MOJ ISSN: 2374-6920MOJPB

Proteomics & Bioinformatics
Proceeding
Volume 1 Issue 1 - 2014
Proteomics Detection of Endothelial Cell Surface Proteins Following Irradiation as Potential Targets for Brain Arteriovenous Malformations Molecular Therapy
Margaret Simonian1,2*, Rachel R Ogorzalek Loo2, Joseph A Loo2, Marcus A Stoodley1 and Mark P Molloy3
1Australian School of Advanced Medicine, Macquarie University, Australia
2David Geffen School of Medicine, Department of Biological Chemistry, University of California Los Angeles (UCLA), USA
3Department of Chemistry and Bimolecular Sciences, Macquarie University, Australia
Received: April 25, 2014 | Published: April 30, 2014
*Corresponding author: Margaret Simonian, David Geffen School of Medicine, Department of Biological Chemistry, University of California Los Angeles (UCLA), 611 Charles E. Young Drive East, CA. 90095, USA, Tel: +1-310-794-7308; Email: margaret@chem.ucla.edu
Citation: Simonian M, Ogorzalek Loo RR, Loo JA, Stoodley MA, Molloy MP (2014) Proteomics Detection of Endothelial Cell Surface Proteins Following Irradiation as Potential Targets for Brain Arteriovenous Malformations Molecular Therapy. MOJ Proteomics Bioinform 1(1): 00002. DOI: 10.15406/mojpb.2014.01.00002

Abstract

Arteriovenous malformations (AVMs) in the brain are vascular neurological lesions consist of abnormal collection of arteries and veins. They are the most common cause of brain haemorrhage in children and young adults. Over 90% of large AVMs (>3 cm) are not curable, or curable with unacceptable risks. Radiosurgery is the treatment option for lesions (< 3cm) in diameter and located in eloquent areas where surgery can cause neurological defecit. However vascular occlusion post radiosurgery can takes up to 3 year to complete while patients remain at risk of haemorrhage. This study aims to develop a molecular therapy for human brain AVMs. Specifically we aim to identify unique protein targets of AVMs that are up-regulated post irradiation. These proteins will then be used to develop a ligand-directed vascular treatment that promotes rapid thrombosis in AVM vessels post radiosurgery.

Introduction

AVMs consist of abnormal collection of arteries and veins. They are the most common cause of haemorrhagic stroke in children and young adults. Radiosurgery is the treatment option recommended for lesions less than 3cm in diameter and located in eloquent areas where surgery can cause neurological deficits. This study aims to identify endothelial protein targets of AVMs that are differentially expressed compared to normal vessels post radiosurgery, to develop a ligand directed vascular treatment that promotes rapid thrombosis in AVM vessels. This is the first time that proteomics approach has been used in AVMs study.

Objectives

• Assess membrane protein changes in the murine endothelial cell culture (bEnd.3) in response to radiation over time using proteomics analysis.
• Determine candidate proteins location with immunocytochemistry.
• Protein candidates will be used for a ligand-directed vascular targeting treatment to promote rapid thrombosis in AVM vessels post radiosurgery.

Methods

In vitro biotinylation and iTRAQ mass spectrometry was used to assess membrane protein changes in the murine endothelial cell cultures (bEnd.3). Cells were irradiated with 25Gy and surface biotinylation was performed using NHS-LC-Biotin at 6h, 24h, 48h and 72 hours post radiation. Biotinylated proteins were captured on streptavidin resin, digested with trypsin, and then labelled with iTRAQ 8-plex reagents kit (Applied Biosystems). Peptides were separated by SCX and analysed by nanoLC/ MS on Qstar Elite (AB Sciex).
Immunocytochemistry was used to validate candidate proteins location and expression in bEnd.3 cells.
In vivo biotinylation of the rat model of AVM was also conducted. The rat was perfused with NHS-LC-biotin, the fistula was harvested, membrane proteins were extracted and captured on streptavidin resin, digested with trypsin and analysed by LC/MS.

Results

NanoLC ESI MS/MS data were submitted to ProteinPilot V4.0 (AB Sciex). iTRAQ MS identified on average 112 proteins from 1120 distinct peptides from 3 independent biological replicates. The detected protein threshold (unused ProtScore) was set as > 1.3 (better than 95%confidence). 11 proteins were significantly differentially expressed in at least 2 out of 3 experiments. The most extensive changes were observed after 48h post radiation, e.g. cadherin 5 & CD109.
CADH5 protein; Expression level in C is higher than R sample at 48h post irradiation (117:118 ratio = C:R) increased 1.2614 fold
CD109 protein; Expression level in R is higher than C sample at 6h post irradiation (114 :113 ratio = C:R) decreased 0.7817 fold
bEnd.3 cells stained for CADH5 and PECAM proteins. IRR=irradiated; C=controls
Mass spectrometry identified 120 proteins in the rat model of AVM, 32 of them were known as membrane proteins, e.g. in the table below

Annexin A2

CD99

Biglycan

ACO3

GP3

ATA1

Lumican

PAT3

PTPRC

CD36

AT1B1

Prolargin

VCAM2

PECAM

OME2

CAD13

Rab1B

Annexin A1

Rat AVM model closely resembles human AVMs, it has an arterial feeder, the nidus and adraining vein.
In vivo biotinylation perfusion of the rat model of AVM.
P: proteins; B: biotin; STA: streptavidin sepharose (Modified image from Rybak et al. [11])

Conclusion

Cell surface protein biotinylation and mass spectrometry of murine endothelial cells identified protein targets in response to irradiation.

References

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