Candidate’s name : PRISCILLA A/P DAS

Supervisor’s name & Programme: Dr Yvonne-Tee Get Bee (Biomedicine program, School of health sciences, Universiti Sains Malaysia, Kelantan,

Malaysia)

Co-supervisor’s name & Programme: Dr Rapeah Suppian & Dr Tan Suat

Cheng (Biomedicine program, School of health sciences, Universiti Sains

Malaysia, Kelantan, Malaysia)

Association between Endothelial Progenitor Cells and von Willebrand Factor in Astrocytic Glioma

INTRODUCTION

BRAIN TUMOR PATIENTGLIOBLASTOMA

• Astrocytic gliomas-brain tumors of glial origin (Schlang et al., 2009)

• Accounts for 2/3 of all tumours in glial origin

• Astrocytomas are typically classified as:

a) Pilocytic (grade I- less aggressive)

b) Diffuse (grade II)

c) Anaplastic (grade III)

d) Glioblastoma multiforme (grade IV- most aggressive)

• Tumors- 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 angiogenicity8,11,12

• The 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

OBJECTIVES

• To determine the association between circulating and tissue resident endothelial progenitor cells ( EPCs) with vWF in astrocytic glioma patients

METHODOLOGYStudy 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 sampling20ml Tissue sampling

Brain tumour

Adjacent normal brainCount of circulating

EPCs

Statistical analysis- Differences in each study outcome and its correlations

FLOW CYTOMETRY

DURING THE SURGERY

Immunofluorescence

Count of tissue Resident EPCs

ELISA

vWF

Measurement of circulating EPCs Flowcytometry

• The EPC was characterised using CD133+/VEGFR2+ markers.

• Peripheral blood mononuclear cells were prepared by gradient centrifugation using Ficoll-Hypaque

• 100 μl of PBMCs (containing 10 6

• cells) was incubated with:– FcR-blocking reagent – PE conjugated anti–human CD133

mAb – APC- conjugated VEGFR-2 mAb– FITC-conjugated annexin V mAb (to

identify the apoptotic cells).

• Isotype-matched IgG1 antibodies were negative controls.

• All experiments were carried out in triplicates and about 50,000 events were collected.

-FCR-Annexin-FITC-CD133-PE-VEGFR2-APC

-FCR-FITC-IgG1-PE-IgG1-APC-IgG1

SAM

PLE

CON

TRO

L

• Endothelial progenitor cell counts are expressed as percentage of total PBMCs in each patient.

• The frequency of cEPCs in peripheral blood was determined by a 2D side-scatter/fluorescence dot-plot analysis of the samples, after exclusion of annexin V+ cells and appropriate gating.

• The exclusion of annexin V+ is performed to rule out contamination with apoptotic cells in positive population. cEPC

CD13

3

VEGFR2+

APC-IgG1

APC-VEGFR2+

PE-Ig

G1

PE-C

D13

3

FITC-IgG1

FITC-ANNEXIN

EPCs

Detection of circulating EPCs (CD133+ and VEGFR2+) markers, in the population of peripheral blood mononuclear cells (PBMCs) of astrocytic glioma.

Immunofluorescence microscopy•The EPC was characterised using CD133+/VEGFR2+ markers.•Tissue fixed in 10% paraformaldehyde embedded in tissue block. 8 serial dissections/thickness=3μm

•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-2• incubated overnight at 4°C • 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.

Immunofluorescence staining. The figure demonstrates the presence of tissue resident EPCs (CD133+/VEGFR2+) in brain tumour and adjacent normal brain of astrocytic glioma

Enzyme-linked Immunosorbant Assay (ELISA)

• Assay involves the use of antibody that is immobilised onto a microtitre plate

• Patient plasma and a series of diluted reference samples were added to the wells and the vWF in the samples is bound by the antibody

• The 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

• The mean percentage of circulating EPCs was 0.01±0.01%

Increased in the percentage of tissue EPCs (mean±SD) 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 (Spearman’s rho r = 0.452, p=0.035)

Brain tumour EPCs (%)

• However no correlation was found between plasma vWF with EPCs in adjacent normal brain (Spearman’s rho r =0. 210, p=0. 349

• The 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 1–5% of normal activity have moderately severe bleeding or have prolonged bleeding after injuries

• Patients 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

DISCUSSION• Previous 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

 

REFERENCES• Janis 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.

• Gallinaro, L., Cattini, M. G., Sztukowska, M., Padrini, R., Sartorello, F., Pontara, E., Bertomoro, A., Daidone, V., Pagnan, A. & Casonato, A. (2008). A shorter von Willebrand factor survival in O blood group subjects explains how ABO determinants influence plasma von Willebrand factor. Blood, 111(7), 3540-3545.

• Greenfield, J. P., Jin, D. K., Young, L. M., Christos, P. J., Abrey, L., Rafii, S. & Gutin, P. H.

Surrogate markers predict angiogenic potential and survival in patients with glioblastoma multiforme. Neurosurgery(10), 2009 May;2064(2005):2819-2026; discussion 2826-2007.

• Rumbaut RE, T. P. S. R. C. M. C. L. S. (2010). Platelet-Vessel Wall Interactions in Hemostasis

and Thrombosis. J Clin Invest. The American Society for Clinical Investigation. , 87(1 ), 229-236. doi: 10.1172/JCI114976

• Schlag P. M., S. H. J. (2009). Gliomas. . Cancer Research 1-259. • Spiel, A. O., Gilbert, J. C. & Jilma, B. (2008). Von Willebrand Factor in Cardiovascular

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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,Cécile 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 Platelet–Endothelial Interactions in VivoJ Exp Med. Apr 17,; 191(8): 1413–1422.

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