In Vitro and In Vivo Biotinylation of Endothelial Cell Surface Proteins in the Pursuit of Targets for Vascular Therapies for Brain AVMs

Abstract

Identification of membrane proteins that are expressed on the endothelium after radiosurgery is of fundamental importance in developing a new treatment for brain vascular formations. We optimized then employed in vitro and in vivo biotinylation methodology to label membrane proteins in a murine cerebral endothelial cell cultures (bEnd.3) and a rat model of arteriovenous malformation (AVM). Membrane proteins were then captured on streptavidin resin and identified using proteomics analysis. Surface biotinylation was performed on the endothelial cell cultures using a modified protocol [13,14]. Each 75 cm 2 flask containing approximately 1×10 6 cells was washed four times with PBS pH 7.4. Twenty millilitres of PBS containing 67 µM EZ-link (Sulfo-NHS-LC-Biotin) (Pierce, IL, USA) were added to the flasks and incubated for 5 min at room temperature. The biotinylation reaction was terminated by adding Tris-Hcl pH 7.5 to a final concentration of 670 µM. After 5 min incubation the cells

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

“…At day 6 after irradiation or sham, cells in T75 flasks were prepared for biotin labelling, using a modification of published methods [ 23 , 24 , 68 ]. Briefly, cells were washed four times with cold 10 mL phosphate-buffered saline (PBS, pH 7.4) to remove all medium and secreted proteins.…”

Proteomics identification of radiation-induced changes of membrane proteins in the rat model of arteriovenous malformation in pursuit of targets for brain AVM molecular therapy

“…After 24 h of irradiation, all six rats were narcotized, prepared and perfused per Simonian et al [9] protocol, with a slight modification. A Gilson Minipuls 3 perfusion pump attached to a tube and needle was used to perfuse the rats with 1 mL of saline (NaCl) with a flow rate of 50 mL/min to wash away the blood, immediately followed with 100 mL of freshly prepared biotinylation solusion [1.5 mg/mL of Sulfo-NHS-LC-Biotin in pre warmed PBS at 37 °C + 10% Dextran 40] by pressing the syringe plug with a flow rate of 25 mL/min while monitoring the pressure and keeping it constant at ~ 100 mm Hg.…”

“…To achieve this goal, we have developed in vitro and in vivo biotinylation perfusion methodology to label endothelial cell surface proteins in murine bEnd. 3 cell cultures and the animal model of AVM [9, 10] and we have shown the feasibility of employing proteomics methods on the arteriovenous fistula (AVF) tissues harvested from the animal model [9, 10]. Here, we employ our in vivo methodology to label, then identify and quantify the differentially expressed membrane proteins in the irradiated and non irradiated animals using a label-free quantitative mass spectrometry of expression (MS E ) technique.…”

Radiation-induced expression of platelet endothelial cell adhesion molecule-1 in cerebral endothelial cells

“…It is a cell-cell adhesion glycoprotein constitutively expressed at the lateral junction of endothelial cells, platelets and circulating leukocytes, and is involved in a plethora of cell adhesion processes (42)(43)(44)(45) . PECAM -1's role in maintaining the structural integrity of the endothelial cell layer, and its luminal location on the AVM endothelium, makes it a potential vascular target (35,46,47) . In this study, we measured the temporal expression of PECAM -1 following irradiation with 5, 15, and 25 Gy in cultured endothelial cells to elucidate its suitability as a marker for vascular targeting in AVM treatment.…”