Molecular Regulation of Vascular Abnormalization and Its Role in Glioma

62 Pages·2015·1.06 MB·English

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1103
Molecular Regulation of Vascular
Abnormalization and Its Role in
Glioma
LEI ZHANG
ACTA
UNIVERSITATIS
ISSN 1651-6206
UPSALIENSIS
ISBN 978-91-554-9239-7
UPPSALA
urn:nbn:se:uu:diva-248713
2015
Dissertation presented at Uppsala University to be publicly examined in Fåhraeussalen,
Rudbecklaboratoriet, Uppsala, Saturday, 30 May 2015 at 09:00 for the degree of Doctor of
Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty
examiner: Professor Rolf Bjerkvig (University of Bergen).
Abstract
Zhang, L. 2015. Molecular Regulation of Vascular Abnormalization and Its Role in Glioma.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine
1103. 61 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9239-7.
Glioblastoma, grade IV glioma, is the one of the deadliest cancers, with a median survival of
only 12-15 months despite aggressive treatment including surgery, chemotherapy and radiation.
One hallmark of glioblastoma is the morphological and functional abnormalization of tumor
blood vessels. The molecular mechanisms involved in this process and their functional and
pathological implications are not yet fully understood. Indentification of molecular mechanisms
that underlie vascular abnormalization in GBM is necessary to develop efficient treatment
regimens for normalizing vascular function.
By analyzing the RNA-content of laser microdissected vessels from human biobank
specimens using affymetrix microarray analysis, we found that the abnormal glioblastoma
vessels have a distinct gene expression signature. We found 95 genes which were differentially
expressed in grade IV glioma vessels as compared to vessels in low grade tumors and control
brain. 78 of which were up-regulated while 17 were down-regulated. Many of these genes are
regulated by VEGFA or TGFβ signaling. In addition, we show a significant increase in Smad
signaling complexes in the vasculature of human glioblastoma in situ, suggesting that TGFβ
signaling may play important role in vessel abnormalization.
CD93 is a single-pass transmembrane glycoprotein, which we found to be up-regulated in
high grade glioma. Vascular expression of CD93 correlates to tumor grade in human glioma.
Moreover, high grade glioma patients with high CD93 expression in the vasculature are associate
with poor prognosis. We found that knocking down CD93 in endothelial cells with siRNA
clearly impaired endothelial cell adhesion, migration and tube formation due to defects in
cytoskeletal rearrangement. In addition, tumor growth was severely delayed in the CD93-/- mice.
Pleiotrophin, a multi-functional heparin-binding growth factor, promotes glioma growth in
several ways. Here, we identify pleiotrophin as a driver of vascular abnormalization in glioma.
We found that high pleiotrophin expression correlates with poor survival of patients with
astrocytomas. Pleiotrophin overexpression in orthotopic GL261 gliomas increases microvessel
density, enhances tumour growth and decreases survival. Vessels in pleiotrophin-expressing
gliomas are poorly perfused and display a high degree of abnormality, coinciding with elevated
levels of vascular endothelial growth factor (VEGF) deposited in direct proximity to the
vasculature. In addition to its role in vessel abnormalization, pleiotrophin enhanced PDGF-
B-induced gliomagenesis. Taken together, our results indicate that PTN has an important role
in glioma initiation and establishment of the characteristic abnormal tumor vasculature in
glioblastoma, identifying PTN as a potential target for therapy.
Keywords: glioma, vasculature, pleiotrophin, PTN, CD93, angiogenesis, GL261, RCAS
Lei Zhang, Department of Immunology, Genetics and Pathology, Rudbecklaboratoriet,
Uppsala University, SE-751 85 Uppsala, Sweden.
© Lei Zhang 2015
ISSN 1651-6206
ISBN 978-91-554-9239-7
urn:nbn:se:uu:diva-248713 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-248713)
To my family
致我的家人

List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.

I Dieterich L.C., Mellberg S., Langenkamp E., Zhang L., Zieba
A., Salomäki H., Teichert M., Huang H., Edqvist P.H., Kraus
T., Augustin H.G., Ologsson T., Larsson E., Söderberg O.,
Molema G., Pontén F., Georgii-Hemming P., Alafuzoff I.,
Dimberg A. (2011) Transcriptional profiling reveals a distinct
gene expression signature of vessels in high grade human glio-
ma. Journal of Pathology. 2012 Aug 31; 228(3): 378-390.
II Langenkamp E., Zhang L., Lugano R., Huang H., Elhassan
T.E.A., Bazzar W., Lööf J., Terndelenburg G, Pontén F., Smtis
A., Dimberg A. (2015) High expression of the C-type lectin
CD93 in glioblastoma vasculature enhances vessel function by
regulating cytoskeletal rearrangements and leads to poor sur-
vival. Manuscript under revision
III Zhang L., Kundu S., Feenstra T., Li X., Jin C., Elhassan
T.E.A., Ohlin K.E., Yu D., Olofsson T., Olsson A.K., Pontén
F., Magnusson P.U., Forsberg-Nilsson K., Essand M., Smits A.,
Dieterich L.C., Dimberg A. (2015) Pleiotrophin promotes vas-
cular abnormalization in glioma and correlates with poor sur-
vival in human astrocytomas. Manuscript under revision
IV Zhang L., Laaniste L., Jiang Y., Uhrbom L., Dimberg A.
(2015) Pleiotrophin enhances PDGF-B-induced gliomagenesis
and promotes angiogenesis in high-grade tumors in mouse ex-
perimental glioma. Manuscript
Reprints were made with permission from the respective publishers.
Related work by the author
Ringvall M., Thulin Å., Zhang L., Cedervall J., Tsuchida-Straeten N., Jah-
nen-Dechent W., Siegbahn A., Olsson A.K. (2011) Enhanced platelet activa-
tion mediates the accelerated angiogenic switch in mice lacking Histidine-
rich glycoprotein. PLOS One. 2011 Jan 17; 6(1): e14526
Femel J., Huijbers E.J., Saupe F., Cedervall J., Zhang L., Roswall P., Lars-
son E., Olofsson H., Pietras K., Dimberg A., Hellman L., Olsson A.K.
(2014) Therapeutic vaccination against fibronectin ED-A attenuates progres-
sion of metastatic breast cancer. Oncotarget. 2014 Dec 15; 5(23): 12418-27
Cedervall J., Zhang Y., Huang H., Zhang L., Femel J., Dimberg A., Olsson
A.K. (2015) Neutrophil extracellular traps impair peripheral vessel function
in individuals with cancer. Manuscript under revision
Contents
Introduction ................................................................................................... 11 
Glioma ...................................................................................................... 11 
Epidemiology ....................................................................................... 11 
Diagnosis and histological classification ............................................. 12 
Molecular markers ............................................................................... 14 
Glioma pathways ................................................................................. 15 
Cell of origin for malignant gliomas ................................................... 17 
Glioma stroma and vasculature ........................................................... 18 
Glioma treatment ................................................................................. 20 
Experimental models of glioma ........................................................... 23 
Angiogenesis ............................................................................................ 24 
Blood vessel structure .......................................................................... 24 
Induction of angiogenesis .................................................................... 25 
Pro-angiogenic factors ......................................................................... 25 
Molecular mechanisms of angiogenesis .............................................. 27 
Tumor angiogenesis ............................................................................. 28 
Tumor vessels ...................................................................................... 28 
Anti-angiogenic therapy ...................................................................... 29 
CD93 ........................................................................................................ 31 
General function .................................................................................. 31 
Role of CD93 in angiogenesis ............................................................. 32 
Pleiotrophin .............................................................................................. 32 
PTN receptors ...................................................................................... 33 
PTN gene expression ........................................................................... 34 
Role of PTN in angiogenesis ............................................................... 35 
Role in tumor development ................................................................. 35 
Present Investigations ................................................................................... 37 
Paper I ...................................................................................................... 37 
Paper II ..................................................................................................... 39 
Paper III .................................................................................................... 40 
Paper IV ................................................................................................... 42 
Concluding Remarks and Outlook ................................................................ 44 
Acknowledgments......................................................................................... 46 
Bibliography ................................................................................................. 48

Abbreviations
ARF p14/p19 Arf
BBB Blood-brain barrier
CDKNA2 Cyclin-dependent kinase inhibitor 2A
CNP 2’,3’-cyclic nucleotide 3’-phosphodiesterase
CNS Central nervous system
CSF Colony stimulating factor
ECM Extracellular matrix
FACS Fluorescence activated cell sorting
FDA Food and Drug Administration
FGF Fibroblast growth factor
GBM Glioblastoma
GFAP Glial fibrillary acidic protein
HIF Hypoxia inducible factor
HSPG Heparan sulfate proteoglycan
IFN Interferon
IL Interleukin
MAPK Mitogen activated protein kinase
MDSC Myeloid derived suppressor cell
MMP Matrix metalloproteinase
NF1 Neurofibromatosis type I
PCR Polymerase chain reaction
PDGF Platelet derived growth factor
PIGF Placental growth factor
PTEN Phosphatase and tensin homolog
RB Retinoblastoma
RTK Receptor tyrosine kinase
SVZ Subventricular zone
TAM Tumor associated macrophage
TNF Tumor necrosis factor
TP53 Tumor suppressor 53
VEGF Vascular endothelial growth factor
WHO World Health Organization