Mechanisms and functions of lymphangiogenesis in regulating the immune response and inflammation resolution 1

LYMPH NODE LYMPHATICS UNDERGO DIFFERENTIAL REMODELING DURING THE COURSE OF INFLAMMATION 53 3.1 Introduction………...……….………...…..53 3.2 Results……….…………...…55 3.2.1 Immunization induces pr

MECHANISMS AND FUNCTIONS OF LYMPHANGIOGENESIS IN REGULATING THE IMMUNE RESPONSE AND INFLAMMATION RESOLUTION

TAN KAR WAI (B.Sc (Pharm), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE

NATIONAL UNIVERSITY OF SINGAPORE

Acknowledgements

I want to thank my supervisor, Dr Veronique Angeli for her guidance in the last 4 years You have been generous in support, immovable in faith and a beacon in stormy waters

We had our differences, I am very grateful that you were magnanimous enough to allow

me the freedom and space to pursue my research interests

I would also like to thank Dr Jean-Pierre Abastado for his invaluable advice and discussions on the projects A heartfelt shout of thanks also goes out to Dr Anne-Laure Puaux and Dr Jo Keeble for their help and suggestions at various points in the 4 years

I am thankful to the various friends who I have met in the last 4 years, without whom the journey would not have been filled with great memories Thank you Shuzhen, for the support and corridor memories Thank you Fiona, for being my long-suffering accomplice in crime and food Thank you Fei Chuin, for being putting up with my rantings And understanding Thank you Hazel, for being my first friend in the program Thank you Victoria, for showing me that courage comes in many forms Thank you Jocelyn, you are the stone that I missed and that turned out to be a diamond

I wish to thank my colleagues and friends in the VA lab, Angeline, Kim, Jun Xiang, Michael, Serena, Lawrence, Ivan and Sandra for the help and meaningful discussions in the 4 years Yes, thanks JX, SY and MT for the clowning and the insanity

I want to thank my friends outside of science for their tremendous support You may not understand what I am doing but you guys have not dismissed me as a geek

Lastly, I want to dedicate this journey and thesis to my family I want to thank my parents who have been loving, supportive and understanding in their usual understated and unwavering manner And to my siblings, thank you for being different and yet the same

Table of Contents

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

ABSTRACT ix

PUBLICATIONS xi

LIST OF TABLES xii

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xvi

CHAPTER 1 INTRODUCTION 1 1.1 The lymphatic vasculature and its functions……… … 1

1.2 Lymphatic vessels during development ……….4

1.2.1 Morphogenesis of lymphatics during development……… 4

1.3 Lymphatic vessels during inflammation……… …… 4

1.3.1 Molecular control of inflammation-associated lymphangiogenesis……… 7

1.3.2 Cellular mediators of inflammatory lymphangiogenesis……….10

1.3.3 Biological roles of lymphatics during inflammation………11

1.3.3.1 Lymphatics and immune cells trafficking……… 11

1.3.3.2 Lymphatics and inflammation resolution……… 13

1.3.3.3 Lymphatics and immune tolerance………15

1.4 The biology of VEGFs and their receptors……… 16

1.4.1 Introduction……….16

1.4.2 Molecular and functional diversity of VEGFs……… 17

1.4.2.1 VEGF-A………17

1.4.2.2 VEGF-C……….21

1.5 The microanatomy of the lymph node……… 22

1.5.1 The building blocks of the lymph node……….22

1.5.2 Blood endothelial cells………24

1.5.3 Fibroblastic reticular cells……… 24

1.6 Lymph node remodeling during inflammation……… 26

1.7 Neutrophils and immunity ……… 27

1.7.1 Regulation of neutrophil homeostasis during basal conditions ………27

1.7.2 Mobilization of neutrophils from BM during stress………28

1.7.3 Activation of neutrophils………30

1.7.4 Neutrophils and their protein cargoes ……….………30

1.7.4.1 Granules and granule proteins……… 17

1.7.4.2 Cytokines, chemokines and angiogeneic factors……… 31

1.8 Aims and rationale ……… 32

CHAPTER 2 MATERIALS AND METHODS 33 2.1 Mice……… ……… 33

2.2 Immunization of mice with complete Freund’s adjuvant/ keyhole limpet hemocyanin……… ………33

2.3 Elicitation of chronic cutaneous hypersensitivity in mice……… … 33

2.4 Cells isolation……… … 34

2.4.1 Isolation of stromal cells from lymph nodes……….34

2.4.2 Isolation of dendritic cells from lymph nodes……….…….35

2.4.3 Isolation of cells from spleen and lymph nodes………….……… 35

2.4.4 Isolation of neutrophils from bone marrow……… … 35

2.4.5 Isolation of peritoneal macrophages……… … 36

2.5 Neutrophil and macrophage cultures and stimulation ……… 37

2.6 Labelling of cells with carboxyfluorescein diacetate succinimidyl

ester (CFSE)……… 38

2.7 Adoptive cell transfers ……… 39

2.8 Culture of hybridoma cells and purification of antibodies… 39

2.9 Treatment of mice with antibodies and FTY720… 40

2.10 Dendritic cell migration assay ……… 41

2.11 Disruption of lymphatic flow through the LN ……… 41

2.12 Flow cytometry ……… 42

2.13 Immunofluorescence analysis………… 44

2.14 Hematoxylin and Eosin (H &E) staining………… …… 47

2.15 Enzyme-Linked Immunoabsorbent Assay (ELISA) … … 47

2.16 Real time-PCR……… … ……… 48

2.17 Western Blot ……… ……… …… …… 50

2.17 Statistical analysis ……… ……… … … 52

CHAPTER 3 LYMPH NODE LYMPHATICS UNDERGO DIFFERENTIAL REMODELING DURING THE COURSE OF INFLAMMATION 53 3.1 Introduction……… ……….……… … 53

3.2 Results……….………… …55

3.2.1 Immunization induces prolonged inflammation and lymphatic vessel expansion in draining lymph nodes……… 55

3.2.2 Lymph node lymphangiogenesis is initiated and sustained by lymphangiogenic factors derived from the inflamed footpad and lymph node ………… 60

3.2.3 Lymph node lymphatics undergo differential remodeling during the course of inflammation………69

3.3 Summary……….……… ……76

CHAPTER 4 DIFFERENTIAL LYMPHATICS

4.1 Introduction……… ……… … 78 4.2 Results……….…79 4.2.1 Expansion of the subcapsular sinuses augmented dendritic cell migration into

draining lymph nodes during early inflammation……… 79

4.2.2 Expansion of cortical and medullary sinuses during prolonged inflammation supported nạve lymphocyte egress out of draining lymph node…… …… 81 4.2.3 Expansion of cortical and medullary sinuses supports egress of antigen-

activated and nạve T cells from stimulated lymph nodes……….…96 4.3 Summary ………101

MEDULLARY SINUSES DURING LATE INFLAMMATION

IS DRIVEN BY A DISTINCTIVE SPATIAL-TEMPORAL DISTRIBUTION OF VEGF-A WITHING ACTIVATED

5.1 Introduction……… ……… 103 5.2 Results……… …104 5.2.1 Spatial differences in VEGF-A distribution accompany the remodeling of

cortical and medullary sinuses during prolonged inflammation …… … 104 5.2.2 Interstitial flow is required for the differential distribution of VEGF-A in

lymph node during inflammation……….… 111 5.3 Summary ……….116

CHAPTER 6 NEUTROPHILS CAN DRIVE LYMPH NODE

6.1 Introduction……… ……… 118 6.2 Results……… …119 6.2.1 Lymph node lymphangiogenesis in µMT mice is greater than WT mice during later phases of inflammation……… …… ….119 6.2.2 Lymph node lymphangiogenesis in µMT mice is accompanied by accumulation

of neutrophils and monocytes… ……….… 122 6.2.3 Neutrophils are critical for lymph node lymphangiogenesis in the absence of B cells … ……… ….… 127 6.2.4 Increased accumulation of neutrophils within µMT lymph nodes is mediated

by increased expression of neutrophil chemoattractants… ……… ….… 131 6.2.5 Neutrophils and non-granulocytic myeloid cells cooperate to drive

lymphangiogenesis in the absence of B cells… ……… ….… 134

6.3 Summary ……….137

LYMPHANGIOGENESIS IN THE INFLAMED PERIPHERY

7.1 Introduction……… ……… 139 7.2 Results……… …140 7.2.1 Neutrophils are required for lymphangiogenesis in the inflamed periphery of

WT mice……….……… …… ….140 7.2.2 Neutrophils are required for lymphangiogenesis and inflammation resolution

in chronic skin inflammation……… ……… …… ….153 7.3 Summary ……….157

CHAPTER 8 NEUTROPHILS MEDIATE LYMPHANGIOGENESIS BY SECRETION OF VEGF-C

8.1 Introduction……… ……… 158 8.2 Results……… …160 8.2.1 VEGF-A and VEGF-C are present in similar levels in immunized footpads of NIMP-R14 and control rat IgG-treated mice ……… …… ….140 8.2.2 Neutrophils are the main source of matrix metalloproteinase-9 (MMP-9) within inflamed footpads and MMP-9 expression is decreased in the absence of neutrophils ……… ……… …… ….163 8.2.3 Neutrophils secrete TIMP-1 free MMP-9 and VEGF-C in vitro following stimulation…… ……… ………….… ….168 8.3 Summary ……….174

9.1 Differential lymphatics remodeling regulates immune cell trafficking through the inflamed lymph node ……… 176 9.1.1 Counter-regulating lymph node expansion during inflammation.…… ….177 9.1.2 Mechanisms driving differential lymphatics remodeling in the lymph node during inflammation.…… ….179 9.1.3 Cross-talk between lymph node stromal cells and immune cells during inflammation may modulate lymph node remodeling.……….….181 9.1.4 Why should we understand lymph node lymphangiogenesis? …… ….….184 9.1.5 Future Work.…… ….185

lymphangiogenesis………… ……… 186

9.2.1 Neutrophils regulate lymphangiogenesis in inflamed sites by modulating

bioavailability of VEGF-A and producing VEGF-C.……….….187

9.2.2 Neutrophils act as a regulator to balance angiogenesis and lymphangiogenesis.……… ….191

9.2.3 Why should we understand the role that neutrophils play in driving lymphangiogenesis.……… ….193

9.2.4 Future Work ……… ……… 196

REFERENCES 198 APPENDICES 231 Appendix 1 Buffers and media 231

Appendix 2 List of antibodies used for flow cytometry 233

Appendix 3 List of antibodies used for immunofluorescence 234

Appendix 4 List of antibodies used for western blots 234

Appendix 5 List of primers used for RT-PCR 235

In an earlier study, we showed that B cells were critical for initiating lymphangiogenesis within the draining LN during early inflammation induced by footpad immunization

Here we discovered that during later phases of inflammation, neutrophils accumulate in inflamed B cell deficient (µMT) LNs and can substitute for B cells in driving lymphangiogenesis Neutrophils do not migrate to wild type LNs during inflammation and hence did not play a role in mediating LN lymphangiogenesis in these mice However neutrophils accumulate in immunized WT footpads and in sensitized skin and were found

in these primary sites of inflammation, to be critical for organizing lymphangiogenesis and consequently, inflammation resolution We noted that the absence of neutrophils did not seem to alter the amount of VEGF-A and VEGF-C in the inflamed footpad Instead, the absence of neutrophils resulted in an obvious reduction in MMP-9, a proteolytic enzyme involved in extracellular matrix remodeling Subsequent experiments revealed that neutrophils were the principal source of MMP-9 in the inflamed footpads Furthermore, such neutrophil-derived MMP-9 was likely to be constitutively active as it

was not associated with TIMP-1, an inhibitor of MMP activity In vitro experiments

further revealed the unexpected finding that neutrophils are able to produce, in addition to MMP-9, VEGF-C upon activation

Jean-2) Neutrophils are novel players in driving lymphangiogenesis and inflammation

resolution (manuscript in preparation)

Kar Wai Tan, Shu Zhen Chong, Sandra Tan, Jo Keeble, Jean-Pierre Abastado, Véronique Angeli

List of tables

Table 1.1 Knockout or mutant mouse models and their phenotypes according to stages in lymphatic vessel morphogenesis 5, 6

List of figures

Figure 1.1 Schematic overview of the structure and function of the lymphatic

vasculature 3

Figure 1.2 VEGF receptor-binding properties and signalling complexes 19

Figure 1.3 DNA, RNA and protein products of human VEGF-A families 20

Figure 1.4 Organization of the lymph nodes 23

Figure 1.5 Regulation of neutrophil homeostasis 29

Figure 3.1 LN expansion following CFA/KLH immunization 56

Figure 3.2 Immunization induced prolonged lymphangiogenesis in the draining LNs 58

Figure 3.3 Immunization induced expansion of the BEC and FRC populations in DLNs 59

Figure 3.4 Lymphangiogenesis in inflamed LNs is initiated and maintained by continued proliferation of lymphatics 61

Figure 3.5 Angiogenesis in inflamed LNs is most active in the first 30 days after immunization 62

Figure 3.6 Elevated whole LN VEGF-A and VEGF-C protein levels contrasted with decreased whole LN mRNA expression of these factors 63

Figure 3.7 Elevated whole footpad VEGF-A and VEGF-C protein levels is accompanied by increased whole footpad mRNA expression of these factors 64

Figure 3.8 mRNA expression of the various VEGF-A isoforms in whole LN and footpad 65

Figure 3.9 FRCs are closely associated with lymphatics and can produce VEGF-A during inflammation to drive lymph node lymphangiogenesis 68 Figure 3.10 Strategy to distinguish subcapsular sinuses from cortical and medullary sinuses 71, 72 Figure 3.11 Differential expansion of subcapsular, cortical and medullary sinuses in the lymph node during different phases of inflammation 73, 74 Figure 3.12 Quantification of total, subcapsular and cortical-medullary sinuses

density in lymph node sections 75 Figure 4.1 Subcapsular sinuses remodeling during early inflammation promotes dendritic cells migration into inflamed LNs 80 Figure 4.2 Short term lymphocyte homing studies reveal that lymphocyte entry into LNs at day 14 post-immunization is greater than day 4 83 Fig 4.3 Lymphocyte egress from stimulated lymph node is increased during

prolonged inflammation 85, 86 Figure 4.4 Treatment of mice with FTY720 attenuates lymphocyte egress from DLNs

at day 14 after immunization 88, 89 Figure 4.5 T cell egress during later phases of inflammation occurs through cortical and medullary sinuses in the DLNs 92 Figure 4.6 Blocking VEGFR2 and VEGFR3 signaling abrogates

lymphangiogenesis 93 Figure 4.7 Blocking lymphangiogenesis abrogates restoration of T cell egress to

steady state levels 95 Figure 4.8 Approach to investigate egress of antigen-specific T cells compared to their nạve counterparts 98 Figure 4.9 Expansion of cortical and medullary sinuses in LNs during inflammation support similar egress of antigen-activated and nạve T cell 99, 100

Figure 5.1 Spatial differences in VEGF-A distribution accompany the

differential remodeling of cortical and medullary sinuses during prolonged

inflammation 105, 106

Figure 5.3 LV surgery to disrupt lymph flow to the popliteal lymph node 112 Figure 5.4 Disrupting interstitial flow through draining LNs affected VEGF-A

localization 114, 115 Figure 6.1 LN remodeling in WT and µMT mice following CFA/ KLH footpad

immunization 120, 121 Figure 6.2 Myeloid cells and neutrophil populations in WT and µMT LNs at

Figure 6.3 Myeloid cells and neutrophil populations in WT and µMT LNs after immunization 124 Figure 6.4 Myeloid cells and neutrophil populations in WT and µMT LNs at day 4 and 14 after immunization 126 Figure 6.5 Neutrophils are critical for LN lymphangiogenesis in the absence of

B cells 129, 130 Figure 6.6 mRNA expression of various neutrophil chemoattractants in WT and µMT LNs 132 Figure 6.7 mRNA expression of Th1 (T-bet), Th2 (Gata-3), Treg (FOXP3) and Th17 (RORγ) specific transcriptional factors in WT and µMT LNs 133 Figure 6.8 Non-granulocytic myeloid cells are critical for LN lymphangiogenesis in the absence of B cells 135, 136 Figure 7.1 Neutrophils are not critical for LN lymphangiogenesis in normal

inflammatory conditions 141, 142 Figure 7.2 Neutrophils accumulate in the inflamed footpads following CFA/ KLH immunization 143

Figure 7.3 Neutrophils are critical for lymphangiogenesis in the inflamed

footpads 145, 146 Figure 7.4 Area density covered by blood vessels 148 Figure 7.5 mRNA expression of various neutrophil chemoattractants in immunized

WT footpads 149 Figure 7.6 Inhibition of lymphangiogenesis and delayed inflammation resolution despite restoration of circulating neutrophils to basal levels 151, 152 Figure 7.7 Neutrophils are critical for lymphangiogenesis in chronic skin

inflammation 155, 156 Figure 8.1 VEGF-A is present in similar amounts in immunized footpads of mice treated with NIMP-R14 MAb or control rat IgG 161 Figure 8.2 VEGF-C is present in similar amounts in immunized footpads of mice treated with NIMP-R14 MAb or control rat IgG 162 Figure 8.3 MMP-9 is present in higher levels in immunized footpads of mice treated with control rat IgG compared to NIMP-R14 MAb 165

Figure 8.6 Stimulation of neutrophils results in release of TIMP-1 free proMMP-9 and MMP-9 171

Figure 9.1 A proposed model for the dynamic remodeling of LN lymphatic vessels during inflammation 183 Figure 9.2 A proposed model for how neutrophils may work to drive

lymphangiogenesis 190

CFA complete Freund’s adjuvant

CCL Chemokine (C-C motif) ligand

CFSE Carboxyfluorescein diacetate succinimidyl ester CXCL Chemokine (C-X-C motif) ligand

DC dendritic cell

DLNs draining LNs

DNCB 1,2 chloro-dinitrobenzene

DPTH dibutyl phthalate

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

ELISA Enzyme Linked Immunosorbent assay

FACS fluorescence activated cell sorting

FCS fetal calf serum

Fig Figure

FITC fluorescein-5-isothiocyanate

fMLP N-formyl-methionine- leucine-phenylalanine FRCs fibroblastic reticular cells

G-CSF granulocyte colony-stimulating factor

G-CSFR granulocyte colony-stimulating factor receptor

H&E Hematoxylin and Eosin

HBSS Hanks Buffered Saline Solution

HSPGs heparan sulphate proteoglycans

HEVs high endothelial venules

ICAM intercellular adhesion molecule

IFA incomplete Freund’s adjuvant

mAb monoclonal antibody

MCSF macrophage-colony stimulating factor

MCSF-R macrophage colony stimulating factor receptor

MACS magnetic activated cell sorting

mRNA messenger ribonucleic acid

NRPs neuropilins

OVA ovalbumin

PBS phosphate buffered saline

PE phycoerythrin

PMA phorbol 12-myristate-13-acetate

PRRs pattern recognition receptors

RA rheumatoid arthritis

RPMI Roswell Park Memorial Institute medium

RT-PCR real-time polymerase chain reaction

rpm round per minute

TNF-α tumor necrosis factor-α

Treg regulatory T cells

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor

VCAM vascular adhesion molecule

VHD VEGF homology domain

WT wild type

µl microlitre

µMT transgenic mice that are unable to express the µ heavy chain hence lacking B cells

Chapter 1 Introduction

1.1 The lymphatic vasculature and its functions

Lymphatic vessels were discovered by Gaspar Asellius in 1627 at virtually the same time William Harvey described the blood circulation Yet the study of this ‘alternate’ circulatory system has been relatively neglected compared with the extensive study of the blood circulation

The lymphatic system is a characteristic feature of higher vertebrates, whose complex cardiovascular system and large body size require the presence of a secondary vascular system for the maintenance of fluid balance The adult lymphatic system is composed of peripheral capillaries, collecting vessels, lymph nodes, larger trunks and the thoracic duct Lymphatic vessels are present in the skin and in most internal organs except the central nervous system, bone marrow, retina and avascular tissues such as cartilage, hair, nails, cornea and epidermis (Tammela and Alitalo, 2010) Initial lymphatics, the absorptive part

of the lymphatic vasculature, are blind-ended vessels formed by a single layer of lymphatic endothelial cells (LECs) devoid of pericyte coverage and possessing a discontinuous basement membrane (Pflicke and Sixt, 2009) The initial lymphatics connect with collecting lymphatic vessels, which are coated by perivascular smooth muscle cells (SMC) to allow lymph propulsion and contain valves to prevent backflow (Alitalo et al., 2005; Karpanen and Alitalo, 2008; Schulte-Merker et al., 2011; Tammela and Alitalo, 2010) Lymphatic capillaries lack mural cells and connect to the extracellular matrix (ECM) via anchoring filaments, which prevent the collapse of capillaries upon the increase of interstitial pressure In contrast to blood flow, which depends on the pumping action of the heart and skeletal muscle contraction (venous flow), cyclical compression and expansion of lymphatic vessels created by surrounding tissues and the spontaneous

phasic contraction of SMCs generate lymph propulsion (Zawieja, 2009) (Figure 1.1)

Oxygen, nutrients and hormones are delivered to tissues by blood vessels and blood capillaries are involved in the molecular exchange of these compounds with the surrounding tissues Blood pressure causes plasma to filtrate continuously from the capillaries into the interstitial space Approximately 90% of the extravasated fluid is reabsorbed at the venous side of the capillary bed, where the colloid osmotic pressure of the blood exceeds the blood pressure The main function of the lymphatic vasculature is

to return the remaining 10% of this fluid back to the circulating blood Fluid, macromolecules and cells, such as leukocytes and antigen-presenting cells (APCs), enter the blind-ended initial lymphatics and form the lymph From here, lymph is transported towards collecting lymphatic vessels and ends up in the thoracic duct, the lymphatic trunk that runs alongside the aorta and finally connects with the subclavian vein (Alitalo et al., 2005; Karpanen and Alitalo, 2008; Schulte-Merker et al., 2011; Tammela and Alitalo, 2010) On its way, lymph will filter through the lymph nodes (LNs) Recent studies indicate that lymphatic vessels may also play a crucial role in the pathogenesis of hypertension or obesity and undergo major alterations associated with circulatory factors

or fat metabolism (Harvey et al., 2005; Lim et al., 2009; Machnik et al., 2009)

Figure 1.1 Schematic overview of the structure and function of the lymphatic vasculature Fluid containing proteins, lipids and other

solutes leaks from blood vessels (BV), percolates through the interstitial tissue and returns to the circulation by the venous capillary bed and lymphatic vessels (LV) Endothelial cells of lymphatic capillaries are oak shaped with overlapping scalloped edges (flaps) These flaps are only sealed on the sides by discontinuous buttonlike junction allowing fluid entry through these flaps without disturbing cell–cell cohesion Lymph is subsequently transported to collecting lymphatics Immune cells (lymphocytes [L], dendritic cells [DC]) likely enter lymphatic capillaries through the intermingled flaps In contrast to BV, initial lymphatics are made up of a discontinuous thin basement membrane (BM) Anchoring filaments connect lymphatic capillaries to extracellular matrix and modulate vessel diameter by pulling adjacent endothelial cells apart Lymphatic endothelial cells (LEC), blood endothelial cells (BEC), fibronectin (FN), hyaluronan (HA) Figure adapted from (Paupert et al., 2011)

1.2 Lymphatic vessels during development

1.2.1 Morphogenesis of lymphatics during development

Experiments performed by Florence Sabin about a hundred years ago laid the cornerstones for the widely accepted model that the mammalian lymphatic vasculature had venous origins (Sabin, 1916) Modern day experiments performing lineage tracing by Srinivasan et al elegantly demonstrated that the mammalian lymphatic vasculature stem from preexisting blood vessels (Srinivasan et al., 2007) During development, lymphatic vascular genesis requires transdifferentiation of venous endothelial cells toward the lymphatic endothelial phenotype, separation of blood and lymphatic vasculature, sprouting of lymphatic vessels, and lymphatic vascular maturation Over twenty genes orchestrate this process in mice and these are summarized and detailed in Table 1 (Schulte-Merker et al., 2011)

1.3 Lymphatic vessels during inflammation

In adults, both blood and lymphatic endothelial cells are normally in a quiescent state, but possess the capability to respond to a variety of stimuli Lymphatic vessel growth also called lymphangiogenesis, appears to follow that of the blood vessels during tissue regeneration, wound healing, tumor growth and inflammation New lymphatic vessels are believed to grow primarily by sprouting from existing ones (Karpanen and Alitalo, 2008; Tammela and Alitalo, 2010) Although the existence of bone marrow–derived hematopoietic cells (He et al., 2004; Jiang et al., 2008; Lee et al., 2010; Religa et al., 2005; Salven et al., 2003) or circulating macrophages (Maruyama et al., 2005) capable of trans-differentiating into LECs has been suggested, the data has been conflicting and their exact contribution to inflammatory lymphangiogenesis remains controversial in the field

Table 1.1 Knockout or mutant mouse models and their phenotypes according to stages in lymphatic vessel morphogenesis Adapted from (Schulte-Merker et al., 2011)

Table 1.1 (continued) Knockout or mutant mouse models and their phenotypes according to stages in lymphatic vessel morphogenesis Adapted from (Schulte-Merker

et al., 2011)

1.3.1 Molecular control of inflammation-associated lymphangiogenesis

Although extensive evidence supports the link between inflammation and lymphangiogenesis, the molecular mechanisms underlying this association are largely unknown LECs are reported to express an array of toll like receptors (TLRs) including TLR1 to 6 and TLR9 It was revealed that LECs responded to most but not all ligands to TLRs 1 to 9 by increasing expression of inflammatory chemokines, cytokines, and adhesion molecules (Pegu et al., 2008) In addition, LECs at least in some tissues, have been shown to constitutively express nuclear factor kappa B (NF-κB) (Saban et al., 2004) Tumor necrosis factor -α (TNF-α) (Baluk et al., 2009) and lymphotoxin-α (LT-α) (Mounzer et al., 2010) have both been reported to drive remodeling and function of blood and lymphatic vessels in several models of infection and inflammation However, it is not clear whether these inflammatory signals act directly on lymphatic vessels to elicit lymphangiogenesis or recruit and stimulate immune and non-immune cells to induce lymphangiogenesis

Vascular endothelial growth factor A (VEGF-A) was originally described as a master

regulator of blood endothelial cell (BEC) growth in vitro and in vivo, transducing its

effect through vascular endothelial growth factor receptor 2 (VEGFR-2) Vascular endothelial growth factor (VEGF) C and D were identified as prototype lymphatic endothelial growth factors, acting via vascular endothelial growth factor receptor 3 (VEGFR-3) to transduce lymphangiogenesis With growing appreciation that the biology

of the VEGFR-2 and VEGFR-3 ligands overlap quite extensively, the clear distinction in roles between the VEGF ligands in regulating angiogenesis versus lymphangiogenesis has given way to a more complex picture Gene profiling studies performed on LECs

versus BECs showed differential expression of only a small percentage of all genes

investigated Indeed, it has been shown that like BECs, LECs also express VEGFR2, and VEGF-A potently promotes their survival in vitro (Hirakawa et al., 2003; Petrova et al., 2002; Podgrabinska et al., 2002)

Inflammatory signals have been described in an early study, to induce the up-regulation

of VEGF-C, suggesting a role for this growth factor in mediating inflammatory lymphangiogenesis (Ristimaki et al., 1998), akin to its established role in lymphatic vascular development during embryogenesis This up-regulation presumably occurs through NFκB signaling, which has a putative binding site in the VEGF-C promoter (Chilov et al., 1997) Interestingly, activation of the NF-κB pathway in LECs upregulates Prox1 and VEGFR-3, which renders the pre-existing lymphatic vessels more sensitive to VEGF-C and VEGF-D produced by leukocytes (Flister et al., 2010) Many studies have attributed lymphangiogenesis during inflammation to be principally driven by VEGF-C

or VEGF-D Blocking these factors with either anti-VEGFR-3 neutralizing antibody or the soluble VEGF-C ligand trap suppressed lymphangiogenesis and provided compelling evidence that inflammatory lymphangiogenesis is driven by VEGF-C and to some extent, VEGF-D (Baluk et al., 2005; Kim et al., 2009; Kubo et al., 2002; Watari et al., 2008)

The tenet that VEGF-A and VEGFR-2 signaling play a negligible role in lymphatic vessel remodeling during development and hence by induction during inflammation too, was first challenged in an earlier report (Nagy et al., 2002) In this study, the authors made the unexpected finding that, in addition to angiogenesis, VEGF-A over-expression also induced lymphangiogenesis, resulting in the formation of greatly enlarged and persistent lymphatics These findings raised the possibility that lymphangiogenesis may also be

expected in situations characterized by VEGF-A over-expression such as chronic inflammation and infection This concept has since been validated in various models of inflammation in which blocking VEGFR-2 signaling allowed efficient ablation of lymphangiogenesis (Halin et al., 2007; Hong et al., 2004; Kunstfeld et al., 2004; Wuest and Carr, 2010)

While lymphatic vascular commitment and remodeling during development is tightly regulated in a stepwise manner, inflammatory lymphangiogenesis may be a more

‘promiscuous’ process driven by the pleiotropy of growth factors, pro-inflammatory cytokines and chemokines released during inflammation These growth factors and inflammatory cytokines would not be present or at least not in sufficient quantities in basal conditions to drive lymphangiogenesis It is likely that both VEGF-A and VEGF-C are upregulated and act in a synergistic manner to drive lymphangiogenesis during inflammation Indeed, both VEGF-A and VEGF-C expression have been reported to be simultaneously increased during inflammation and blockade of signaling using a combination of anti-VEGFR-2 and anti-VEGFR-3 signaling antibodies have proven to be more efficacious in suppressing lymphangiogenesis (Angeli et al., 2006; Huggenberger et al., 2010; Kataru et al., 2009) An alternative explanation is that some of the effects of VEGF-A on lymphangiogenesis may be secondary to its chemotactic recruitment of the inflammatory cells that produce VEGF-C and VEGF-D (Baluk et al., 2005; Cursiefen et al., 2004b) In the latter scenario, blockade of both VEGFR-2 and VEGFR-3 signaling to inhibit lymphangiogenesis would continue to be an attractive approach

1.3.2 Cellular mediators of inflammatory lymphangiogenesis

Both non-immune and immune cells have been described to orchestrate lymphangiogenesis in inflamed peripheral sites and the draining LNs (DLNs) Epithelial cells have long been implicated, though not directly proven to be involved in driving lymphangiogenesis during inflammation (Baluk et al., 2005; Halin et al., 2007; Yao et al., 2010) and infection (Baluk et al., 2005; Yao et al., 2010) In an elegant study employing VEGF-A reporter transgenic mice, Wuest and colleagues identified herpes simplex virus-infected epithelial cells as the primary source of VEGF-A that induced corneal lymphangiogenesis (Wuest and Carr, 2010)

Amongst immune cells, the most rigorous evidence for a role in lymphangiogenesis probably exists for macrophages They have been demonstrated to be indispensable in driving lymphangiogenesis in the inflamed periphery (Baluk et al., 2005; Cursiefen et al., 2004b; Cursiefen et al., 2011; Kang et al., 2009; Kataru et al., 2009; Kim et al., 2009; Kubota et al., 2009; Maruyama et al., 2005; Yao et al., 2010) and LNs draining the inflamed sites (Kataru et al., 2009) These seminal studies employing various systems to deplete macrophages such as liposomal clodronate, anti-macrophage colony stimulating factor receptor (MCSF-R) antibody, small molecule tyrosine kinase inhibitors specific for MCSF-R signaling (Ki20227) and osteopetrotic (op/op) mutant mice (mice that possess a mutant non-funtional M-CSF gene) have established that pathological lymphangiogenesis including that arising from inflammation, is efficiently ameliorated in the absence of macrophages Although dendritic cells (DCs) have been linked to the induction of lymphangiogenesis in various models of tissue inflammation (Baluk et al., 2005; Hamrah

et al., 2003; Kanao and Miyachi, 2006; Muniz et al., 2011), a direct causal role for DCs in

lymphangiogenesis has not been established T cells have also been reported to induce de novo lymphangiogenesis when recruited into murine thyroids over-expressing chemokine (C-C motif) ligand 21 (CCL21) (Furtado et al., 2007) Interestingly, T cells have recently been described to play a counter-regulatory role in inhibiting lymphangiogenesis during inflammation through a paracrine secretion of interferon-γ (Kataru et al., 2011) T cells are therefore proposed to maintain a homeostatic balance of LN lymphatic vessels during inflammation Different subsets of T cells may be recruited into secondary (Kataru et al., 2011) and tertiary (Furtado et al., 2007) lymphoid organs during inflammation and this may account for the differences in the two studies Our group (Angeli et al., 2006) and others (Liao and Ruddle, 2006; Shrestha et al., 2010) have shown that B cells are critical for LN lymphangiogenesis occurring early after immunization and sensitization Although neutrophils have been described to express VEGF-C in a murine model of chronic airway inflammation (Baluk et al., 2005), the observation was casual and a direct involvement in lymphangiogenesis has not been carefully examined

1.3.3 Biological roles of lymphatics during inflammation

1.3.3.1 Lymphatics and immune cells trafficking

During inflammation, lymphatic vessels provide routes for lymph containing activated antigen presenting cells (APCs), inflammatory cytokines or antigens to drain from the periphery into LNs where they prime the LNs for an impending immunological response

Expansion of lymphatic vessels within the immunized LNs and peripheral tissues have

been shown to support a more robust migration of DCs from the periphery into the draining LNs Interestingly, such increased migration of DCs into the draining LNs includes an influx from both inflamed and distal uninvolved sites (Angeli et al., 2006) Afferent lymphatic vessels provide routes for nạve and memory T lymphocytes to migrate from peripheral tissues into afferent lymph and DLNS CCL21 which is secreted

by lymphatic endothelium, is suggested to provide molecular cues to guide DCs and T lymphocytes expressing the chemokine receptor CCR7 to enter afferent lymphatic vessels (Bromley et al., 2005; Debes et al., 2005)

Under resting conditions, preformed portals in afferent lymphatic vessels allows DCs to intravasate lymphatic vessels without any requirement for cell-integrins or cell-matrix interaction (Pflicke and Sixt, 2009), although this situation is likely to be confined to non-inflamed lymphatic transmigration Inflammation induces lymphatic endothelium to up-regulate expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin (Johnson et al., 2006) and CCL21 (Johnson and Jackson, 2010) These molecules were shown to be important for facilitating the entry of DCs and lymphocytes into afferent lymphatic vessels during inflammation (Johnson et al., 2006; Johnson and Jackson, 2010) Interestingly, a recent study suggests expression of inflammatory chemokines in LECs in vivo may be modulated in a stimulus-dependent manner (Vigl et al., 2011), underpinning that LECs possess a complex control mechanism to coordinate immune cells entry during inflammation

Collectively, this points towards an active role for lymphatics in coordinating immune cells traffic into the specific LN as a means to enhance encounters between DCs and

antigen-specific lymphocytes

1.3.3.2 Lymphatics and inflammation resolution

Lymphangiogenesis has been observed to occur in chronic inflammatory diseases, such as psoriasis (Kajiya et al., 2006; Kunstfeld et al., 2004), inflammatory bowel disease (IBD) (Fogt et al., 2004; Geleff et al., 2003; Kaiserling et al., 2003; Pedica et al., 2008) or rheumatoid arthritis (RA) (Wilkinson and Edwards, 1991; Xu et al., 2003; Zhang et al., 2007) Studies of human kidney transplants show that transplant rejection is frequently associated with lymphangiogenesis (Kerjaschki et al., 2006; Kerjaschki et al., 2004) Thus, lymphangiogenesis has been proposed to support the establishment of an inflammatory loop by promoting immune cell trafficking to the DLNs Much as this argument makes intuitive sense, it has not been formally addressed in any controlled studies

Although selective blockade of VEGFR-3 signaling decreased rejection in murine corneal transplantation, this effect was attributed to impaired DC trafficking to draining lymph nodes rather than inhibition of lymphangiogenesis (Chen et al., 2004) Subsequent studies

in murine corneal transplantation utilizing various strategies to block both hemangiogenesis and lymphangiogenesis (Bachmann et al., 2008; Bachmann et al., 2009; Cursiefen et al., 2004a) or lymphangiogenesis selectively (Dietrich et al., 2010) improved graft survival and this was primarily attributed to the inhibition of lymphangiogenesis (Dietrich et al., 2010)

On the other hand, lymphangiogenesis might be beneficial for the resolution of chronic

inflammation since lymphatic vessels drain and remove accumulated fluid, immune cells and inflammatory cytokines from the sites of inflammation Indeed, inhibition of VEGFR-3 signaling and hence lymphangiogenesis, has been found to aggravate mucosal edema in a mouse model of chronic airway inflammation (Baluk et al., 2005) In recent years, there has also been growing recognition that targeted strategies to promote lymphangiogenesis and improve lymphatic drainage in skin inflammation (Huggenberger

et al., 2011; Huggenberger et al., 2010; Kajiya et al., 2009; Kataru et al., 2009) and rheumatoid arthritis (Guo et al., 2009; Polzer et al., 2008; Proulx et al., 2007) may represent more effective modes of therapy for these chronic inflammatory disorders than blocking lymphangiogenesis

While blockade of lymphangiogenesis in the corneal transplantation model is associated with favorable outcomes, results from other chronic inflammatory models seem to indicate the contrary Conflicting data from these studies may be more easily reconciled if

we were to relate them to the model of inflammation in which lymphangiogenesis occurs The cornea is considered an immune privileged site and one chief mechanism conferring this immunological privilege is that the normal cornea is devoid of blood and lymphatic vessels In normal circumstances, this prevents access of APCs to corneal antigens and the traffic of immune cells and inflammatory mediators to the regional LNs where an immune response may be primed (Patel and Dana, 2009) During inflammation, neovascularization involving both blood and lymphatic vessels disrupts the immune privileged barriers of the cornea, leading to undesirable immunological sequelae On the other hand, lymphatic drainage at inflamed sites acts as a regulatory mechanism to accelerate transport of fluid bearing inflammatory mediators and cells out of the ‘hot zone’, thereby limiting further tissue damage Clearly, the occurrence of

lymphangiogenesis may mediate differing biological effects with vastly varying outcomes depending on the system of inflammation Overall, these data highlight the need for more studies to elucidate and clarify the biological role of lymphangiogenesis in chronic inflammation

1.3.3.3 Lymphatics and immune tolerance

CD8 T cell peripheral tolerance has been shown to be induced by LN stromal cells that directly express otherwise tissue-restricted proteins (Gardner et al., 2008; Lee et al., 2007; Nichols et al., 2007) Of late, LN LECs were revealed to express multiple peripheral tissue antigens and presentation of one particular antigen led to deletion of antigen-specific CD8 T cells (Cohen et al., 2010) These results establish like other LN stromal

cells, LECs can be mediators of peripheral immune tolerance In vitro and in vivo studies

from another group demonstrated that contact of DCs with an inflamed lymphatic endothelium reduced expression of the costimulatory molecule CD86 by DCs and suppressed the ability of DCs to induce T cell proliferation These effects dependent on interactions between Mac-1 on DCs and ICAM-1 on LECs were observed only in the absence of pathogen-derived signals (Podgrabinska et al., 2009) Together, these data imply that LECs can play a direct role in the modulation and curtailing of immune responses to prevent undesirable immunological reactions

1.4 The biology of VEGFs and their receptors

1.4.1 Introduction

The VEGF family of growth factors includes five members in mammals: VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D VEGFs belong to the platelet-derived growth factor (PDGF)/VEGF superfamily of secreted dimeric glycoprotein growth factors that contain a cysteine knot motif, that is, the regularly spaced eight cysteine residues characteristic of each monomer (Holmes and Zachary, 2005; Yamazaki and Morita, 2006)

The VEGF ligands bind with differing specificities to 3 endothelial transmembrane tyrosine kinase receptors, VEGFR-1/fms-like tyrosine kinase 1, VEGFR-2/human kinase insert domain receptor (KDR)/mouse foetal liver kinase 1 and VEGFR-3/fms-like tyrosine kinase 4 All VEGFRs have a conserved intracellular tyrosine kinase domain and

a series of immunoglobulin-like domains in the extracellular part Neuropilins (NRPs), originally identified as semaphorin receptors important in axon guidance, function as co-receptors for specific VEGFs In addition, some forms of VEGFs bind to ECM and some integrins are also thought to form complexes with VEGFs and VEGFRs (Lohela et al., 2009; Olsson et al., 2006) We shall be focusing on 2 VEGF members, VEGF-A and VEGF-C as they have been established as the key players orchestrating lymphangiogenesis during inflammation

1.4.2 Molecular and functional diversity of VEGFs

1.4.2.1 VEGF-A

The first VEGF to be discovered, VEGF-A, is known to induce proliferation and

migration of endothelial cells, resulting in strong angiogenesis in vivo (Lohela et al.,

2009; Olsson et al., 2006) VEGF-A binds to both VEGFR-1 and VEGFR-2, though most

of the major biological activities of VEGF-A are mediated by VEGFR-2 (Fig 1.2) VEGFR1 is believed to function as a “decoy” receptor for secreted VEGF during embryogenesis by restricting its accessibility to VEGFR-2 on developing blood vessels (Fong et al., 1995; Fong et al., 1999) VEGFR-1 is also expressed in endothelial cells, monocytes/ macrophages and pericytes, and its tyrosine kinase activity is required for cell migration towards VEGF or PlGF (Barleon et al., 1996; Clauss et al., 1996) This is in contrast to the dispensable function of VEGFR-1 tyrosine kinase for vascular development (Hiratsuka et al., 1998; Hiratsuka et al., 2005)

The biology of the VEGF-A protein is complex Alternative splicing of the VEGF-A family members on chromosome 6 gives rise to at least 9 isoforms with differences in biological activities The human isoforms are denoted as VEGFA121, VEGFA145, VEGFA165, VEGFA189 and VEGFA206 and so forth The mouse isoforms are one amino acid residue shorter than the corresponding human isoform and they are denoted as VEGFA120 and so forth Human VEGF-A121, VEGFA165, VEGFA189 and their equivalents

in other species are the predominant isoforms in mammals (Ferrara et al., 2003; Woolard

et al., 2009) All transcripts contain exons 1-5 and 8, with diversity generated through the alternative splicing of exons 6 and 7 Exon 6 encodes a heparin-binding domain, while exons 7 and 8 encode a NRP/ heparin-binding domain VEGF-A121 lacks exons 6 and 7,

VEGF-A165 lacks exon 6 and VEGF-A189 contains both exons 6 and 7 (Fig 1.3) With the exception of VEGF-A121, all isoforms bind to heparan sulphate proteoglycans (HSPGs), polysaccharides abundantly found on endothelial cells and the extracellular matrix (Ferrara et al., 2003; Woolard et al., 2009) There is a wealth of evidence indicating that differences in the expression of the NRPs and heparin-binding domains give rise to the diverse biochemical and functional properties of the VEGF-A isoforms such as binding to ECM (Houck et al., 1992; Park et al., 1993), plasmin processing of matrix-bound VEGF-

A (Keyt et al., 1996; Roth et al., 2006), NRP1-enhanced affinity to VEGFR-2 binding (Gluzman-Poltorak et al., 2000; Soker et al., 1998) and vascular branch formation morphogenesis (Carmeliet et al., 1999; Ruhrberg et al., 2002; Stalmans et al., 2002)

Figure 1.2 VEGF receptor-binding properties and signalling complexes Mammalian vascular endothelial growth factors (VEGFs) bind

to the three VEGF receptor (VEGFR) tyrosine kinases, leading to the formation of VEGFR homodimers and heterodimers Proteolytic

processing of VEGFC and D allows for binding to VEGFR2 Adapted from (Olsson et al., 2006)