes in an animal model of brain arteriovenous malformations treated with Gamma Knife radiosurgery.

OBJECT: Brain arteriovenous malformations (AVMs) are a major cause of stroke. Many AVMs are effectively obliterated by stereotactic radiosurgery, but such treatment for lesions larger than 3 cm is not as effective. Understanding the responses to radiosurgery may lead to new biological enhancements to this treatment modality. The aim of the present study was to investigate the hemodynamic, morphological, and histological effects of Gamma Knife surgery (GKS) in an animal model of brain AVM. METHODS: An arteriovenous fistula was created by anastomosing the left external jugular vein to the side of the common carotid artery in 64 male Sprague-Dawley rats (weight 345 ± 8.8 g). Six weeks after AVM creation, 32 rats were treated with a single dose of GKS (20 Gy); 32 animals received sham radiation. Eight irradiated and 8 control animals were studied at each specified time point (1, 3, 6, and 12 weeks) for hemodynamic, morphological, and histological characterization. RESULTS: Two AVMs showed partial angiographic obliteration at 6 weeks. Angiography revealed complete obliteration in 3 irradiated rats at 12 weeks. Blood flow in the ipsilateral proximal carotid artery (p < 0.001) and arterialized jugular vein (p < 0.05) was significantly lower in the irradiated group than in the control group. The arterialized vein's external diameter was significantly smaller in GKS-treated animals at 6 (p < 0.05) and 12 (p < 0.001) weeks. Histological changes included subendothelial cellular proliferation and luminal narrowing in GKS-treated animals. Neither luminal obliteration nor thrombus formation was identified at any of the time points in either irradiated or nonirradiated animals. CONCLUSIONS: GKS produced morphological, angiographic, and histological changes in the model of AVM as early as 6 weeks after treatment. These results support the use of this model for studying methods to enhance radiation response in AVMs.