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

Abstract

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.

Correlation of Appearance of MRI Perinidal T2 Hyperintensity Signal and Eventual Nidus Obliteration Following Photon Radiosurgery of Brain AVMs: Combined Results of LINAC and Gamma Knife Centers

“…Accordingly, the smaller nidal vessels will receive a much greater radiation dose compared with the larger perinidal vessels. Such a proposed explanation is further supported by the findings of other researchers 22,42,47 who reported that, in contrast to the radiation-induced obliteration cascade of the smaller nidal vessels, the radiation-induced damage of the larger perinidal vessels will hinder the vessel wall nourishment and lead to subsequent increased vessel wall permeability.…”

“…17,41 The thrombus formation is promoted by intimal injury, increased platelet aggregation, and turbulent blood flow that play an important role in the obliteration cascade of the small pathologic vessels of the AVM nidus. 35,41,42 Also, the subendothelial and the periadventitial proliferation and the contraction of the myofibroblasts, particularly the A phenotype, play an important role in shrinkage and eventual luminal closure of the thrombosed AVM after SRS. The replacement of the contracted thrombosed AVM by scar tissue is probably attributable to the proliferation of fibrocytes and V phenotype fibroblasts in the connective tissue stroma.…”

Ionizing radiation reduces ADAM10 expression in brain microvascular endothelial cells undergoing stress-induced senescence

“…Identification of novel senescence-associated surface proteins may identify potential markers amenable to vascular targeting to treat or remove senescent cells as a therapeutic approach to treat age-associated disease [25-27]. Our own studies are investigating the use of radiation as a priming agent to induce novel marker expression for vascular targeting in brain arteriovenous malformations, of which radiation-induced senescence markers could be of use [51-53]. Identifying markers at the endothelial surface may have many applications for understanding and subsequently attenuating age-associated disease.…”

“…Samples were analyzed using data-dependent and SWATH LC/MS/MS procedures described previously, with modifications [53, 69]. LC/MS/MS was performed using a TripleTOF 6600 mass spectrometer (AB SCIEX) equipped with a NanoLC™ 400 liquid chromatography system (Eksigent) and cHiPLC unit (Eksigent).…”

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

“…Hence, we aim to use radiosurgery to prime the AVM endothelium to induce potential targets. Our previous studies demonstrated that radiation can induce upregulation 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.…”