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The mean percentage of circulating EPCs was 0.010.01%INTRODUCTION

BRAIN TUMOR PATIENTGLIOBLASTOMAAstrocytic gliomas-brain tumors of glial origin (Schlang et al., 2009)Accounts for 2/3 of all tumours in glial originAstrocytomas are typically classified as:Pilocytic (grade I- less aggressive) Diffuse (grade II)Anaplastic (grade III)Glioblastoma multiforme (grade IV- most aggressive)

2Tumors- requirements for oxygen and nutrients lead to the growth of new blood vessels (angiogenesis) Additional angiogenic and vasculogenic mechanisms : the recruitment of endothelial progenitor cells (EPCs) (Janis et al., 2010)

The circulating EPCs homing the sites of tumor -used as a biomarker for diagnosis and monitoring tumor aggressiveness and angiogenicity8,11,12The von Willebrand factor (vWF) is a high molecular weight plasma protein, produced and released by endothelial cells and circulates in the blood plasma (Spiel et al., 2008) (Verma et al., 2003)

vWF function in the platelets adhesion to the vessel wall (colagen fibrils) and enhances the interaction of platelet to platelet (Rumbaut et al., 2010)

Platelets interact with EPCs - mediated by P-selectin on EPCs and P-selectin glycoprotein ligand-1 (PSGL-1) on platelets (Paul et al., 2000; Frenette et al.,1995)

vWF involved in the blood vessel formation (Gallinaro et al., 2008).vWF

vWF is a mediator of platelet adhesion to the vessel wall and enhance the platelet-platelet interactions (Rumbaut et al., 2010)

Irregular vasodilatation and vasoconstriction of blood vessels in the tumour leads to - blood vessels injury recruitment of more vWF, platelets and EPCs- to repair the injured endothelium

OBJECTIVESTo determine the association between circulating and tissue resident endothelial progenitor cells ( EPCs) with vWF in astrocytic glioma patientsMETHODOLOGYStudy design : Cross sectional studies Sampling : Random consecutive sampling (n=22)

Inclusion criteria: Patients identified by radiological appearance of astrocytic gliomas with subsequent histological confirmation

Exclusion criteria:Patients identified with non gliomas, brain metastases, infective lesions (e.g brain tuberculosis) Ruptured cases Pre-surgical embolization

Astrocytic Glioma cases that undergo surgery (n=22) Peripheral blood sampling20mlTissue sampling Brain tumourAdjacent normal brainCount of circulating EPCsStatistical analysis- Differences in each study outcome and its correlationsFLOW CYTOMETRYDURING THE SURGERYImmunofluorescenceCount of tissue Resident EPCs ELISA vWF

APC-IgG1APC-VEGFR2+PE-IgG1PE-CD133FITC-IgG1FITC-ANNEXINEPCs

Detection of circulating EPCs (CD133+ and VEGFR2+) markers, in the population of peripheral blood mononuclear cells (PBMCs) of astrocytic glioma.Immunofluorescence microscopyThe EPC was characterised using CD133+/VEGFR2+ markers.Tissue fixed in 10% paraformaldehyde embedded in tissue block. 8 serial dissections/thickness=3m

Deparafanization: - 0.1% Triton X-100 was added - BSA 0.5%- Washed with PBS- Tissue stained with:- (PE) anti-human CD133- (FITC) anti-human VEGFR-2incubated overnight at 4C counterstained with 4',6-diamidino-2-phenylindole (DAPI)

Accessed using BX41 Leica microscopy at 200x magnifications & analysed using Image J software

About 606.50 mm2 of the tissue area investigated.

Enzyme-linked Immunosorbant Assay (ELISA) Assay involves the use of antibody that is immobilised onto a microtitre platePatient plasma and a series of diluted reference samples were added to the wells and the vWF in the samples is bound by the antibodyThe plates then was rinsed and a second antibody was added conjugated to horseradish peroxidase (HRP). -A second wash step was performed and a substrate for the HRP was added (tetramethylbenzidine) followed by hydrogen peroxide. The absorbance at 450nm was measured From the reference curve the concentration of antigen in the patient plasma sample was determined.

vWF standard curve

results

Increased in the percentage of tissue EPCs (meanSD) in tumour tissue compared to adjacent normal tissue EPCs (tested using paired t-test, p=0.001)

There was a significant correlation between brain tumor EPCs and plasma vWF (Spearmans rho r = 0.452, p=0.035)

Brain tumour EPCs (%)However no correlation was found between plasma vWF with EPCs in adjacent normal brain (Spearmans rho r =0. 210, p=0. 349The normal level of vWF/FVIII is 200 ng/ml (100%). The vWF/FVIII level was categorised into three levels of severity:-severe (factor level less than 1%)-moderate (factor level of 1-5%)-mild (factor level of >5%)Previous studies has categorised the patient with factor level less than 1% as those who frequently bleed spontaneously and excessively after injuries, surgery. Patients with 15% of normal activity have moderately severe bleeding or have prolonged bleeding after injuriesPatients with >5% of normal activity usually bleed only with surgery or trauma or prolonged bleeding after a serious injury, trauma or surgery

Correlation between von Willebrand factor protein ( vWF/FVIII) and circulating endothelial progenitor cells (EPCs) of glioma patients with mild vWF/FVIII level

DISCUSSIONPrevious studies have reported that the newly formed blood vessels in the tumor were less disorganised, tortuous, dilated, leaky and hemorrhagic (Benedetta Bussolati, 2010; Chi et al., 2009; Greenfield et al.; Rafat ., 2010).

In our current study, the data showed that the plasma vWF associated with the tissue resident EPCs in brain tumor and not with the adjacent normal brain EPCs in the astrocytic glioma.

The patient with mild level of vWF had a significant correlation between plasma vWF and EPCs in circulation.

Therefore we postulate that the glioma tumor may have severe impairment in the vasculature system as it is not observed in the adjacent normal brain tissues.

Conclusion

The study suggest the circulating EPCs and vWF attracted to the brain tumour site to form new blood vessels in the tumour.

Targeting circulating EPCs, plasma vWF and tissue resident EPCs may be useful in the treatment of astrocytic glioma

REFERENCESJanis B., A.A.S., The role and therapeutic potential of endothelial progenitor cells in tumor vascularization. Scientific World Journal 2010. 10: p. 1088-1099. .Benedetta Bussolati, M.C.D., and Giovanni Camussi. , Characterization of molecular and functional alterations of tumor endothelial cells to design anti-angiogenic strategies. Current vascular pharmacology, 2010(doi:10.2174/157016110790887036).Rafat N., B.G.C.H., Schulte J., Tuenttenberg J., Vajkoczi P. , Circulating endothelial progenitor cells in malignant gliomas. J Neurosurg 2010. 12: p. 43-49. .

Chi, A.S., et al., Angiogenesis as a therapeutic target in malignant gliomas. The Oncologist, 2009. 14(6): p. 621-636.Varma, N.R., et al., Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma. BMC Medical Imaging, 2013. 13(1): p. 17.Malgorzata Frontczak-Baniewicz, D.C., Jaroslaw Andrychowski, Michal Walski, The immature endothelial cell in human glioma.Ultrastructural features of blood capillary vessels. Folia Neuropathol 2008. 46 (1): p. 49-56.

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Frenette PS, Johnson RC, Hynes RO, Wagner DD (1995). Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin.Proc Natl Acad Sci USA ;92

Paul S. Frenette,Ccile V. Denis, Linnea Weiss, Kerstin Jurk, Sangeetha Subbarao, Beate Kehrel, John H. Hartwig, Dietmar Vestweber, and Denisa D. Wagner (2000)P-Selectin Glycoprotein Ligand 1 (Psgl-1) Is Expressed on Platelets and Can Mediate PlateletEndothelial Interactions in VivoJ Exp Med. Apr 17,; 191(8): 14131422.

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