1. No category

Krista Honkonen
Adenoviral Gene Therapy
for the Stimulation of
Neovessel Growth
New Treatment Strategies for Lymphedema
and Peripheral Ischemia
Publications of the University of Eastern Finland
Dissertations in Health Sciences
KRISTA HONKONEN
Adenoviral Gene Therapy for the
Stimulation of Neovessel Growth
New Treatment Strategies for Lymphedema and Peripheral
Ischemia
To be presented by permission of the Faculty of Health Sciences,
University of Eastern Finland for public examination in Mediteknia Auditorium, Kuopio,
on Friday, May 29th 2015, at 12 noon
Publications of the University of Eastern Finland
Dissertations in Health Sciences
Number 280
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
University of Eastern Finland
Kuopio
2015
Grano Oy
Kuopio, 2015
Series Editors:
Professor Veli-Matti Kosma, M.D., Ph.D.
Institute of Clinical Medicine, Pathology
Faculty of Health Sciences
Professor Hannele Turunen, Ph.D.
Department of Nursing Science
Faculty of Health Sciences
Professor Olli Gröhn, Ph.D.
A. I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
Professor Kai Kaarniranta, M.D., Ph.D.
Institute of Clinical Medicine, Ophthalmology
Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy)
School of Pharmacy
Faculty of Health Sciences
Distributor:
University of Eastern Finland
Kuopio Campus Library
P.O. Box 1627
FI-70211 Kuopio, Finland
http://www.uef.fi/kirjasto
ISBN (print): 978-952-61-1778-2
ISBN (pdf): 978-952-61-1779-9
ISSN (print): 1798-5706
ISSN (pdf): 1798-5714
ISSN-L: 1798-5706
III
Author’s address:
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
University of Eastern Finland
P.O. Box 1627, FI-70211
KUOPIO
FINLAND
Email: [email protected]
Supervisors:
Professor Seppo Ylä-Herttuala, M.D., Ph.D.
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
University of Eastern Finland
KUOPIO
FINLAND
Johanna Lähteenvuo, M.D., Ph.D.
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
Faculty of Health Sciences
University of Eastern Finland
KUOPIO
FINLAND
Reviewers:
Professor Yulong He, M.Sc., Ph.D.
Cyrus Tang Hematology Center
Soochow University
SUZHOU, JIANGSU
CHINA
Professor Juha-Pekka Salenius, M.D., Ph.D.
Division of Vascular Surgery
Department of Surgery
Tampere University Hospital
TAMPERE
FINLAND
Opponent:
Docent Mikko Savontaus, M.D., Ph.D.
Turku Centre for Biotechnology
Åbo Akademi and University of Turku
TURKU
FINLAND
IV
V
Honkonen, Krista
Adenoviral Gene Therapy for the Stimulation of Neovessel Growth – New Treatment Strategies for
Lymphedema and Peripheral ischemia
University of Eastern Finland, Faculty of Health Sciences
Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 280. 2015. 102 p.
ISBN (print): 978-952-61-1778-2
ISBN (pdf): 978-952-61-1779-9
ISSN (print): 1798-5706
ISSN (pdf): 1798-5714
ISSN-L: 1798-5706
ABSTRACT
In this thesis, stimulation of the growth of both lymphatic and blood vessels was studied in
large animal models using adenovirally (Ad) delivered vascular endothelial growth factors
(VEGFs) and hypoxia-inducible factors (HIFs), which transcriptionally regulate a plethora
of factors involved in angiogenesis, including VEGF-A isoforms.
Two recognized lymphangiogenic growth factors, VEGF-C and VEGF-D, were employed
to induce lymphatic vessel growth in a new large animal model of lymphedema.
Lymphedema, which often develops as a consequence of the surgical removal of cancer or
radiation therapy, lacks curative treatments, thus making the application of gene therapy to
repair lymphatic damage an attractive new treatment option for the patients suffering from
this condition. Both AdVEGF-C and AdVEGF-D were shown to induce lymphatic vessel regrowth and, consequently, improve lymphatic flow in the pig model of lymphedema,
where lymphatic damage was first caused by excising all of the lymphatic vessels around
an inguinal lymph node. Interestingly, AdVEGF-D was found to evoke more pronounced
blood vascular effects than AdVEGF-C. Therefore, VEGF-C gene therapy was selected for
further evaluation and optimization in the same large animal model of lymphedema. A
subsequent comparison was performed of VEGF-C gene transfer into the lymph node
versus into the fat surrounding the lymph node in terms of efficacy and adverse effects. The
regeneration of lymphatic vessels was equally effective regardless of the gene transfer site.
However, gene transfer into the fat surrounding the lymph node was found to have less
adverse effects on the node.
In the final part of this thesis, HIF-1α and HIF-2α, which are upstream regulators of
VEGF-A expression, were adenovirally delivered to mouse myocardium and ischemic
rabbit hindlimbs to evaluate their capability to induce blood vessel growth. Patients with
cardiovascular disease are becoming increasingly older and suffering additional comorbid
conditions, i.e. they may not withstand the current invasive procedures but would be
suitable for these emerging alternative treatment options. Both of the transcription factors
increased blood capillary size and perfusion in skeletal muscles. Additionally, HIF-1α gene
transfer enhanced the recovery of ischemic muscles from energy depletion induced by
electrical stimulation.
In conclusion, adenovirally delivered VEGF family members VEGF-C and VEGF-D
efficiently induce lymphangiogenesis, which results in improved lymphatic flow, whereas
AdHIF-1α triggers angiogenesis, which is sufficient to improve energy metabolism of
ischemic muscles.
National Library of Medicine Classification: QU 107, QU 560, QZ 170, WG 500, WH 700
Medical Subject Headings: Lymphangiogenesis; Blood Vessels/growth & development; Angiogenesis
Inducing Agents; Genetic Therapy; Adenoviridae; Gene Transfer Techniques; Vascular Endothelial Growth
Factors; Basic Helix-Loop-Helix Transcription Factors; Lymphedema/therapy; Ischemia/therapy; Therapies,
Investigational; Disease Models, Animal
VI
VII
Honkonen, Krista
Adenovirusvälitteinen geeniterapia uudissuonten kasvattamiseksi – uusia hoitomenetelmiä lymfedeemaan ja
alaraajaiskemiaan
Itä-Suomen yliopisto, terveystieteiden tiedekunta
Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 280. 2015. 102 s.
ISBN (print): 978-952-61-1778-2
ISBN (pdf): 978-952-61-1779-9
ISSN (print): 1798-5706
ISSN (pdf): 1798-5714
ISSN-L: 1798-5706
TIIVISTELMÄ
Tässä väitöskirjassa tutkittiin adenovirusvälitteisesti (Ad) yliekspressoitujen verisuonen
endoteelin kasvutekijäperheen (vascular endothelial growth factor, VEGF) jäsenten sekä
hapenpuutteessa aktivoituvien transkriptiotekijöiden (hypoxia-inducible factor, HIF) kykyä
saada aikaan imu- ja verisuonten uudiskasvua isoeläinmalleissa. HIF-1α ja HIF-2α
säätelevät useita verisuonten uudismuodostukseen osallistuvia tekijöitä, mukaan lukien
VEGF-A:ta.
Väitöskirjan ensimmäisessä osatyössä kehitettiin uusi lymfedeeman isoeläinmalli, jonka
avulla tutkittiin tunnettujen lymfangiogeneettisten kasvutekijöiden VEGF-C:n ja VEGF-D:n
kykyä aikaansaada imusuonten uudismuodostusta imunestekierron parantamiseksi.
Lymfedeema eli imunesteturvotus kehittyy usein syövän kirurgisen poiston tai sädetyksen
seurauksena, eikä siihen ole olemassa parantavaa hoitoa. Imusuonten uudismuodostus
geeniterapian avulla voisi tarjota uuden hoitovaihtoehdon lymfedeemasta kärsiville
potilaille. Lymfedeema aiheutettiin tuhoamalla sian nivusen imusolmuketta ympäröivät
imutiet, minkä jälkeen imusolmukkeeseen tehtiin AdVEGF-C- tai AdVEGF-D-geeninsiirto.
Molemmat kasvutekijät saivat aikaan voimakasta imusuonten muodostusta ja paransivat
imunestekiertoa, mutta VEGF-D:n havaittiin aiheuttavan lisäksi huomattavia
verisuonivaikutuksia. Tästä johtuen VEGF-C valikoitui toisen osatyön kohteeksi, jossa
verrattiin imusolmukkeen sisään tehtyä AdVEGF-C-geeninsiirtoa imusolmuketta
ympäröivään rasvaan tehtyyn geeniinsiirtoon tehokkuuden ja haittavaikutusten osalta.
Imusuonten uudiskasvu oli yhtä tehokasta geeninsiirtopaikasta riippumatta, mutta rasvaan
tehdyn geeninsiirron havaittiin olevan suotuisampi imusolmukkeen kannalta.
Väitöskirjan viimeisessä osatyössä tutkittiin AdHIF-1α:n ja AdHIF-2α:n kykyä saada
aikaan verisuonten uudiskasvua hiiren sydämessä ja kanin hapenpuutteesta kärsivässä
takaraajassa. Sydän- ja verisuonitautipotilaat ovat yhä vanhempia ja kärsivät usein
liitännäissairauksista, mikä rajoittaa nykyisten kajoaviavien hoitotoimenpiteiden käyttöä
heidän kohdallaan ja luo tarpeen vaihtoehtoisten hoitomuotojen kehittämiselle. Molemmat
transkriptiotekijät HIF-1α ja HIF-2α saivat aikaan verisuonikapillaarien laajentumista ja
verenkierron lisääntymistä luurankolihaksessa. Lisäksi AdHIF-1α-geeninsiirron osoitettiin
parantavan iskeemisen lihaksen energiavarastojen palautumista sähköisesti tuotetun
lihassupistelun aiheuttamasta energiavajeesta.
Yhteenvetona voidaan todeta, että adenovirusvälitteinen geeninsiirto VEGF-C:llä tai
VEGF-D:llä johtaa voimakkaaseen imusuonten uudismuodostukseen sekä parantuneeseen
imunestekiertoon, kun taas AdHIF-1α aikaansaa verisuonten uudiskasvua, joka on riittävää
parantamaan hapenpuutteesta kärsivän lihaksen palautumista rasituksesta.
Luokitus: QU 107, QU 560, QZ 170, WG 500, WH 700
Yleinen Suomalainen asiasanasto: imusuonisto; verisuonet; kasvutekijät; angiogeneesi; geeniterapia;
adenovirukset; hoitomenetelmät; lymfedeema; iskemia; koe-eläinmallit
VIII
IX
Acknowledgements
This thesis work was carried out in the Department of Biotechnology and Molecular
Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland
during the years 2008-2014. Overall, it was immensely exciting to be able to contribute
something to this branch of science, but this was made even more so by the great people
who mentored and assisted me along the way.
First and foremost I wish to thank Professor Seppo Ylä-Herttuala for giving me the
chance to work in this group back in 2008 as a first-year medical student. Despite me being
practically a “tabula rasa” in the academic world, you allowed me to find my own path and
orient towards the research topics that appealed to me. Your enthusiasm and reassurances
kept my spirits up at times when desperation was raising its head. I also want to
acknowledge Anne Saaristo for her hands-on mentoring, thoughtful insights and prompt
solutions to whatever problems arose with the first and second publications of this thesis.
You introduced me to the world of lymphatics and included me into your projects, for
which I will be forever grateful.
I wish to thank Henna Niemi for taking me under her wing when I first arrived in the
group and for teaching me a large part of the laboratory methods employed in this thesis,
thus aiding me to become more independent as a researcher. I would also like to thank
Johanna Lähteenvuo for her supervision of this thesis, but above all, for letting me take part
in her large animal studies and her ever-so-helpful attitude towards me. I owe my gratitude
to Markku Lähteenvuo for being one of my closest collaborators during my years in AIVI.
Our discussions, whether about science or something completely different, always gave me
a lot to think about.
I was privileged to have Professor Yulong He and Professor Juha-Pekka Salenius as
thesis pre-examiners and wish to formally acknowledge them for their time and valuable
advice in improving this thesis. I would also like to thank Dr. Ewen MacDonald for his
excellent services in English-language proofreading.
My sincere gratitude goes to all the co-authors and colleagues without whom this thesis
would not have been possible. I would specifically like to thank Tomi Tervala for his
contributions to the execution of the two lymphatic studies and Mikko Visuri for his
meticulous refining of the second publication. Suvi Jauhiainen and Jari Lappalainen are
appreciated for their assistance with the experiments required for the revision of the first
publication. In addition, it was an honor to be able to collaborate and learn from such
enthusiastic and goal-oriented researchers as Tuomas Rissanen and Petra KorpisaloPirinen. Special thank-yous are sent to the summer workers Heikki Junkkari and Nina
Knuutinen for their help with the practical work, as well as for my previous summer
worker, now a PhD student, Paavo Halonen for his participation in the lymphatic studies.
Completing a thesis requires input from a veritable army of people and none of it would
have been possible without the sublime expertise of the technical staff. I am indebted to
Heikki Pekonen, Heikki Karhunen and Minna Törrönen for their invaluable assistance in
the experiments conducted at the Lab Animal Centre. I extend my gratitude to all the
technicians in AIVI and in particular to the following people: Seija Sahrio and Anneli
Miettinen for all the technical help in the laboratory over the years; Pekka Alakuijala, Jouko
Mäkäräinen and Jari Nissinen for assisting with computer- and workroom- related issues;
and Tiina Koponen and Sari Järveläinen for providing the adenoviruses used in these
studies. I also wish to express my heartfelt gratitude to Helena Pernu, Marja Poikolainen
and Jatta Pitkänen for their kind and patient guidance in various practical and
administrative issues over the years.
X
I hold in high regard all the past and present members of the SYH-group, most of whom
I’ve gone to for assistance at least once during my time in the group. Line LottonenRaikaslehto, thank you for becoming my friend as our paths intertwined both in the
research world and in medical school.
Aside from research colleagues, I wish thank my parents Anne-Marie and Ari for their
constant support, encouragement and confidence in me. Without you, I would have never
enjoyed so many opportunities in life. Katrine and Riina, you are the best little sisters
anyone could hope for. Thank you, Kati, for staying a close godmother through my life, and
Airi, my grandmother, for always inviting us over when we are nearby.
Finally, my deepest gratitude goes to Henri, my partner in life and co-worker in the
SYH-group. You have been a never-ending source of support to me and an amazing father
to our son. The last five years with you have been the most rewarding of my life and I hope
there are many great years ahead for us. Linus, my darling son, you brought a whole new
meaning to my life when you arrived a year ago. You are the biggest source of joy and
pride and put a smile on my face every day.
Kuopio, April 2015
Krista Honkonen
This work was supported by grants from the Graduate School of Molecular Medicine
(University of Eastern Finland), TEKES, Ark Therapeutics Ltd, Academy of Finland,
Finnish Medical Foundation, Sigrid Juselius Foundation, Emil Aaltonen Foundation,
Instrumentarium Science Foundation, Ida Montini Foundation, Aarne and Aili Turunen
.
Foundation.
XI
List of the original publications
This dissertation is based on the following original publications:
I
Lähteenvuo M*, Honkonen K*, Tervala T*, Tammela T, Suominen E, Lähteenvuo
J, Kholová I, Alitalo K, Ylä-Herttuala S, Saaristo A. Growth factor therapy and
autologous lymph node transfer in lymphedema. Circulation 123: 613-620, 2011.
II
Honkonen KM, Visuri MT, Tervala TV, Halonen PJ, Koivisto M, Lähteenvuo MT,
Alitalo KK, Ylä-Herttuala S, Saaristo AM. Lymph node transfer and perinodal
lymphatic growth factor treatment for lymphedema. Annals of Surgery 257: 961967, 2013.
III
Niemi H, Honkonen K, Korpisalo P, Huusko J, Kansanen E, Merentie M, Rissanen
TT, André H, Pereira T, Poellinger L, Alitalo K, Ylä-Herttuala S.
HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery.
European Journal of Clinical Investigation 44: 989-99, 2014.
* Authors with equal contribution
The publications were adapted with the permission of the copyright owners.
XII
XIII
Contents
1 INTRODUCTION ........................................................................................................................... 1
2 REVIEW OF THE LITERATURE ................................................................................................. 3
2.1 DISEASES OF THE BLOOD AND LYMPHATIC VASCULAR SYSTEM ............................ 3
2.1.1 Atherosclerosis .................................................................................................................... 3
2.1.2 Peripheral arterial disease ................................................................................................. 3
2.1.3 Coronary artery disease ..................................................................................................... 4
2.1.4 Lymphedema....................................................................................................................... 5
2.1.4.1 Primary lymphedema .............................................................................................. 7
2.1.4.2 Secondary lymphedema .......................................................................................... 8
2.1.5 Tumor lymphangiogenesis and metastasis ................................................................... 10
2.1.6 Other lymphatic vasculature-associated diseases ........................................................ 11
2.2 THE BLOOD VASCULAR SYSTEM AND BLOOD VESSEL GROWTH ........................... 12
2.2.1 Structure and function of the blood vascular system .................................................. 12
2.2.2 Blood vessel components................................................................................................. 13
2.2.3 Vasculogenesis .................................................................................................................. 14
2.2.4 Angiogenesis ..................................................................................................................... 15
2.2.5 Arteriogenesis.................................................................................................................... 16
2.3 THE LYMPHATIC SYSTEM AND LYMPHATIC VESSEL GROWTH............................... 17
2.3.1 Anatomy and function of the lymphatic system .......................................................... 17
2.3.2 Lymphangiogenesis.......................................................................................................... 19
2.3.3 Lymphatic vessel markers ............................................................................................... 21
2.3.4 Lymph nodes ..................................................................................................................... 21
2.4 MOLECULAR REGULATION OF BLOOD AND LYMPHATIC VESSEL GROWTH ..... 23
2.4.1 Hypoxia-inducible factors ............................................................................................... 23
2.4.2 Vascular endothelial growth factors and their receptors ............................................ 25
2.4.2.1 VEGF-A .................................................................................................................... 26
2.4.2.2 VEGF-B..................................................................................................................... 27
2.4.2.3 VEGF-C .................................................................................................................... 27
2.4.2.4 VEGF-D .................................................................................................................... 28
2.4.2.5 PlGF .......................................................................................................................... 28
2.4.2.6 VEGF-receptors....................................................................................................... 28
2.4.2.7 NRP-receptors ......................................................................................................... 30
2.5 GENE THERAPY ........................................................................................................................ 30
2.5.1 Viral vectors ....................................................................................................................... 31
2.5.1.1 Adenoviral vectors ................................................................................................. 32
2.5.1.2 Other viral vectors .................................................................................................. 33
2.5.2 Non-viral vectors .............................................................................................................. 34
2.5.3 Safety issues in gene therapy .......................................................................................... 34
2.6 PRECLINICAL AND CLINICAL STUDIES ............................................................................ 35
2.6.1 Preclinical studies of lymphedema ................................................................................ 35
2.6.2 Preclinical studies of proangiogenic therapy for PAD ................................................ 36
2.6.3 Clinical studies of proangiogenic therapy for PAD ..................................................... 37
3 AIMS OF THE STUDY ................................................................................................................ 39
XIV
4 MATERIALS AND METHODS ................................................................................................. 41
4.1 ANIMAL MODELS..................................................................................................................... 41
4.1.1 Pig model of lymphedema (I-II) ..................................................................................... 41
4.1.2 Rabbit model of hind limb ischemia (III) ...................................................................... 41
4.2 GENE TRANSFER METHODS ................................................................................................. 42
4.2.1 Intranodal and perinodal gene transfers (I-II) .............................................................. 42
4.2.2 Intramuscular gene transfer (I, III) ................................................................................. 42
4.2.3 Myocardial gene transfer (III) ......................................................................................... 43
4.3 X-RAY IMAGING ....................................................................................................................... 43
4.3.1 Lymphangiography (I-II) ................................................................................................. 43
4.3.2 Digital subtraction angiography (III) ............................................................................. 43
4.4 ULTRASOUND IMAGING ....................................................................................................... 44
4.5 31P-MAGNETIC RESONANCE SPECTROSCOPY................................................................. 44
4.6 VASCULAR PERMEABILITY AND TISSUE EDEMA .......................................................... 46
4.6.1 Seroma fluid evaluation (I-II) .......................................................................................... 46
4.6.2 Modified Miles Assay (I, III) ........................................................................................... 46
4.7 PROTEIN ANALYSES................................................................................................................ 46
4.7.1 Western Blot (I, III) ........................................................................................................... 46
4.7.2 ELISA (I) ............................................................................................................................. 46
4.8 mRNA EXPRESSION ................................................................................................................. 47
4.9 IMMUNOHISTOCHEMISTRY AND HISTOLOGY .............................................................. 47
4.10 ASSESSMENT OF LYMPHATIC FUNCTION ..................................................................... 49
4.11 STATISTICAL ANALYSES...................................................................................................... 49
5 RESULTS ........................................................................................................................................ 51
5.1 THERAPEUTIC LYMPHANGIOGENESIS FOR LYMPHEDEMA (I) ................................ 51
5.1.1 Lymphatic vessel regrowth induced by AdVEGF-C and AdVEGF-D ..................... 51
5.1.2 Lymphatic transport 2 months after AdVEGF-C and AdVEGF-D treatment ......... 52
5.1.3 Increased seroma formation following AdVEGF-D gene transfer ............................ 53
5.1.4 Histopathological evaluation of safety samples ........................................................... 54
5.2 OPTIMIZING ADVEGF-C THERAPY FOR LYMPHEDEMA (II) ....................................... 54
5.2.1 Lymphatic neovessel growth induced by intranodal and perinodal AdVEGF-C .. 54
5.2.2 Effects of intranodal and perinodal AdVEGF-C on lymph node preservation and
seroma formation ....................................................................................................................... 57
5.2.3 Macrophage accumulation inside the lymph node following intranodal GT ......... 57
5.3 THERAPEUTIC ANGIOGENESIS VIA OVER-EXPRESSION OF HIFS (III) ..................... 58
5.3.1 Angiogenic response following AdHIF-1α and AdHIF-2α GTs ............................... 58
5.3.2 Effects of AdHIFs on muscle energy metabolism ........................................................ 60
5.3.3 Upregulation of HIF target genes following AdHIF-1α and AdHIF-2α GTs .......... 60
6 DISCUSSION ................................................................................................................................ 61
6.1 LYMPHANGIOGENIC GENE THERAPY FOR LYMPHEDEMA (I-II) ............................. 61
6.2 PHYSIOLOGICAL ANGIOGENESIS INDUCED BY HIFS (III) .......................................... 64
7 CONCLUSIONS AND FUTURE PERSPECTIVES ................................................................ 67
8 REFERENCES ................................................................................................................................ 69
APPENDIX: ORIGINAL PUBLICATIONS I-III
XV
Abbreviations
31
Phosphorus-magnetic
resonance spectroscopy
ELISA
Enzyme-linked
immunosorbent assay
AAV
Adeno-associated virus
eNOS
ABI
Ankle-brachial index
Endothelial nitric oxide
synthase
Ad
Adenoviral
EPC
Endothelial progenitor cell
ANG
Angiopoietin
FDA
ANOVA
Analysis of variance
The U.S. Food and Drug
Administration
APC
Antigen-presenting cell
FGF
Fibroblast growth factor
ApoE
Apolipoprotein E
FIH
ATP
Adenosine triphosphate
Factor inhibiting hypoxia-inducible
factor 1
BM
Basement membrane
FOXC2
BM-EPC
Bone marrow-derived
endothelial progenitor cell
Forkhead box C2
transcription factor
fps
Frames per second
GATA2
GATA binding protein 2
GJC2
Gap junction protein,
gamma 2
31
P-MRS
CABG
Coronary artery bypass
grafting
CAD
Coronary artery disease
CAR
Coxsackie-adenovirus
receptor
GMP
Good manufacturing practice
GT
Gene transfer
Collagen and calcium binding
EGF domains 1
HAM
Human alveolar macrophage
HDL
High-density lipoprotein
CD
Cluster of differentiation
HGF
Hepatocyte growth factor
cDNA
Complementary DNA
HIF
Hypoxia-inducible factor
CLEC-2
C-type lectin domain family 2
HLA
Human leukocyte antigen
CLI
Critical limb ischemia
HLT
cm
Cubic centimetre
Hypotrichosis-lymphedematelangiectasia
CMV
Cytomegalovirus
HRE
Hypoxia-response element
COUP-TFII Chicken ovalbumin upstream
promoter transcription factor
2
HRP
Horseradish peroxidase
HSPG
Heparan sulfate
proteoglycans
CPS
Contrast pulse sequence
HUVEC
DAB
Diaminobenzidine
Human umbilical vein
endothelial cell
DNA
Deoxyribonucleic acid
IgG
Immunoglobulin G
DSA
Digital subtraction
angiography
i.v.
Intravenous
KAT
Kuopio Angiogenesis Trial
E10.5
Embryonic day 10.5
kbp
Kilobase pair
EC
Endothelial cell
KIF11
Kinesin family member 11
EF%
Ejection fraction
KSHV
Kaposi sarcoma herpes virus
CCBE1
3
XVI
LAM
Lymphangioleiomyomatosis
Pi
Inorganic phosphate
LacZ
β-Galactosidase
PlGF
Placental growth factor
LD
Lymphedema-distichiasis
ppm
Parts per million
LDL
Low-density lipoprotein
PROX1
LEAD
Lower extremity arterial
disease
Prospero-related homeobox
transcription factor 1
PTPN14
Protein tyrosine phosphatase,
non-receptor type 14
pVHL
Von Hippel-Lindau tumor
suppressor protein
LEC
Lymphatic endothelial cell
LV
Lymphatic vessel
LVAW
Left ventricle anterior wall
LYVE-1
Lymphatic vascular
endothelial hyaluronan
receptor-1
Q-Q
Quantile-quantile
RCT
Reverse cholesterol transport
RNA
Ribonucleic acid
M.
Musculus
SCID
mL
Milliliter
Severe combined
immunodeficiency
MLD
Manual lymphatic drainage
SD
Standard deviation
mm
Millimeter
SEM
Standard error of mean
MOI
Multiplicity of infection
siRNA
Small interfering RNA
mRNA
Messenger RNA
SLP76
NFATc1
Nuclear factor of activated T
cells, cytoplasmic, calcineurin
dependent 1
SH2 domain-containing
leukocyte protein of 76kDa
SMC
Smooth muscle cell
SOX18
Sex determining
region Y box 18 transcription
factor
sVEGFR
Soluble vascular endothelial
growth factor receptor
NF-κB
Nuclear factor kappa-lightchain-enhancer of activated
B cells
nm
Nanometer
NO
Nitric oxide
SYK
Spleen tyrosine kinase
NRP
Neuropilin
TIE1
ns
Non-significant
O2
Oxygen
Tyrosine kinase with
immunoglobulin-like and
EGF-like domains 1
PAD
Peripheral arterial disease
TSA
Tyramide signal amplification
PCI
Percutaneous coronary
intervention
VEGF
Vascular endothelial growth
factor
PCr
Phosphocreatine
VEGFR
PCR
Polymerase chain reaction
Vascular endothelial growth
factor receptor
PDGF
Platelet-derived growth factor
vp
Viral particles
PDK1
Pyruvate dehydrogenase
kinase, isozyme 1
μL
PECAM-1 Platelet/endothelial cell
adhesion molecule 1
pfu
Plaque-forming units
pH
Power of hydrogen
Microliter
μm
2
Square micrometer
1 Introduction
Gene therapy comprises a set of strategies which modify the expression of specific genes in an
attempt to treat or prevent diseases. Although still in its infancy, it holds promise for treating a
wide range of diseases, including several cardiovascular diseases. Cardiovascular diseases are
still the leading cause of death globally, despite major progress in current treatment practices
(WHO 2014). The pathophysiology of these diseases involves a process called atherosclerosis, in
which lipid accumulation and inflammation lead to a gradual occlusion of arteries and
consequently to ischemia (Libby 2002). Cardiovascular gene therapy aims at stimulating
vascular growth in order to ensure sufficient blood flow to the tissue suffering from ischemia
(Yla-Herttuala, Martin 2000). This can be achieved by therapeutically over-expressing proteins
that are essential for the formation of new blood vasculature. Vascular endothelial growth
factors (VEGFs) govern the sprouting of new blood vessels from pre-existing vessels, termed as
angiogenesis, and they have been widely used for vascular therapeutic purposes (Ribatti,
Crivellato 2012). A single administration of VEGF-A evokes intense angiogenic effects, but also
can evoke severe side effects such as tissue edema (Rissanen et al. 2005). Therefore, it has been
postulated that upstream regulators of VEGF expression, such as hypoxia-inducible factors
(HIFs), would represent a better therapeutic target than a single angiogenic factor since HIFs are
able to activate multiple angiogenic growth factors as well as regulating many of the genes
involved in energy metabolism and recovery of ischemic tissues (Semenza 2012). As a result of
multiple growth factors working in harmony, the angiogenic response may be more
physiological than anything that can be achieved by one individual factor.
Parallel to blood vascular circulatory system in mammals there exists another vascular
network that transports a plasma-derived, milk-like fluid called lymph from the peripheral
tissues back to the central blood circulation (Breslin 2014). Insufficiency of this lymphatic
vascular system may lead to lymphedema, which is a debilitating condition characterized by
gross swelling of the affected part of the body (Warren et al. 2007). In the industrialized
countries, lymphedema often develops as a consequence of surgical or radiotherapeutic
interventions for cancer (Rockson, Rivera 2008). Apart from cardiovascular gene therapy, where
angiogenic growth factors are administered to achieve therapeutic blood vessel growth in
ischemic tissues, gene therapy may also be employed to stimulate the growth of new lymphatic
vessels and thus to ameliorate the symptoms of lymphedema. Two vascular endothelial growth
factor family members, VEGF-C and VEGF-D, are known to be stimulators of
lymphangiogenesis; these factors have been demonstrated to restore lymphatic drainage to a
site of lymphatic damage in experimental rodent models (Tammela et al. 2007, Yoon et al. 2003,
Szuba et al. 2002).
The aim of this thesis was to utilize large animal models to clarify the lymphangiogenic
potential of two adenovirally delivered single growth factors, VEGF-C and VEGF-D, and
moreover, to study whether the adenoviral application of a “master-switch” agent, such as HIF1α/-2α, which is responsible for the coordinated induction of multiple angiogenic factors,
would lead to a more physiological response than could be achieved with the administration of
a single growth factor, such as VEGF-A.
2
3
2 Review of the literature
2.1 DISEASES OF THE BLOOD AND LYMPHATIC VASCULAR SYSTEM
2.1.1 Atherosclerosis
Atherosclerosis is a disease of large arteries and the underlying pathogenic process behind
ischemia affecting the lower extremities, the heart and the brain. There are several recognized
risk factors for atherosclerosis including high serum LDL (low-density lipoprotein) and low
HDL (high-density lipoprotein) levels, hypertension, diabetes, smoking, obesity and lack of
exercise. The atherosclerotic process involves the accumulation of lipids into the subendothelial
space, the infiltration of inflammatory cells, intracellular lipid accumulation in macrophages
and smooth muscle cells (SMCs), the migration and proliferation of medial SMCs within the
inflamed intima, fibrous cap formation, and finally calcification and neovascularization within
the plaque. (Libby 2002, Lusis 2000.) Initially, the lesion grows towards the adventitia until,
after a certain point, it begins to protrude into and obstruct the vessel lumen (Lusis 2000). The
progressive narrowing of the arterial lumen impairs blood flow and leads to the first clinical
manifestations: ischemic pain at stress and later also at rest. Rupture of an atherosclerotic lesion
and the concomitant thrombosis are responsible for major cardiovascular events, such as
sudden death, myocardial infarction or stroke. (Libby 2002.) Although the root cause of the
inflammatory process remains as yet undefined, endothelial dysfunction is considered to be the
preceding step prior to LDL entry within the arterial intima, and thus, it can be considered as
the basis for the development of angiopathy (Badimon, Vilahur 2012).
2.1.2 Peripheral arterial disease
Peripheral arterial disease (PAD) refers to the narrowing of arteries distal to the arch of aorta,
including carotid, vertebral, upper extremity, mesenteric, renal and lower extremity vascular
sites (Figure 1) (European Stroke Organisation 2011). The total disease prevalence ranges
between 3 and 15% increasing up to 20% with age (Norgren et al. 2007). The risk factors for
PAD include the typical risk factors for atherosclerotic disease, such as smoking, hypertension,
diabetes and dyslipidemia (European Stroke Organisation et al. 2011). The lower extremity
artery disease (LEAD) has different presentations that can be categorized according to the
Fontaine (stages I-IV) or the Rutherford classification (grades 0-III). The most typical symptom
is intermittent claudication: pain in the calves that increases with walking and disappears
quickly at rest (Fontaine stage II, Rutherford grade I). In more severe cases, pain is present at
rest (Fontaine stage III, Rutherford grade II). Severe ischemia is evidenced by ulcers and
gangrene, typically presenting in the distal part of the limb (Fontaine stage IV, Rutherford
grade III). Critical limb ischemia (CLI) is defined as the presence of ischemic rest pain for more
than 2 weeks or ischemic skin lesions, either ulcers or gangrene. Acute limb ischemia may occur
following an embolic event or local thrombosis and has a 10 - 30% 30-day amputation rate.
(Norgren et al. 2007.)
The primary test for the diagnosis of LEAD is the ankle-brachial index (ABI), i.e. the ratio of
ankle and brachial systolic blood pressures (European Stroke Organisation 2011). A value of
< 0.9, indicative of a hemodynamically-significant arterial stenosis, is 95% sensitive and 99%
specific for the diagnosis of LEAD (McDermott et al. 2002). Moreover, a reduced ABI has
prognostic value in identifying persons at risk for suffering cardiovascular morbidity and
4
mortality (Hirsch et al. 2006). A very high ABI (> 1.4) in relation to stiff (calcified) arteries is also
associated with increased mortality (Criqui et al. 2010).
The conservative treatment of PAD includes smoking cessation, dietary therapy aiming at
weight reduction, exercise and pharmacotherapy comprising antiplatelet agents, lipid-lowering
agents and antihypertensive drugs (European Stroke Organisation 2011). In addition,
vasodilators such as cilostazol and naftidrofuryl, may have a mild-to-moderate effect to
improve walking distance in patients with intermittent claudication (Pande et al. 2010, De
Backer et al. 2009). Revascularization is considered in patients with CLI or severely debilitating
claudication when the response to conservative therapy has been inadequate (O'Donnell et al.
2011). Angioplasty is preferred for single short stenoses in the common iliac artery, whereas
endovascular or open vascular surgery utilizing vein grafts or synthetic prostheses are best
suited for diffuse occlusive disease (European Stroke Organisation 2011). Bypass surgery
appears to have a better long-term patency than endovascular interventions, although the risks
of surgery are greater in terms of mortality rates, major complications and technical success rate
(European Stroke Organisation 2011, Norgren et al. 2007). Amputation remains the final option
to resolve irreversible limb ischemia, particularly in the case of infectious gangrene or extensive
necrosis. One year after the onset of CLI, 25% of patients will have died and 25% will have
required a major amputation. (Norgren et al. 2007.)
Figure 1. Magnetic resonance angiography of a patient with PAD. The double-ended arrow marks the
superficial femoral artery that is severely stenosed on the right and occluded on the left. Small
arrows point to collaterals arising from the deep femoral artery. Reprinted by permission from
Macmillan Publishers Ltd: Nature Reviews Cardiology, Lau et al., “Peripheral artery disease. Part 1:
clinical evaluation and noninvasive diagnosis” © 2011.
2.1.3 Coronary artery disease
Cardiovascular diseases are the leading cause of death in both high- and low-income countries,
with 7.2 million people dying of coronary artery disease (CAD) alone in 2002 (WHO 2014). With
respect to all the cardiovascular diseases, CAD is the most prevalent manifestation with clinical
presentations including silent ischemia, stable and unstable angina pectoris, myocardial
5
infarction, heart failure and sudden cardiac death (Kumar, Abbas & Fausto 2005). These are
generally caused by a progressive encroachment of the arterial lumen leading to stenosis or
acute plaque rupture with thrombosis, with or without concomitant vasoconstriction. A 75%
reduction in the cross-sectional area of the lumen induces symptoms during exertion, whereas
an occlusion exceeding 90% leads to insufficient coronary flow at rest. (Kumar, Abbas & Fausto
2005, Grech 2003.) Primary interventional therapies for coronary insufficiency are percutaneous
coronary intervention (PCI) and coronary artery bypass grafting (CABG). CABG remains the
treatment of choice for patients with severe CAD, i.e. those with left main coronary artery
disease or three-vessel disease. (Levine et al. 2011, Serruys et al. 2009.) For PCI, a focal stenosis
on a straight artery without involvement of major side branches is ideal (O'Toole, Grech 2003).
Patients unsuitable for revascularization procedures form a heterogenous group with
annualized mortality rates ranging from 3% to 21%. Nonsuitability for revascularization may be
due to anatomical reasons, such as diffuse thread-like coronary atherosclerosis, or nonanatomical factors including excessive comorbidities, procedural risk and poor compliance to
medication. (Jolicoeur et al. 2012.)
2.1.4 Lymphedema
The lymphatic vessels play a key role in the regulation of tissue fluid homeostasis and immune
functions of the body (Alitalo 2011, Witte, Witte 1987). Blockage of lymphatic drainage due to
infection, surgery, cancer or a genetic defect leads to lymphedema, which manifests as
disfiguring and gross swelling of the extremities (Figure 2) (Warren et al. 2007). Edema
develops as plasma proteins leak into the interstitium raising the colloid osmotic pressure of the
interstitial fluid, which draws even more fluid out of the capillaries (Guyton, Hall 2006).
Lymphedema is usually a progressive, chronic condition that is often complicated by recurrent
lymphangitis, cellulitis, or in rare cases, cutaneous malignant tumors (Szuba, Rockson 1998). In
the stage I clinical classification of lymphedema, the swelling, which consists of protein-rich
fluid, is considered to be reversible simply by limb elevation, whereas in stage II, the affected
tissue hardens as a result of inflammation, fibrosis, adipose tissue hypertrophy and scarring,
eventually becoming irreversible. Advanced lymphedema (stage III) refers to lymphostatic
elephantiasis, cartilaginous hardening with hyperkeratosis and papillomatous outgrowths of
the skin. (International Society of Lymphology 2009.) Lymphedemas are etiologically divided
into two categories: Primary lymphedemas are caused by rare congenital defects and can
develop in utero, neonatally, or years or decades after birth, whereas the more common
secondary lymphedemas are caused by disruption of a normal lymphatic system by disease or
an iatrogenic process (Warren et al. 2007). Different types of lymphedema, mutated genes
identified as being involved or other causes, as well as clinical manifestations are listed in table
1.
6
Figure 2. Lymphedema of the lower limbs. Reprinted by permission from Macmillan Publishers Ltd:
Nature Genetics, Ostergaard et al., “Mutations in GATA2 cause primary lymphedema associated with
a predisposition to acute myeloid leukemia (Emberger syndrome)”, © 2011.
Table 1. Various lymphedema syndromes, their causes or causative mutations and clinical findings.
Disease
Cause/Mutation
Manifestations
Lymphedema-distichiasis
FOXC2
Lymphedema of lower extremities, double row of
eyelashes, additional complications, e.g. ptosis
Primary congenital
lymphedema / Nonne-Milroy
lymphedema
VEGFR3
Congenital lymphedema of lower limbs
Milroy-like disease
VEGFC
Congenital lymphedema of lower limbs
Hypotrichosis-lymphedematelangiectasia syndrome
SOX18
Lymphedema, alopecia, telangiectasias
Meige disease / lymphedema
praecox
GJC2
Lymphedema affecting all 4 extremities, onset
typically at <15 years of age
Hennekam syndrome
CCBE1
Lymphedema, intestinal lymphangiectasias,
mental retardation, dysmorphic facial features
Lymphedema-choanal atresia
PTPN14
Lymphedema of lower extremities in children,
blockage of choanae
Emberger syndrome
GATA2
Lymphedema of lower extremities and genitals,
hematological abnormalities, deafness
MCLMR
KIF11
Microcephaly with or without chorioretinopathy,
lymphedema, or mental retardation
Filariasis
Parasite infection
Lymphedema of lower extremities and genitals
Cancer-related lymphedema
Surgical or radiation
therapy of cancer
Lymphedema of extremities or trunk depending
on the location, type and treatment of cancer
7
2.1.4.1 Primary lymphedema
The prevalence of primary lymphedema has been estimated at 1.15 per 100000 persons less than
20 years of age (Smeltzer, Stickler & Schirger 1985). To date, at least 9 genes responsible for
different isolated or syndromic forms of primary lymphedema have been identified with the
help of genetic studies and genetically modified mouse models (table 1). Intriguingly, most of
the proteins encoded by these genes are linked to the VEGF-C/VEGFR-3 (Vascular endothelial
growth factor receptor-3) signaling axis. Overall, genetic mutations in 8 specific genes have
been estimated to account for the etiology of 40% of familial lymphedema and 10% of the
sporadic occurrences, which together account for less than 25% of patients with lymphedema.
(Brouillard, Boon & Vikkula 2014, Mendola et al. 2013.)
The most common form of primary lymphedema is lymphedema-distichiasis (LD), which is
an autosomal-dominant disorder caused by loss-of-function mutations affecting the forkhead
transcription factor (FOXC2) (Fang et al. 2000). Transcription factors, such as FOXC2, cause
syndromic forms of lymphedema by acting downstream of VEGFR-3 and affecting several
target genes (Brouillard, Boon & Vikkula 2014). LD is characterized by late-onset lymphedema
of the lower extremities and a congenital double row of eyelashes (distichiasis) occurring in 94100% of the patients, with additional complications including varicose veins, ptosis, extradural
cysts, cardiac defects, and cleft palate. In this disease, lymphedema develops as a result of
lymph reflux, which is likely due to a primary valve failure. (Connell, Brice & Mortimer 2008.)
Foxc2-/- mice have been found to exhibit abnormal pericyte recruitment to the lymphatic
capillaries and valve agenesis in the collecting vessels, indicating that FOXC2 is essential for the
formation of a pericyte-free lymphatic network and lymphatic valve development (Petrova et
al. 2004). Moreover, patients with LD experience venous valve failure in addition to
lymphedema, suggesting a common mechanism for the morphogenesis of venous and
lymphatic valves (Mellor et al. 2007). Gain-of-function mutations in FOXC2 have also been
shown to occur in patients with lymphedema, although their association with distichiasis has
not been confirmed (van Steensel et al. 2009).
There is another form of hereditary lymphedema called primary congenital lymphedema,
also known as Nonne-Milroy lymphedema, which is an early-onset congenital lymphedema
linked to heterozygous missense mutations inactivating the kinase activity of VEGFR3 in about
70% of cases (Gordon et al. 2013b, Karkkainen et al. 2000). In addition, a homozygous recessive
mutation in VEGFR3 has been reported in a lymphedema patient (Ghalamkarpour et al. 2009).
A recent study identified a novel mutation in VEGFC as a cause for Milroy-like primary
lymphedema (Gordon et al. 2013a). Mutations have been reported in more than 100 families to
date (Brouillard, Boon & Vikkula 2014). The phenotype of Milroy patients is characterized by
uni- or bilateral edema of the lower limbs with more infrequent manifestations including
cellulitis, large calibre leg veins, papillomatosis and upslanting toenails (Brice et al. 2005).
Lymphatic abnormalities are only present at sites of swelling, and the lymphatic drainage
routes of the upper limb are normal despite the germline mutation (Connell, Brice & Mortimer
2008). The vast majority, 90%, of patients with VEGFR3 mutations were found to exhibit
saphenous vein reflux, regardless of whether lymphedema was present or not, implying a dual
role of the gene in venous and lymphatic development (Mellor et al. 2010). Contrary to the
former belief that Nonne-Milroy lymphedema follows from aplasia, skin biopsies from the
swollen feet of patients with VEGFR3 mutations have revealed the presence of abundant yet
dysfunctional skin lymphatic vessels, attributing lymphedema rather to the functional failure of
initial lymphatics than their absence (Mellor et al. 2010). However, knockout mice with similar
Vegfr3 mutations display hypoplasia of the cutaneous lymphatics due to defective
lymphangiogenesis, and this condition can be rescued by therapeutic delivery of VEGF-C
(Karkkainen et al. 2001).
Hypotrichosis-lymphedema-telangiectasia (HLT) is a very rare syndrome associated with
mutations in the Sex determining region Y box 18 transcription factor (SOX18), and inherited
8
either autosomal dominantly or autosomal recessively (Irrthum et al. 2003). It is characterized
by a combination of sparse hair, dilation of small blood vessels particularly in the palms, and
lymphedema of the legs, which is likely due to lymphatic capillary hypoplasia (Schulte-Merker,
Sabine & Petrova 2011). SOX18 has been demonstrated to promote lymphatic endothelial cell
(LEC) differentiation from venous precursors and to act in several developmental systems,
including hair follicle development, vasculogenesis, and lymphangiogenesis (Francois et al.
2008).
Meige disease, also termed lymphedema praecox, manifests as lymphedema in all four
extremities and it is usually detected at less than 15 years of age, but a causative genetic
mutation is yet to be confirmed. However, missense mutations in the GJC2 (Gap junction
protein, gamma 2) gene encoding connexin 47 have been found to alter gap junction function,
leading to impaired pulsatile lymphatic flow in patients with dominantly inherited
lymphedema. (Ostergaard et al. 2011a, Ferrell et al. 2010.) Thus, GJC2 is considered as a
candidate gene to explain the lymphedema of some patients previously labeled as having
Meige disease (Connell et al. 2013). Hennekam syndrome, which is inherited autosomal
recessively, comprises lymphedema, intestinal and renal lymphangiectasias, mental retardation
and dysmorphic facial appearance (Alders et al. 2009). The genetic mutation responsible for
lymphatic vessel dysplasia in this syndrome is located in CCBE1 (Collagen and calcium binding
EGF domains 1), which normally potentiates the effects of VEGF-C on VEGFR-3 and has been
shown to regulate LEC precursor budding and angiogenic sprouting in zebrafish (Jeltsch et al.
2014, Bos et al. 2011, Alders et al. 2009, Hogan et al. 2009). PTPN14 (Protein tyrosine
phosphatase, non-receptor type 14) is an intracellular phosphatase that interacts with VEGFR-3
upon activation by VEGF-C. Ptpn14-deficient mice display postnatal lymphedema due to
hyperplastic lymphatic vessels. (Au et al. 2010.) A mutation in PTPN14 gene leads to
lymphedema-choanal atresia syndrome in humans, which is characterized by blockage of nasal
passage (choana) and lymphedema of the lower extremities in children (Schulte-Merker, Sabine
& Petrova 2011). Emberger syndrome presents as co-occurring primary lymphedema and
myelodysplasia progressing to acute myeloid leukaemia, with or without congenital deafness.
The mutation responsible for lymphedema and predisposition to leukaemia in this syndrome is
in GATA2 (GATA binding protein 2), which is a transcription factor controlling PROX1
(Prospero-related homeobox transcription factor 1) and FOXC2 expression. (Brouillard, Boon &
Vikkula 2014, Ostergaard et al. 2011b.) Mutations in KIF11 (Kinesin family member 11) cause a
specific syndrome named MCLMR, consisting of microcephaly with or without
chorioretinopathy, lower limb lymphedema, or mental retardation. A definitive link between
KIF11 and VEGF-C/VEGFR-3 pathway is yet to be established, but inhibition of EG5, which is a
kinesin motor protein involved in mitotic spindle assembly and encoded by KIF11, activates a
downstream signaling pathway of VEGFR-3. (Brouillard, Boon & Vikkula 2014, Ostergaard et
al. 2012.) In addition, lymphedema occurs in several other syndromes including Turner
syndrome, Noonan syndrome, and Aagenaes’ syndrome (cholestasis-lymphedema syndrome)
(Ferrell, Finegold 2008, Loscalzo et al. 2005).
2.1.4.2 Secondary lymphedema
The most prevalent etiology of acquired lymphedema is filariasis (elephantiasis) secondary to
Wuchereria bancrofi (~95%), Brugia malay or Brugia timori infection, which is estimated to affect
120 million people worldwide with 1.2 billion people at risk of infection (Ottesen et al. 2008).
The mosquito-borne parasitic nematodes reside in the lymphatic system; this triggers an
inflammatory reaction and activates the production of VEGF-A, VEGF-C and VEGF-D, thus
leading to hyperplasia and obstruction of the lymphatic vessels usually in the lower extremities
and genitals (Pfarr et al. 2009). The Global Programme for the Elimination of Lymphatic
Filariasis is attempting to eliminate the disease as a public health problem by the year 2020, by
conducting mass drug administrations with the aim of stopping transmission in endemic areas
(Ottesen et al. 1997). Reductions in infection prevalence and even certified elimination of
9
lymphatic filariasis have been achieved in some isolated areas by the use of microfilaricidal
drugs, i.e. drugs that kill the first larval stage, the microfilariae (Hoerauf 2008). However, these
drugs provide no help to already infected individuals, as it is the death of the adult-stage worm
that causes the pathological changes to occur (Pfarr et al. 2009). Instead, doxycycline has shown
strong macrofilaricidal activity in affected patients by killing worm symbiotic bacteria
(Wolbachia), consequently leading to a reduction in plasma concentrations of VEGF-C/sVEGFR-3
(soluble vascular endothelial growth factor receptor-3), which precedes the amelioration of
lymphatic vessel dilation and lymphedema (Debrah et al. 2006).
In the developed countries, the most common cause of secondary lymphedema is surgical or
radiation therapy of a malignancy, especially breast cancer but also pelvic cancer and malignant
melanoma (Oremus et al. 2012). Tumor cells often metastasize to lymph nodes, necessitating
radical surgery with lymph node dissection and adjuvant radiotherapy, which damage the
collecting lymphatic vessels and impair lymphatic flow (Norrmen et al. 2011). The remaining
lymphatic vessels need to distend to accommodate the increased fluid load. Collecting
lymphatic vessels may regenerate and spontaneously recanalize to some extent, but typically an
adequate formation of new lymphatic vessels is not observed in lymphedema patients. (Tobbia
et al. 2009, Ikomi et al. 2006, Abe 1976.) The pathophysiology of breast cancer-related
lymphedema is not yet fully understood. The traditional view is the so-called stopcock
hypothesis, according to which the surgically traumatized axilla impairs the lymphatic drainage
from the entire arm, as if a stopcock had been partially closed. It does not, however, explain the
delayed onset of swelling, the non-uniform distribution of swelling along the arm, sparing of
some regions, e.g. the hand, and the lowering of plasma protein levels in the affected arm.
(Stanton et al. 2009b.) The edema mainly accumulates in the highly compliant subcutis and skin,
whereas there is very little swelling observed in the muscle compartment (Collins et al. 1995).
The literature does not provide reliable prevalence rates, although there is an estimate that
there are 10 million patients suffering from secondary lymphedema in the US alone (Szuba et al.
2003). The incidence of upper-limb lymphedema following mastectomy ranges between 24%
and 49%, with lower rates of 4 - 28% reported for lumpectomy, although there has been a
reduction in the reported incidence since the development of lymphatic mapping and sentinel
lymph node biopsy to assess metastatic spread of cancer (DiSipio et al. 2013, Warren et al. 2007).
Approximately 75% of breast cancer-related lymphedema cases develop within 2 years of
surgery and 90% within 3 years, but it can remain latent for as long as 20 years (Stanton et al.
2009b). Risk factors for developing post-mastectomy lymphedema include the extent of axillary
surgery, adjunctive radiotherapy, postoperative seroma, obesity, infection or trauma to the
ipsilateral limb, and most novelly, a high peripheral blood vascular filtration rate, i.e. higher
demand for lymphatic drainage (Stanton et al. 2009a, Warren et al. 2007, Herd-Smith et al.
2001). Recently, a genetic predisposition has been implicated in the development of
lymphedema following breast cancer treatment (Newman et al. 2012, Finegold et al. 2012).
Acquired lymphedema can also be secondary to peripheral arterial disease, varicose vein
surgery, saphenous vein harvesting for bypass surgery, circumcision, intrathecal pump
insertion for analgesia, recurrent bacterial lymphangitis, burns, and the spread of malignant
tumors obstructing the lymphatics or regional lymph nodes (Rockson, Rivera 2008).
There is currently a paucity of effective treatment strategies for lymphedema. The availabe
therapy is based on conservative methods, e.g. compression garments, manual lymphatic
drainage (MLD) and complex decongestive therapy (MLD, limb compression, skin care and
exercise combined), or mechanical treatments such as intermittent pneumatic compression
devices and low-level laser therapy, or occasionally surgery. In addition, pharmacological
therapy in the form of benzopyrones has been attempted, as benzopyrones are thought to
increase proteolysis in the edematous tissue. (Warren et al. 2007.) Problems associated with
conservative treatments are minor, and they are likely to have little impact on the clinical
outcome (Oremus et al. 2012). Surgical approaches, including resection procedures,
microsurgical interventions and liposuction, are considered when lymphedema is refractory to
10
non-operative treatment. Resection surgery involves the excision of subcutaneous tissue, and it
is applied in extreme cases, when massive limb changes or fibrotic induration are observed.
(Murdaca et al. 2012.) These ‘debulking’ operations may, however, result in extensive scar
formation and substantial morbidities of the skin, including ulceration, keloids and
papillomatosis (Warren et al. 2007). Circumferential liposuction combined with lifelong
compression, on the other hand, is a minimally invasive, novel technique to remove the
hypertrophied adipose tissue and it is claimed to achieve a consequential improvement in skin
microcirculation, reduced cellulitis, and a significant reduction of edema with no recurrence
during 7 years’ follow-up (Damstra et al. 2009, Brorson 2003). Microsurgical techniques,
creating lymphaticovenous or lymphaticolymphatic anastomoses in order to directly correct the
underlying pathology, appear to promote the long-term alleviation of edema (Felmerer et al.
2012, Campisi, Boccardo 2004, Baumeister, Siuda 1990). Although no consensus prevails with
regard to what is the optimal surgical method, a minimally invasive lymphovenous shunt
operation utilizing microsurgical techniques is the most popular at present (Suami, Chang
2010). Overall, surgical treatment modalities have met with limited success due to the
difficulties in identifying, preserving and anastomosing lymphatic vessels to pre-existing
networks (Saaristo et al. 2012).
Microvascular lymph node transfer, a technique in which composite soft tissue including
lymph nodes is transplanted to the axilla or wrist of the lymphedematous limb, has been
reported to decrease perimeter and improve lymph drainage in the affected arm (Lin et al. 2009,
Becker et al. 2006). Lymph node transfer has also been combined with standard breast
reconstruction surgery in the simultaneous reconstruction of the missing breast and lymphatic
network in the operated axilla (Saaristo et al. 2012). However, in microvascular lymph node
transfer, new lymphatic vessels are believed to sprout spontaneously from the transferred
lymph nodes and form anastomoses with the recipient site (Suami, Chang 2010). It is essential
that there is a viable connection with lymphatic vessels for the viability and function of the
lymph nodes, and experimental studies have shown that incorporation of the lymph nodes into
the existing lymphatic vasculature may fail, resulting in a deterioration of the transferred lymph
node (Hadamitzky, Blum & Pabst 2009, Tammela et al. 2007, Mebius et al. 1991). Therefore, the
combination of surgical lymph node transfer with simultaneous administration of prolymphangiogenic growth factors may promote the reconstruction of the lymphatic vasculature
and alleviate postoperative lymphedema. This approach will be further reviewed in chapter
2.6.1 “Preclinical studies of lymphedema”.
2.1.5 Tumor lymphangiogenesis and metastasis
The lymphatic vasculature serves as a conduit for tumor cells, as they spread via lymphatic
vessels to colonize lymph nodes. In some respects, tumor cells resemble leukocytes by
exploiting many of the same trafficking pathways, chemokines, and adhesion molecules to gain
access to lymphatic vessels. (Holopainen et al. 2011, Muller et al. 2001.) Several cancers, breast
cancer and melanoma in particular, are known to metastasize to lymph nodes; this often
determines their prognosis and predicts patient survival. The metastatic status of lymph nodes
has an important role in tumor staging and clinical decision-making. (Sleeman, Thiele 2009,
Scoggings et al. 2005.) Many tumors and tumor-associated macrophages express two
lymphangiogenic growth factors, VEGF-C and VEGF-D, and this property has been shown to
correlate with tumor-associated lymphangiogenesis, lymphatic invasion, distant metastasis, and
poor clinical outcomes (Holopainen et al. 2011). Interestingly, lymphangiogenesis has also been
reported to occur in sentinel lymph nodes even prior to the arrival of metastatic tumor cells, as
well as in metastatically non-involved, remote lymph nodes, indicating that primary tumors
prepare their future metastatic site in advance by producing sufficient concentrations of
lymphangiogenic growth factors to induce lymphatic vessel growth in the target tissue
(Hirakawa et al. 2007, Van den Eynden et al. 2006). Antilymphangiogenic strategies using
11
neutralizing antibodies against VEGFR-3, the neuropilin 2 (NRP2) co-receptor, VEGF-C, and
VEGF-D, as well as soluble VEGFR-3 fusion proteins, have been employed in the battle against
tumor lymphangiogenesis and metastasis, and a VEGFR-3-targeting monoclonal antibody has
entered phase 1 clinical trials (Alitalo 2011). Blocking of VEGFR-3 exerts no effect on preexisting vessels, but leads to inhibition of lymphatic vessel growth in tumors and thus has
achieved a reduction of lymph node metastases in preclinical animal models (Karpanen et al.
2006b, Lin et al. 2005, He et al. 2005). The involvement of lymphangiogenic growth factors in
tumor progression and metastasis is an important consideration regarding the safety of prolymphangiogenic gene therapy, and will be further discussed in chapter 2.5.3 “Safety issues in
gene therapy”.
2.1.6 Other lymphatic vasculature-associated diseases
Lymphatic hyperplasia or enhanced lymphangiogenesis have been associated with chronic
inflammation in psoriasis, colitis ulcerosa, rheumatoid arthritis, Mycoplasma pulmonis infection
and kidney transplant rejection (Cueni, Detmar 2008). Recent research has shed light into the
molecular mechanisms behind inflammation-induced lymphangiogenesis, suggesting that
induction of the NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells)
pathway by inflammatory stimuli promotes VEGFR-3 expression in LECs, thus leading to
increased sensitivity of the pre-existing lymphatics to VEGF-C and VEGF-D and enhanced
lymphangiogenesis (Flister et al. 2010). At sites of tissue inflammation, macrophages contribute
to lymphatic vessel growth by producing VEGF-C and VEGF-D, and furthermore, they have
been reported to transdifferentiate into LECs in inflamed mouse cornea (Maruyama et al. 2005,
Cursiefen et al. 2004). In addition, VEGF-A appears to have a role in inflammation-induced
lymphangiogenesis, as it was found to induce leaky, functionally impaired lymphatic vessels in
the murine epidermis after ultraviolet B-irradiation, and this could be prevented by
administration of a VEGF-A neutralizing antibody (Kajiya, Hirakawa & Detmar 2006). VEGF-Amediated inflammatory lymphangiogenesis may be the result of VEGF-A’s chemotactic
recruitment of macrophages, which in turn supply VEGF-C and -D (Cursiefen et al. 2004).
Enhanced lymphangiogenesis in inflammatory conditions facilitates the resolution of edema
and inflammatory infiltrate, making stimulation of lymphangiogenesis an attractive treatment
option for certain inflammatory conditions (Cueni, Detmar 2008). In contrast, blockade of
VEGFR-3 signaling has been reported to suppress dendritic cell migration and concomitant
corneal transplant rejection in mice, as well as adaptive immune responses towards heart
transplants in a rat model, which may be evidence in favor of inhibition of lymphangiogenesis
in the context of heterologous tissue transplantation (Alitalo 2011, Nykanen et al. 2010, Chen et
al. 2004).
Lymphatic dysfunction has been proposed to be connected to disturbances in lipid
homeostasis, as patients with chronic lymphedema gradually accumulate subcutaneous fat in
edematous sites (Dixon 2010a). Similar observations have been made in mice with secondary
lymphedema, where impaired lymphatic drainage led to abnormal lipid accumulation that
persisted even after the lymphatic drainage was restored (Rutkowski et al. 2006). In Prox1+/mice, abnormal leakage of lymph from mesenteric lymphatic vessels appears to promote lipid
accumulation, and these mice also possess higher levels of leptin and fatty livers, which are
features of late-onset obesity in humans (Harvey et al. 2005). The role of lymphatic vessels in
atherosclerosis remains as yet undefined, although increased lymphangiogenesis has been
observed in progressive atherosclerotic lesions and stenotic aortic valves (Syvaranta et al. 2012,
Kholova et al. 2011). Lymphatic vessels are rare in the intima and abundant in the adventitia of
atherosclerotic coronary arteries, but the functional consequences of lymphangiogenesis in the
adventitia are unknown (Nakano et al. 2005). ApoE-/- (Apolipoprotein E) mice display not only
tertiary lymphoid organs in the adventitia of aortas but also degeneration and loss of function
of lymphatic vessels in response to hypercholesterolemia (Lim et al. 2009, Grabner et al. 2009).
12
Altogether, it has been proposed that a compromised lymphatic system may contribute to the
increased plasma cholesterol levels and the accumulation of cholesterol and inflammatory cells
in atherosclerotic lesions (Vuorio et al. 2014, Syvaranta et al. 2012, Lim et al. 2009). However,
VEGF-C and VEGF-D were shown to exert neither anti- nor pro-atherogenic effects in a mouse
model of atherosclerosis (Leppanen et al. 2005). Lymphatic vessels were recently found to play
a significant role in reverse cholesterol transport (RCT), which is involved in the removal of
excess cholesterol from peripheral tissues (Lim et al. 2013, Martel et al. 2013). Mice transplanted
with atherosclerotic aortas displayed impaired RCT when lymphatic drainage was blocked
(Martel et al. 2013). Conversely, VEGF-C treatment to restore lymphatic function improved RCT
(Lim et al. 2013).
LECs give rise to various lymphovascular tumors, such as lymphangiosarcoma, which is a
rare malignant tumor reported in the context of chronic lymphedema due to mastectomy,
inguinal lymphadenectomy, trauma, or infection (Krishnamoorthy et al. 2012).
Lymphangioleiomyomatosis (LAM), on the other hand, is a benign neoplasm affecting young
females that is characterized by the proliferation of SMC-like cells and lymphatic vessels, as
well as pulmonary cysts that impair lung function over time, finally resulting in respiratory
failure. LAM cells secrete high levels of VEGF-D and spread through the lymphatic vessels to
distant sites, where they may disrupt lymphatic function. (Seyama et al. 2010.) Kaposi sarcoma,
caused by the Kaposi sarcoma herpesvirus (KSHV), is an angiogenic tumor predominantly seen
in people with acquired immunodeficiencies. Kaposi sarcoma cells express both blood
endothelial and LEC markers, and VEGFR-3 antibody treatment has been claimed to be
effective in reducing lymphangiogenic sprouting of KSHV-infected LECs in three-dimensional
culture. (Alitalo 2011, Tvorogov et al. 2010.)
Interstitial lymphangiogenesis due to increased production of VEGF-C by macrophages has
been linked to extrarenal maintenance of normal blood pressure in conditions of interstitial salt
accumulation in the skin. Blockade of VEGF-C signaling has led to salt-induced hypertension in
rats, suggesting that the immune system regulates volume and blood pressure homeostasis via
lymphangiogenic growth factor activation. (Machnik et al. 2009.) Furthermore, lymphatic vessel
dysfunction has been implicated in the failure of neonatal lung inflation (Jakus et al. 2014).
2.2 THE BLOOD VASCULAR SYSTEM AND BLOOD VESSEL GROWTH
2.2.1 Structure and function of the blood vascular system
The blood vascular system exists to deliver oxygen, nutrients and hormones to the tissues, as
well as to remove carbon dioxide and metabolic waste products from the tissues (Guyton, Hall
2006). The vascular system is the first organ system to develop in the embryo, and a failure to
establish a functional circulation leads to death during early organogenesis (Copp 1995). The
cardiovascular system comprises the heart, which is responsible for circulating blood; the
arteries and veins which transport blood to and from the peripheral tissues; and the capillaries,
which allow the diffusion of substances between the blood and the surrounding tissues. The
rate of blood flow is regulated intrinsically by tissues and organs to meet their metabolic and
functional needs, as well as extrinsically via neural or hormonal influences. Ultimately, it is the
balance between these regulatory mechanisms that determines the vascular tone and blood flow
in the tissue. (Guyton, Hall 2006.)
The heart pumps blood first through large elastic arteries such as the aorta, subclavian, and
iliac arteries, which then branch into medium-sized muscular arteries and subsequently into
small arteries and arterioles, which are the main regulators of physiologic resistance to blood
flow (Kumar, Abbas & Fausto 2005). Capillaries originate from the point of metarteriolecapillary junctions, where precapillary sphincters act as shunts by opening and closing the
entrance to the capillaries based on local O2 (oxygen) concentrations (Guyton, Hall 2006). The
capillaries with their thin walls as well as the pores in their walls when combined with the tiny
13
diameter of a single erythrocyte and slow flow, create the ideal conditions for the exchange of
fluid, nutrients, electrolytes and hormones between the blood and the interstitial fluid (Kumar,
Abbas & Fausto 2005). Filtration of fluid in and out of the capillaries is powered by hydrostatic
pressure forcing fluid out of the arterial side of the capillary bed, and colloid osmotic pressure
causing reabsorption of extravasated fluid into the venous side of the capillary bed (Guyton,
Hall 2006). The capillaries are located within 200 μm of any cell, except in avascular tissues such
as cartilage and cornea of the eye, and they are highest in density in metabolically active tissues,
such as the myocardium (Kumar, Abbas & Fausto 2005, Carmeliet, Jain 2000). The O2-deprived
blood flows from the capillaries into postcapillary venules, which are the main site of vascular
leakage and leukocyte infiltration in inflammatory reactions. The larger veins, which are
formed progressively from mergers of venules, transport the blood back to the heart at a slower
rate and under lower pressure conditions than those prevailing in the arterial system. Venous
valves and surrounding skeletal muscle contractions augment blood flow particularly in the
extremities to overcome gravity and prevent reverse flow. The veins are able to retain large
quantities of blood: approximately 66% of all blood is in the veins at any given time. (Guyton,
Hall 2006, Kumar, Abbas & Fausto 2005.)
2.2.2 Blood vessel components
The main constituents of blood vessel walls are endothelial cells (ECs), SMCs, and extracellular
matrix, including elastin, collagen and glycosaminoglycans. Three layers - intima, media, and
adventitia - separated by the internal and external laminas, can be defined in arteries and veins,
although the proportions of these layers vary dramatically between these two vessel types. In
comparison to veins, arteries have thicker SMC layers of media, and round, open lumens even
when not filled with blood. In contrast, veins display larger diameters and lumens, as well as
thinner media but thicker adventitia to protect them from external trauma. (Kumar, Abbas &
Fausto 2005.) The Notch signaling pathway controls the commitment of angioblasts to arterial
or venous lineages in the embryo before the appearance of the circulation, but arterial-venous
differentiation is also driven by hemodynamic forces (le Noble et al. 2004, Lawson, Vogel &
Weinstein 2002). Vascular cells display a high degree of plasticity with respect to environmental
factors, and the predetermined identity can be remolded even in the adult (Fann et al. 1990).
Capillary walls consist of merely a monolayer of ECs adjoined by different types of
intercellular junctions, such as tight junctions or the more dynamic adherens junctions, which
control endothelial permeability (Dejana, Corada & Lampugnani 1995). ECs play a central role
in vascular biology and pathology, particularly in vasotonus modulation, blood-tissue
interactions, fibrinolysis and coagulation, blood cell activation and migration, and the
vascularization of normal and neoplastic tissues. The microvascular endothelium exhibits
different phenotypes based on anatomic location and adaptation to environment. For example,
the endothelium in the central nervous system, which is a part of the blood-brain barrier, is
continuous with complex tight junctions and allows only selective movement of molecules
across, whereas the fenestrated endothelium promotes a high rate of absorption in the
gastrointestinal tract and secretion in the endocrine glands. (Risau 1995.) Lymph nodes contain
special postcapillary venules called high endothelial venules in reference to their plump
phenotype, which permit lymphocyte entry from the blood circulation to the lymph node
(Girard, Springer 1995). Endothelial activation happens in response to various
pathophysiological stimuli, e.g. there are inducers such as cytokines, bacterial products or
hemodynamic stresses which activate ECs to express adhesion molecules, and to produce other
cytokines, chemokines, growth factors and vasoactive molecules (Kumar, Abbas & Fausto 2005).
Endothelial dysfunction is characterized by impaired vasoreactivity and an abnormally
thrombogenic and proinflammatory endothelial surface; these pathological changes are
associated with atherosclerosis, thrombus formation, and cardiovascular diseases in general
(Endemann, Schiffrin 2004). The EC layer of capillaries, excluding the discontinuous
14
endothelium type found in the liver, spleen and bone marrow, is lined abluminally by a
basement membrane (BM) and encircled by microvascular pericytes embedded in the
membrane (Hirschi, D'Amore 1996, Risau 1995). Pericytes are multipotent mesenchymal cells
that make specific focal contacts with ECs and play a role in vascular growth, stabilization and
remodeling, as well as in the regulation of capillary blood flow (Armulik, Abramsson &
Betsholtz 2005).
2.2.3 Vasculogenesis
The development of the vascular system occurs via two processes, vasculogenesis and
angiogenesis (Figure 3A). Vasculogenesis is the first step in the formation of the embryonic
cardiovascular system, involving the de novo differentiation of blood vessels from vascular stem
cells. EC lineages as well as hematopoietic cells arise from mesoderm-derived stem cells. These
precursors, called hemangioblasts, form blood islands, in which the centrally located cells
differentiate into hematopoietic stem cells, whereas the peripheral cells develop into angioblasts
that further differentiate into vessel wall components, i.e. ECs and mural cells. (Risau, Flamme
1995.) The blood islands fuse to form a primary capillary plexus, which then undergoes
complex remodeling to generate the mature vasculature (Coultas, Chawengsaksophak &
Rossant 2005). Organs developing from the mesoderm and endoderm, e.g. liver, spleen and
myocardium, are vascularized by vasculogenesis, whereas organs that are not initially
colonized by angioblasts, e.g. the brain and the spinal cord, develop their vasculature via
angiogenesis (Kassmeyer et al. 2009). The genetic control of normal vascular development
includes activation of fibroblast growth factor-2 (FGF-2) to induce production of vascular
progenitors, as well as VEGF signaling that plays a decisive role in the further development of
the vascular system (Coultas, Chawengsaksophak & Rossant 2005, Risau, Flamme 1995).
The default state of the adult vasculature is quiescence, i.e. physiological formation of new
blood vessels is rare, except in cases of EC proliferation during the estrus cycle or in exerciseinduced muscular hyperplasia (Gielen, Schuler & Adams 2010, Reynolds, Killilea & Redmer
1992). Neovascularization of this kind occurs primarily via angiogenesis, which was long
believed to be the only mechanism by which new vessels could be formed in adults. However,
the term postnatal vasculogenesis has emerged; this refers to de novo recruitment of bone
marrow-derived endothelial progenitor cells (BM-EPCs) and circulating EPCs for blood vessel
formation in adults. (Eguchi, Masuda & Asahara 2007, Kopp, Ramos & Rafii 2006.) EPCs are
believed to migrate, proliferate and differentiate into ECs at their destined location (Kassmeyer
et al. 2009). In the late 1990s, EPCs were isolated from peripheral blood and shown to be
recruited at sites of ischemia, thus implying that they would be capable of promoting
vasculogenesis (Asahara et al. 1997). Since their discovery, it has been observed that the EPC
population actually consists of several different species of endothelial progenitors with varying
phenotypic profiles and biological functions (Zampetaki, Kirton & Xu 2008, Lin et al. 2000).
Subsequently, EPCs have been shown to contribute to blood vessel development in wound
healing and in tumor growth, as well as to re-endothelialization of injured vessels and vascular
prostheses (Kassmeyer et al. 2009, Ranjan et al. 2009, Xiao et al. 2006). Low levels of EPCs
appear to predict the likelihood of major cardiovascular events and to promote the progression
of atherosclerosis (Fadini et al. 2010, Werner et al. 2005, Hill et al. 2003). Over the years,
contradictory findings regarding the physiological relevance of EPCs in postnatal
neovascularization have emerged, and it has been proposed that EPCs do not actually
proliferate and differentiate into ECs, but derive from mononuclear cells and induce neovessel
growth indirectly by secreting growth factors (Larrivee, Karsan 2007, Kinnaird et al. 2004,
Rehman et al. 2003).
15
2.2.4 Angiogenesis
Angiogenesis is the mechanism by which embryonic organs that are not initially colonized by
angioblasts, e.g. the brain and the spinal cord, develop a vasculature, as well as the principal
mechanism of blood vessel growth in adults (Risau 1997, Baldwin 1996). Furthermore,
pathological angiogenesis is a prerequisite for tumor growth beyond 1-2 mm in diameter and
the concept of treating tumors by inhibiting angiogenesis was first postulated in the 1970s
(Folkman 1971). Tumor vessels are irregular and immature with increased leakiness and
susceptibility to thrombosis and rupture (Carmeliet, Jain 2011). Conversely, insufficient vessel
growth and regression contribute to disorders like myocardial infarction, stroke,
and neurodegeneration (Potente, Gerhardt & Carmeliet 2011). Angiogenesis can occur by
sprouting from pre-existing capillaries, which comprises proliferation, migration, threedimensional organization and tube formation of ECs, or by intussusception, in which
transluminal pillars are formed within capillaries and divide the vessel lumen into two parallel
vessels, or by bridging, where the vessels split to form daughter vessels via the formation of EC
bridges (Figure 3A) (Kassmeyer et al. 2009, Burri, Djonov 2002, Conway, Collen & Carmeliet
2001).
‘The angiogenic switch’ is a widely accepted term used to describe the balance between the
inducers of angiogenesis and the countervailing inhibitors: the ‘switch’ is ‘off’ when the
proangiogenic molecule concentration is balanced by that of the antiangiogenic molecules, and
‘on’ when the balance shifts towards a proangiogenic state (Bergers, Benjamin 2003). Hypoxia,
low pH, hypoglycemia, mechanical stress, inflammatory conditions, and malignancies are all
triggering events of the switch (Carmeliet, Jain 2000). Tissue hypoxia is the main physiological
stimulus for angiogenesis, leading to increased expression of proangiogenic growth factors,
most importantly VEGF-A, via activation of the hypoxia-inducible factor, HIF (Hickey, Simon
2006). After the release of growth factors, a cascade of events takes place in order for vascular
sprouting to occur: the pre-existing vessels become dilated and hyperpermeable in response to
nitric oxide (NO) and VEGF-A respectively, the extracellular matrix is remodeled and the BM
degraded, plasma proteins leak from the angiogenic vessels and serve as a provisional matrix,
activated ECs proliferate and migrate towards the growth factor gradient, a capillary tube is
formed, and finally, the sprouts anastomose to form a functional loop, thereby establishing a
perfusing vessel (Jain 2003). The immature vessel network is then remodeled and pruned by
tissue-derived signaling molecules and blood flow conditions, e.g. pressure and wall-shear
stress, to adequately oxygenate the perfused organ (Wacker, Gerhardt 2011, Resnick et al. 2003).
In order to achieve vessel stabilization and maturation, pericyte coverage is acquired and the
BM is formed (Jain 2003). ECs with three distinct phenotypes have been described for the
different roles in vessel branching: non-proliferative, filopodia-rich ’tip cells’ respond to
environmental cues and lead the way for the growing vessel, ‘stalk cells’ follow behind
elongating the sprout, and ‘phalanx cells’ are responsible for vessel maturation and stabilization
(Figure 3B) (De Smet et al. 2009). Tip cells share similar features with the axon growth cone
guiding neuronal growth, and they both respond to signals from some of the same molecular
families, such as Slits, Netrins, Semaphorins, and Eph receptors and their ephrin ligands
(Adams, Eichmann 2010). In contrast to sprouting, intussusception does not require cell
proliferation and BM degradation, and is usually driven by higher levels of shear stress rather
than angiogenic factors (Styp-Rekowska et al. 2011).
Although VEGF-A is the most important driver of vascular formation, other growth factors
are involved in the process as well. Angiopoietin 1 (ANG1) and ephrin-B2 are required for
further remodelling and maturation of the immature vasculature. Ephrin-B2 has a crucial role in
distinguishing arterial and venous identity of developing vessels. (Yancopoulos et al. 2000.)
Platelet-derived growth factor B (PDGF-B) mediates pericyte differentiation and migration in
the maturation process of vessels (Lindahl et al. 1997). FGFs are able to initiate angiogenesis,
where they act not only on ECs but also on other cell types (Hanahan, Folkman 1996).
16
Extracellular edema due to increased vascular permeability is strictly associated with
angiogenesis, and it can be encountered during proangiogenic gene therapy as a potentially
serious side effect (Rissanen et al. 2003b, Dvorak et al. 1995). The development of edema in
skeletal muscle, for example, may worsen ischemia by physically compressing blood vessels.
An attempt to limit tissue edema by inducing lymphatic vessel growth simultaneously with
angiogenesis resulted in reduced edema in rabbit skeletal muscles (Lähteenvuo 2009). Thus, a
combination of proangiogenic and prolymphangiogenic gene therapy may confer added benefit
in comparison to angiogenic monotherapy alone.
2.2.5 Arteriogenesis
Arteriogenesis denotes the postnatal remodeling of small arterioles into larger caliber
conducting arteries (Schaper, Scholz 2003). This process is known to occur in ischemic
conditions, when gradually developing stenoses stimulate the outgrowth of pre-existing
collateral arteries to circumvent the arterial occlusion (Figure 3C) (Troidl, Schaper 2012).
Arteriogenesis takes place in the vicinity of the occluded artery, as opposed to the angiogenic
response to ischemia, which occurs relatively far and downstream from the occlusion (Schaper,
Scholz 2003, Ito et al. 1997). The key forces driving arteriogenesis are fluid shear stress acting on
the endothelium, and circumferential wall stress caused by the tramsmural pressure gradient
across the vessel wall (Schaper, Scholz 2003, Price et al. 2002). When an arterial occlusion
develops, a pressure gradient is created between the high-pressure area upstream of the
occlusion and the low-pressure downstream area. In order for arteriogenesis to successfully
restore circulation to the affected area, an interconnecting arteriolar network between the
preocclusive and the postocclusive microcirculation must exist. (Heil, Schaper 2004.) The
formation of a pressure gradient is followed by a redistribution of perfusion, an increase in
flow, and consequently, an increase in fluid shear stress, which activates the endothelium
(Persson, Buschmann 2011). The ECs swell and genes coding for chemoattractant cytokines,
growth factors, and adhesion molecules are upregulated (Lee et al. 2004, Deindl et al. 2003,
Schaper et al. 1976). This attracts monocytes to adhere to the endothelium, which then mature
into macrophages and these cells begin to remodel the vascular wall by secreting proteases
(Bergmann et al. 2006). Mitosis of ECs and SMCs follows, the neo-intima develops, and a
collateral artery with a typical corkscrew-like morphology is formed (Scholz et al. 2000).
Collateral growth is terminated early when the maximal conductance has reached 35 - 40% of
normal, because the fluid shear stress decreases due to collateral diameter enlargement (Hoefer
et al. 2001). A few large vessels mature and smaller vessels are pruned in the course of
establishing an effective collateral circulation, as small vessels have high resistance and vascular
adaptation always aims at the most economical solution (Troidl, Schaper 2012). Significant
variation in the ability to grow collateral vessels has been observed between individuals
(Rehman 2008). A minority (20 - 25%) of patients with functional collaterals seem to be able to
prevent myocardial ischemia completely during coronary occlusion, which indicates that some
people may possess a genetic predisposition that favors adaptive collateral growth (Wustmann
et al. 2003).
17
Figure 3. Schematic representation of the mechanisms of vessel formation. A, Endothelial-cell
precursors (angioblasts and hemangioblasts) differentiate into ECs which coalesce to form primitive
vasculogenic networks (vasculogenesis). These networks are remodeled through angiogenic
processes involving sprouting, intussusception and bridging. Adapted by permission from Macmillan
Publishers Ltd: Nat Rev Cancer, Hendrix et al., “Vasculogenic mimicry and tumour-cell plasticity:
lessons from melanoma” © 2003. B, Tip cells lead the way for a growing vessel with stalk cells
following behind, while phalanx cells are involved in vessel maturation. Adapted with permission
from Wolters Kluwer Health: Arterioscler Thromb Vasc Biol., De Smet et al., “Mechanisms of Vessel
Branching: Filopodia on Endothelial Tip Cells Lead the Way, 2009. C, Arteriogenesis occurs in
response to gradually developing occlusion of an artery to resolve ischemia of the target tissue.
Adapted from Integr Biol (Camb)., Korpisalo & Ylä-Herttuala, “Stimulation of functional vessel
growth
by
gene
therapy”
(2010),
with
permission
of
The
Royal
Society
of
Chemistry
(http://dx.doi.org/10.1039/b921869f).
2.3 THE LYMPHATIC SYSTEM AND LYMPHATIC VESSEL GROWTH
2.3.1 Anatomy and function of the lymphatic system
The lymphatic system consists of lymph that is a protein-rich fluid derived from plasma,
lymphatic vessels that transport lymph, lymphoid cells, and organized lymphoid tissues, i.e.
lymph nodes, spleen, thymus, tonsils, Peyer’s patches in the intestine, as well as lymphoid
18
tissue in the liver and lungs (Szuba et al. 2003). Lymphatics are dispersed throughout the body,
particularly at sites that come into direct contact with the external environment, but are absent
from avascular tissues such as epithelia and cartilage, as well as from the central nervous
system and bone marrow (Tammela, Alitalo 2010, Drake, Vogl & Mitchell 2005). The lymphatic
system parallels and supplements the cardiovascular system, but differs from it in several
aspects. In contrast to the blood vasculature, the lymphatic system is not a circulatory system
but a one-way transport system. (Norrmen et al. 2011.) Blind-ended lymphatic capillaries
absorb excess fluid containing pathogens, certain leukocytes, hormones, and cell debris, from
the interstitial space (Alitalo 2011, Drake, Vogl & Mitchell 2005). Lymph is conveyed to
progressively larger lymphatic vessels, first to precollectors which then coalesce into collecting
lymphatic vessels, ultimately to be returned back to the systemic circulation at the
lymphaticovenous junctions between the thoracic or right lymphatic duct and the subclavian
veins (Norrmen et al. 2011). The thoracic duct drains the left side of the body, abdomen, lower
limbs, left arm and shoulder, and the left side of the head and neck, whereas lymph from the
upper right quadrant of the body wall, right upper limb, and the right side of the head and neck
is collected into the right lymphatic duct (Drake, Vogl & Mitchell 2005, Jeltsch et al. 2003). The
overall lymph flow in humans has been estimated to range between 2 and 8 liters per day
(Levick, Michel 2010, Guyton, Hall 2006). Lymph nodes are located along the course of the
larger lymphatic vessels; the lymph passes through these nodes before entering the venous
system. In the lymph nodes, the lymph is screened for pathogens and activated immune cells
and antibodies are released into the general circulation. (Young et al. 2006.) Thus the lymphatic
system also plays an integral role in immune responses of the body by serving as a trafficking
route for immune cells. Specialized lymphatic vessels called lacteals inside the intestinal villi
absorb dietary fats and fat-soluble vitamins and transport them to the venous circulation (Dixon
2010b).
The lymphatic system of the extremities is divided into a superficial (epifascial) compartment
that drains lymph from the skin and subcutis, and a deeper (subfascial) compartment that
drains muscle, bone and the deeper blood vessels. These two systems fuse in the pelvic area,
whereas those of the upper limb merge in the axilla. (Szuba et al. 2003.) In cases of obstruction,
the epifascial and subfascial compartments can function in an interdependent manner such that
the healthy compartment is able to compensate for the obstructed counterpart by accelerating
transport (Brautigam et al. 1998). In normal conditions, subfascial drainage is slower than the
epifascial form (Mostbeck, Partsch 1999, Brautigam et al. 1998).
Microstructurally lymphatic capillaries are composed of a monolayer of oak-leaf-shaped
LECs that are connected by discontinuous button-like junctions, have little to no BM, and lack
pericytes and SMCs (Schulte-Merker, Sabine & Petrova 2011). Fluid and leukocyte entry is
enabled by a discontinuous BM containing portals that facilitate cell migration into the vessel,
and by the presence of specialized flap-like openings between LECs that function as primary
valves (Pflicke, Sixt 2009, Baluk et al. 2007, Trzewik et al. 2001). LECs are attached to
extracellular matrix by elastic anchoring filaments, which prevent collapse of the capillary
should there be an increase in interstitial pressure (Leak, Burke 1968). In contrast to the
lymphatic capillaries, the LECs of the collecting lymphatic vessels are connected by continuous
zipper-like junctions and ensheathed by a SMC layer as well as a BM (Tammela et al. 2007).
Lymphangions, i.e. the functional units of the collecting lymphatic vessels, are separated by
intraluminal valves, which are particularly numerous per vessel segment in organs with high
hydrostatic pressure, such as legs (Schulte-Merker, Sabine & Petrova 2011). The collecting
lymphatic vessels are analogous to the veins in the respect that lymph is propelled forward by
the intrinsic contractility of SMCs, contractions of adjacent skeletal muscles, and arterial
pulsations, and backflow is prevented by valves (Muthuchamy, Zawieja 2008).
19
2.3.2 Lymphangiogenesis
The lymphatic system in the human embryo begins to develop about a month later than the first
blood vessels, at around embryonic week 6 or 7 (Wilting, Neeff & Christ 1999). It is assumed
that lymphatic vessels sprout from veins when a subpopulation of ECs in the cardinal vein
starts to express PROX1, signifying their commitment to the LEC lineage (Figure 4) (Wigle,
Oliver 1999, Kaipainen et al. 1995). Prox1 is a lymphatic hallmark gene and its expression is
induced by Sox18 and COUP-TFII (the orphan nuclear receptor chicken ovalbumin upstream
promoter transcription factor 2), as evidenced by experiments performed in mice (Srinivasan et
al. 2010, Francois et al. 2008). COUP-TFII further interacts with PROX1 by forming a protein
complex, which is known to control the expression of several LEC genes, including VEGFR3
(Lin et al. 2010, Lee et al. 2009). Notch signaling has been proposed to function as a negative
regulator of LEC specification, as loss of Notch1 results in a surplus of LEC progenitors in
mouse embryos (Murtomaki et al. 2013). As a consequence of PROX1 expression, LEC
progenitors bud off from the cardinal vein while starting to express podoplanin and then
migrate along a gradient formed by VEGF-C, concomitantly creating the first lymphatic
structures, the lymph sacs, which further give rise to a primary lymphatic capillary plexus
(Yang, Oliver 2014). In addition to VEGF-C, CCBE1 is required for the budding of LEC
progenitors from the cardinal vein (Bos et al. 2011). In fact, Ccbe1 has been shown to regulate
Vegfc-mediated induction of Vegfr3 signaling during developmental lymphangiogenesis in
zebrafish (Le Guen et al. 2014). Several factors influencing lymph sac formation have also been
identified lately, including components of adrenomedullin signaling, TIE1 (Tyrosine kinase
with immunoglobulin-like and EGF-like domains 1) which is an angiopoietin receptor, and
GATA2 (Yang, Oliver 2014). Vessels spread by sprouting throughout the embryo from the
primary lymphatic capillary plexus (Norrmen et al. 2011). Lymphatic sprouting requires an
interaction between NRP2 and VEGFR-3 in response to VEGF-C, and this process can be
inhibited in vivo by preventing VEGF-C from binding to NRP2 (Xu et al. 2010). The formation of
a hierarchical network of capillaries and collecting vessels is finalized by remodeling and
maturation (Adams, Alitalo 2007). A small population of LEC progenitors remains inside the
cardinal vein and forms the lymphovenous valve, which is needed to return lymph fluid back
to systemic blood circulation (Srinivasan, Oliver 2011).
Much of the current knowledge of the embryonic development of the lymphatic system has
emerged from studies performed in knockout mouse models, which have revealed a number of
the genes responsible for lymphatic development. Mice with inactivated Sox18 or COUP-TFII
suffer a total blockade of LEC differentiation (Lin et al. 2010, Francois et al. 2008). Prox1- and
Vegfc-deficient mice do not develop any lymphatic structures either due to failed budding and
sprouting of LECs (Karkkainen et al. 2004, Wigle, Oliver 1999). The number of LECs is reduced
in Prox1+/- embryos because of decreased progenitors in the cardinal vein (Srinivasan, Oliver
2011). Interestingly, conditional deletion of Prox1 at any developmental or postnatal stage leads
to the reversal of LEC identity to a blood endothelial cell-like fate, demonstrating that LECs
require the continual expression of PROX1 in order to maintain their phenotypic identity
(Johnson et al. 2008). Vegfc+/- mice are viable but suffer from a lymphatic deficiency and
lymphedema (Karkkainen et al. 2004). Ccbe1-/- mice lack lymphatic vessels, develop edema, and
die prenatally (Bos et al. 2011). Additionally, VEGFR-3 kinase activity is indispensable for
lymphatic sprouting (Zhang et al. 2010). Vegfr3-/- mouse embryos die due to cardiovascular
defects before the emergence of lymphatic vessels, which has prevented direct assessment of the
requirement for VEGFR-3 in the migration of LEC progenitors from the early veins (Dumont et
al. 1998). Intriguingly, Vegfc-/-; Vegfd-/- double-knockout embryos do not phenocopy the Vegfr3-/embryos, perhaps evidence for some ligand-independent VEGFR-3 signaling in the early
embryo or that yet-to-be-determined ligands of the receptor exist (Haiko et al. 2008). VEGFR-3
and PROX1 are closely related as it was recently shown that VEGFR-3 regulates PROX1
expression via a positive feedback loop in LECs (Srinivasan et al. 2014). Lymphatic
development is not compromised in Vegfd-deficient mice, but exogenous VEGF-D can rescue
20
the phenotype of Vegfc-/- mice (Baldwin et al. 2005, Karkkainen et al. 2004). The separation of
lymphatic vessels from the blood vasculature in the mouse embryo is controlled by SYK (Spleen
tyrosine kinase) and the adaptor protein SLP76 (SH2 domain-containing leukocyte protein of
76kDa), and a deficiency of either one leads to abnormal lymphaticovenous connections, bloodfilled lymphatic vessels and chylous ascites (Abtahian et al. 2003). Platelets are also a
prerequisite for lymphatico-venous separation, as they block the entry of blood into the
developing lymphatic vessels (Zheng, Aspelund & Alitalo 2014). Podoplanin, which apart from
being an early marker of LEC differentiation, also binds to platelet CLEC-2 (C-type lectin
domain family 2) receptor and activates downstream SYK-SLP76 signaling (Finney et al. 2012,
Bertozzi et al. 2010).
Several molecules have been implicated in the maturation process of the lymphatics. FOXC2
regulates the differentiation of the lymphatic capillary from the collecting lymphatic vessel
phenotype through an interaction with NFATc1 (Nuclear factor of activated T cells,
cytoplasmic, calcineurin dependent 1), which is present in LECs during the maturation of
collecting lymphatic vessels (Norrmen et al. 2009). Foxc2-/- mice display agenesis of lymphatic
valves in the collecting vessels as well as ectopic coverage of SMCs and BM in capillaries
(Petrova et al. 2004). Many of the axonal guidance molecules, which play significant roles
during blood vessel growth, also participate in lymphangiogenesis. Ephrin-B2, a member of the
Eph receptor tyrosine kinase family, is essential for the maturation of collecting lymphatic
vessels and valves. (Eichmann, Makinen & Alitalo 2005, Makinen et al. 2005.) Signaling of
another axon guidance molecule, semaphorin 3A, via an interaction of its NRP1 and plexin A1
receptors, is required for lymphatic valve formation (Jurisic et al. 2012, Bouvree et al. 2012).
Furthermore, mice lacking Angpt2 (angiopoietin 2) display defective lymphatic remodeling and
agenesis of valves, which can be genetically rescued with Angpt1 (Dellinger et al. 2008, Gale et
al. 2002). After the first few postnatal weeks, signaling via VEGFR-3 is no longer required for
the maintenance of pre-existing vessels, and the lymphatic vessels regenerate even should there
be sustained inhibition of VEGFR-3, indicating that other factors in addition to VEGF-C/-D are
required for the growth and stability of lymphatic vessels during adulthood (Karpanen et al.
2006b). Potential postnatal factors responsible for the VEGFR-3-independent pathway include
VEGF-A, hepatocyte growth factor (HGF), and the angiopoietins, which are all known to act as
stimulators of lymphangiogenesis (Karpanen et al. 2006b, Kajiya et al. 2005, Tammela et al. 2005,
Hong et al. 2004, Gale et al. 2002).
Physiological formation of new lymphatic vessels in adult tissues is infrequent, occurring
mainly during ovarian follicle growth and wound healing by sprouting from pre-existing
vessels (Brown, Robker & Russell 2010, Paavonen et al. 2000). Pathological lymphangiogenesis
is associated with tumor growth and metastasis, as well as with inflammation (Karpanen,
Alitalo 2008). It remains unclear whether lymphatic vessel growth in adults is solely due to
sprouting, or whether it involves incorporation of circulating or BM-EPCs, capable of
transdifferentiating into LECs (see chapter 2.2.3 “Vasculogenesis”). Their role has been claimed
to have been demonstrated in mice models as well as in human kidney transplants, but it
remains a topic of some controversy (Kerjaschki et al. 2006, Buttler et al. 2006, Religa et al. 2005).
21
Figure 4. Developmental lymphangiogenesis. Expression of PROX1 marks the first LECs within
embryonic veins. VEGFR-3 signaling is focal throughout lymphangiogenic growth and is enhanced by
NRP2. FOXC2, Ephrin-B2, NRP1 and ANG2 among other factors are involved in the regulation of the
late stages of lymphatic differentiation. Adapted by permission from Macmillan Publishers Ltd: Nat
Rev Mol Cell Biol., Adams & Alitalo, “Molecular regulation of angiogenesis and lymphangiogenesis” ©
2007.
2.3.3 Lymphatic vessel markers
The identification of several lymphatic vascular specific molecular markers has made possible
the distinction between blood and lymphatic vessels and facilitated research into finding targets
for selectively promoting or inhibiting lymphangiogenesis in pathological conditions.
Lymphatic capillaries and the collecting vessels differ in their pattern of lymphatic endothelial
marker expression (Norrmen et al. 2011). The earliest marker indicating lymphatic endothelial
competence during embryonic development, as well as one of the most widely exploited
lymphatic markers, is lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) (Banerji
et al. 1999). This protein is highly expressed in lymphatic capillaries but mostly absent from
collecting lymphatics (Tammela et al. 2007, Makinen et al. 2005). Other markers expressed in
mature lymphatic capillaries include PROX1, VEGFR-3, and the transmembrane glycoprotein,
podoplanin (Johnson et al. 2008, Wigle, Oliver 1999, Breiteneder-Geleff et al. 1999, Kaipainen et
al. 1995). In mature collecting vessels, expression of VEGFR-3, PROX1, and FOXC2 is
downregulated from the high levels observed during development (Norrmen et al. 2011). One
further distinction between lymphatic capillaries and collecting lymphatics can be made with
SMCs: collecting lymphatics are covered by SMCs unlike lymphatic capillaries (Jurisic, Detmar
2009). Interestingly, intraluminal collecting vessel valves display a different molecular
expression pattern from the nearby cells of the collecting vessel trunk, with high levels of
VEGFR-3, PROX1, and FOXC2 (Norrmen et al. 2011). Both lymphatic and blood vascular
endothelium express the adhesion molecule CD31 (PECAM-1) (Jurisic, Detmar 2009).
2.3.4 Lymph nodes
Lymph nodes are discrete, bean-shaped organs composed of fibrovascular tissue and
surrounded by a collagenous capsule (Figure 5) (Young et al. 2006). They act as filters to trap
22
infectious agents, as well as hubs for lymphocytes and antigen-presenting cells (APCs) (Kumar,
Abbas & Fausto 2005). Humans have approximately 450 identifiable lymph nodes, which exist
mostly in groups at circulation bottlenecks, e.g. in axillae, groins and the neck (Young et al.
2006, Willard-Mack 2006). Lymph nodes are highly plastic and enlarge greatly when mounting
an immunological response (Young et al. 2006). Lymph flows into the lymph node via afferent
lymphatics that pierce the capsule, subsequently passing through the nodal medullary sinuses,
and finally exiting through the hilum into efferent lymphatic vessels (Guyton, Hall 2006). The
uninterrupted influx of lymph and APCs through the afferent lymphatic vessels is a
prerequisite for the maintenance of lymph nodes, and without constant flow lymph nodes will
atrophy and regress in size (Tammela et al. 2007, Mebius et al. 1991). Atrophy is characterized
by lymphoid depletion, i.e. a decrease in the number of germinal centers and depletion of
paracortical lymphocytes, and it can either be focal, multifocal or diffuse within a lymph node
(Elmore 2006).
Lymphocytes, the cells which are responsible for acquired immunity, are exposed to a variety
of antigens in lymph nodes. They circulate continually between the lymphatic and blood
vasculature, and enter the lymph nodes mainly by migrating across the walls of special
postcapillary venules called high endothelial venules. (Young et al. 2006.) The T and B cells
interact with APCs and undergo clonal expansion in their respective areas inside the node: B
cells proliferate within the follicles to form distinctive germinal centers, which are subsequently
referred to as secondary follicles, whereas T cells proliferate in the paracortex concomitantly
enlarging it (Willard-Mack 2006). B cells mature into plasma cells that secrete antibodies and
exit the node along with the efferent lymph in order to circulate to the site of damage (Young et
al. 2006). Tissue macrophages line the sinusoids of lymph nodes and phagocytize any foreign
particles that might enter the sinus by way of the lymph (Guyton, Hall 2006). Tingible body
macrophages are a type of macrophages containing apoptotic cell debris; these cells are found
predominantly in the germinal centers (Willard-Mack 2006).
It was believed for over a century that lymph nodes arise from embryonic primitive lymph
sacs, when a mesenchymal bud protrudes into the sac and begins to acquire endothelial
coverage in tandem with its expansion (Mebius 2003). However, recent research has revealed
that lymph node formation is at least initiated normally in mice devoid of lymph sacs, although
the following hematopoietic cell clustering within the lymph nodes is attenuated (Vondenhoff
et al. 2009). Nevertheless, a highly coordinated series of interactions between hematopoietic
cells and stromal cells residing at sites of future lymphoid organs is crucial for the development
of secondary lymphoid organs (van de Pavert, Mebius 2010). They do not all develop at the
same time, but sequentially, with the mesenteric lymph nodes forming first and Peyer’s patches
last (Mebius 2003). The genes regulating lymphoid organogenesis differ from those regulating
lymphangiogenesis, for example, lymphotoxin beta receptor signaling is pivotal for lymph node
development but not essential for lymphatic vessel development (Furtado et al. 2007, Futterer et
al. 1998). New lymph nodes do not form after embryogenesis, although the development of socalled isolated lymphoid follicles occurs in the small intestine of adults (Randall, Carragher &
Rangel-Moreno 2008). Moreover, tertiary lymphoid tissues develop in adult non-lymphoid
organs in response to chronic inflammation, infection, and autoimmunity (Carragher, RangelMoreno & Randall 2008). Intriguingly, artificial lymph nodes composed of stromal and
dendritic cells embedded in collagenous scaffolds have been shown to induce potent immune
responses in immune-deficient mice, pointing to a novel approach for strengthening the
immune system or replacing damaged lymph nodes (Okamoto et al. 2007).
23
Figure 5. The structure of a lymph node. Lymph from the afferent lymphatic vessels flows through
the subcapsular, trabecular and ultimately medullary sinuses, which converge at the hilum. Lymph
then exits the lymph node via the efferent lymphatic vessel. Valves in the lymphatic vessels prevent
backflow. Public domain image, courtesy of Wikimedia Commons.
2.4 MOLECULAR REGULATION OF BLOOD AND LYMPHATIC VESSEL
GROWTH
2.4.1 Hypoxia-inducible factors
HIFs are highly-conserved transcription factors that regulate the hypoxic response in all
metazoan species examined to date (Loenarz et al. 2011). They activate transcriptionally 100-200
genes that are involved in erythropoiesis, angiogenesis, autophagocytosis and energy
metabolism (Kaelin, Ratcliffe 2008). HIF-1 is a heterodimer composed of a constitutively
expressed beta subunit, and an alpha unit that is also continually translated but subjected to
rapid proteasomal degradation under normoxic conditions (Huang et al. 1996, Wang et al.
1995). Degradation occurs when two proline residues of the alpha subunit are hydroxylated by
prolyl-hydroxylases in the presence of O2, followed by the binding of the von Hippel-Lindau
tumor suppressor protein (pVHL) and concomitant recruitment of the E3 ubiquitin-protein
ligase, which eventually leads to ubiquitylation and degradation of HIF-1α by a proteasome
(Figure 6) (Jaakkola et al. 2001, Kamura et al. 2000, Maxwell et al. 1999). Conversely, the rate of
HIF hydroxylation is suppressed by hypoxia. When oxygen is not readily available, HIF-1α
accumulates and is transported to the nucleus, where it dimerizes with HIF-1β and binds to
hypoxia responsive element (HRE) of the target gene promoter (Semenza et al. 1996, Wang et al.
1995). HIF-1 has many target genes, e.g. erythropoietin, VEGF-A, VEGFR-1, and PDK1
(Pyruvate dehydrogenase kinase, isozyme 1), which is capable of reducing oxygen consumption
of cells, maintaining ATP (adenosine triphosphate) levels under hypoxia, and attenuating
mitochondrial reactive oxygen species production to rescue cells from hypoxia-induced
apoptosis (Papandreou et al. 2006, Kim et al. 2006, Gerber et al. 1997, Jiang et al. 1996, Forsythe
et al. 1996). The transcriptional activity of HIF is further regulated by the factor inhibiting HIF-1
(FIH-1), which represses its transactivation function in normoxic and moderately hypoxic
conditions (Mahon, Hirota & Semenza 2001). The pivotal role of HIF-1 in the development of
the circulatory system is underlined by the fact that Hif1α-/- mice die by E10.5 due to cardiac
malformations, vascular defects and impaired erythropoiesis (Yoon et al. 2006, Iyer et al. 1998).
Increased HIF-1α expression contributes to tumor progression, whereas loss of HIF activity
24
results in decreased tumor growth, thus making it a potential therapeutic target (Semenza
2010). Conversely, HIF-1 can mediate protective physiological responses in ischemic diseases by
activating genes encoding multiple angiogenic growth factors, and it has been shown to
promote arteriogenesis and to increase muscle perfusion in occluded rabbit hindlimbs (Patel et
al. 2005). Due to its role as a master regulator of hypoxic angiogenesis, HIF-1 has been proposed
to represent a better therapeutic target than a single angiogenic factor (Semenza 2012).
Interestingly, recent findings have suggested that HIF-1α may also have a central role in the
initiation and regulation of lymphangiogenesis in response to hypoxia, inflammation and
lymphatic stasis (Zampell et al. 2012, Bridges et al. 2012).
HIF-2α has a similar structure to HIF-1α but a different expression pattern, as HIF-2α is only
expressed in certain tissues in contrast to the ubiquitously expressed HIF-1α. HIF-2α expression
is prevalent in endothelium, as well as in well-vascularized tissues like kidney, lung, heart, and
small intestine. (Wiesener et al. 2003, Tian, McKnight & Russell 1997, Ema et al. 1997.) Hif2α-/mice die by E16.5 because of bradycardia, vascular defects, and incomplete lung maturation
(Compernolle et al. 2002, Tian et al. 1998). The two HIF-α subunits regulate both common and
unique target genes. HIF-2α is unable to activate HIF-1α regulated glycolytic enzymes such as
lactate dehydrogenase, but enhances the expression of some of the same angiogenic target
genes as HIF-1α, e.g. VEGF-A and VEGFR-1 (Takeda et al. 2004, Hu et al. 2003). HIF-2α has
been shown to induce angiogenesis in a mouse model of wound healing (Takeda et al. 2004).
Moreover, it has been found to promote tumorigenesis, particularly in neuroblastoma and renal
cell carcinoma (Holmquist-Mengelbier et al. 2006, Kondo et al. 2003). HIF-3α, another HIF
isoform, is claimed to be an inhibitory regulator of angiogenesis and hypoxic response, and it is
highly expressed in the cornea, where there is a low level of hypoxia-inducible VEGF-A
expression and an avascular phenotype (Makino et al. 2001).
25
Figure 6. HIF signaling under normoxic and hypoxic conditions. Under normal oxygen supply, proline
residues of HIF-1α are hydroxylated by prolyl hydroxylase, followed by the binding of the Von
Hippel-Lindau protein (pVHL), which is a component of a ubiquitin ligase complex. This leads to HIF1α ubiquitination and proteasomal degradation. When there is a poor oxygen supply, hydroxylation
is inhibited leading to accumulation of HIF-1α, which is transported to the nucleus where it dimerizes
with HIF-1β and then binds to the hypoxia-response element (HRE) of target genes, activating their
transcription. © 2013 Paula Province, Corinne E. Griguer, Xiaosi Han, Nabors Louis B. and Hassan
Fathallah Shaykh. Adapted from “Evolution of the Molecular Biology of Brain Tumors and the
Therapeutic
Implications”,
originally
published
under
CC
BY
3.0
license.
Available
from:
http://dx.doi.org/10.5772/53298
2.4.2 Vascular endothelial growth factors and their receptors
VEGFs are secreted glycoproteins with several isoforms generated by alternative splicing and
post-translational processing (Jeltsch et al. 2013). They are the main regulators of blood and
lymphatic vessel formation during embryological development as well as in physiological and
pathological conditions in adults. The VEGF family comprises VEGF-A, placental growth factor
(PlGF), VEGF-B, VEGF-C and VEGF-D. (Lohela et al. 2009.) In addition to these five members
found in mammals, viral VEGF homologues (VEGF-E) produced by Orf viruses and snake
venom VEGF (VEGF-F) have been identified (Yamazaki, Morita 2006, Ogawa et al. 1998).
VEGFs bind to cellular receptor tyrosine kinases VEGFR-1, VEGFR-2 and VEGFR-3 as well as to
co-receptors NRP1 and NRP2 with varying affinities and selectivities (Lohela et al. 2009).
Additionally, there is a heparin binding domain in VEGF that interacts with heparan sulfate
proteoglycans (HSPG), which are also able to serve as co-receptors (Koch et al. 2011). The
VEGFs and their binding to VEGF and NRP receptors are illustrated in Figure 7.
26
Figure 7. VEGFs and their receptors. VEGF-A binds to both VEGFR-1 and VEGFR-2, whereas VEGF-B
and PlGF bind to only VEGFR-1. VEGF-C and -D bind to both VEGFR-3 and VEGFR-2. NRP1 and NRP2
act as co-receptors and modulate VEGFR activation and signaling. All VEGFs bind to both NRP1 and
NRP2, except for VEGF-B, which binds to only NRP1. Different VEGF-A isoforms have different
binding properties towards the NRP receptors. Adapted by permission from The American
Physiological Society: Physiol Rev., de Almodovar et al., “Role and Therapeutic Potential of VEGF in
the Nervous System” © 2009.
2.4.2.1 VEGF-A
VEGF-A binds to VEGFR-1 and -2 and is upregulated under hypoxic conditions (Ferrara,
Gerber & LeCouter 2003, Minchenko et al. 1994). It is produced in at least eight different
isoforms due to alternative splicing of mRNA from a single VEGFA gene (Dehghanian, Hojati &
Kay 2014). VEGF-A165 is the most abundant and biologically active isoform that binds to
VEGFR-1, VEGFR-2, NRP1 and NRP2. In addition, VEGF-A165 is able to bind HSPGs. Different
VEGF-A isoforms vary in their affinities towards NRP and HSPG co-receptors. (Koch et al.
2011.) VEGF-A has a dominant role in embryonic vasculogenesis and angiogenesis, as Vegfa+/embryos die between E11 and E12 due to defects in blood vessel formation (Carmeliet et al.
1996). In addition, VEGF-A has been shown to increase vascular permeability, to promote EC
proliferation and migration, to inhibit apoptosis, and to induce vasodilation indirectly via NO
release. Excessive levels of VEGF-A contribute to several pathological conditions, such as tumor
angiogenesis, diabetic retinopathy and polycystic ovary syndrome. (Ferrara, Gerber & LeCouter
2003.) Consequently, VEGF-A has become the primary drug target for antiangiogenic therapy
with several VEGF-A–based inhibitors approved for clinical use by the U.S. Food and Drug
Administration (FDA) (Crawford, Ferrara 2009). However, only a fraction of patients appear to
benefit from the therapeutic blockade of angiogenesis, as tumors are capable of developing
27
resistance mechanisms to VEGF-A inhibitors (Bergers, Hanahan 2008). In addition to being the
key regulator of angiogenesis, VEGF-A has been shown to induce the growth of lymphatic
vessels, which although abnormal in shape and functionality, become VEGF-A independent
once formed and persist indefinitely (Nagy et al. 2002). However, the lymphangiogenic effects
of VEGF-A have been attributed to the recruitment of VEGFR-1 expressing inflammatory cells
that secrete lymphangiogenic factors (Cursiefen et al. 2004). The weak lymphangiogenic
potential of VEGF-A is supported by the observation that deletion of Vegfr2 from lymphatic
vessels results in hypoplastic but functional lymphatic vessels in embryos and adult mice
(Dellinger et al. 2013).
2.4.2.2 VEGF-B
VEGF-B is a ligand for VEGFR-1 and NRP1, and unlike VEGF-A, it is not regulated by hypoxia
(Makinen et al. 1999, Enholm et al. 1997). It is produced in two isoforms: freely soluble VEGFB186 and heparin-binding VEGF-B167 (Olofsson et al. 1996b). VEGF-B is abundantly expressed in
tissues with active energy metabolism needs, such as myocardium, striated muscle and brown
fat, indicating a role in supporting metabolic functions. In many tissues, VEGF-B is coexpressed
with VEGF-A, and the two can also join to form heterodimers which then bind to VEGFR-2.
(Bry et al. 2014, Olofsson et al. 1996a.) Although the ability of VEGF-B to induce angiogenesis in
most tissues is weak, it has been shown to be a promising candidate for both therapeutical
angiogenesis in myocardium as well as in the prevention of heart failure (Huusko et al. 2012,
Lahteenvuo et al. 2009). Vegfb-/- mice have been reported to have small hearts and impaired
recovery from ischemia, but they are viable and fertile in contrast to Vegfa knockout mice. Thus,
VEGF-B is not essential for the development of the cardiovascular system. (Aase et al. 2001,
Bellomo et al. 2000.)
2.4.2.3 VEGF-C
VEGF-C is a primarily lymphangiogenic factor that binds to VEGFR-3 and NRP2 with high
affinity in its full length, unprocessed form, and with increasing affinity to VEGFR-2 in its
proteolytically processed form (Xu et al. 2010, Joukov et al. 1997). The full length form is mainly
lymphangiogenic, which is in agreement with its strong binding to VEGFR-3, whereas the
mature short form promotes angiogenesis and vascular permeability via VEGFR-2 (Veikkola et
al. 2001, Cao et al. 1998, Joukov et al. 1997). CCBE1 has been shown recently to enhance the
proteolytic cleavage of inactive VEGF-C into a potent form, which is able to activate its cognate
receptor VEGFR-3 (Jeltsch et al. 2014). During embryogenesis, VEGF-C is highly expressed in
regions of lymphatic vessel development (Kukk et al. 1996). In Vegfc-/- mice, ECs cannot sprout
to form lymphatic vessels, whereas Vegfc+/- mice display lymphatic hypoplasia and lymphedema
(Karkkainen et al. 2004). The expression of VEGF-C decreases in most fully developed tissues
but remains high in lymph nodes (Saaristo et al. 2012, Lymboussaki et al. 1999). Overexpression of VEGF-C has proven effective in the induction of lymphatic vessel growth in
several animal models (Tammela et al. 2007, Saaristo et al. 2002a, Enholm et al. 2001). Although
VEGF-C does not appear to increase the growth of primary tumors, it has been shown to
promote lymphatic metastasis of tumor cells (Hirakawa et al. 2007, Skobe et al. 2001). The
expression of VEGF-C and VEGF-D directly correlates with lymphatic invasion, lymph node
involvement, distant metastasis, and sometimes poor prognosis of survival (Achen, Stacker
2008). An engineered isoform of VEGF-C, VEGF-C156S, has been shown to be a selective
agonist of VEGFR-3 and capable of inducing lymphangiogenesis but it does not exert any
effects on the blood vasculature (Rissanen et al. 2003b, Saaristo et al. 2002b, Joukov et al. 1998).
28
2.4.2.4 VEGF-D
VEGF-D, like VEGF-C, is considered to be a lymphangiogenic factor, although it is not essential
for the formation of the lymphatic system and its physiological role remains unclear. Vegfd-/mice are healthy and do not diplay any obvious abnormalities in their phenotype. (Baldwin et
al. 2005.) Endogenous VEGF-D is not able to compensate for the lack of VEGF-C in embryonic
lymphatic development, although in Vegfc-/- mice, exogenous VEGF-D can rescue sprouting of
ECs committed to the lymphatic lineage, at least to some extent (Karkkainen et al. 2004). The
expression of VEGF-D in the adult is strongest in the heart, lung, skeletal muscle, and the
intestine (Achen et al. 1998). The binding affinity of VEGF-D to its receptors is very similar to
that of VEGF-C, with the full-length form binding primarily to VEGFR-3 and NRP2, and the
mature, proteolytically processed form (VEGF-DΔNΔC) preferring VEGFR-2 (McColl et al. 2007,
Achen et al. 1998). The fully processed form of VEGF-D has a 290-fold greater affinity towards
VEGFR-2 than the full-length VEGF-D (Stacker et al. 1999). Proteolytic prosessing is required
for the angiogenic activity of VEGF-D, and in concordance with this, VEGF-DΔNΔC has been
found to induce strong angiogenesis in rabbit hind limb skeletal muscle (Rissanen et al. 2003b).
Over-expression of full-length VEGF-D and VEGF-DΔNΔC alike has been shown to induce
lymphatic vessel growth in mice (Tammela et al. 2007, Veikkola et al. 2001). Importantly, with
regard to safety considerations, VEGF-D has been implicated in tumor metastasis and
spontaneous tumor formation (Karkkainen et al. 2009, Ishii et al. 2004).
2.4.2.5 PlGF
PlGF is expressed in placenta, thyroid and lungs in the adult, and binds VEGFR-1 as well as
NRP1 and NRP2 (Fischer et al. 2008, Ziche et al. 1997, Clauss et al. 1996). PlGF has four different
isoforms in humans, of which PlGF-1 and -3 are freely soluble and -2 and -4 bind to heparin
(Yang et al. 2003, Cao et al. 1997). The deletion of PlGF does not cause any overt vascular
defects or infertility in mice. Although PlGF is redundant for angiogenesis during development,
it appears to be angiogenic in pathological settings, as Plgf-/- mice exhibit impaired angiogenesis
and arteriogenesis during ischemia, inflammation, wound healing and tumor growth.
(Carmeliet et al. 2001.) Over-expression of PlGF has been shown to induce revascularization of
ischemic limb and myocardium and enhanced healing of wounds in diabetic mice (Cianfarani et
al. 2006, Luttun et al. 2002). Its angiogenic properties are at least in part explained by its ability
to enhance VEGF-A signaling. Firstly, PlGF can displace VEGF-A from VEGFR-1, thus allowing
VEGF-A to bind VEGFR-2, resulting in angiogenic signaling (Fischer et al. 2008). Secondly, PlGF
can upregulate the expression of VEGF-A and other angiogenic growth factors (Roy et al. 2005).
Furthermore, an intermolecular crosstalk between VEGFR-1 and -2 occurs when VEGFR-1 is
activated by PlGF, which consequently promotes VEGF-A mediated angiogenesis (Autiero et al.
2003). PlGF is also able to signal through VEGFR-1 independently of VEGF-A (Fischer et al.
2008). However, there are a few studies indicating that PlGF might be a weak EC mitogen in
vitro and capable of inducing only a weak signal via VEGFR-1 (Migdal et al. 1998, Park et al.
1994).
2.4.2.6 VEGF-receptors
VEGFs bind to their cognate tyrosine kinase receptors, VEGFRs, on the cell surface, causing
them to dimerize and become activated through transphosphorylation, which then leads to
coordination of the cellular responses regulated by downstream signaling molecules. VEGFRs
comprise an extracellular, ligand-binding domain consisting of seven immunoglobulin-like
domains, a transmembrane domain, a juxtamembrane domain, an intracellular split tyrosine
kinase domain, and a C-terminal tail. (Koch et al. 2011.) Receptor activity is largely controlled
by trafficking mechanisms, e.g. regulating the numbers of receptors at the cell surface, the rate
at which these receptors are internalized, and the degradation vs. recycling of receptors (Zhang,
Simons 2014).
29
VEGFR-1 is necessary for the development of the embryonic vasculature, as Vegfr1-/- mice
form abnormal vascular structures and die in utero. The disorganization of vasculature was
found to be a consequence of excess EC progenitors, evidence of an inhibitory role for VEGFR-1
in the regulation of angiogenesis. (Fong et al. 1999, Fong et al. 1995.) VEGFR-1 displays a high
afiinity towards VEGF-A, VEGF-B and PlGF, the latter two of which share structural similarities
(Tammela et al. 2005). VEGFR-1 also has a naturally occurring soluble form (sVEGFR-1)
comprising the extracellular part of VEGFR-1; this is thought to act as a VEGF-A trap, further
implying an inhibitory role for this receptor in VEGF-A induced angiogenesis (Hiratsuka et al.
1998, Kendall, Thomas 1993). In contrast, VEGFR-1 is capable of promoting proangiogenic
signals by binding PlGF and thereby indirectly activating VEGFR-2 (Autiero et al. 2003). The
expression of VEGFR-1 is directly upregulated by hypoxia via a HIF binding enhancer element
in the VEGFR-1 promoter, and it is expressed not only in ECs but also in macrophages,
pericytes, renal mesangial cells, placental trophoblasts and many other cell types (Tammela et
al. 2005, Gerber et al. 1997). VEGFR-1 is able to transmit only weak mitogenic signals in ECs by
itself. However, it can form heterodimers with VEGFR-2 that possess a stronger signaling
capacity. (Huang et al. 2001.) VEGFR-1 has also been shown to engage in macrophage
recruitment to promote tissue remodeling (Pipp et al. 2003).
VEGFR-2 is the main mediator of EC differentiation, proliferation and migration, as well as
vascular tube formation and vascular permeability (Koch et al. 2011, Gille et al. 2001). Ligand
binding to VEGFR-2 leads to a robust angiogenic response. VEGFR-2 binds VEGF-A, the mature
forms of VEGF-C and VEGF-D, VEGF-E, and also VEGF-F. (Koch et al. 2011, Yamazaki et al.
2003.) It is highly expressed in the vascular endothelium during embryogenesis (Quinn et al.
1993). VEGFR-2 is pivotal for the development of the vascular system, as Vegfr2-/- mice die
between E8.5-9.5 after failing to develop endothelial and hematopoietic cells (Shalaby et al.
1995). Recent research has pointed to a role for VEGFR-2 in lymphatic vessel sprouting as well
(Dellinger et al. 2013). After birth, VEGFR-2 expression decreases but is upregulated again in
times of physiological and pathological angiogenesis in the adult (Jeltsch et al. 2013). VEGFR-2,
like VEGFR-1, has a soluble form, which acts as a VEGF-C trap, preventing VEGF-C from
binding to VEGFR-3 and consequently inhibiting its lymphangiogenic effects (Albuquerque et
al. 2009).
VEGFR-3 has an important role in the formation of the lymphatic system and it is activated
by both the full-length and the processed forms of VEGF-C and VEGF-D (Jeltsch et al. 2013).
VEGF-C binding to VEGFR-3 triggers signals to promote proliferation, migration and survival
of LECs (Salameh et al. 2005, Makinen et al. 2001). VEGFR-3 activation can also occur
independently of ligand-binding and in response to mechanical signals. An elevation in the
interstitial pressure in mice embryos was found to be sufficient to phosphorylate VEGFR-3,
thereby promoting LEC proliferation and lymphatic vessel growth. (Planas-Paz et al. 2012.) The
key role of VEGFR-3 in lymphatic function is highlighted by the fact that missense mutations in
this receptor lead to hereditary lymphedema in humans and a similar condition in mice
(Karkkainen et al. 2001, Karkkainen et al. 2000, Irrthum et al. 2000). VEGFR-3 is expressed in all
endothelia at the beginning of development but it becomes restricted to LECs, with the
exception of some fenestrated capillaries e.g. those found in endocrine organs, as the lymphatic
system starts to develop (Partanen et al. 2000, Kaipainen et al. 1995). In addition to being
expressed in normal lymphatics, VEGFR-3 appears as a positive marker in some pathological
conditions such as vascular tumors and neovascularization of nonvascular tumors (Partanen,
Alitalo & Miettinen 1999). VEGFR-3 is known to play a crucial role in the formation of the
vascular system already before the appearance of lymphatics, as indicated by the finding that
Vegfr3-/- mice die in utero due to the defective formation of vasculature and cardiovascular
failure (Dumont et al. 1998). VEGFR-3 is able to heterodimerize with VEGFR-2, and these
heterodimers appear to be active in sprouting angiogenesis (Nilsson et al. 2010). Moreover,
antibodies against VEGFR-3, which inhibit both VEGFR-3 homodimerization and VEGFR2/VEGFR-3 heterodimerization, lead to decreased vascular and lymphatic EC migration and
30
vessel sprouting (Tvorogov et al. 2010). Similarly as in the case of VEGFR-2, the soluble form of
VEGFR-3 appears to limit the local activity of VEGFR-3 and subsequent lymphangiogenesis in
some tissues, such as the cornea (Singh et al. 2013).
2.4.2.7 NRP-receptors
NRP1 and NRP2 are receptors for class 3 semaphorins, which mediate inhibitory signals during
neuronal axon guidance (Koch et al. 2011). They also engage in angiogenesis and VEGFR
signaling where they act as co-receptors, with NRP1 binding VEGF-A, VEGF-B, VEGF-C,
VEGF-D, PlGF and VEGF-E, and NRP2 binding VEGF-A, VEGF-C, VEGF-D and PlGF
(Uniewicz, Fernig 2008, Karpanen et al. 2006a, Makinen et al. 1999, Soker et al. 1998). NRPs have
been postulated to play a role in determining the arterial or venous fate of blood vessels, since
in the course of embryonic development, NRP1 expression is mostly prevalent in arteries and
NRP2 primarily marks veins (Herzog et al. 2001).
NRP1 contributes to EC migration and survival owing to its effects on VEGF-A-VEGFR-2
interactions (Koch et al. 2011). Additionally, it has been linked to VEGFR-2 mediated vascular
permeability and VEGF-A induced sprouting and branching of ECs (Kawamura et al. 2008,
Becker et al. 2005). The deletion of the Nrp1 gene is lethal, causing a wide range of vascular and
neuronal defects in mouse embryos, whereas over-expression of NRP1 leads to embryonic
lethality in mice due to excessive formation of vessels, hemorrhages and cardiac malformations
(Gu et al. 2003, Kawasaki et al. 1999, Kitsukawa et al. 1995). The blockade of NRP1 function
with a specific antibody has reduced angiogenesis and vascular remodeling in vivo and
rendered vessels more susceptible to anti-VEGF-A therapy in the battle against tumor growth
(Pan et al. 2007).
NRP2 is expressed not only in veins but also in lymphatic vessels and it can interact with
VEGFR-2 and VEGFR-3 to promote the migration and the survival of ECs (Koch et al. 2011,
Favier et al. 2006). Nrp2-/- mice are characterized by the absence of small lymphatic vessels and
capillaries at birth, with blood vessels and larger collecting lymphatics developing normally.
No signs of edema were observed in the nullizygous Nrp2 mice despite a reduced number of
lymphatic vessels in the skin and the internal organs. Lymphatic vessel growth occurred
postnatally in these mice, albeit abnormally patterned, particularly in the heart and the
intestine. (Yuan et al. 2002.) The mechanism by which NRP2 could modulate
lymphangiogenesis was later shown to be through an interaction with VEGFR-3 to mediate
VEGF-C-induced lymphatic sprouting (Xu et al. 2010). Administration of an anti-NRP2
antibody was able to reduce the metastatic spread of tumor cells, tumoral lymphangiogenesis
and functional lymphatics in tumors without affecting established lymphatics in mice (Caunt et
al. 2008).
2.5 GENE THERAPY
A gene, consisting of a sequence of DNA, is the fundamental unit of heredity that determines a
particular characteristic in an organism (Nelson, Cox 2008). Gene therapy is a form of molecular
medicine that utilizes genes in treating or preventing diseases. One can take different
approaches to gene therapy, e.g. replacing a mutated disease-causing gene with a healthy copy
of the gene, inactivating a mutated gene that is functioning undesirably, or introducing a new
gene into target cells to help fight disease. (Wally, Murauer & Bauer 2012, Drude et al. 2007, YlaHerttuala, Alitalo 2003.) The possibility of using endogenous proteins to locally treat only the
affected tissues as well as the potential long-term therapeutic effect achieved by a single
application are considered as advantages of gene therapy over conventional therapeutic
modalities (Yla-Herttuala, Alitalo 2003).
31
For gene therapy to achieve its goals, a sufficient amount of the therapeutic gene and/or its
product should be delivered to the target cells without causing significant toxicity. The effect
profile of gene transfer (GT) is determined by the chosen therapeutic gene, the vector carrying
the gene, as well as the route of administration of the GT. Different diseases present different
requirements for gene therapy in terms of the locality of the treatment and the duration of the
gene expression. For example, in the case of some genetic disorders, long-term expression in a
small proportion of cells might be sufficient, whereas inhibition of cancer growth would require
gene transfer of tumor suppressor genes to a large number of abnormal cells. (Kay, Glorioso &
Naldini 2001.)
Carriers called vectors are engineered to deliver the therapeutic gene into target cells. At
present, three types of gene delivery methods are available: viral vector based, non-viral vector
based, and physical methods. Certain genetically modified viruses are able to deliver the
desired gene by infecting the target cell, and viral vectors in general have the advantage of high
delivery efficiency to a variety of cells. In contrast, non-viral vectors, such as naked DNA
plasmids, are less toxic and immunogenic than viral vectors but exhibit lower delivery
efficiencies. Physical methods make use of a physical force to overcome the membrane barrier
of the cell, thereby enabling intracellular GT. There are different types of physical methods e.g.
gene delivery by needle injection and sonoporation, which utilizes ultrasound to permeabilize
the cell membrane. For the time being, both non-viral and physical methods of gene delivery
are generally considered unacceptable for clinical use due to their significantly lower delivery
efficacy in comparison to viral vectors. (Kamimura et al. 2011, Verma, Weitzman 2005.)
The duration of transgene expression in the target cell depends on the vector. Some vectors
e.g. retroviral vectors, have the ability to integrate into the genome of the host cell leading to
long-term gene expression that can be passed onto daughter cells. In contrast, transient
expression of only 2-4 weeks can be achieved by the use of adenoviruses. (Kamimura et al.
2011.)
Vectors carrying the therapeutic gene can be delivered to the desired tissue by a range of
different delivery routes. In the case of therapeutic angiogenesis, commonly used routes of
delivery include local, intramuscular injections and systemic, intravascular delivery.
Intravenous (i.v.) delivery is straightforward to execute in small animal models, but becomes
impractical when applied to larger animals and humans due to their great blood and tissue
volumes. (Karvinen, Yla-Herttuala 2010.) Furthermore, following the systemic spreading of the
gene drug via i.v. administration, viral particles may tend to accumulate in unwanted
destinations, mainly the liver, rather than in the target area (Lozier et al. 2002, Alemany, Suzuki
& Curiel 2000). Local injections into the heart or skeletal muscles have proven to be most
effective in achieving high expression of transgenes, and they also appear to be well tolerated
(Karvinen, Yla-Herttuala 2010, Harvey et al. 2002).
2.5.1 Viral vectors
Viruses have an inherent ability to hijack the machinery of a host cell in order to produce
copious amounts of their own genome. This characteristic is exploited in gene therapy, where
viral vectors are employed to deliver therapeutic genetic material into target cells. Viral vectors
are derived from either RNA or DNA viruses and designated as either integrating or nonintegrating vectors. In the first phase of engineering a viral vector, the viral sequences required
for replication, the assembly of viral particles, the packaging of the viral genome, and the
delivery of the transgene, are identified. Next, inessential genes are deleted to make room for
the transgene and to prevent replication, as well as to reduce pathogenity and the expression of
immunogenic viral antigens. Subsequently, the transgene cassette is inserted into the vector
construct, which is then introduced into a packaging cell line. In a packaging cell, a separate
packaging construct or a helper virus supplies the proteins needed for replication and assembly
of the virion. Finally, the recombinant viral vector is produced following the encapsidation of
32
the replicated vector genomes into virus particles. (Vannucci et al. 2013, Verma, Weitzman
2005.)
Transduction denotes the delivery of genetic material into a precise cell via a viral vector. In
this process, the viral vector itself does not replicate but merely carries the inserted genetic
information to its destination. Retroviral and lentiviral vectors are able to integrate into the host
genome holding the promise of lifelong expression, whereas DNA-based viral vectors are
normally present as episomes in the host cell nucleus. (Vannucci et al. 2013, Kamimura et al.
2011.) An ideal vector would be one that could be produced in high titres, would be capable of
targeting the appropriate cell type in each case, and would lead to a stable, sustained gene
expression without eliciting adverse effects such as undesirable immune responses. No such
vector exists as yet, but several approaches have been adopted to overcome obstacles related to
production, targeting the desired tissues, efficient gene delivery and pathogenity. (Verma,
Weitzman 2005.)
2.5.1.1 Adenoviral vectors
Adenovirus was originally isolated from human adenoids in 1953 (Rowe et al. 1953). Its genome
consists of a double-stranded linear DNA, and it is on average 36 kbp (kilobase pairs) in size
(Davison, Benko & Harrach 2003). Usually the adenovirus infection in humans is mild but may
pose life-threatening risks to immunocompromized individuals (Hendrickx et al. 2014). There
are more than 100 Ad serotypes, including 51 identified human Ad serotypes; these are
unevenly prevalent and commonly infect the lung epithelium and the gastrointestinal tract but
also other organs (Sharma et al. 2009a). Several cell surface molecules are involved in the entry
of the adenovirus into the cell, the most common being the Coxsackie-adenovirus receptor
(CAR) for the majority of serotypes (Sharma et al. 2009a, Bergelson et al. 1997).
Adenoviral vectors are mostly derived from the Ad5 serotype and to a lesser extent from
serotype 2 (Verma, Weitzman 2005). They can be produced in high titres, they display tropism
towards a variety of cell types, and they express the transgene at high levels, all of which make
the adenovirus an attractive candidate for gene therapy. However, there is extensive preexisting immunity against the adenovirus in the population, and furthermore the adenoviral
proteins are highly immunogenic in humans, which impact on the use of common serotypes
and limit the number of administrations which can be given to the same individual. (Vannucci
et al. 2013.) The expression of the transgene is transient and peaks in 1-7 days, waning to an
undetectable level by 2-4 weeks due to both innate and acquired immune responses against
viral proteins (Yang et al. 1996, Yang et al. 1994). The advantages and disadvantages of
adenoviral vectors are listed in Table 2.
Circumventing the antiadenovirus immunity has been a topic of intensive research. So-called
“gutless” adenovirus vectors have been developed, where all the adenovirus genes are removed
and the essential genes needed to create the vector are provided by the producer cells
(Kamimura et al. 2011). Another approach is the concept of “seroswitch”, denoting the use of a
different serotype adenovirus vector for the second administration, which provides another 1-2
weeks of expression (Mastrangeli et al. 1996). The short-term expression elicited by the
immunogenicity of the adenovirus can also be exploited to advantage. Transient expression is
suitable for applications aimed at building new biologic structures, such as that of VEGF gene
transfer for the generation of neovasculature (Crystal 2014). The anti-adenovirus immunity
limits the expression of the growth factor to a few weeks, but this can be sufficient for initiating
angiogenesis (Mack et al. 1998). Moreover, the short-term expression of the transgene caters for
the needs of cancer gene therapy, where the goal is to destroy tumoral cells (Vannucci et al.
2013). The strong immune response elicited by Ad can help augment the otherwise diminished
immunity against tumor cells, further asserting its position as the most widely used oncolytic
vector. Nevertheless, the lack of specific targeting due to widespread distribution of CAR and
poor transduction of many tumor types because of low levels of CAR expression has warranted
the development of several retargeting approaches. (Sharma et al. 2009b.) An example of this
33
retargeting is the use of hybrid Ad vectors, which are directed to other target-specific cellular
receptors and have shown efficient transduction of CAR-deficient cells, reduced liver tropism
and improved transduction of cancer cells in many different cancer types (Zheng et al. 2007,
Suominen et al. 2006, Breidenbach et al. 2004, Kanerva et al. 2002). The potent anti-adenoviral
immunity has also been harnessed to create vaccines against several pathogens and tumors
(Krause, Worgall 2011). In summary, adenovirus vectors are the number one choice for highlevel, short-term expression or for deliberately evoking immunity.
Table 2. Advantages and disadvantages of adenovirus vectors. Adapted by permission from
Macmillan Publishers Ltd: Mol Ther., Rissanen & Ylä-Herttuala, “Current Status of Cardiovascular
Gene Therapy”, © 2007.
Advantages
Disadvantages
High transduction efficiency
Strongly immunogenic
Transduce both dividing and non-dividing cells
Prevalence of pre-existing anti-Ad immunity in the
population
High capacity to accomodate foreign DNA (~8
kbp)
Predominant hepatotropism following systemic
administration
Well suited as oncolytic vector
Transient expression (~14 days)
Easy to produce in high titers
2.5.1.2 Other viral vectors
Adeno-associated viruses (AAV) are small DNA viruses that belong to the Parvoviridae family.
The wild type AAV requires co-infection with adenovirus or herpesvirus in order to replicate.
In the absence of a helper virus, wild type AAV is able to integrate into chromosome 19, where
it remains latent until rescued by a helper virus. However, current AAV vectors do not possess
this property. The advantages of AAV vectors include their low immunogenicity, their potential
for site-specific integration, their wide tropism, their long expression time, and their ability to
transduce both dividing and quiescent cells. The two main handicaps of using AAVs as vectors
are their limited cloning capacity that is unsuited for most therapeutic genes, and the difficulty
to achieve high vector titers. Most gene therapy trials employing AAV as gene transfer vector
are aimed at treating monogenic diseases. (Vannucci et al. 2013, Daya, Berns 2008.)
Lentiviruses and retroviruses are RNA viruses capable of integrating into the genome of the
host cell, thus inducing stable transgene expression. Following entry into the target cell, the
reverse transcriptase converts the genomic RNA into double-stranded DNA, which is then
integrated into the host chromosome. Lentiviruses are capable of transducing both nondividing and dividing cells, whereas retroviruses transduce only replicating cells. (Vannucci et
al. 2013.) Lentiviral vectors have been successfully used to treat β-thalassemia as well as
progressive cerebral demyelination in X-linked adrenoleukodystrophy patients in clinical trials
(Cartier et al. 2009, Kaiser 2009). Retroviral vectors have been applied in the treatment of severe
combined immunodeficiency (SCID), which hampers T cell development and function, utilizing
ex vivo gene transfer into autologous hematopoietic stem cells. Unfortunately, the use of firstgeneration retroviral vectors was associated with the occurrence of acute leukemia in clinical
trials, which will be further discussed in chapter 2.5.3 “Safety issues in gene therapy”. (Touzot
et al. 2014.)
Other more uncommon viral vectors include poxviruses, also known as Vaccinia viruses,
which have been mostly used for the production of recombinant proteins, oncolytic cancer
therapy, and vaccination; baculoviruses which are convenient tools for recombinant protein
34
production in insect cells but they can also transduce a plethora of mammalian cell types; and
Sindbis virus, which is an alphavirus capable of reaching metastatic tumors when systemically
administered due to its natural tropism for tumor cells (Vannucci et al. 2013, Airenne et al. 2013,
Quetglas et al. 2010).
2.5.2 Non-viral vectors
Non-viral gene delivery methods can be categorized into natural vectors, such as plasmid DNA
or small interfering RNAs (siRNAs), and synthetic carriers, such as liposomes or cationic
polymers. Another classification categorizes non-viral vectors into either organic or inorganic
delivery systems. (Dizaj, Jafari & Khosroushahi 2014.) The simplest non-viral delivery method
is the naked DNA plasmid injection (Kamimura et al. 2011). DNA-based transposon vectors,
such as the leading non-viral vector of gene therapy called Sleeping Beauty, are based on
integration of transposon DNA, which contains a transgene, into chromosomal DNA by a
transposase enzyme (Aronovich, McIvor & Hackett 2011). The advantages of non-viral methods
over viral vectors include simple large scale production, lower immunogenicity, lack of preexisting immunity in patients, and unlimited therapeutic gene packaging capacity. The main
limitation of non-viral vectors is their lower gene transfer efficiency in relation to viral vectors.
(Yin et al. 2014.) That is why at present, the vast majority of gene therapy clinical trials have
been conducted using viral vectors (http://www.abedia.com/wiley/vectors.php). Recently, a
plasmid DNA encoding a major histocompatibility complex class I heavy and light chains of
HLA-B7 and β2-microglobulin complexed with a lipid-based delivery system failed to reach its
efficacy points in a Phase III clinical trial for the treatment of advanced metastatic melanoma
(Hersey, Gallagher 2014). Some minor setbacks regarding the safety of non-viral vectors have
arisen over the years, as plasmid DNA was found to cause prominent inflammation in skeletal
muscle and humans receiving intracoronary plasmid liposomes suffered a transient fever.
(Hedman et al. 2003, McMahon et al. 1998). However, no major adverse effects have been
reported and non-viral vectors in general are considered to have a fairly unblemished safety
profile.
2.5.3 Safety issues in gene therapy
Worldwide, adenoviral vectors are the most commonly used vectors in gene therapy clinical
trials,
being
utilized
in
22.5%
of
all
gene
therapy
trials
(http://www.abedia.com/wiley/vectors.php). Consequently, extensive safety data from
adenovirus vectors in several different clinical applications have accumulated, and a uniformly
good safety profile has been outlined, suggesting that adenoviral gene therapy, when
administered with relevant doses and the optimal route of administration, is safe and well
tolerated (Muona et al. 2012, Hedman et al. 2009, Harvey et al. 2002, Crystal et al. 2002).
Notwithstanding the generally good safety profile, adenoviral gene therapy resulted in the
death of a young patient with ornithine transcarbamylase deficiency in 1999. The patient
suffered multiple organ failure four days after intraportal delivery of a high-dose adenovirus
vector, and a severe anti-Ad immune response was later determined as the cause of death.
(Raper et al. 2003.) This tragic incident warranted efforts to circumvent the anti-Ad immunity
with various approaches, such as progressive deletion of the genes causing inflammation and
stimulation of the host immune response (Vannucci et al. 2013). Another death of a genetherapy recipient occurred in an AAV trial for the treatment of rheumatoid arthritis in 2007,
when a 36-year-old woman died 3 weeks after she had received a second intra-articular
injection of the AAV-vector. However, her death was considered not to have been caused by
AAV but by an infection made possible due to the strong immunosuppressive medication.
(Williams 2007.)
35
Another major safety issue in viral vector gene therapy concerns the disruption of important
genes in target cells. With integrating viruses, there is a risk of insertional mutagenesis which
means that a mutation has been created by the insertion of new genetic material into a normal
gene. Integration of the retrovirus into the host genome may have different outcomes, the most
probable being integration outside a cellular coding sequence or inside an irrelevant gene.
However, in worse case scenarios, integration may lead to the death of transduced cells if it
disrupts a vital gene, or neoplastic proliferation if the integration activates a proto-oncogene.
(Vannucci et al. 2013.) In 2000, it was reported that children suffering from a fatal form of SCID
had been cured with retroviral gene therapy (Cavazzana-Calvo et al. 2000). Sadly, after the
initial promising results, acute leukemias occurred in four of the nine patients. It was found that
the transgene had integrated in proximity to different proto-oncogenes, leading to uncontrolled
division of T cells and consequent leukemia. (Hacein-Bey-Abina et al. 2008, Hacein-Bey-Abina
et al. 2003.) This tragedy prompted efforts to target the integration of transgenes to
predetermined genomic areas (Wirth, Parker & Yla-Herttuala 2013).
In the case of proangiogenic gene therapy, the most commonly used vectors are adenoviruses
and plasmids, which express the transgene transiently and do not integrate into the host
genome. More than 1000 patients have taken part in angiogenic gene therapy trials over the
years and so far no major adverse effects have been reported. (Gupta, Tongers & Losordo 2009.)
However, only a few long-term follow-up studies have been conducted. After the Kuopio
Angiogenesis Trial (KAT), patients were followed for eight years and no increases in the
incidence of cardiovascular events or other diseases were detected in patients receiving
adenoviral VEGF-A therapy as compared to placebo (Hedman et al. 2009). Similarly, in a 10year follow-up study of patients receiving a local AdVEGF-A GT into an ischemic lower limb,
the incidence of cancer, diabetes and retinopathy did not differ between the treatment and the
control groups (Muona et al. 2012).
One other aspect that needs to be considered in regard to safety of gene therapy is the
transgene and its possible harmful effects. VEGF-C and VEGF-D, the factors that have been
studied in this thesis, have been implicated in tumor formation and metastasis (Karkkainen et
al. 2009, Hirakawa et al. 2007, Ishii et al. 2004, Skobe et al. 2001). Over-expression of VEGF-C
has been shown to induce tumor reactive lymphadenopathy, which means that the lymphatic
networks within the lymph node were enlarged even before the onset of metastasis, most likely
facilitating the spreading of tumor cells (Hirakawa et al. 2007). Apart from tumor
lymphangiogenesis, the proteolytically processed forms of VEGF-C and VEGF-D may
contribute to tumor angiogenesis by binding VEGFR-2, as they have been shown to induce
angiogenesis in many pathological settings, including cancer (Achen, Stacker 2008). However,
normal lymph nodes constantly produce low levels of VEGF-C, and in the model used in this
thesis, only transient over-expression of VEGF-C or VEGF-D is induced in the lymph node as a
result of the adenoviral delivery (Saaristo et al. 2012). Nonetheless, for patient safety, it would
be crucial to screen potential recipients of this kind of therapy very carefully, particularly since
they often are individuals with a history of malignant disease. Thus far, no promotion of
spontaneous tumor growth has been reported after therapeutic delivery of lymphangiogenic
growth factors in experimental models.
2.6 PRECLINICAL AND CLINICAL STUDIES
2.6.1 Preclinical studies of lymphedema
Lymphedema has been previously studied mostly in small animal models, although some large
animal studies exist as well (Baker et al. 2010, Blum et al. 2010, Tammela et al. 2007, Yoon et al.
2003, Szuba et al. 2002, Saaristo et al. 2002b, Karkkainen et al. 2001). The most widely used
lymphedema animal models comprise the canine hind limb model, the rabbit ear model, the
36
rodent limb model and the rodent tail model; in all of these models lymphedema is induced by
a surgical procedure and in some cases, additional irradiation. However, all of the
aforementioned models suffer from some major disadvantages. The previously used canine
models demanded complex procedures, a long latency time before chronic lymphedema
developed, and all in all they would be considered unethical today. Small animal models are
appropriate for molecular studies, but do not sufficiently resemble the conditions encountered
in human lymphedema, thus casting doubts about their clinical translation to the therapy of
human patients. (Hadamitzky, Pabst 2008.) The major hurdle in the development of successful
animal models has been the inability to replicate the chronic disease in a stable, reproducible
format (Shin, Szuba & Rockson 2003).
Recombinant VEGF-C, naked plasmid VEGF-C and adenoviral VEGF-C have all been shown
to induce the growth of functional lymphatics and to ameliorate lymphedema in mice, rabbits
or sheep (Baker et al. 2010, Cheung et al. 2006, Yoon et al. 2003, Szuba et al. 2002, Karkkainen et
al. 2001). VEGF-C or VEGF-D in conjunction with lymph node transfer has helped rebuild the
vascular anatomy after lymph node excision, reduce lymphedema, and even reconstitute the
immunological barrier function of the transferred lymph nodes in a mouse model of
lymphedema (Tammela et al. 2007). Recently, the approach of combining autologous lymph
node transfer with the application of exogenous VEGF-C was further investigated in rats, with
promising results regarding transplant regeneration and recovery of the lymphatic integrity
(Schindewolffs et al. 2014, Sommer et al. 2012). Apart from VEGFs, other therapeutic agents
used to treat secondary lymphedema in the experimental setting have included retinoic acids,
anti-inflammatory drugs such as ketoprofen, as well as combined autologous bone marrow
stromal cell and VEGF-C protein injections (Choi et al. 2012, Zhou et al. 2011, Nakamura et al.
2009). As yet, there are no clinical studies of gene therapy for lymphedema.
2.6.2 Preclinical studies of proangiogenic therapy for PAD
In the context of cardiovascular diseases, proangiogenic therapies for the revascularization of
tissues have been studied in animal models of myocardial and peripheral ischemia. In the most
commonly used animal models of peripheral ischemia, ischemia is induced by ligation, excision
or ameroid constriction of a large artery in the hindlimb of a mouse, rat or rabbit (Limbourg et
al. 2009, Tang et al. 2005, Rissanen et al. 2005, Masaki et al. 2002, Baffour et al. 2000). Several
proangiogenic factors have been tested using these models, including VEGFs, members of the
FGF family, HIF-1α, and HGF (Dragneva, Korpisalo & Yla-Herttuala 2013). Both plasmid and
adenoviral gene transfers of VEGF-A resulted in angiogenesis, increased muscle perfusion, and
the formation of collateral arteries in ischemic rabbit hind limbs (Korpisalo et al. 2008a,
Rissanen et al. 2005, Takeshita et al. 1996). Furthermore, AdVEGF-A gene transfer induced
positive effects on tissue energy metabolism and exercise tolerance in ischemic rat hind limbs
(Gowdak et al. 2000). However, local VEGF-A gene transfer has been demonstrated to evoke
unfavorable effects as well, such as increased vascular permeability, edema, and disorganized,
immature vascular structures (Karvinen et al. 2011, Weis, Cheresh 2005, Lee et al. 2000).
Therefore, combinations of growth factors and factors modulating the expression of multiple
downstream targets, such as HIF-1α, have been evaluated in attempts to elicit more
physiological angiogenesis (Vincent, Feron & Kelly 2002). Increased angiogenesis and muscle
perfusion were observed in ischemic rabbit hind limbs following an injection of naked DNA
encoding HIF-1α (Vincent et al. 2000). Adenovirally delivered, constitutively active HIF-1α
promoted collateral vessel formation and improved tissue perfusion in ischemic rabbit hind
limbs (Li et al. 2011, Patel et al. 2005). In contrast to VEGF-A, HIF-1α delivered via AAV did not
result in vascular leakiness in transduced normoxic mouse skeletal muscles (Pajusola et al.
2005). Importantly, HIF-1α was found to induce marked capillary sprouting in the non-ischemic
muscles, whereas VEGF-A only induced endothelial proliferation without the formation of
proper capillary structures (Pajusola et al. 2005). It has been claimed that the approach of
37
inducing angiogenesis at its onset by utilizing hypoxia-induced signaling would harness the
innate mechanism that naturally generates angiogenesis in physiological as well as pathological
states (Hadjipanayi, Schilling 2013).
2.6.3 Clinical studies of proangiogenic therapy for PAD
The first effort to treat PAD in a human patient was made in 1996 with the administration of
plasmid DNA encoding VEGF-A (Isner et al. 1996). In a randomized, double-blind, placebocontrolled study conducted in patients with PAD manifesting as claudication and critical limb
ischemia, intravascular delivery of adenoviral or plasmid liposome VEGF-A resulted in a
significant increase in vascularity but not in any significant clinical improvements in
comparison to controls (Makinen et al. 2002). Dose-dependent peripheral edema was observed
in patients receiving intramuscular injections of low-dose or high-dose VEGF-A, providing
evidence for bioactivity of adenoviral VEGF-A. Disappointingly, the efficacy end points, such as
ABI, claudication onset time, and quality-of-life measures, showed no improvement following
administration of AdVEGF-A. (Rajagopalan et al. 2003.) In another trial of intramuscularly
administered plasmid VEGF-A, somewhat more promising results were obtained, as VEGF-A
treated patients experienced fewer amputations, better skin ulcer healing, and hemodynamic
improvement in comparison with controls (Kusumanto et al. 2006). Plasmid VEGF-A165
(Neovasculgen®) was approved for the treatment of PAD in Russia in 2011
(http://www.genetherapynet.com/january-2012.html). Intramuscular administration of AdHIF1α has resulted in a good safety profile, pain relief and ulcer healing in a small trial of 38 PAD
patients, but a larger trial of 289 patients with intermittent claudication revealed no differences
in peak walking time (exercise treadmill test), claudication onset time, ABI, or quality-of-life
measurements between HIF-1α and placebo groups (Creager et al. 2011, Rajagopalan et al.
2007). A meta-analysis derived from a total of 12 randomized, controlled trials of proangiogenic
gene therapy for PAD detected no benefit from gene therapy for all-cause mortality,
amputations, or ulcer healing (Hammer, Steiner 2013).
Several explanations have been offered as to why angiogenic gene therapy clinical trials have
failed to produce evidence of benefit despite ample, encouraging preclinical data. The animals
used in preclinical models are usually healthy, whereas patients are typically older. Older
people may exhibit deficiencies in the recruitment of angiogenic cells and growth factor
expression, and often suffer from multiple comorbidites, which may impede the response to
ischemia. (Gupta, Tongers & Losordo 2009.) It has been proposed that the age-dependent
reduction in VEGF-A expression may be attributable to reduced HIF-1 activity, which would
explain the impairment in angiogenesis in the elderly (Bosch-Marce et al. 2007, Rivard et al.
2000). Other factors possibly accounting for the modest results obtained from clinical trials
include inadequate dosing of the therapeutic drug, insufficient duration of exposure to the
angiogenic agent, compromised delivery and poor vector transduction efficiency. In addition,
the obvious differences between animals and humans with respect to physiology, body size,
and the distance which collateral arteries or lymphatics need to span, should be taken into
account. (Dragneva, Korpisalo & Yla-Herttuala 2013.) Thus, before embarking on clinical trials,
it is essential that the therapeutic agent should be evaluated in a large animal model to support
data obtained from small rodents.
38
39
3 Aims of the Study
The aim of this thesis was to study the potential of two adenovirally delivered single growth
factors, VEGF-C and VEGF-D, to achieve therapeutic lymphangiogenesis for the treatment of
lymphedema, and furthermore, instead of administering only a single growth factor, to study
the potential of influencing several angiogenic growth factors via adenoviral over-expression of
HIF-1α/-2α in the treatment cardiovascular diseases. Moreover, the aim was to evaluate
whether adenovirally induced, transient over-expression of growth factors would be sufficient
to result in measurable functional improvements, such as improved lymphatic flow and
enhanced energy metabolism in ischemic tissue in large animal models.
The specific aims of each study were as follows:
I
To develop a novel large animal model of lymphedema which would better
resemble the condition in human patients, and, then to use this model to test the
possibilities of adenoviral VEGF-C and VEGF-D gene transfers in the induction of
lymphangiogenesis to improve lymphatic flow.
II
To optimize adenoviral VEGF-C gene transfer in the large animal model of
lymphedema for prospective clinical translation.
III
To compare the angiogenic effects of a single adenovirally delivered growth factor,
VEGF-A, to the effects of adenoviral HIF-1α/-2α, which activate several
proangiogenic growth factors, in normoxic and ischemic rabbit hindlimbs as well as
in normoxic mouse myocardium.
40
41
4 Materials and Methods
4.1 ANIMAL MODELS
A total of three animal models were used in this thesis: a novel pig model of lymphedema
(Studies I and II), a previously developed model of rabbit hindlimb ischemia (Study III)
(Rissanen et al. 2003a), and a murine myocardium model, where gene transfer was performed
into the normoxic cardiac muscle of a mouse (Study III). All animal experiments were approved
by the National Animal Experiment Board of Finland.
4.1.1 Pig model of lymphedema (I-II)
As a continuation to previous small animal models of lymphedema, a novel large animal model
was developed which would better resemble the features of lymphedema in human patients,
such as the distance which lymphatic neovessels need to span and the volume of edema
formation. The model was used in this thesis to test the lymphangiogenic potential of AdVEGFC and AdVEGF-D in combination with autologous lymph node transfer. After anesthetizing the
pigs, the normally invisible lymphatic vessels of the inguinal area were rendered visible to the
naked eye by the Patent Blue (Guerbet) tracer dye. To block lymphatic drainage from the lower
limbs, the lymphatic vasculature surrounding the inguinal lymph node was damaged
surgically, leaving the blood vascular pedicle intact to simulate microsurgical anastomosis of
blood vessels to the lymph node (Figure 8A). Next, gene transfer was performed as described
later, after which the pedicular lymph node was attached into the remaining tissue 4 cm lateral
from its original position. This procedure mimicks lymph node transfer in human lymphedema
patients in that the lymph node is vascularized but lacks all lymphatic connections. The animals
were followed for 2 or 6 months postoperatively. The sizes of the transferred lymph nodes as
well as those of unoperated contralateral lymph nodes were measured in three dimensions at
euthanasia which occurred 2 months after the surgical procedure. In addition, samples from
internal organs (heart, lung, liver, kidney, spleen, gonads) were collected for safety examination
(I).
4.1.2 Rabbit model of hind limb ischemia (III)
A rabbit model of acute hind limb ischemia was used to study the angiogenic and metabolic
effects of AdHIFs in peripheral muscles. To induce ischemia in the calf region, the superficial
part of the femoral artery was excised (Figure 8B). Additionally, two re-entry branches were
ligated to block collateral growth from the lateral circumflex and deep femoral arteries. As a
result, the calf region became severely ischemic but the thigh remained normoperfused. The
animals were euthanized 7 days after the procedure and 6 days after GT.
42
Figure 8. A, The pig model of lymphedema and autologous lymph node transfer. The afferent and
efferent lymphatic vessels surrounding the inguinal lymph node were excised, leaving only the main
artery and vein supplying the node intact. Gene transfer was made either intranodally under the
capsule of the node or perinodally into the fat surrounding the node. Reprinted by permission from
Ann Surg.: May 2013 - Volume 257(5) - p 961-967, Wolters Kluwer Health Lippincott Williams &
Wilkins ©. B, The rabbit model of hindlimb ischemia. The superficial femoral artery and two re-entry
branches were ligated to induce ischemia in the calf region. Black bars mark ligations. Adapted with
permission from Johanna Lähteenvuo © 2001.
4.2 GENE TRANSFER METHODS
Human clinical grade first generation serotype 5 replication deficient (E1, partial E3-deletion)
adenoviruses produced under GMP conditions in 293 cells were used as gene transfer vectors
throughout the studies. All of the used viruses have the cytomegalovirus (CMV) promoter. The
functionality of the viral constructs was tested in vitro before using them in animal experiments.
4.2.1 Intranodal and perinodal gene transfers (I-II)
Intranodal gene transfer was performed under the capsule of the inguinal lymph node of pigs
with a series of injections using a needle and a syringe (I, II) (Figure 8A). Perinodal gene
transfer was conducted in a similar manner into the fat surrounding the lymph node (II) (Figure
8A). A total dose of 1 x 1011 viral particles (vp) encoding full-length VEGF-C, full-length VEGFD, or control gene β-galactosidase (LacZ) was injected per animal (I,II). In addition, a lower
dose of 1 x 1010 vp encoding full-length VEGF-D was administered for a subset of animals (I).
Gene transfer was performed immediately after the surgical excision of lymphatic vessels.
4.2.2 Intramuscular gene transfer (I, III)
Intramuscular gene transfer was performed by manual injections into the hindlimb skeletal
muscles of New Zealand White rabbits using a needle and a syringe (III). Adenoviruses
encoding the stabilized forms of mouse HIF-1α and HIF-2α were used as therapeutic drugs,
AdLacZ as a negative control virus, and AdVEGF-A as a positive control virus. A dose of 1 x
43
1011 vp was injected into the non-ischemic semimembranosus muscles, whereas in ischemic
hindlimbs, a total dose of 3 x 1011 vp was administered to the whole hindlimb covering the
semimembranosus, quadriceps femoris, abductor cruris cranialis and tibialis anterior muscles.
Gene transfer into ischemic muscles was performed 1 day after the ischemia procedure.
Intramuscular gene transfer was also performed on the right hind limbs (m. gastrocnemius)
of C57/Bl/6J mice (I). The mice received a dose of 5 x 108 plaque-forming units (pfu) of AdVEGFC, AdVEGF-D, or AdLacZ.
4.2.3 Myocardial gene transfer (III)
Adenoviruses encoding HIF-1α, HIF-2α, and LacZ were used for echocardiography-guided
myocardial gene transfers in C57Bl/6J mice. Following induction of isoflurane inhalation
anesthesia, 1 x1010 viral particles in a volume of 10 μL were injected into the anterior wall of of
the left ventricle as described previously (Huusko et al. 2010, Springer et al. 2005).
4.3 X-RAY IMAGING
X-ray radiography was used in all of the studies either to assess the growth of collecting
lymphatic neovessels in pigs (I,II) or collateral artery formation in rabbits (III).
4.3.1 Lymphangiography (I-II)
X-ray lymphangiographies were recorded with Siemens Siremobil 3000 C-arm (I) or Innova
3100 IQ Excellence X-ray machine (GE Healthcare) (II) from the inguinal region of pigs before
and after the excision of lymphatics (Figure 9), and at the time of euthanasia. Lipiodol (Guerbet)
served as a radio-opaque contrast agent. Lymphangiograms were used perioperatively to
confirm that the lymphatic defect was of a standard width, as well as to evaluate the therapeutic
effect of gene transfers after a predetermined follow-up period of 2 or 6 months. For this, the
numbers of Lipiodol-positive lymphatic vessels connecting to and draining past the lymph
node were quantified from still images and dynamic X-ray sequences (30 fps, frames per
second).
4.3.2 Digital subtraction angiography (III)
Digital subtraction angiography (DSA) is a type of fluoroscopy technique that permits the
visualization of vascular structures without superimposed bone and soft tissue densities.
Images of blood vessels are produced by subtracting a pre-contrast image from subsequent
images taken with contrast medium. In this thesis, DSA was used to visualize collateral blood
vessel formation following gene transfer into ischemic rabbit hindlimbs. A 5 French catheter
(Cordis) was introduced into the abdominal aorta of rabbits for the injection of contrast agent
(Hexabrix, Guerbet). Serial DSA images (30 fps) were recorded immediately after the injection
of contrast agent bolus with Innova 3100 IQ Excellence X-ray machine. Collateral vessel
numbers and diameters were quantified from images with the best arterial filling.
44
Figure 9. Perioperative lymphangiograms before and after lymphatic vessel excision. Arrows mark
excision sites and the asterisk shows the lymph node. Reprinted by permission from Ann Surg.: May
2013 - Volume 257(5) - p 961-967, Wolters Kluwer Health Lippincott Williams & Wilkins ©.
4.4 ULTRASOUND IMAGING
Ultrasound imaging was used to evaluate the effects of gene transfer on mouse myocardium
and rabbit skeletal muscle (III). Myocardial imaging was performed as described previously
(Huusko et al. 2010) with a high-resolution imaging system (Vevo 770, VisualSonnics Inc) using
a high-frequency ultrasound probe (RMV-707B) on the day of the GT and 6 days after GT. The
determined parameters included ejection fraction (EF%) and left ventricle anterior wall
thickness (LVAW; mm).
Native Doppler ultrasound and contrast pulse sequence (CPS) imaging performed with
Acuson Sequoia 512 (Siemens) were used to assess perfusion in gene-transduced hindlimb
muscles of rabbits. CPS is a rather new ultrasound perfusion imaging technique associated with
better spatial resolution and tissue penetration than previous perfusion imaging modalities, and
has proven to provide an accurate evaluation of real-time perfusion in small laboratory animals
(Rissanen et al. 2008, Phillips, Gardner 2004). A 0.5 mL bolus of SonoVue (Bracco) contrast
microbubbles was administered via the ear vein and transversal images of semimembranosus
muscles were captured immediately thereafter. Imaging was performed one day after ischemia
procedure and six days after GT.
Skeletal muscle perfusion was also measured during physical activity. In that assessment, the
sciatic nerve was electrically stimulated for 6 minutes to induce maximal contraction in the calf
muscles. Perfusion imaging was performed after the stimulation ceased. The signal intensities
were quantified with DataPro software (version 2.13, Noesis).
4.5
31
P-MAGNETIC RESONANCE SPECTROSCOPY
Phosphorus-magnetic resonance spectroscopy (31P-MRS) is an established method for the
assessment of metabolic correlates of tissue perfusion and was employed as a time-dependent,
non-invasive measurement of changes in muscle metabolites, i.e. phosphocreatine (PCr) and
inorganic phosphate (Pi), during rest, exercise and recovery from exercise (III) (Gowdak et al.
31
45
2000). At the beginning of exercise, hydrolyzed ATP is resynthesized from PCr breakdown.
Thus, PCr decreases and Pi increases, while ATP levels remain constant (Figure 10). During
recovery, PCr is restored and Pi levels normalize. (Prompers et al. 2006.) The ratio of PCr to Pi
(PCr/(PCr+Pi)) can be used to assess the energy balance of the muscle (Korpisalo et al. 2008b).
31P-MRS was performed on ischemic rabbit skeletal muscles before GT and 6 days after GT
with a UNITYINOVA imaging console (Varian) as described previously in detail (Korpisalo et al.
2008b). Physical activity in the muscles was achieved through stimulation of the sciatic nerve in
a similar manner as during ultrasound imaging. Muscle recovery was followed for 12 minutes
with 31P-MRS after the cessation of stimulation. The PCr/(PCr+Pi) ratios were calculated and
used as a measure of aerobic capacity and recovery rate.
Figure 10. Representative
31
P-MRS spectra of normoxic and ischemic muscles during rest, exercise
and recovery. At rest, high PCr and ATP peaks are visible. During exercise, PCr decreases and Pi
peaks. PCr reservoirs are then replenished during recovery. In ischemic muscles, the high Pi peak
and low PCr during exercise indicate a shift towards anaerobic metabolism. Korpisalo et al.,
“Therapeutic angiogenesis with placental growth factor improves exercise tolerance of ischaemic
rabbit hindlimbs”, Cardiovasc Res., 2008, 1;80(2):263-70, by permission of Oxford University Press.
46
4.6 VASCULAR PERMEABILITY AND TISSUE EDEMA
4.6.1 Seroma fluid evaluation (I-II)
To evaluate the amount of seroma fluid that accumulated in the operated area of pigs following
the excision of lymphatic vessels, an observer blinded to the study groups visually estimated
the volume of fluid collection in deciliters every day for 2 weeks (I) or until the edema had
dissipated (II). The seroma sacs were drained with a needle in case the volume was deemed
harmful for the well-being of the animal.
4.6.2 Modified Miles Assay (I, III)
Extravasation of plasma proteins and the leakiness of neovessels in rabbit transduced muscles
were assessed by modified Miles Assay (III) (Rissanen et al. 2003a). Evans Blue (Sigma Aldrich)
dye, which binds mostly to plasma albumin, was injected intravenously 30 minutes before
sacrifice. Following sacrifice, the animals were perfusion fixed with 1% paraformaldehyde in
citrate buffer (pH 3.5) to rinse off the remaining dye from the blood vessels. The extravasated
dye from muscle samples was then eluted in formamide, after which it was quantified with a
spectrophotometer at a wavelength of 610 nm.
Modified Miles Assay was also performed in C57/Bl/6J mice receiving a gene transfer of
AdVEFG-C, AdVEGF-D, or AdLacZ to determine the effects of VEGF-C and VEGF-D on
vascular permeability (I).
4.7 PROTEIN ANALYSES
4.7.1 Western Blot (I, III)
Seroma fluid samples were collected from pigs 11 days after excision of lymphatics and snap
frozen in liquid nitrogen (I). To assess the amount of VEGF-C and VEGF-D in seroma fluid,
Western Blot protein expression analysis was performed with antibodies against VEGF-D (AF286, R&D Systems) and VEGF-C (AF-752, R&D Systems). Horseradish-peroxidase-conjugated
Donkey anti-goat IgG (Santa Cruz Biotechnology) served as a secondary antibody. The blots
were scanned, and densitometric ratios of short isoforms of growth factors versus full-length
isoforms were quantified with Adobe Photoshop CS4.
AdHIF-1α and AdHIF-2α constructs were tested with Western Blot for their protein
production in vitro prior to their usage in vivo (III). Monoclonal antibodies against HIF-1α
(NB100-123, Novus Biologicals) and HIF-2α (NB100-132, Novus Biologicals) were used.
4.7.2 ELISA (I)
The enzyme-linked immunosorbent assay (ELISA) was performed to test the in vitro protein
production of AdVEFG-C and AdVEGF-D. Human umbilical vein endothelial cells (HUVECs)
were transduced with AdVEGF-C, AdVEGF-D or AdLacZ. Cell culture media were harvested
72 hours after transduction, and VEGF-C and VEGF-D protein levels were measured from these
media with the appropriate ELISA kits (DVEC00 and DVED00, respectively, R&D Systems).
47
4.8 mRNA EXPRESSION
Quantitative PCR was used to verify the expression of mouse HIF-1α and HIF-2α mRNA in
transduced rabbit hindlimb muscle (III). Total RNA was extracted from snap-frozen muscle
samples with an appropriate commercial kit (RNeasy Fibrous Tissue Midi Kit, #75742, Qiagen)
and cDNA was synthetized from 2 μg of total RNA using random primers (Promega).
Expression levels of HIF-1α and HIF-2α mRNA were measured with StepOnePlusTM Real-Time
PCR System (Applied Biosystems).
Quantitative PCR was also used to examine the in vitro upregulation of HIF target genes
VEGF-A and PDK1 (III). HUVECs were transduced with AdHIF-1α, AdHIF-2α, AdLacZ, or an
empty adenovirus vector (AdCMV). Fortyeight hours after transduction, the cells were lysed in
TriReagent (Invitrogen), RNA was isolated with chloroform, and cDNA was synthesized.
VEGF-A and PDK1 mRNA levels were measured with StepOnePlusTM Real-Time PCR System.
4.9 IMMUNOHISTOCHEMISTRY AND HISTOLOGY
Immunohistochemical stainings were used to evaluate lymphangiogenesis (I, II), angiogenesis
(I, III), inflammatory cell numbers (I, II), and the expression of specific proteins (HIFs, VEGF-A,
eNOS) (III). Hematoxylin-eosin stainings were used to characterize the tissue morphology (IIII). All the stainings were performed on sections cut from paraffin embedded tissues. The
avidin-biotin HRP (horseradish peroxidase) system with diaminobenzidine (DAB; Vector
Laboratories) and the alkaline phosphatase system with Vector Blue (Vector Laboratories) were
used for signal detection. The antibodies used are listed in table 3 and the protocols are
described or referenced in the original publications.
48
Table 3. Antibodies used in immunohistochemistry
Antibody
Structure stained
Dilution
Pretreatment
Manufacturer
Study
PECAM-1
endothelium (pig)
1:100
-
R&D Systems
I
CD31
endothelium (rabbit)
1:50
-
DAKO
III
Griffonia
simplicifolia
lectin I
endothelium (mouse) 1:100
-
Vector Laboratories
III
α-SMA
smooth muscle cells,
pericytes
1:250
-
Sigma Aldrich
I
LYVE-1
lymphatic
endothelium
1:100
-
R&D Systems
I
PROX-1
lymphatic
endothelium
1:100
Citrate buffer
boiling, TSA
R&D Systems
I
VEGFR-3
lymphatic
endothelium
1:100
Citrate buffer
boiling
non-commercial
(Jussila et al. 1998)
II
CD3
T cells
1:25
Target Retrieval
Solution (DAKO)
DAKO
I, II
CD20cy
B cells
1:200
Target Retrieval
Solution (DAKO)
DAKO
I, II
CD23
Dendritic cells
1:10
Target Retrieval
Solution (DAKO)
DAKO
I, II
HAM56
Macrophages
1:100
-
DAKO
II
HIF-1α
HIF-1α
1:250
-
Novus Biologicals
III
HIF-2α
HIF-2α
1:250
-
Novus Biologicals
III
VEGF-A
VEGF-A
1:250
-
Santa Cruz
Biotechnology
III
eNOS
eNOS
1:50
-
BD BioSciences
III
TSA = tyramide signal amplification
To evaluate lymphatic and blood vessel growth from the immunostainings, mean
capillary/vessel areas (μm2), capillary/vessel perimeters (μm), and numbers of
capillaries/vessels were quantified with AnalySIS software (Soft Imaging System) from the
microscopic images (Olympus AX-70) of VEGFR-3-, PROX-1 + α-SMA-, PECAM-1 + α-SMA-,
CD31- and lectin-stained sections (I-III). In addition, the numbers of inflammatory cells inside
the lymph nodes were counted from sections stained for B cells, T cells, dendritic cells (I, II) and
macrophages (II). The stage of lymph node atrophy following disruption of lymph flow to the
node was evaluated from the hematoxylin-eosin stained specimens (I, II).
49
4.10 ASSESSMENT OF LYMPHATIC FUNCTION
The functionality of the lymphatic neovessels was evaluated from Lipiodol X-ray
lymphangiograms as described in chapter 4.3.1 (I, II) and by an adaptation of the modified
Miles assay (I). In the modified version of the assay, a mix of Patent Blue and Evans Blue dyes
was injected distally into the pig hindlimb and serum samples were collected 13 minutes after
the injection. The marker dyes were then eluted from the serum in formamide, and their
amounts quantified with a spectrophotometer. The assay is based on the expectation that the
lymphatic neovasculature in the operated area will collect the dye and transport it to the
systemic circulation. Thus, the more dye there is in the collected serum samples, the better is the
lymph flow in the operated area.
4.11 STATISTICAL ANALYSES
Results are expressed as means ± standard deviation (SD) (I, II) or means ± standard error of
mean (SEM) (III) for normally distributed data, and medians with interquartile ranges for nonnormally distributed data (II). The normality of distribution was assessed visually by plotting
data from individual experiments to a quantile (Q-Q) plot and residuals of data to a histogram.
Data estimated to approximately follow normal distribution were tested using parametric tests.
The independent-samples Student’s t-test was used when comparing only two groups against
each other. For comparing multiple groups, the one-way analysis of variance (ANOVA) was
performed, and in the case of a statistical significance, followed by Dunnett’s Multiple
Comparison test or Tukey’s Multiple Comparison test, or pairwise comparison and Bonferroni
correction for multiple comparisons. Two-way ANOVA for repeated measures with
Bonferroni’s post hoc test was used when comparing multiple groups with two independent
variables. In case of non-normally distributed data, the nonparametric analysis of variance
(Kruskal-Wallis) was performed, followed by the Mann-Whitney U test for pairwise
comparison and the Bonferroni adjustment for multiple testing. Linear mixed-effect models
were used to analyze group differences at baseline and during follow-up in the evaluation of
seroma fluid accumulation (I, II). Benjamini-Hochberg false discovery rate was used to adjust
results for multiple comparisons. Throughout, corrected p-values of < 0.05 were considered
statistically significant.
50
51
5 Results
5.1 THERAPEUTIC LYMPHANGIOGENESIS FOR LYMPHEDEMA (I)
Therapeutic lymphangiogenesis in the form of adenoviral VEGF-C and VEGF-D gene transfers
was studied in a novel pig model of lymphedema. The inguinal lymphatic vascular network
was destroyed and adenovirally over-expressed therapeutic genes were administered into the
lymph node with an intact vascular pedicle simulating microvascular anastomosis of
transferred lymph nodes. AdLacZ served as control.
5.1.1 Lymphatic vessel regrowth induced by AdVEGF-C and AdVEGF-D
The numbers of collecting lymphatic vessels detectable by Lipiodol x-ray lymphangiography
were counted 2 months after lymphatic vessel excision and gene transfer. Both AdVEGF-C and
AdVEGF-D induced a statistically significant increase in the number of functional lymphatic
vessels spanning the operated area as compared to the AdLacZ control group (Figure 11A).
Most of the lymphatic neovessels were connected to the lymph node, although vessels
bypassing the node were also detected in many animals regardless of the given treatment.
Prominent lymphatic vessel growth following AdVEGF-C and AdVEGF-D GT was observed
in lymph node tissue sections stained for LYVE-1 (lymphatic capillaries) (Figure 11B-D). Dense
LYVE-1 positive lymphatic capillary networks were concentrated around the pericapsular area
in AdVEGF-C-treated pigs, and in the connective tissue surrounding the lymph node in
AdVEGF-D treated pigs. The numbers of PROX-1 + α-SMA positive vessels (collecting
lymphatic vessels) were higher in both the AdVEGF-C (157 ± 41, P < 0.01) and AdVEGF-D
groups (134 ± 36, p < 0.05) than in the AdLacZ group (75 ± 22).
As a consequence of the reconstituted lymphatic connections to the lymph node, the lymph
node structure, as evaluated from the hematoxylin-eosin staining, was better preserved in the
AdVEGF-C and AdVEGF-D groups than in the control group (Figure 11E). Hematoxylin-eosin
staining of the lymph nodes revealed that the normal follicular structure was retained in most
of the AdVEGF-C and AdVEGF-D transduced lymph nodes, whereas control lymph nodes had
been more often infiltrated with adipose and connective tissue, as well as groups of
lymphocytes and siderophages detected mainly around vessels (Figure 11F-H).
52
Figure 11. A, The numbers of lymphatic vessels in the inguinal area 2 months after the procedure as
counted
from
Lipiodol
lymphangiograms.
B-D,
LYVE-1
immunostaining
revealing
robust
lymphangiogenesis characterized by dilated sinuses in the AdVEGF-C-treated lymph nodes and more
discrete lymphangiogenesis in the AdVEGF-D-treated lymph nodes. E, Quantification of the amount
of connective and adipose tissue in the lymph node sections. F-H, Hematoxylin-eosin staining of the
lymph nodes. The asterisk in G points to the slight deterioration of the nodal tissue, and the
asterisks in H point to the grouped lymphocytes and siderophages. Magnifications: B,C,D,F,G:
12,5X; H: 100X. ** = p < 0.01, *** = p < 0.001.
5.1.2 Lymphatic transport 2 months after AdVEGF-C and AdVEGF-D treatment
The lymphatic neovessels detected by lymphangiography mainly in the AdVEGF-C- and
AdVEGF-D-treated animals carried Lipiodol contrast agent in a distal-to-proximal direction,
thus indicating restored lymphatic flow in the operated area (Figure 12A-C). To further assess
improvement in lymph transport across the damaged area, the accumulation of Evans Blue +
Patent Blue dye mix in the systemic blood following distal injection of the dyes was quantified.
AdVEGF-C and AdVEGF-D increased dye transport into the systemic circulation by 50% and
40%, respectively, as compared to AdLacZ controls (Figure 12D).
Figure 12. A-C, Lipiodol lymphangiograms of the inguinal area 2 months after operation with
arrowheads pointing to lymphatic neovessels and asterisks marking the lymph nodes. D,
Quantification of the accumulated Patent Blue + Evans Blue dyes in the systemic blood after a dye
mix injection distal to the lymphatic excision site. *** = p < 0.001.
53
5.1.3 Increased seroma formation following AdVEGF-D gene transfer
The surgical excision of lymphatic vessels led to an immediate accumulation of seroma fluid in
the inguinal ring area. During follow-up, the volume of seroma was visually estimated by an
observer blinded to the study groups. The amounts of seroma in each treatment group
increased at a similar rate for the first 6 days after the surgical procedure (Figure 13A). On the
7th day postoperation, the curve depicting the volume of seroma in the AdVEGF-D treatment
group started to diverge upwards from those of the AdVEGF-C and the AdLacZ groups.
Seroma fluid volumes in the AdVEGF-D-treated animals continued to rise for the remainder of
the 2-week follow-up period, whereas fluid volumes in AdVEGF-C and AdLacZ groups
remained low and at comparable levels. Furthermore, AdVEGF-D-treated animals required
drainage of excessive fluid due to animal welfare considerations, whereas spontaneous
dissipation of seroma without the need for drainage was observed in the other two groups by
two weeks after the procedure.
In an attempt to clarify the causes of the dissimilar seroma fluid buildup between groups, a
few possible explanations were explored. First, the adenoviral constructs were tested for their
protein production in HUVEC cells. ELISA analysis of the cell culture media revealed negligible
differences in protein expression between constructs (Figure 13B). Next, a new set of pigs
receiving a 10-fold lower dose (1 x 1010 vp) of AdVEGF-D was operated to study whether a
lower viral dose would reduce seroma formation. However, a similar trend in the seroma fluid
accumulation was observed as previously with a higher dose of AdVEGF-D (Figure 13A).
Finally, the proteolytic processing of the administered growth factors was examined in order to
determine whether angiogenesis and increased vascular permeability known to be induced by
the processed short forms could be involved in the fluid buildup. For this, a Western Blot
analysis of collected seroma samples was performed. It was observed that AdVEGF-D animals
displayed high levels of proteolytically processed human VEGF-D, whereas AdVEGF-C
animals had only small amounts of processed VEGF-C in their seroma (Figure 13C). A
subsequent densitometric analysis from the Western Blots showed significantly higher ratios of
processed versus full-length forms of VEGF-D (0.53 ± 0.11) than VEGF-C (0.30 ± 0.025) in the
AdVEGF-D and AdVEGF-C transduced animals, respectively, implying that there had been
increased processing of VEGF-D into its shorter isoform (p = 0.03).
To investigate potential angiogenic effects of the short isoform of VEGF-D that was found to
be present in significant amounts in seroma fluid, lymph node sections were immunostained for
PECAM-1 + α-SMA marking blood capillaries. There was a two-fold increase in mean capillary
area in AdVEGF-D-treated nodes in comparison to AdLacZ controls (p < 0.05). Furthermore, a
modified Miles assay performed in C57/Bl/6J mice revealed an almost 9-fold increase in the
vascular permeability in AdVEGF-D transduced mouse hindlimbs as compared to mice
receiving AdLacZ (p < 0.001). Extravasation was 3.5-fold higher but not statistically significantly
higher in AdVEGF-C-treated mice than in control mice.
54
Figure 13. A, Seroma fluid buildup as a function of time. For both AdVEGF-D groups vs. AdLacZ
p < 0.001. ‘Dr’ marks drainage in AdVEGF-D higher dose group on postoperative day 11 and 13, and
in AdVEGF-D lower dose group on postoperative day 11. B, ELISA analysis of the in vitro protein
production of AdVEGF-C and AdVEGF-D constructs. C, Western Blot analysis of VEGF-C and VEGF-D
proteins in seroma fluid. Upper band represents the full-length form and the lower band is the
processed short form, P = positive control, L = LacZ, C = VEGF-C, D = VEGF-D.
5.1.4 Histopathological evaluation of safety samples
Hematoxylin-eosin-stained sections of the collected safety samples (heart, lung, liver, kidney,
spleen, gonads) were examined by a pathologist. No signs of pathological changes were
observed in these organs in either of the treatment groups or in the control group when
examined two months after the procedure.
5.2 OPTIMIZING ADVEGF-C THERAPY FOR LYMPHEDEMA (II)
Adenoviral VEGF-C gene transfer was established as a promising lymphangiogenic agent in the
first study and was therefore selected for further evaluation and optimization of delivery in the
same pig model of lymphedema as used previously. AdVEGF-C gene transfer under the
capsule of the lymph node was compared to gene transfer into the fat surrounding the lymph
node in terms of efficacy and injurious effects. Control animals received injections of AdLacZ or
saline.
5.2.1 Lymphatic neovessel growth induced by intranodal and perinodal AdVEGF-C
Lymphatic vessel regrowth in the operated area two months after GT was evaluated from
lymphangiograms recorded with a new Innova 3100 IQ Excellence X-ray machine with superior
resolution than that used in the earlier experiment. Dense lymphatic networks and perfused
lymph nodes were observed in animals treated with AdVEGF-C regardless of whether GT was
55
performed intranodally or perinodally (Figure 14A and C). In control pigs, markedly fewer
functional vessels were observable, although some isolated lymphatic vessels also spanned the
damaged area in those animals (Figure 14B, D and E). Lymphangiograms taken from four
animals at six months after the procedure revealed similar differences in lymphatic vessel
growth between pigs receiving either intranodal AdVEGF-C or intranodal AdLacZ (Figure 14F
and G). However, no quantitative comparison could be made at the 6-month timepoint due to
the small number of animals.
Figure 14. Lymphangiogenic effects of intranodally and perinodally administered AdVEGF-C versus
control groups as illustrated by lymphangiograms taken two (A-E) and six (F-G) months after
operation and GT. Perfused lymph nodes (asterisk) can be seen in the AdVEGF-C-treated animals.
Scale bars = 10 mm.
Lymphatic vessels connected to and bypassing the lymph node were counted from the twomonth-timepoint lymphangiograms. The total number of lymphatic vessels and the number of
lymph node-connected vessels were higher in the intranodal and perinodal AdVEGF-C groups
than in their respective AdLacZ control groups and saline control group (Figure 15). Some
lymphatic vessels bypassing the node were observed in most animals but their numbers did not
differ significantly between the groups.
56
Figure 15. Quantification of lymphatic vessels from the lymphangiograms two months after the
procedure. *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = non-significant.
Lymphangiogenic effects of AdVEGF-C were further examined at the microscopic level from
VEGFR-3 immunostained sections of lymph nodes and adjacent tissues (Figure 16). Perinodal
AdVEGF-C gene transfer induced lymphangiogenesis most prominently in the pericapsular
area and in the connective tissue surrounding the lymph node, whereas intranodal AdVEGF-C
appeared to stimulate lymphatic capillary growth also inside the lymph node with capillary
clusters replacing small areas of normal follicular structure. VEGFR-3 positive lymphatic vessel
lumens in both AdVEGF-C groups were significantly larger than in their respective control
groups (intranodal VEGF-C vs. intranodal LacZ: p < 0.001; perinodal VEGF-C vs. perinodal
LacZ: p = 0.001).
Figure 16. Robust lymphangiogenic effect in the connective tissue surrounding the lymph node in
AdVEGF-C-treated
animals
as
visualized
Magnification: 100X, scale bars: 100 μm.
by
immunohistochemical
staining
for
VEGFR-3.
57
5.2.2 Effects of intranodal and perinodal AdVEGF-C on lymph node preservation and seroma
formation
Lymph nodes were excised and measured in three dimensions after euthanizing the animals
two months after the procedure. The lymph node sizes in the intranodal LacZ group (12 ± 6 cm3,
p = 0.009), the perinodal LacZ group (14 ± 7 cm3, p = 0.003), and the saline control group (12 ± 5
cm3, p < 0.001) were smaller than the nonoperated lymph nodes of the left limb (24 ± 8 cm 3).
Lymph nodes receiving either intranodal (30 ± 7 cm3) or perinodal AdVEGF-C (28 ± 12 cm3)
retained their original size and exhibited no statistical difference to the intact left side lymph
nodes.
Lymph node structure was examined from hematoxylin-eosin-stained sections and atrophy
was given a grade between 0 and 4, with 0 representing an intact lymph node structure and 4
representing extensive atrophy. The median values with interquartile ranges for different
groups were as follows: intranodal AdVEGF-C 1.0 (0.0 - 2.0), perinodal AdVEGF-C 1.0 (1.0 1.5), and NaCl control 3.0 (2.0 - 4.0). In general, local decreases in cellularity and minor fibrofatty formations were observed in AdVEGF-C-treated lymph nodes, whereas confluent areas of
fibro-fatty tissue were found in NaCl control nodes (Figure 17).
Seroma formation during follow-up was evaluated in a similar manner as in Study I. Despite
differences in volumes between study groups at certain time points, the overall seroma volumes
did not differ statistically between groups (p = 0.062).
Figure 17. Hematoxylin-eosin staining of the lymph nodes. Asterisks mark atrophied areas.
Magnification: 12.5X, scale bars: 100 μm.
5.2.3 Macrophage accumulation inside the lymph node following intranodal GT
HAM56-antibody was used to label macrophages inside the lymph nodes. Clusters of tingible
body macrophages were observed in the germinal centers of the intranodally injected lymph
nodes, whereas fewer macrophages were found lining the subcapsular and trabecular sinuses of
lymph nodes receiving perinodal GT or saline injections (Figure 18). There were 2.3-fold more
HAM56-positive cells present in the intranodal AdVEGF-C-treated lymph nodes than in the
perinodal AdVEGF-C-treated lymph nodes (p < 0.05), and 2.7-fold more in the lymph nodes
receiving intranodal AdLacZ than in the lymph nodes receiving perinodal AdLacZ (p < 0.01).
No treatment-related differences were observed in the numbers of B cells, T cells and dendritic
cells inside the lymph node.
58
Figure 18. Immunohistochemical staining for the macrophage marker HAM56 showing intense
accumulation of macrophages in the germinal centers of lymph nodes following intranodal
administration of AdVEGF-C. Arrowheads point out HAM56-positive cells. Magnifications: 12,5X for
large images, 200X for insets. Scale bars: 100 μm for bigger images, 10 μm for insets.
5.3 THERAPEUTIC ANGIOGENESIS VIA OVER-EXPRESSION OF HIFS (III)
The effects of transcription factors HIF-1α and HIF-2α on angiogenesis and muscle energy
metabolism were studied in normoxic and ischemic rabbit hindlimbs, as well as in normoxic
mouse myocardium. Gene transfer was performed by intramuscular injections. The effects of
adenoviral delivery of HIFs were evaluated 6 days after GT. AdVEGF-A served as a positive
control and AdLacZ as a negative control.
5.3.1 Angiogenic response following AdHIF-1α and AdHIF-2α GTs
Both AdHIF-1α and AdHIF-2α induced enlargement of blood capillaries in rabbit skeletal
muscles and mouse myocardium, as evidenced by the immunostainings for CD31 and Griffonia
simplicifolia lectin (Figure 19A-H). The increase in capillary size evoked by AdHIF-1α and
AdHIF-2α was approximately four-fold in normoxic rabbit hindlimbs and two-fold in normoxic
mouse myocardium as compared to AdLacZ. AdVEGF-A induced a much more robust
enlargement of the capillaries, which, on the downside, led to substantial tissue edema and
vascular leakage that were not associated with AdHIFs. Vascular leakage, as evaluated with the
modified Miles assay, was 35-fold (p = 0.006) greater in the AdVEGF-A transduced muscles
than in the intact control muscles.
Native Doppler and CPS imaging were used to quantify perfusion of the transduced
normoxic hindlimb muscles at rest (Figure 19I-P). Perfusion measurements revealed a 2- to 5fold increase in AdHIF-1α/-2α transduced muscles 6 days after GT compared to AdLacZ.
AdVEGF-A transduced muscles showed an 18- and 34-fold higher perfusion than controls, as
determined from CPS and Doppler images, respectively. In ischemic muscles, AdHIF-2α but
not AdHIF-1α, statistically significantly improved perfusion after physical activity from day 0
to day 6, as quantified from CPS images.
Digital subtraction angiography was used to assess collateral artery formation in ischemic
hindlimbs. AdHIF-1α and AdHIF-2α increased the number of opened collateral vessels as
compared to AdLacZ, albeit not statistically significantly. The mean diameters of collateral
vessels were similar between all groups.
.
59
Figure 19. Effects of AdHIFs and AdVEGF-A on normoxic rabbit skeletal muscles and normoxic
mouse myocardium. A-D, CD31 staining demonstrating notably enlarged capillaries in AdVEGF-A
transduced rabbit skeletal muscle and moderate enlargement of capillaries following AdHIF-1α and
AdHIF-2α GTs. E-H, Similar pattern of capillary enlargement evident in the lectin-stained mouse
myocardium. I-L, Native Doppler imaging showing blood flow in large vessels of the m.
semimembranosus. M-P, CPS imaging demonstrating muscle perfusion at the capillary level.
Asterisks in J and N point to increased tissue edema between m. gracilis and m. semimembranosus
after AdVEGF-A GT.
The effects of AdHIFs on cardiac function in mice were evaluated by ultrasound imaging.
AdHIF-2α maintained cardiac function at the basal level, as concluded from ejection fraction
percentages (Figure 20). A decrease in this parameter was observed after AdHIF-1α and
AdLacZ GT. In contrast, AdHIF-1α was associated with an increased thickness of the left
ventricle anterior wall at diastole.
60
Figure 20. Parameters depicting left ventricle function in mice before and 6 days after GT. EF% =
ejection fraction, LVAW;d = left ventricle anterior wall thickness. * = p < 0.05, *** = p < 0.001.
5.3.2 Effects of AdHIFs on muscle energy metabolism
In addition to the angiogenic response, the effects of HIFs on skeletal muscle metabolism were
also a major subject of interest. 31P-MRS was used to measure energy metabolism in the
ischemic hindlimbs of rabbits. Aerobic capacity, as depicted by the ratio PCr/(PCr+Pi) at the end
of the exercise period, declined in all the study groups (Figure 21). At d0, only intact muscles
were able to properly recover from exercise and restore PCr, while the PCr/(PCr + Pi) ratios in
the ischemic muscles remained at low and comparable levels during the course of recovery. Six
days after GT, AdHIF-1α transduced muscles replenished PCr storage markedly better than
AdLacZ transduced muscles. By d6, recovery of AdHIF-2α transduced muscles from exercise
had returned back to baseline but did not significantly differ from the recovery of AdLacZ
muscles.
Figure 21. PCr/(PCr+Pi) ratios representing aerobic capacity and recovery rate one day after the
ischemia procedure (d0) and six days after gene transfer (d6). AdHIF-1α improved recovery of
ischemic muscles from exercise 6 days after GT.
5.3.3 Upregulation of HIF target genes following AdHIF-1α and AdHIF-2α GTs
At protein level, VEGF-A was detectable in immunohistochemically stained sections of AdHIF1α and AdHIF-2α transduced muscles, whereas AdLacZ muscles expressed no VEGF-A.
Moreover, eNOS was markedly upregulated in AdHIF-2α transduced muscles and to a lesser
extent also in AdHIF-1α muscles. At the mRNA level in vitro, AdHIF-1α upregulated PDK1
more vigorously than AdHIF-2α. In contrast, VEGF-A mRNA expression in ECs was higher
after transduction with AdHIF-2α than with AdHIF-1.
61
6 Discussion
6.1 LYMPHANGIOGENIC GENE THERAPY FOR LYMPHEDEMA (I-II)
Secondary lymphedema remains a common clinical challenge after oncologic surgery and
radiation therapy in the industrialized countries. Breast cancer as well as melanoma of the
extremities are good examples of high prevalence tumors with a tendency to develop lymphatic
metastasis (Scoggings et al. 2005). Removal or irradiation of draining lymph nodes is a key
prognostic factor when treating these malignancies (Chang et al. 2013). Alas, such treatments
often lead to seconday lymphedema. A recent meta-analysis revealed that approximately one in
five women with breast cancer will develop lymphedema (DiSipio et al. 2013). Autologous
lymph node transfer, which is intended to rebuild the normal lymphatic vascular anatomy and
function after cancer treatment, represents as a promising novel approach to treat this condition
that has no curative treatment at present (Saaristo et al. 2012, Becker et al. 2006). A shortcoming
of this approach is a prerequisite that lymphatic anastomoses form spontaneously and
incorporate the transferred lymph node flap into the existing lymphatic vasculature at the
lymphedema-subjected area. Lymph nodes left without lymphatic vasculature are known to
regress, necessitating the formation of lymphatic connections between the transplant and the
recipient site for successful result of the treatment (Mebius et al. 1991).
In this thesis, a gene therapeutic approach was adopted to promote integration of the lymph
node graft and restore damaged lymphatic function. A novel large animal model of
lymphedema was developed to better mimick the features of the human lymphatic circulation,
the most important features being the similar hydrostatic conditions and lymphatic vessel
dimensions shared by humans and pigs. Previous studies of therapeutic lymphangiogenesis
have been mostly performed using rodent lymphedema models, where the regenerating
lymphatics only need to grow over an area measurable in millimeters to repair the damage in
lymphatic network (Tammela et al. 2007, Yoon et al. 2003, Szuba et al. 2002, Saaristo et al. 2002a,
Karkkainen et al. 2001). In the pig model used in this thesis, lymphatic neovessels need to span
a length of 5 centimeters if they are to form anastomoses with the distal and proximal ends of
the existing lymphatic vascular tree. Although some spontaneously growing lymphatic vessels
of this magnitude were detected in control animals, the stimulation of lymphatic growth with
adenoviral VEGF-C and VEGF-D proved to be effective in inducing robust growth of functional
collecting lymphatic vessels to the site of surgical damage. These lymphatic neovessels were
still observable after a two-month follow-up, even though the adenoviral over-expression of
delivered growth factors lasts for only up to two weeks. Hence, long-term growth factor
expression may not be required to achieve a maintained therapeutic effect. Importantly, the
newly formed lymphatic vessels were also found to improve lymphatic flow in the damaged
area.
It seems likely that due to the successful integration of the lymphatic neovessels into the preexisting lymphatic network and consequential improvement in lymphatic flow, the lymph node
size and histological architecture were best preserved in those animals transduced with
AdVEGF-C and almost as well in animals receiving AdVEGF-D. Animals receiving control gene
AdLacZ or saline displayed smaller lymph nodes than those in the therapeutic treatment
groups, with fibro-fatty formations replacing parts of the normal follicular structure. Due to
immune cell trafficking through the lymph node, the preservation of histological architecture is
crucial to its function. The use of Lipiodol as a radio-opaque contrast agent in this thesis may
have affected the size measurements and histology evaluations, as Lipiodol has been shown to
influence lymph node size and to be retained in lymph nodes for 12 months on average (McIvor
62
1980). This might explain why the AdVEGF-C-treated lymph nodes were slightly, albeit not
statistically significantly, larger than the intact left-side lymph nodes not perfused with
Lipiodol. However, all the operated right-side lymph nodes were imaged using Lipiodol in a
similar manner and treatment-related differences in size and histology were observed,
warranting the conclusion that growth factor treatment may enhance the prognosis of the
transferred lymph nodes.
Despite offering a platform to study therapeutic interventions for lymphedema with an
adequate scale, the pig model developed in this thesis possessed its own limitations. Unlike in
chronic lymphedema, this model is based on acute lymphatic damage in otherwise young and
healthy animals. In the clinical situation, lymphedema occurs mostly in older patients, where
the initial accumulation of protein-rich fluid in the subcutaneous tissue is often complicated by
fibrosis, increased lipid deposition, inflammation and attenuated immune responses (Rockson
2001). No such changes were observed in the present model, as seroma fluid naturally
dissipated within a few weeks after lymphatic excision even in the control groups. It would be
preferable to develop a model of chronic lymphedema when investigating the potential of
adenoviral growth factor therapy in severe states of the disease. However, autologous lymph
node transfer can easily be performed in combination with breast reconstruction surgery 1 - 2
years after oncological treatments of breast cancer without the need for a separate operation
(Viitanen et al. 2013). At this stage, the secondary changes of lymphedema have not occurred
yet and the patients are likely to benefit most from lymph node transfer. From this perspective,
the pig model of acute lymphatic damage investigated in this thesis serves its purpose as a
proof-of-concept model to test the efficacy of lymph node transfer in combination with
lymphangiogenic gene therapy before conducting clinical trials.
Intriguingly, despite possessing similar lymphangiogenic effects, AdVEGF-D appeared to
provoke more blood vascular effects than AdVEGF-C. Blood capillaries were more dilated, their
permeability increased, and seroma volumes were greater after application of AdVEGF-D in
comparison to AdVEGF-C and control. Proteolytically processed short forms of VEGF-C and
VEGF-D are known to bind with high affinity to VEGFR-2, and these forms can induce
angiogenesis and vessel hyperpermeability (Anisimov et al. 2009, McColl et al. 2007, Achen et
al. 1998, Joukov et al. 1997). The short isoform of VEGF-D, VEGF-DΔNΔC, has been linked to an
increase in pericardial effusion when injected into the heart at high doses, as well as in notable
vascular permeability and subsequent edema in transduced hindlimbs (Rutanen et al. 2004,
Rissanen et al. 2003b). Thus, hyperpermeability of angiogenic vessels induced by VEGF-DΔNΔC
could account for the observed increase in seroma fluid in the AdVEGF-D group, at least to
some extent. Western Blot analysis of seroma fluids provided evidence of more extensive
processing of the full-length VEGF-D protein into its short isoform in comparison to VEGF-C,
indicating that there were substantial amounts of VEGF-DΔNΔC present in the seroma fluid.
However, in the Western Blot analysis, human antibodies were used to detect VEGF-C and
VEGF-D proteins, and even though these had originated from human transgenes, they had been
processed into their short isoforms by endogenous porcine proteases. There is no certainty that
the recognizion of these processed isoforms by human antibodies was complete. Another
possible factor interfering with the accuracy of this assay could be cross-reactivity between
human-origin short isoforms and endogenous porcine short isoforms.
VEGF-C was selected for further study due to its slightly superior lymphangiogenic and
lesser angiogenic effects than VEGF-D. Using the same pig model of lymphedema, AdVEGF-C
was injected under the capsule of the lymph node similarly as in the first study, or into the fat
surrounding the node, with the objective to determine the efficacy and potential adverse effects
of both administration routes. Perinodally administered AdVEGF-C proved to be as effective in
inducing growth of new lymphatic vessels as intranodally injected AdVEGF-C. The architecture
as well as the size of lymph nodes were equally well preserved in both treatment groups.
However, gene transfer into the perinodal fat appeared to be gentler to the grafted lymph node.
Pigs receiving intranodal injections of AdVEGF-C displayed increased macrophage infiltration
63
inside the lymph node two months after gene transfer. Since a similar increase in the numbers
of macrophages was also observed in animals receiving intranodal AdLacZ, it would appear
that the macrophage accumulation was due to the adenoviral vector or mechanical damage
caused by the injections. However, this result should be viewed with caution since the HAM56
antibody used here to identify macrophages labels ECs as well, which may hamper the reliable
quantification of the pure macrophage population (Gown, Tsukada & Ross 1986). The
adenovirus vector per se has been associated with a prominent inflammatory reaction, but the
innate inflammatory response mediated by macrophages against the adenovirus would be
expected to have subsided two months after vector administration (Schnell et al. 2001, Zhang et
al. 2001, Worgall et al. 1997, Simon et al. 1993). One possible explanation for the increased
macrophage number at the site of vector distribution could be that the high local concentrations
of the adenoviral vector inside the lymph node had elicited cytotoxicity, leading to loss of
transduced cells, which would then be removed by macrophages. High doses of the adenoviral
vector are known to cause cytotoxicity of the target cells and chronic inflammation, at least in
neuronal tissue (Thomas et al. 2001). Moreover, macrophages have been shown to phagocytose
adenovirus vector-transduced cells in mouse brain (Zirger et al. 2012). Phagocytosis marks a
late stage in the process of transduced cell death. In general, local administrations of reasonable
doses (109 - 1011 vp) of the adenoviral vector have been claimed to be well tolerated (Harvey et
al. 2002). In the perinodally treated lymph nodes, sparse macrophages lined the subcapsular
and trabecular sinuses and no such clustering was observed as in the intranodally treated
lymph nodes. These findings favor the administration of AdVEGF-C into the perinodal fat of
the lymph node graft.
One important aspect when one considers future clinical trials is the ability of lymphatic
growth factors to promote lymphatic metastasis of tumor cells, even spontaneous tumor
formation (Karkkainen et al. 2009, Hirakawa et al. 2007, Ishii et al. 2004, Skobe et al. 2001). It is
possible that the viral construct or the protein product could diffuse from the injection site to
other organs and cause undesirable side effects. Although the safety samples collected from
internal organs in the first study of this thesis showed no signs of pathological changes, the
toxicity of AdVEGF-C will need to be more comprehensively evaluated in a separate study
prior to clinical testing. Patient safety is a key issue when identifying recipients for this
treatment in clinical trials. However, VEGF-C, which is constantly produced by normal human
lymph nodes in small amounts, was only transiently (~ 1 week) over-expressed in the treatment
strategy explored in this thesis. Nonetheless, the risks and benefits of combined lymph node
transfer and AdVEGF-C over-expression will need to be carefully weighed especially in patients
with a history of malignant disease. Lymph node transfer in itself may provide added benefit
for these patients in their battle against cancer recurrence, since grafted lymph nodes have been
shown to maintain the ability to trap tumor cells and mount a cytotoxic immune response
against cancer cells (Tammela et al. 2007, Rabson et al. 1982).
The ultimate selection of the growth factor to be taken into clinical testing requires a
thorough examination of the advantages and disadvantages of each candidate. VEGF-C was
proven to be the preferred growth factor over VEGF-D in this thesis. In addition to these two
contenders, an engineered variant of VEGF-C (VEGF-C156S) exists, which is a selective agonist
of VEGFR-3 and believed to induce only lymphangiogenesis without any angiogenic effects.
Nonetheless, this growth factor exhibited inferior lymphangiogenic potential compared to
VEGF-C in a recent study conducted in the same pig model of lymphedema as employed in this
thesis (Visuri et al., unpublished results). A similar observation was also made in a mouse
model of lymphedema and lymph node transfer (Tervala et al. 2015). Based on these findings
and the results obtained in this thesis, VEGF-C can be considered to represent the primary
candidate for lymphangiogenic gene therapy trials due to its superior lymphangiogenic efficacy
and negligible vascular effects as compared to VEGF-D. A summary of the effects of these
growth factors observed in this thesis is presented in figure 22.
64
Figure 22. A summary of the effects of VEGF-C and VEGF-D on blood and lymphatic vasculature
observed in this thesis.
6.2 PHYSIOLOGICAL ANGIOGENESIS INDUCED BY HIFS (III)
The aim of the third study of this thesis was to examine the activation of several factors
involved in angiogenesis via AdHIFs in order to achieve a more physiologigal angiogenic
response than could be achieved by over-expressing a single proangiogenic factor. VEGF-A, for
example, is known to induce robust angiogenesis, which is unfortunately accompanied by side
effects such as notable tissue edema and the formation of immature, leaky vascular structures
(Karvinen et al. 2011, Weis, Cheresh 2005). Hypoxia-inducible factors function as master
regulators of angiogenesis and vascular remodeling by coordinately regulating the expression
of several downstream target genes that play key roles in mediating vascular responses to
ischemia (Semenza 2014). Thus, it has been hypothesized that gene transfer of these
transcription factors may lead to more physiological angiogenesis by turning on several genes
simultaneously.
The effects of AdHIF-1α and AdHIF-2α gene transfers on angiogenesis were studied in
normoxic mouse myocardium as well as in normoxic and ischemic rabbit skeletal muscles. The
use of stabilized forms of HIF-1α and HIF-2α in viral constructs decreased their normal
proteolytic degradation under normoxic conditions. Indeed, both AdHIFs induced capillary
enlargement in all target tissues when they were assessed under the miscroscope; this effect was
moderate compared to the effect of AdVEGF-A. The capillaries appeared to have a uniform
shape after AdHIF-1α/-2α GTs in contrast to the disorganized and greatly enlarged capillaries
induced by AdVEGF-A. Tissue edema, which commonly accompanies severe capillary
65
dilatation, was markedly evident in AdVEGF-A transduced muscles, whereas no harmful
edema was observed after AdHIF-1α and AdHIF-2α gene transfers. Taken together, these
findings support the working hypothesis that HIFs lead to more physiological angiogenesis
than VEGF-A on its own. Additionally, AdHIFs appeared to enhance collateral vessel opening
in comparison to AdLacZ as determined by DSA in ischemic rabbit hindlimbs.
Perfusion measurements by ultrasound imaging techniques were performed with the
objective to answer the question of whether the angiogenic effect induced by HIFs would be
adequate to improve muscle circulation at rest and, furthermore, to improve perfusion recovery
after exercise. Perfusion at rest 6 days after GT was approximately 4-fold higher in the AdHIF
treatment groups than in controls, and even several-fold higher than that in the AdVEGF-A
transduced muscles, which is in accordance with the histological capillary measurements.
Contrast pulse imaging (CPS), which was used here to quantify perfusion, has been shown to be
more accurate at the capillary level than the traditional native Doppler technique, which
revealed no significant difference in perfusion between AdHIFs and negative control in this
study (Rissanen et al. 2008). Interestingly, AdHIF-2α but not AdHIF-1α resulted in enhanced
perfusion recovery from exercise in ischemic limbs from day 0 to day 6 after GT, as quantified
by CPS. This study was the first to report improved blood flow by HIF-2α in large animal
skeletal muscle.
AdHIF-2α also appeared to exert cardioprotective effects in the mouse myocardium. The
contractility of the left ventricle was maintained at the basal level 6 days after GT in AdHIF-2α
transduced mice hearts, whereas a decrease in the ejection fraction was observed in AdHIF-1α
and AdLacZ transduced hearts. The reduced cardiac function in these groups may have been
attributable to the gene transfer procedure itself possibly inflicting mechanical damage to the
tiny hearts of mice, as well as the inflammation and edema caused by the adenoviral vector
(Merentie et al., unpublished results). These interfering factors, tissue damage and
inflammation, may also hamper reliable quantification of cardiac function, which will warrant
further confirmation of the observation that AdHIF-2α could protect the myocardium. The
stable HIF expression achieved by genetic modification or siRNA transfection has been shown
previously to protect mouse myocardium from an ischemic insult (Holscher et al. 2011,
Hyvarinen et al. 2010, Natarajan et al. 2006). However, there are also reports indicating that
chronic activation of HIFs could result in heart failure that mimicks ischemic cardiomyopathy
(Holscher et al. 2012, Moslehi et al. 2010). Hence, the role of HIFs in cardioprotection versus
cardiac injury remains to be fully characterized.
An ideal gene therapy construct would not only increase vascularity and perfusion but also
improve energy metabolism of the target tissue as an indicator of the functionality of the
induced vessels. In this study, changes in muscle energy metabolism in the context of exercise
following AdHIF gene transfers were evaluated using 31P-MRS, which can be used to assess the
metabolic correlates of tissue perfusion in a noninvasive manner. AdHIF-1α, in particular,
improved the recovery of ischemic limbs from the energy loss induced by electrical stimulation
in comparison to AdLacZ. This is in line with previous findings from a study performed in a
transgenic mouse model that revealed an unchanged cellular energy state after ischemia as a
result of stable HIF-1 and HIF-2 expression (Hyvarinen et al. 2010). However, neither of the
AdHIFs improved exercise tolerance of the ischemic limbs in this study, which has been
previously shown for AdPlGF and AdVEGF-A by 31P-MRS using a similar rest-exerciserecovery protocol (Korpisalo et al. 2008b, Gowdak et al. 2000). It is possible that different
growth factors and stimulators of angiogenesis influence different phases in muscle energy
metabolism. Altogether, it was observed that the angiogenic effect induced by AdHIF-1α
resulted in metabolic improvements, indicative of normal function of the angiogenic vessels.
31P-MRS is a relevant method for obtaining objective information about the energy state and
function of muscles and its values have been shown to correlate with several clinical parameters
such as the ankle-brachial index in patients with PAD (Greiner et al. 2006). In future studies of
proangiogenic gene therapy, muscle metabolism could also be measured by 18FDG PET, which
66
utilizes radiolabeled glucose to reveal the metabolic status of tissue (Pappas, Olcott & Drace
2001).
In summary, the dilation of capillaries, increased muscle perfusion, and the elevated number
of opened collaterals observed after gene transfer with AdHIFs resulted in enhanced recovery
of ischemic muscles from exercise in the experimental ischemic animal model employed in this
study. It should be noted, however, that this study was performed in young, healthy animals,
and the ischemia was caused by ligating an artery causing an abrupt change in blood flow. The
pathophysiology involved in the gradual occlusion of arteries in chronic human atherosclerotic
disease differs considerably from the acute ligation of healthy arteries. Indeed, the therapeutic
potential of AdHIF-1α has been called into question by a recent clinical trial that showed no
benefit from AdHIF-1 GT in the treatment of intermittent claudication of PAD patients (Creager
et al. 2011). There has been a trend among angiogenic gene therapy studies that the promising
results obtained in experimental models cannot be replicated in clinical trials. Several reasons
have been postulated to account for this discrepancy, such as inadequate dosing, insufficient
duration of exposure to the angiogenic agent, compromised delivery and poor vector
transduction efficiency (Dragneva, Korpisalo & Yla-Herttuala 2013). The species-, age-, and
health-related differences between animal models and patients, as discussed above, may also
account for the failure in the translation of techniques optimized in animals to the human
clinical population. In future preclinical studies of proangiogenic gene therapy, it would be
advisable to favour large animals rather than rodents, and, instead of using young and healthy
animals, to employ disease models such as atherosclerotic or diabetic models that better mimick
ischemic disease in humans.
Taken together, the three studies included in this thesis revealed that adenoviral gene
transfer as a tool to induce neovessel growth, whether it be angiogenic or lymphangiogenic, is
sufficient to achieve effects that lead not only to histological changes in the target tissue, but
also achieve functional improvements. AdVEGF-C and Ad-VEGF-D improved lymphatic flow
through the induction of lymphangiogenesis, whereas angiogenesis triggered by AdHIF-1α in
ischemic muscles led to improved energy recovery after exercise. Importantly, these effects
were demonstrated using large mammals, which adds to their relevance with regard to their
potential clinical translation. Notable angiogenic and lymphangiogenic effects were observed
both when the expression of only a single growth factor, such as VEGF-C or VEGF-D in the first
two studies or VEGF-A in the third study, was altered, and when transcription factors HIF-1α/2α, which govern the expression of several growth factors, were administered. The concept of
employing a “master-switch” agent rather than a single growth factor to yield a more
physiological induction of neovessel growth is not a new concept in therapeutic angiogenesis.
Already in the year 2000, a consensus statement by a panel of researchers in this field proposed
that a combination of angiogenic cytokines or the use of transcription factors that upregulate
several genes may be optimal for clinically beneficial therapeutic angiogenesis (Simons et al.
2000). Experimental support for this strategy had been obtained previously from studies in
transgenic mice: the over-expression of VEGF-A was found to induce formation of
hyperpermeable blood vessels and subsequent edema in the skin of mice, whereas
simultaneous over-expression of VEGF-A and ANG1 produced vessels that were not leaky
(Thurston et al. 1999, Larcher et al. 1998). Although the direct over-expression of VEGF-C via
adenoviral vectors studied in this thesis was found to be effective without side-effects, the
modulation of VEGF-C activity as a way of stimulating therapeutic lymphangiogenesis only
emerged recently, when CCBE1 was shown to be a strong activator of VEGF-C. In one
experiment, mouse skeletal muscles displayed significantly enhanced lymphangiogenesis when
transduced with AAV vectors expressing both CCBE1 and VEGF-C, as compared to
transduction with AAV expressing VEGF-C alone. (Jeltsch et al. 2014.) Hence, the
implementation of several factors may also confer added benefit in the field of therapeutic
lymphangiogenesis.
67
7 Conclusions and future perspectives
The following conclusions can be drawn from the individual studies (I-III) of this thesis:
I
Short-term adenoviral over-expression of both VEGF-C and VEGF-D led to
pronounced lymphangiogenesis which was sufficient to improve lymphatic flow
and to retain the architecture of the grafted lymph node. AdVEGF-D increased
seroma formation as compared to AdVEGF-C and appeared not only to have
lymphatic effects but also blood vascular effects were noted.
II
AdVEGF-C gene transfer performed into the fat surrounding the lymph node graft
is the preferred method of administering lymphangiogenic gene therapy in
prospective clinical trials of lymphedema.
III
Transient adenoviral over-expression of HIFs resulted in the enlargement of
capillaries and an increase in muscle perfusion that was sufficient to improve the
metabolic recovery of rabbit ischemic skeletal muscle from exercise. Influencing
several angiogenic factors at a more upstream level by using HIFs leads to a more
physiological angiogenic response than the use of a single, strong angiogenic
growth factor such as VEGF-A.
In summary, adenovirus-mediated, transient over-expression of growth factors is sufficient to
induce lymphatic and blood vessel growth, which results in measurable functional effects, such
as improvements in lymphatic flow and enhanced energy metabolism in ischemic muscles.
Although the over-expression of a single growth factor may be sufficient to achieve these
effects, the upregulation of several growth factors rather than simply one factor might represent
an alternative therapeutic target since it may achieve a more physiological response and thus be
less likely to cause side-effects.
Specifically, the adenoviral VEGF-C gene therapy studied in this thesis may provide a
treatment option for lymphedema patients when administered in conjunction with the lymph
node transfer procedure. AdVEGF-C could easily be injected into the edges of a lymphatic
tissue flap harvested from the patient’s abdominal wall and inserted to the recipient site in the
lymphedematous area. A phase I/II clinical trial to evaluate the safety and efficacy of adenoviral
VEGF-C in the treatment of secondary lymphedema is being designed and scheduled to begin
in the near future.
For the transcription factor HIF-1α/2α gene therapy, further pre-clinical studies are needed,
and the adequate dosing and duration of therapy, end-points, and potential patient populations
will have to be re-evaluated before drawing any final conclusions about the potential benefits of
this therapy in the treatment of cardiovascular diseases.
.
68
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Krista Honkonen
Adenoviral Gene Therapy
for the Stimulation of
Neovessel Growth
New Treatment Strategies for Lymphodema
and Peripheral Ischemia
In this thesis, gene therapeutic
approaches for the treatment
of lymphedema and peripheral
ischemia, which result from
insufficiency of lymphatic and blood
vessels, respectively, are described.
Adenoviral VEGF-C and VEGF-D
were shown to induce robust
lymphangiogenesis and, subsequently,
improve lymphatic flow. Furthermore,
the angiogenic response triggered
by adenovirally delivered hypoxiainducible factors was shown to
enhance the recovery of ischemic
muscles from energy depletion
induced by exercise.
Publications of the University of Eastern Finland
Dissertations in Health Sciences
isbn 978-952-61-1778-2