LYMPHANGIOGENIC SIGNALING IN THE EPICARDIUM
by
GANGA HASINI KARUNAMUNI
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisor: Dr Michiko Watanabe
Department of Anatomy
CASE WESTERN RESERVE UNIVERSITY
January 2011
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
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candidate for the ______degree *.
(signed)______(chair of the committee)
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*We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION
To Charles Palihawadena, my grandfather and role model, my guiding light, and the best
person I ever knew
To my parents and brother, who have always encouraged me and given me so much love
and support
1 TABLE OF CONTENTS
LIST OF TABLES…………………………………………………...………...... 5
LIST OF FIGURES………………………………………………. ...………...... 6
ACKNOWLEDGEMENTS…….……...………………………………………...... 9
LIST OF ABBREVIATIONS………………………………………………...... 11
ABSTRACT………………………………………………………………………...... 14
CHAPTER 1: BACKGROUND…………………………………………………...... 16
Historical Perspective on the lymphatics………………....…...……………... 16 The Function and Morphology of the lymphatics………...…..……………... 16 Lymphatic Markers…………………………………………………………… 17 Origins of the Lymphatic System…………..………………………………… 19 Formation of the Lymphatic System in Mammals…….……...…………….. 21 Lymphatic Gene Knockout Models…...………...…...…………….…...... 22 Lymphangiogenic Factors and Receptors…...………………………………. 25 Clinical Significance of the Lymphatics……….…………………….……...... 30 The Lymphatics of the Heart...…………………………………………...... 33 Statement of Purpose………………………………………………………...... 36
CHAPTER 2: Expression of lymphatic markers during avian and mouse cardiogenesis……………………………………………….……………………………52
Abstract…………………………………………….……………………...... 52 Introduction……………………………………….………………………...... 54 Materials and Methods………………………….………………………...... 57 Results…………………...………………………………………………...... 61 The formation of the Prox-1-positive epicardial lymphatic network in quail…...…………………………………………………………...... 61 Co-localization of Prox-1 with other lymphatic markers in
2 mouse...……………………………………………………………...... 63 Lymphatic markers in epicardial cells in vitro…….………………...... 65 Prox-1 Western Blots....……………………………………………...... 65 Discussion……………...………………………………………..………...... 67 The three sets of cardiac cells with lymphatic phenotype……………..... 67 The lymphangiogenic potential of the epicardium…...………………..... 71 Summary……………………………...... 72
CHAPTER 3: Activation of epicardial cells by VEGF-C signaling………...... 100
Abstract………………………………………………………………...... 100 Introduction…………………………………………………………...... 102 Materials and Methods……………………………………………...... 104 Results………………………………………………………………...... 108 Lymphatic progenitors are present in the epicardium…...... 108 VEGF-C induced Prox-1 nuclear accumulation ex vivo via the VEGFR-3 pathway…….…...... 109 VEGF-C specifically induced Prox-1 nuclear accumulation in the adult epicardium…...... 110 The MEK inhibitor UO126 blocked Prox-1 nuclear accumulation both in vitro and ex vivo...... 112 Discussion……………………………………………………………...... 113 Summary……………………………...... 115
CHAPTER 4: SUMMARY, SUPPLEMENTARY DATA AND FUTURE DIRECTIONS……………………………….…………………………………...... 139 Summary……………………………………………………………...... 139 The lymphatic network of the embryonic and adult heart………...... 139 The epicardium as a source of lymphatic cells………………….…...... 141 Supplementary Data…………………………………………………...... 143
3 Oxygen tension and the epicardial lymphatics…………………...... 143 HIF-1 and Prox-1…………………………………………..…...... 146 TCDD and its effect on Prox-1 protein levels………....…………...... 149 Genes regulated by Prox-1 in the epicardium…...…………………...... 152 Future Directions……………………………………………………...... 154 Final Words…………………………………………………………...... 156
REFERENCE LIST……………………………………………………………...... 170
4 LIST OF TABLES
Table 3-1. Prox-1 nuclear accumulation in the ARECs at different time points of
VEGF-C treatment...... 137
5 LIST OF FIGURES
Figure 1-1. Model for the differentiation of the mammalian lymphatic vasculature...... 38
Figure 1-2. Prox-1 expression in the mouse embryo.……………………………...... 40
Figure 1-3. The thoracic duct and the right lymphatic duct.…………..………...... 42
Figure 1-4. The VEGF Family.………………...... 44
Figure 1-5. Branching patterns of the lymphatic vasculature in the adult heart (as determined by dye injection studies)...... 46
Figure 1-6. The proepicardial organ (arrow) in an HH stage 17 chick embryo...... 48
Figure 1-7. Tracing the path of the proepicardial organ...... 50
Figure 2-1. Lymphatic markers in the early quail embryonic heart...... 74
Figure 2-2. Lymphatic markers in the septated quail embryonic heart...... 76
Figure 2-3. Diagram of the progressive development of the Prox-1-positive lymphatic network in the avian embryonic heart from HH Stage 24 to 38...... 78
Figure 2-4. Prox-1 immunofluorescence in quail heart sections...... 80
Figure 2-5. Lymphatic markers in the ED 9.5 mouse heart...... 82
Figure 2-6. Prox-1 and LYVE-1/CD44 were initially not co-localized in the mouse or chick embryo heart...... 84
Figure 2-7. Confocal microscopy of LYVE-1-positive cells (green) in the ED 13.5 mouse heart (441X magnification)…...... 86
Figure 2-8. Lymphatic markers in the ED 15 mouse heart...... 88
Figure 2-9. Lymphatic markers in the adult mouse heart...... 90
Figure 2-10. LYVE-1 and CD31/PECAM co-localization in adult mouse heart sections...... 92
6 Figure 2-11. Diagram of the three different types of cells/vessels with lymphatic phenotype in the mouse heart arranged according to location...... 94
Figure 2-12. Lymphatic markers in vitro...... 96
Figure 2-13. Prox-1 Western Blots...... 98
Figure 3-1. Prox-1 staining in ED 16.5 sections of WT1-cre mice…………………….117
Figure 3-2. LYVE-1 staining in ED 16.5 sections of WT1-cre mice…………………..119
Figure 3-3. VEGF-C treatment in explanted hearts for 30 minutes…………………….121
Figure 3-4. Adult rat epicardial cells (ARECs) with FGF, VEGF-C and VEGF-A treatment for 24 hours…………………………………………………………………..123
Figure 3-5. Adult rat epicardial cells treated with VEGF-C for 30 minutes……………125
Figure 3-6. Comparing Prox-1 protein levels via Western Blot………………………..127
Figure 3-7. Adult rat epicardial cells treated with nuclear export inhibitor leptomycin B for 2 hours……………………………………………………………….129
Figure 3-8. The MEK inhibitor UO126 induced a down-regulation of Prox-1 in the nucleus………………………………………………………………………………….131
Figure 3-9. Smooth muscle actin and phallodin staining in DMSO/UO126 and
VEGF-C-treated ARECs………………………………………………………………..133
Figure 3-10. The effects of inhibiting VEGFR-3 activation in quail hearts in vivo…....135
Figure 4-1. The lymphatics and coronary arteries in the HH stage 30 quail heart...... 158
Figure 4-2. Myocardial vessels with venous and lymphatic phenotype in the adult mouse heart……………………………………………………………...... 160
Figure 4-3. Comparison of great vessel lymphatics in normoxic and hyperoxic avian embryonic hearts...... 162
7 Figure 4-4. Comparison of ventricular lymphatics in normoxic and hyperoxic avian embryonic heart...... 164
Figure 4-5. Comparison of Prox-1 protein levels under hypoxic and hyperoxic conditions in the developing heart...... 166
Figure 4-6. Comparison of Prox-1 protein levels with TCDD exposure in the developing heart...... 168
8 ACKNOWLEDGEMENTS
I should first thank my friend and former co-worker Jamie Wikenheiser for
suggesting that I rotate through Dr. Michiko Watanabe’s lab. If not for him, I probably
would not have met Mich at all. As my PI, Mich has taught me so much over the years
that it can scarcely be put into words. I will always be grateful for her guidance and
infinite patience throughout my stay with her. She has been my mentor in the truest sense
of the word, both in research and in life, and I sincerely appreciate her special brand of
wisdom in all things scientific. What she has been to me truly transcends the title of
“Research Advisor”.
I would also like to thank all the wonderful people I have met in Mich’s lab. Our
lab manager Yong Qiu Doughman has always been extremely supportive and patient with me ever since I first showed up as an inexperienced rotating student. Jamie, as the senior graduate student, showed me all the ropes. Ke Yang, a postdoctoral associate who worked with us, taught me so many cell culture and molecular techniques, for which I will be forever thankful.
I am very grateful to all the other members of my thesis committee: Dr. Thomas
Hering, Dr. Patricia Parsons-Wingerter, Dr. Monica Montano, and Dr. Barbara Freeman.
They have been a constant source of encouragement during my graduate years, and I
strongly appreciate all their advice and feedback.
I have had a great experience with the Anatomy Department as well. Our
Administrator Christine Marshall continues to amaze me with her resourcefulness and
efficiency. In my humble opinion, the Anatomy professors, including Dr. Freeman, Dr.
Joe Miller, Dr. Charles Maier, Dr. Darin Croft, Dr. Scott Simpson, Dr. John Fredieu, Dr.
9 William Bligh-Glover, and Dr. Yohannes Haile-Selassie, are some of the best teachers I
have ever had the pleasure of encountering. My research rotations with Dr. Croft and Dr.
Nicole Ward were truly enlightening, and I credit them for starting me on the road to
becoming a research scientist myself. I especially want to thank D’Arbra Blankenshipp
for kindly taking me under her wing and training me in all the inner workings of a lab.
Finally, I want to acknowledge my wonderful family. None of this would have been possible had it not been for the unconditional love and unwavering support of my parents. I have always tried to be the best I can be, and a large part of who I am today is very much thanks to them. I strive to live my life with the same compassion and kindness that they have shown me. My brother has taught me so much, such as the importance of facing the world with good-natured humor. He is my rock, my confidant, and my friend.
And of course, to the Cleveland gang – Lindsy, Mike, Arvind, Wei, Sid, Big Zhao, Little
Zhao – I thank you for making my life that much brighter.
It’s been a great ride.
10 LIST OF ABBREVIATIONS
4’,6-Diamidino-2-Phenylindole DAPI
Adult Rat Epicardial Cell AREC
Angiopoietin-1 Ang-1
Angiopoietin-2 Ang-2
Aorta Ao
Atrioventricular Junction AVJ
Atrium At
Cluster of Differentiation 44 CD44
Dimethyl Sulfoxide DMSO
Dulbecco’s Modified Eagle’s Medium DMEM
Embryonic Day ED
Embryonic Mouse Epicardial Cell EMEC
Endocardium endo
Epicardial Mesothelium EM
Epicardium epi
Extracellular Signal-Regulated Kinase ERK
Fetal Bovine Serum FBS
Fibroblast Growth Factor-2 FGF-2
Forkhead box C2 FOXC2
Hamburger and Hamilton HH
Hypoxia Inducible Factor 1 HIF-1
Hypoxia Response Element HRE
11 Insulin-like Growth Factor-1 IGF-1
Insulin-like Growth Factor-2 IGF-2
Insulin-like Growth Factor-1 Receptor IGF-1R
Lymphatic Endothelial Cell LEC
Lymphatic Vascular Hylauronan Receptor 1 LYVE-1
Lymphocyte Cystosolic Protein 2 SLP-76
Myocardium myo
Neuropilin 2 Nrp2
Nuclear Factor of Activated T-cells cytoplasmic NFATc1
calcineurin-dependent 1
Outflow Tract OFT
Phosphate Buffered Saline PBS
Platelet Endothelial Cell Adhesion Molecule PECAM
Proepicardial Organ PEO
Prospero-related Homeobox 1 Prox-1
Pulmonary Trunk PT
Quail Epicardial Cell QEC
Smooth Muscle Actin SMA
Smooth Muscle Cell SMC
Spleen tyrosine kinase Syk
SRY-related HMG box 18 SOX18
Subepicardial Mesenchyme SM
Vascular Endothelial Growth Factor VEGF
12 Vascular Endothelial Growth Factor A VEGF-A
Vascular Endothelial Growth Factor C VEGF-C
Vascular Endothelial Growth Factor D VEGF-D
Vascular Endothelial Growth Factor Receptor 2 VEGFR-2
Vascular Endothelial Growth Factor Receptor 3 VEGFR-3
Ventricle Vent
13 Lymphangiogenic Signaling in the Epicardium
Abstract
by
GANGA HASINI KARUNAMUNI
The epicardial lymphatic system of the heart is responsible for eliminating excess fluid in
the interstitium. Its failure to function properly results in edema or tissue fibrosis. Despite
its physiological importance, the development of the cardiac lymphatics as well as the
cardiac lymphangiogenic process have not been thoroughly investigated. In this study, we
used the nuclear-localized transcription factor Prox-1 as a lymphatic marker, given that it
is frequently used to label adult lymphatic endothelial cells and can be used to induce
various cell types to transform into a more lymphatic phenotype. We tracked the
progressive formation of the cardiac lymphatic network in both avian and mouse models,
respectively from HH Stages 24-40 and from ED 9.5 to adult stages. Additional
lymphatic markers such as LYVE-1, VEGFR-3, and podoplanin were used to verify
lymphatic identity. We thus observed that there were three types of cells with lymphatic
phenotype in the mammalian heart: (1) incoming Prox-1-positive cells, (2) LYVE-1-
positive epicardial cells, and (3) LYVE-1-positive/VEGFR-3-positive myocardial
cells/vessels. Adult and embryonic epicardial cell lines and primary cultures of epicardial
cells also expressed Prox-1, LYVE-1, and VEGFR-3 in a subset of cells, but not in the subcellular localization seen for mature lymphatics. We explored the mechanisms that may push these pluripotent epicardial cells into the lymphatic phenotype. Adult epicardial
14 cells were treated with the lymphangiogenic growth factor VEGF-C, resulting in a
dramatic increase in Prox-1 and phosphorylated ERK expression in the nuclei of a subset
of cells. The nuclear accumulation of Prox-1 appeared to be factor specific. The ERK inhibitor UO126 induced a decrease in Prox-1 expression in the cell nuclei in both
untreated and VEGF-C treated cells, suggesting that phosphorylated ERK may regulate
Prox-1 localization and transcriptional function. Similar findings were observed with
explanted embryonic epicardial cells. Tissue oxygen levels, mediated by the hypoxia
inducible factor HIF-1, may also regulate lymphangiogenic signaling in the epicardium.
Targeting components of the VEGFR-3/ERK or HIF pathways could therefore be
instrumental in developing therapeutic strategies aimed at stimulating controlled
lymphangiogenesis in cases of cardiac edema, or even inhibiting the process during
tumor metastasis.
15 CHAPTER 1: BACKGROUND
Historical Perspective on the Lymphatics
The lymphatic system has been present in the literary record since the 5th century
BC, when Hippocrates referred to the lymph nodes in his work. Lymphatic vessels were first mentioned by the Greek anatomist Herophilus in the 3rd century BC during his observation of the intestines (Ambrose, 2006). His findings were later corroborated by
Galen’s dissection of the intestinal lymphatic vessels and mesenteric lymph nodes in apes and pigs in the 2nd century AD (Ambrose, 2006; Fanous et al, 2007). Investigation of the lymphatic system thereafter fell by the wayside until the 17th century when Aselli
rediscovered the lymphatics in the intestines and named them the lacteals (Flourens,
1859; Ambrose, 2006). In 1651, Jean Pecquet’s studies in the dog model allowed him to
trace the flow of fluid through the lacteals into the venous system via the thoracic duct
(Flourens, 1859; Ambrose, 2006). In 1652-1653, Olaus Rudbeck and Thomas Bartholin
separately published their findings on a comprehensive systemic lymphatic circulatory
network in the body (Flourens, 1859; Eriksson, 2004).
The Function and Morphology of the Lymphatics
The lymphatics are essential in regulating homeostasis in many species (Alitalo et al, 2005). Blood vessels deliver oxygen, nutrients, and hormones to the body while capillaries are involved in the molecular exchange of these compounds with the surrounding tissues. Blood pressure causes plasma to leak from the capillaries into the interstitial space. The lymphatic vasculature is then responsible for returning this protein-
16 rich fluid (lymph) back to the circulating blood. The lymph enters the lymphatic capillaries to be transported towards the collecting lymphatic vessels. On its way lymph is filtered through the lymph nodes, and is eventually returned to the blood circulation through lymphatico-venous junctions in the jugular area. Thus, the main functions of the lymphatics include the maintenance of the fluid balance of the internal environment, the absorption of fat from the small intestine, and immune surveillance. In addition, the lymphatics serve as one of the major routes by which tumor cells can metastasize to distant organs.
Lymphatic capillaries are blind-ended vessels with a single layer of non- fenestrated endothelial cells to facilitate the uptake of fluid, macromolecules, and cells. In contrast to blood capillaries, lymphatic capillaries have an incomplete basement membrane and are not invested by pericytes, so they are often partially or fully collapsed
(Pepper and Skobe, 2003). They also have poorly developed junctions with frequent large inter-endothelial gaps. An increase in interstitial fluid pressure causes these junctions to open to allow the passage of particles and fluid into the lymphatic capillaries. Lumen patency is maintained by anchoring filaments that attach the lymphatic endothelial cells to the extracellular matrix (Pepper, 2001). The lymph is then collected into the larger lymphatic vessels which may be surrounded by pericytes and have unidirectional, semi- lunar valves which prevent backflow. The flow of lymph is dependent on peristalsis, valvular operation, and the contraction of surrounding skeletal muscles or tissues.
Lymphatic Markers
17 Until recently, it has been difficult to distinguish between blood vessels and
lymphatic vessels because they share several markers such as the endothelial label
CD31/PECAM. However, with the discovery of relatively specific lymphatic endothelial
cell markers such as Prox-1, LYVE-1, podoplanin and VEGFR-3, it has been easier to
visualize the lymphatic system.
Prox-1 is a nuclear-localized transcription factor which is expressed in adult
lymphatic endothelium, and in non-endothelial cells of the lens, liver, kidney, pancreas, and nervous system (Wigle et al, 1999; Sosa-Pineda et al, 2000). It is thought to be involved in the budding and elongation of lymphatic sprouts during embryonic development (Wigle and Oliver, 1999; Wigle et al, 2002).
LYVE-1 is a membrane glycoprotein that is found in lymphatic endothelial cells,
as well as in hepatic sinusoidal endothelial cells (Mouta Carreira et al, 2001) and
placental synctiotrophoblasts (Sleeman et al, 2001). It is a receptor for the extracellular
glycosaminoglycan hyaluronan (Banerji et al, 1999; Prevo et al, 2001), which regulates
cell migration during embryonic morphogenesis and several adult processes such as
wound healing and tumor metastasis (Edward et al, 2005).
Podoplanin is a glomerular podocyte membrane mucoprotein that is required for
lymphatic development (Breiteneder-Geleff et al, 1999). It is usually expressed in small
lymphatic capillaries with a single layer of endothelial cells, but is also found in lung
alveolar type I epithelial cells (Rishi et al, 1995), choroid plexus cells, osteoblasts
(Wetterwald et al, 1996), and embryonic cardiac myocytes (Gittenberger-de Groot et al,
2007).
18 In adults, the cell surface receptor VEGFR-3 is expressed in lymphatic endothelial
cells (Kaipainen et al, 1995), as well as in hepatic and splenic sinusoids, pancreatic duct
epithelium, capillaries of kidney glomeruli and other endocrine glands, macrophages,
monocytes, and certain dendritic cells (Partanen et al, 2000; Schoppmann et al, 2002;
Hamrah et al, 2003). Furthermore, it is also expressed in early embryonic blood vessels and up-regulated in tumor blood vessels (Kubo et al, 2000; Paavonen et al, 2000), thus complicating its suitability as a lymphatic-specific marker.
Currently, more and more molecules are being identified as being important in
lymphatic development and could potentially be used as markers in the future. However,
in most if not all cases, these markers will likely have to be used in combination because
none appear to be totally specific for lymphatic vessels, especially during development.
This underscores the close link and the transforming ability of the blood vessels in
relation to lymphatic precursors, which is highlighted in the venous origin model
discussed in the following section.
Origins of the Lymphatic System
There are two major theories on the origin of lymphatic endothelial cells. The first
was proposed by Florence Sabin in 1902. She performed India ink injections into the
jugular lymph sacs of pig embryos, and suggested that lymphatic endothelial cells budded
off the cardinal vein and thus had a venous origin (Sabin, 1902; 1904; 1909). Her theory
was referred to as the centrifugal model. In 1910, Huntington and his colleagues offered
an alternative model where lymphatic endothelial cells originated from mesenchyme,
giving rise to the centripetal model (Huntington and McClure, 1910; Kampmeier, 1912).
19 Current studies in the field appear to support both theories. For example, in accordance
with the centrifugal model, Wigle and Oliver demonstrated that two lymphatic markers,
Prox-1 and LYVE-1, were expressed in venous endothelial cells before the formation of
the lymph sacs in the murine embryo (Wigle and Oliver, 1999; Wigle et al, 2002).
Similarly, Yaniv et al (2006) showed that in zebrafish, the progenitors for the lymphatic
endothelial cells of the thoracic duct are derived from the cardinal vein. Evidence for the centripetal model of lymphatic development is also strong. Buttler’s work illustrated that in the mouse embryo, there existed LYVE-1-positive mesenchymal cells which down- regulated leukocyte markers such as CD45 and up-regulated lymphatic and endothelial markers such as Prox-1 and CD31 respectively, as they are incorporated into the growing lymphatic system (Buttler et al, 2006). Individual mesenchymal lymphangioblasts also appeared to be incorporated into the developing lymphatic networks in the avian choriollantoic membrane (Parsons-Wingerter et al, 2006) and the avian wing bud
(Schneider et al, 1999; Papoutsi et al, 2001). It is also highly likely that the lymphatics may derive both from embryonic veins and mesenchyme. Wilting et al (2006) showed that intravenous application of the endothelial label DiI-conjugated acetylated low- density-lipoprotein into early avian embryos revealed labeling of the jugular lymph sac at later stages, suggesting that the lymph sac was of venous origin. However, quail-chick grafting of paraxial mesoderm showed that quail-derived lymphatic endothelial cells had been integrated into the superficial parts of the jugular lymph sac, whereas the dermal lymphatics were derived from the dermatomes. Thus in avian embryos, it appears that the lymphatic system has a dual origin: the main parts of the lymph sacs appear to originate
20 from veins, while the peripheral parts of the lymph sacs and the dermal lymphatics are
derived from scattered lymphangioblasts.
Formation of the Lymphatic System in Mammals
In mice, endothelial cells from the anterior cardinal vein start to express LYVE-1
at ED 9-9.5 (Wigle and Oliver, 1999), at which point they are said to have acquired
“lymphatic endothelial cell competence” (Figure 1-1, Wigle et al, 2002; Oliver, 2004). A
specific unknown lymphatic inducing signal then triggers Prox-1 expression in a subset
of LYVE-1-positive endothelial cells in the cardinal vein at approximately ED 10.5 which now have “lymphatic bias”. The Prox-1-positive lymphatic precursor cells then start expressing other lymphatic markers such as VEGFR-3 and podoplanin and move away from the embryonic veins between ED 10.5-11.5, having obtained “lymphatic specification”. These budding cells will migrate away to form the primary lymph sacs at
ED 12.5 (Figure 1-2). The interaction of the mesenchymal growth factor VEGF-C and
VEGFR-3 has been found to be important for the proliferation and migration of these lymphatic precursor cells from the cardinal veins (Veikkola et al, 2001; Karkkainen et al,
2004). Recent evidence suggests that the lymphatic differentiation process is initiated by the promoter fragment SOX18 which is thought to activate Prox-1 in the anterior cardinal vein, thus promoting the commitment of the venous endothelial cells to a lymphatic lineage (Francois et al, 2008; Kiefer and Adams, 2008). Prox-1 along with NFATc1 and the orphan nuclear receptor COUP-TFII may be then required to induce the gene expression profile and morphology characteristic of lymphatic endothelial cells (Kulkarni et al, 2009; Srinivasan et al, 2010).
21 There are eight primary lymph sacs in mammalian embryos: the unpaired
retroperitoneal lymph sac; the paired jugular, posterior, and subclavian lymph sacs; and
the cisterna chyli. In the lymph sacs, the lymphatic precursor cells down-regulate the
expression of blood-specific genes such as CD34 and laminin, while up-regulating the expression of lymphatic-specific genes such as VEGFR-3. The primary lymph sacs will eventually form the primary lymphatic plexus, after which the lymphatic system will continue to mature. At later stages in the human embryo, the blood and lymphatic
vascular networks start to separate, leaving only the right lymphatic duct and thoracic
duct still connected to the venous circulation. The right lymphatic duct drains the right side of the head and neck, the right arm, and the right half of the thorax, and empties into
the right subclavian vein. The thoracic duct drains the rest of the body and empties into
the left subclavian vein (Figure 1-3).
Lymphatic Gene Knockout Models
Knocking out Prox-1 in the mouse embryo resulted in the complete absence of a
lymphatic system and death at ED 14.5 (Wigle and Oliver, 1999). In Prox-1-null mice,
the budding of lymphatic endothelial progenitor cells from the cardinal vein was arrested
at ED 11.5. These cells did not up-regulate lymphatic-specific markers such as VEGFR-
3, but did continue to express blood-specific genes such as CD34. The formation of the
blood vessels or angiogenesis was not affected. Mice heterozygous for a null allele at the
Prox-1 locus developed a lymphatic network but eventually exhibited edema and chylous
ascites, and died 2-3 days after birth. In addition, adenovirally transduced expression of
Prox-1 in blood vessel endothelial cells reprogrammed the cells with a lymphatic
22 endothelial cell phenotype with a higher expression of lymphatic markers and a decreased expression of blood-specific markers (Hong et al, 2002; Petrova et al, 2002). Therefore, it
appears that Prox-1 serves as a master switch that is responsible for turning on the
lymphatic phenotype of endothelial cells. It is also essential for the development of the
lymphatic system, but has no effect on the development of the blood vasculature (Wigle
et al, 1999; Wigle et al, 2002).
Clues to the function of Prox-1 are provided by studies of its role in the
development of the lens system (Wigle et al, 1999). In homozygous Prox-1-null mice,
Prox-1 inactivation caused abnormal cellular proliferation, down-regulated expression of
certain cell cycle inhibitors, mis-expression of E-cadherin, and inappropriate apoptosis.
The lens fiber cells failed to polarize and elongate properly, thus Prox-1 was found to be
essential for the terminal differentiation and elongation of lens fiber cells. In addition,
Duncan et al (2002) established that the Prox-1 protein is predominantly cytoplasmic in
the mouse lens placode as well as the lens epithelium and germinative zone during
development. However, during fiber cell differentiation, the Prox-1 protein is
redistributed to the cell nuclei. Therefore, it appears that Prox-1 function is associated
with changes in its subcellular distribution during development at least in the mouse lens
model. It has not been determined yet whether any of the findings for Prox-1 action in
lens development are applicable to lymphatic development.
Comparatively, little is known about the functions of the membrane protein
podoplanin and LYVE-1, a membrane glycoprotein that is a receptor for the extracellular
glycosaminoglycan hyaluronan (Banerji et al, 1999; Breiteneder et al, 1999; Prevo et al,
2001). LYVE-1-deficient mice are healthy and fertile, with no visible defects in their
23 lymphatic system (Gale et al, 2007). On the other hand, podoplanin knockout mice acquire defects in the patterning of their lymphatic vessels, but not their blood vessels.
Eventually, they develop edema and die of respiratory failure at birth (Schacht et al,
2003). It has been proposed that podoplanin is important for lymphatic capillary
formation, and that it plays a role in LEC migration, adhesion and tube formation
(Schacht et al, 2003; Nakamura and Rockson, 2007). Podoplanin is also believed to
interact with SLP-76 which together with Syk signaling mediates the separation of lymphatic vessels from blood vessels (Abtahian et al, 2003; Bertozzi et al, 2010; Uhrin et al, 2010). However, there is still much to be discovered about the roles of LYVE-1 and podoplanin with respect to the development of the lymphatics.
In mice, the growth factor VEGF-C is expressed in the mesenchyme surrounding
the cardinal veins, where the embryonic lymph sacs will eventually develop (Kukk et al,
1996). In VEGF-C knockout mice, the Prox-1-positive lymphatic precursor cells fail to
migrate away to form the lymph sacs, and may disappear through apoptosis. Thus, the
development of the lymphatic system is brought to a halt. However, the application of
VEGF-C and VEGF-D, both of which bind to VEGFR-3, rescued the sprouting and migration of the lymphatic progenitor cells (Karkkainen et al, 2004). The receptor
VEGFR-3 is expressed in venous endothelial cells which will eventually migrate away
from the cardinal veins to form the endothelial cells of the lymphatic vessels in the
embryo (Kaipainen et al, 1995; Lymboussaki et al, 1998). In the adult, VEGFR-3 is
usually restricted to the lymphatic endothelium (Kaipainen et al, 1995), but has been
detected in hematopoeitic cells of monocytic lineage (Hamrah et al, 2004), and the
endothelium of some fenestrated blood capillaries, but not in the endothelial cells of
24 larger blood vessels (Partanen et al, 2000). VEGFR-3 expression is also up-regulated in
vascular endothelium during inflammation and tumor angiogenesis (Partanen et al, 1999;
Kubo et al, 2000; Paavonen et al, 2000; Skobe et al, 2001). VEGFR-3 knockout mice die
at approximately ED 9.5 before the initial formation of the lymphatics (Dumont et al,
1998), which has made it difficult to analyze the role of the receptor in the development
of the lymphatic system using this model. Recent findings suggest however that VEGFR-
3 ligand-binding and kinase activity are required for lymphangiogenesis but not for
angiogenesis (Zhang et al, 2010).
In summary, it would appear that Prox-1 is essential for the differentiation of the
venous endothelial cells into lymphatic endothelial cells (Wigle and Oliver, 1999; Wigle
et al, 2002), while VEGF-C signaling via VEGFR-3 is required for the sprouting of these
Prox-1-positive cells from the cardinal veins so that they can migrate away to form the primary lymph sacs (Karkkainen et al, 2004).
Lymphangiogenic Factors and Receptors
Lymphangiogenesis refers to the formation of new lymphatic vessels from pre-
existing ones. It has an important role in several pathological processes such as wound
healing, inflammation, lymphedema, and tumor metastasis. Recently, it has been
discovered that many factors and receptors play a part in lymphangiogenesis and the
development of the lymphatic system, in addition to those discussed in the previous
section.
(1) Vascular Endothelial Growth Factor Family
25 VEGF-C is a growth factor that binds to the receptors VEGFR-2 and VEGFR-3
(Figure 1-4). It increases vascular permeability, and stimulates angiogenesis as well as the migration and proliferation of lymphatic endothelial cells in the embryo (Joukov et al,
1996; Joukov et al, 1997; Cao et al, 1998). It contributes significantly to the formation and maintenance of the venous and lymphatic systems (Kaipainen et al, 1995), and has also been found to induce lymphatic vessel growth in numerous experimental models
(Jeltsch et al, 1997; Oh et al, 1997). VEGF-C expression is regulated by pro- inflammatory cytokines (Ristimaki et al, 1998), but it remains to be seen whether it is also regulated by hypoxia (Enholm et al, 1997; Simiantonaki et al, 2008). Both VEGF-C and VEGFR-3 are expressed in active macrophages (Skobe et al, 2001; Schoppmann et al, 2002), so it appears that VEGF-C has some role in inflammation. Mice with targeted
VEGF-C deletion do not develop a lymphatic system, and die at ED 15.5-17.5 due to
edema, but acquire a normal blood vascular system. This indicates that VEGF-C is not
necessary for the development of the blood vasculature but is needed for the formation of
the lymphatics (Karkkainen et al, 2004).
Like VEGF-C, VEGF-D binds to both VEGFR-2 and VEGFR-3 (Stacker et al,
1999). However, VEGF-D binds only to VEGFR-3 in mice, but will bind to VEGFR-2 and VEGFR-3 in humans, so this growth factor may differ functionally from species to
species (Baldwin et al, 2001). In the embryo, VEGF-D is most abundantly expressed in
the lungs and skin, but it is expressed in several adult tissues such as the lung, heart,
skeletal muscle, colon, and small intestines (Farnebo et al, 1999). VEGF-D acts as a
mitogen for endothelial cells, and is thought to regulate the development of the blood and
lymphatic systems (Achen et al, 1998; Marconcini et al, 1999; Stacker et al, 1999).
26 VEGF-D knockout mice exhibit no visible defects in either vascular system so the growth
factor may not have a truly essential role in their formation as other factors may
compensate for it (Baldwin et al, 2005). In experimental models, VEGF-D has also been
found to induce the growth of intratumoral lymphatics and stimulate tumor metastasis
(Stacker et al, 2001; Krishnan et al, 2003).
VEGF-A is an important stimulator of angiogenesis and binds to the receptor
VEGFR-2, which is expressed in the blood and lymphatic vascular endothelium
(Mustonen and Alitalo, 1998). In studies of wound healing in the skin, transgenic mice
were developed where VEGF-A was over-expressed specifically in the epidermis. In this
case, VEGF-A stimulated lymphangiogenesis via VEGFR-2 signaling (Hong et al, 2004).
VEGF165 in particular has also been found to increase the diameter and density of
lymphatic vessels, and induce lymphatic dissociation from blood vessels in the quail
chorioallantoic membrane (Parsons-Wingerter et al, 2006). In other experiments, the
adenoviral over-expression of VEGF-A in rabbit ears resulted in the formation of large, hyperplastic lymphatics (Nagy et al, 2002). In addition, Hirakawa’s group showed that
the transgenic delivery of VEGF-A specifically to the skin promoted skin carcinogenesis,
and induced the growth of VEGFR-2-expressing tumoral lymphatics (Hirakawa et al,
2005). Thus, the VEGF-A stimulated changes in the lymphatics may be due to direct
effect on VEGFR-2 signaling but there may also be an indirect effect through the
recruitment of macrophages which release lymphangiogenic factors (Cursiefen et al,
2004).
(2) Fibroblast Growth Factor Family
27 FGF-2 promotes angiogenesis as well as lymphangiogenesis. In vitro, this growth factor stimulated the proliferation and migration of lymphatic endothelial cells, and their assembly into capillary tube-like structures (Tan, 1998). In the mouse cornea model,
FGF-2 had a dose-dependent effect on lymphangiogenesis since low concentrations of
FGF-2 induced the growth of robust lymphatics but little or no angiogenesis, thus demonstrating that lymphangiogenesis can occur in the absence of angiogenesis (Chang et al, 2004). Furthermore, blocking VEGFR-3 signaling suppressed FGF-2-induced lymphangiogenesis, indicating that FGF-2 induced the growth of lymphatic vessels indirectly via the up-regulation of VEGF-C expression in vascular endothelial cells and perivascular cells (Chang et al, 2004).
(3) Angiopoietins
Angiopoietin-2 (Ang-2) causes the destabilization of the blood vasculature during the sprouting of new vessels throughout angiogenesis through the binding and activation of the Tie-2 receptor (Holash et al, 1999). Ang-2 knockout mice exhibited defects in blood vessel remodeling, and also displayed defects in the function and patterning of the lymphatic vasculature (Gale et al, 2002). The mice developed highly disorganized and hypoplastic intestinal and dermal lymphatic capillaries, as well as larger lymphatic collecting vessels that were poorly invested by smooth muscle cells (Gale et al, 2002;
Shimoda et al, 2007). The mice also suffered from subcutaneous edema and usually died at 2 weeks post-birth due to severe chylous ascites, which is often characteristic of defective lymphatic function. However, overexpression of Ang-1 which also binds to
Tie-2 rescued the lymphatic phenotype in Ang-2 knockout mice, suggesting that
28 activation of Tie-2 is required for normal lymphatic development (Gale et al, 2002). In
mice overexpressing Ang-1, lymphatic vessels were enlarged in concordance with
lymphatic endothelial cell proliferation and new vessel sprouting, similar to what is
observed in mice overexpressing VEGF-C, which infers that there might be crosstalk
between the VEGF and Ang families during lymphangiogenesis.
(4) Neuropilins
Neuropilin-2 (Nrp2) is a receptor for class III semaphorins and for certain
members of the VEGF family, and has been implicated to play a role in neuronal
development via axon guidance. In the vascular system, Nrp2 has been found to be
expressed in veins and lymphatic vessels. In homozygous Nrp2 mutants, small lymphatic
vessels and capillaries were either absent or significantly reduced in number during
development (Yuan et al, 2002). This phenomenon was associated with a reduction in
DNA synthesis in the lymphatic endothelial cells of the mutants. Arteries, veins and
larger collecting lymphatic vessels were seen to develop normally, suggesting that Nrp2
is selectively required for the formation of small lymphatic vessels and capillaries (Yuan
et al, 2002). Studies have shown that both VEGF-C and VEGF-D interact with Nrp2 in
vitro, with VEGF-C doing so in a heparin-independent manner and VEGF-D in a
heparin-dependent manner (Karpanen et al, 2006). Nrp2 was also co-internalized along with VEGFR-3 in endocytic vesicles of lymphatic endothelial cells upon stimulation with
VEGF-C or VEGF-D. Furthermore, Nrp2 was shown to interact with VEGFR-3 in co- precipitation studies, indicating that Nrp2 is directly involved in an active signaling complex with key lymphangiogenic regulators and could be functionally important for
29 the development of the lymphatic vasculature (Karpanen et al, 2006; Xu et al, 2010).
Recent findings suggest that the PDZ domain-containing scaffold protein synectin also genetically interacts with VEGFR-3 and Nrp2 in regulating lymphangigenesis in zebrafish and tadpole models (Hermans et al, 2010).
Thus, there are several factors that are important in regulating the formation of the lymphatic system. However, there is still very little known about the lymphangiogenic effect of most of the factors, or the mechanisms involved, as is the case for Prox-1 which appears to serve as the master switch for turning on the lymphatic phenotype of a cell.
Some factors often have an indirect effect on lymphangiogenesis (FGF-2), while others are not truly essential to the process (VEGF-D) or have not been fully investigated with respect to lymphatic development, such as ephrinB2 whose C-terminal PDZ interaction site is required for lymphatic remodeling (Makinen et al, 2005). Prox-1 and VEGF-C appear to be essential for the lymphangiogenic process, since they are the only two factors whose absence results in the complete lack of a lymphatic system. In view of the critical requirement for these two factors, the relationship between the two warrants further investigation.
Clinical Significance of the Lymphatics
The impaired ability of the lymphatics to collect and transport fluid has been observed across a broad range of acquired and inherited pathological illnesses. Lymphatic vascular incompetence is most often characterized by the swelling of tissues, referred to as lymphedema, which is due to inefficient lymph transport. Edema is often accompanied
30 by tissue fibrosis, susceptibility to infection, and impaired wound healing. Lymphedema can be classified as either a secondary or primary condition.
(1) Secondary Lymphedema
Secondary or acquired lymphedema is the more common form of lymphatic
dysfunction, and is often triggered after surgical and radiotherapeutic interventions for
cancer, such as lymph node dissection and accompanying radiation. Breast cancer-
associated lymphedema of the upper arm is the most frequently observed in patients in the US (Szuba and Rockson, 1998; Rockson, 1998). It is estimated that after such interventions, 20 to 30% of breast cancer survivors will experience clinically relevant lymphedema (Erickson et al, 2001; Moffatt et al, 2003). Secondary lymphedema can also occur as a result of trauma, such as burns or wounds, or infection. Globally, secondary
lymphedema has often been ascribed to filiarisis, which affects more than 129 million
people worldwide (Keiser and Nutman, 2002; Taylor, 2002). Currently, lymphedema treatment comprises mainly the chronic use of physiotherapeutic techniques based on
lymphatic-specific massage methods that alleviate symptoms but do not reverse the
disease.
(2) Primary Lymphedema
Primary or congenital lymphedema is present in approximately 1:6000 to 1:10000
live births (Rockson, 2000). The associated syndromes show significant familial
occurrence with an autosomal pattern of genetic transmission and inexplicable female
predominance. The autosomal dominant form of congenital familial lymphedema was
31 first described in 1892 and has often been referred to as Milroy’s disease (Milroy, 1892).
The disease has been recently linked to a missense inactivating mutation in the flt4 locus
that encodes for the VEGFR-3 receptor (Karkkainen et al, 2000).
Another autosomal dominant cause of familial lymphedema is lymphedema-
distichiasis. The phenotype involves the pubertal or post-pubertal onset of a distally
distributed form of lymphedema accompanied by the development of a supplementary
row of eyelashes (distichiasis) arising from the Meibomian glands. This disease, among
other primary lymphedema disorders, has been associated with truncating mutations in
the forkhead-related transcription factor FOXC2 (Fang et al, 2000). Mutations in the
transcription factor SOX18 have also been linked to a form of congenital lymphedema
known as hypotrichosis-lymphedema-telangiectasia, where both autosomal dominant and
recessive transmission patterns have been observed (Irrthum et al, 2003). SOX18 may
likely play a role in the development or maintenance of the lymphatics perhaps by
directly modulating VEGF-C and VEGF-D expression, since the promoters of these
genes contain DNA-binding sites for SOX proteins, but the underlying mechanics have
not yet been determined (Irrthum et al, 2003).
The molecular regulation of lymphatic vascular development has recently been investigated for its therapeutic potential in modulating postnatal lymphatic vessel growth
and remodeling (An and Rockson, 2004; Nakamura and Rockson, 2007). VEGF-C and
VEGF-D have often been proposed as possible therapeutic agents since both growth
factors stimulate lymphangiogenesis through VEGFR-3 binding and thus mediate the
growth and survival of the lymphatics (Makinen et al, 2001; Veikkola et al, 2001;
32 Karkkainen et al, 2004). For example, in the Chy mouse strain, a murine model of
Milroy’s disease, overexpression of VEGF-C and VEGF-D promoted the development of functional lymphatic vessels (Karkkainen et al, 2001). In animal models of acquired lymphedema, where the lymphatic vasculature was ablated, both the direct administration of recombinant VEGF-C and plasmid-mediated gene therapy induced a significant reversal of the lymphedema pathology (Szuba et al, 2002; Yoon et al, 2003; Cheung et al,
2006). Similarly, in a murine lymphedema model, multipotent mesenchymal stem cells acquired a lymphendothelial phenotype after induction with VEGF-C conditioned supernatant derived from lymphatic cells and subsequently enhanced lymphatic generation in vivo (Conrad et al, 2009). Thus, such specifically designed therapeutic techniques could be used to stimulate controlled lymphangiogenesis to compensate for damaged or dysfunctional lymphatic components in order to prevent edema and tissue destruction (Jussila and Alitalo, 2002). Furthermore, a molecular understanding of the lymphangiogenic process could help in designing therapies aimed at reducing tumor metastasis which has been correlated to lymphangiogenesis.
The Lymphatics of the Heart
The lymphatic network of the adult heart consists of two plexuses: (a) the deep
plexus immediately under the endocardium; and (b) the superficial plexus subjacent to
the visceral pericardium (Gray, 1918; Millard, 1922). The deep plexus opens into the
superficial plexus, whose efferent vessels will form right and left collecting trunks. The
left trunks ascend in the anterior longitudinal sulcus, while receiving vessels from both ventricles. On reaching the coronary sulcus, they are joined by a large trunk from the
33 diaphragmatic surface of the heart, and then unite to form a single vessel that ascends
between the pulmonary artery and the left atrium and ends in one of the tracheobronchial
lymph nodes. The right trunk receives its afferents from the right atrium and from the
right border and diaphragmatic surface of the right ventricle. It ascends in the posterior
longitudinal sulcus and then runs forward in the coronary sulcus, and passes up behind
the pulmonary artery, to end in one of the tracheobronchial nodes.
Although the adult heart is described to have an extensive lymphatic network
(Figure 1-5), the lymphatics of the embryonic heart have not yet been thoroughly
investigated. Presumably, Prox-1-positive lymphatic precursor cells will migrate away
from the cardinal vein towards the developing heart. However, it is possible that some of
these cells may also arrive from another source, such as the epicardium which is derived
from the proepicardial serosa or organ.
The proepicardial organ (Figure 1-6) is an outgrowth of the dorsal body wall, and becomes visible at the sinoatrial pole of the heart in the avian embryo at Hamburger &
Hamilton (HH) Stage 13 (Hamburger and Hamilton, 1951). At HH Stage 17-18, the proepicardial organ (PEO) makes contact with the embryonic heart, and the proepicardial cells migrate over the myocardium to form the pericardium and the epicardial monolayer
(Manner, 1999). Between HH Stages 19-23, some epicardial cells move into the subepicardial space and myocardium, undergoing an epithelial-to-mesenchymal transformation (Reese et al, 2002; Wada et al, 2003). Some cells become vasculogenic,
and differentiate to form tubes that connect to form plexuses. Other mesenchymal cells
are induced to turn into smooth muscle cells and enwrap the endothelial tubes. This
marks the formation of the first definitive arteries, including the left and right coronary
34 vessels, which will finally attach to the aorta at HH Stage 32 (Figure 1-7). Since the
proepicardial organ gives rise to precursor cells in the epicardium that will eventually
differentiate into blood vessel endothelial cells, fibroblasts and smooth muscle cells, it is
reasonable to hypothesize that Prox-1-positive lymphatic precursor cells in the
developing heart may originate from this tissue as well.
Early studies of the developing cardiac lymphatics conducted almost four decades
ago (Klika et al, 1971; Rychter et al, 1971) involved the injection of gelatin or dye into
the embryonic chicken heart. These findings indicated that the lymphatic vessels reached the heart at around HH Stage 35 and spread in the mesenchyme on the anterior surface of the heart in three directions: left, right, and towards the apex. By HH Stage 37, the lymphatics had spread to the posterior aspect of the developing heart and were found to cover the entire ventricular surface within a few days. Lymphatic vessel valves were observed at the base of the heart at HH Stage 40. Furthermore, Rychter and his colleagues noted that the development of the coronary bed was ahead of the development of the lymph bed. More recently, Wilting and colleagues (Wilting et al, 2007) provided further clues regarding the development of the cardiac lymphatics. The proepicardial organs (PEOs) of HH Stage 17 quail embryos were grafted into chick embryos, which were then re-incubated until around HH Stage 40. Double staining with QH-1 and the lymphatic marker Prox-1 revealed that there were no Prox-1-positive/QH-1-positive lymphatic endothelial cells or vessels in the chick host, except for a large lymphatic trunk
at the base of the chick heart, which was presumed to indicate a lympho-venous
anastomosis and a ‘homing’ mechanism of venous endothelial cells into the lymphatic
trunk. Furthermore, Wilting and his collaborators (Wilting et al, 2007) observed a basal
35 to apical gradient of scattered LYVE-1-positive subepicardial cells in the mouse heart at
ED 12.5 that showed the contribution of immigrating lymphangioblasts to the cardiac lymphatic system. In our studies, the cardiac lymphatics of the quail and mouse embryos were investigated in detail throughout development by immunostaining for three lymphatic markers: Prox-1, LYVE-1, and VEGFR-3 wherever possible, in order to verify lymphatic identity. This is in contrast to previous studies where such markers were used separately in the embryonic heart at few stages.
Other studies (Lavine et al, 2006) have reported on the progression of VEGF-C mRNA expression emanating from the atrioventricular and interventricular grooves at ED
12.5 in the mouse heart and extending to cover the ventricles by ED 13.5. The pattern of staining observed in sections suggested expression of VEGF-C in the perivascular cells within the myocardium. At ED 13.5, VEGFR-3 mRNA was expressed within the atrioventricular groove in the embryonic epicardium. This suggests that the epicardial cells are capable of responding to VEGF-C, but whether they respond and how they respond as multipotent precursor cells has not yet been studied.
Statement of Purpose
The heart is invested with an extensive epicardial lymphatic system, suggesting that a causal link may exist between lymphatic dysfunction and the pathogenesis of cardiovascular disease. For example, developmental abnormalities of the lymphatic vasculature appear to be associated with cardiac malformations in children. In the adult, disruption or obstruction of the lymphatic system as a result of surgery or perturbations in myocardial fluid homeostasis following a myocardial infarction causes myocardial
36 edema, triggering an inflammatory response, and subsequently tissue fibrosis (Laine and
Allen, 1991; Laine and Allen, 1998; Geissler and Allen, 1998; Stolyarov et al, 2002;
Kong et al, 2006; Li et al, 2009). This sequence of events may contribute, in part, to heart
failure or cardiac transplant rejection (Li et al, 2009; Soong et al, 2010). Despite such a
central role, the development of the lymphatic system as well as its role in maintaining
the physiological integrity of the cardiovascular tissue during development and in the
adult is poorly understood, and remains to be more thoroughly investigated.
Our studies make use of the developing heart which gives us an opportunity to look at a relatively simple system where vasculogenesis is taking place at a defined time
in an accessible location. An understanding of lymphangiogenesis in this region could
have therapeutic potential, especially since myocardial edema and lymphatic dysfunction
are a common finding in various cardiac disease states. Any interruption in the lymphatic
flow in the thoracic cavity, for example due to infection or surgery or radiation therapy,
will also cause a similar build up of interstitial fluid. In these cases, lymphangiogenesis
will be required to drain away the excess fluid in order to maintain homeostasis within
the internal environment, and Prox-1 may be significantly involved in this process given
its status as a “master switch” that turns on the lymphatic phenotype of cells (Hong et al,
2002; Petrova et al, 2002). In addition, the cardiac lymphangiogenic signaling pathway
involving the lymphangiogenic receptor VEGFR-3 and the lymphatic master gene Prox-1
is currently not known. Our aim therefore is to gain insight into the underlying molecular
mechanisms of this pathway in the epicardium.
37 Figure 1-1. Model for the differentiation of the mammalian lymphatic vasculature
Figure from Oliver (2004).
38
39 Figure 1-2. Prox-1 expression in the mouse embryo. Figure from Wigle and Oliver
(1999). (A) At ED 9.5, Prox-1 (green) is turned on in a population of cells in the anterior cardinal vein. (B) At ED 10.5, the Prox-1-positive lymphatic precursor cells (blue) bud off the cardinal vein and travel as individual cells. (C) At ED 12.5, the Prox-1-positive cells (dark blue) form the primary lymph sacs. (D) At ED 16.5, the Prox-1-positive lymphatic vessels (blue) appear alongside the blood vessels (red). CV = cardinal vein.
40
41 Figure 1-3. The Thoracic Duct and the Right Lymphatic Duct.
42
43 Figure 1-4. The VEGF Family.
44
45 Figure 1-5. Branching Patterns of the Lymphatic Vasculature in the Adult Heart (as determined by dye injection studies). Figure from Feola et al (1977).
46
47 Figure 1-6. The proepicardial organ (arrow) in an HH stage 17 chick embryo. Bar =
100 um.
48
49 Figure 1-7. Tracing the path of the proepicardial organ. Epi = epicardium, myo = myocardium. Figure from Reese et al (2002).
50
51 CHAPTER 2: EXPRESSION OF LYMPHATIC MARKERS DURING AVIAN AND MOUSE CARDIOGENESIS (Karunamuni et al, 2010)
Ganga Karunamuni1,2, Ke Yang1, Yong Qiu Doughman1, Jamie Wikenheiser3, David Bader4, Joey Barnett5, Anita Austin5, Patricia Parsons-Wingerter6, and Michiko Watanabe1
Departments of Pediatrics1 and Anatomy2, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106
Pathology and Laboratory Medicine3, UCLA, Box 951732, Los Angeles, CA 90095
Cell and Developmental Biology4, Vanderbilt University Medical Center, 21st Avenue South and Medical Center Drive, Nashville, TN 37232
Department of Pharmacology5, Vanderbilt University Medical Center, 21st Avenue South and Medical Center Drive, Nashville, TN 37232
Biological Fluid Physics6, John Glenn NASA Research Center, MS 110-3 21000 Brookpark Rd., Cleveland, OH 44135
Abstract
The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored.
The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-
1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the
CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these
LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they
52 may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-
1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in
adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear- localized expression in primary cultures of embryonic avian epicardial cells. Our data
identified three types of cells in the embryonic heart expressing lymphatic markers: (1)
Prox-1-positive cells from an extracardiac source that migrate within the serosa of the
outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive
cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic
vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not
become Prox-1-positive even in the adult heart.
53 Introduction
The main functions of the lymphatics include the maintenance of the fluid balance of the internal environment, the absorption of fat from the small intestine, and the trafficking of antigen-presenting cells from tissues to lymph nodes in immune surveillance (Randolph et al, 2005; Baluk et al, 2007). In addition, the lymphatics serve as one of the major routes by which tumor cells can metastasize to distant organs. Prox-1
(Prospero-related homeobox 1), podoplanin, LYVE-1 (Lymphatic Vascular Endothelial
Hyaluronan Receptor-1), and VEGFR-3 (Vascular Endothelial Growth Factor Receptor-
3) are expressed in lymphatic endothelial cells and have been used as lymphatic markers in previous studies (Kaipainen et al, 1995; Banerji et al, 1999; Breiteneder-Geleff et al,
1999; Wigle and Oliver, 1999). While these lymphatic markers are not totally specific to lymphatic endothelial cells (Rishi et al, 1995; Wetterwald et al, 1996; Wigle et al, 1999;
Partanen et al, 2000; Sosa-Pineda et al, 2000; Mouta Carreira et al, 2001; Schoppmann et al, 2002; Hamrah et al, 2003; Gittenberger-de Groot et al, 2007), they play a significant role in lymphatic development and when used in combination, have been useful in identifying lymphatic precursors and lymphatic endothelial cells during development.
There are two major theories on the origin of lymphatic endothelial cells. In 1902,
Florence Sabin proposed that the lymphatics had a venous origin by suggesting that lymphatic endothelial cells bud off the cardinal vein (Sabin, 1902; Sabin, 1904; Sabin,
1909). Her theory was referred to as the centrifugal model. In 1910, Huntington and his colleagues countered that lymphatic endothelial cells originate from mesenchyme, giving rise to the centripetal model (Huntington and McClure, 1910; Kampmeier, 1912). Recent studies in the field appear to support both hypotheses (Kaipainen et al, 1995; Kukk et al,
54 1996; Schneider et al, 1999; Wigle and Oliver, 1999; Papoutsi et al, 2001; Wigle et al,
2002; Parsons-Wingerter et al, 2006; Yaniv et al, 2006). In mice, Prox-1 is expressed in a
subset of LYVE-1-positive endothelial cells in the anterior cardinal vein at ED
(Embryonic Day) 9.5-10 (Wigle and Oliver, 1999; Oliver and Detmar, 2002; Oliver,
2004). These cells also express other lymphatic markers such as VEGFR-3. The Prox-1-
positive lymphatic precursor cells then migrate away to form the primary lymph sacs.
The interaction of the mesenchymal growth factor VEGF-C and VEGFR-3 plays an
important role in the proliferation and migration of these lymphatic precursor cells from
the cardinal veins (Veikkola et al, 2001; Karkkainen et al, 2004).
Knocking out the gene for Prox-1 in the mouse embryo resulted in the complete
absence of a lymphatic system in all tissues and death at ED 14.5 (Wigle and Oliver,
1999). In Prox-1-null mice, the budding of lymphatic endothelial progenitor cells from
the cardinal vein was arrested at ED 11.5. These cells did not up-regulate lymphatic
markers such as VEGFR-3, but did continue to express blood-specific genes such as
CD34. There was no obvious alteration in heart function in the Prox-1 knockout mice as
assessed by the circulation of injected ink. However, no further physiological assays have been carried out. In addition, adenoviral transduced expression of Prox-1 in cultured
blood vessel endothelial cells re-programmed the cells to express a lymphatic endothelial
cell phenotype with a higher expression of lymphatic markers and a decreased expression
of blood-specific markers (Hong et al, 2002; Petrova et al, 2002). Therefore, Prox-1
appears to be essential for the development of the lymphatic system, but is not required
for the development of the blood vasculature (Wigle and Oliver, 1999; Wigle et al, 2002).
55 Comparatively less is known about the functions of the highly O-glycosylated integral membrane protein podoplanin and LYVE-1, a membrane glycoprotein that is a receptor for the extracellular glycosaminoglycan hyaluronan (Banerji et al, 1999; Prevo et
al, 2001; Edward et al, 2005). LYVE-1-deficient mice are healthy and fertile, with no visible defects in their lymphatic system (Gale et al, 2007). On the other hand, podoplanin knockout mice acquire defects in the patterning of their lymphatic vessels,
but not their blood vessels. Eventually, they develop edema and die of respiratory failure
at birth (Schacht et al, 2003). In VEGF-C knockout mice, the Prox-1-positive lymphatic
precursor cells fail to migrate away from the cardinal veins, and may disappear through
apoptosis. Thus, the development of the lymphatic system is brought to a halt. However,
the application of VEGF-C and VEGF-D, both of which bind to VEGFR-3, rescued the
sprouting and migration of the lymphatic progenitor cells (Karkkainen et al, 2004).
VEGFR-3 knockout mice die at approximately ED 9.5 before the initial formation of the
lymphatics in normal development (Dumont et al, 1998; Jussila and Alitalo, 2002). This
early death precludes analysis of the role of the receptor in the development of the
lymphatic system using this model. Thus, the roles of lymphatic markers in
lymphangiogenesis, even that of Prox-1 that is the most extensively studied, are still not
completely understood.
Despite the extensive nature and importance of the lymphatic system in the adult
heart (Shimada et al, 1989; Shimada et al, 1990), the developmental steps of the
lymphatics in the embryonic heart have not been described in detail. In a recent study
(Wilting et al, 2007), Prox-1 expression was examined at later stages of embryonic cardiac development after morphogenesis was largely complete (from Hamburger-
56 Hamilton (HH) Stage 35 onwards). These investigators also conducted quail-chick
grafting experiments and concluded that grafts of the proepicardial organ, an outgrowth
of the dorsal body wall that eventually gives rise to the epicardium and the precursors of
the coronary arteries (Mikawa et al, 1996; Munoz-Chapuli et al, 2002; Perez-Pomares et
al, 2002), do not give rise to the bulk of lymphatic endothelial cells within the heart. In
their studies, LYVE-1 was also used to demarcate the lymphatic vessels in ED 11.5-13.5
mice but no comparison was made between Prox-1 and LYVE-1 expression at these stages. In our study, we analyzed the expression pattern of Prox-1 at younger stages in the avian embryo than in previous studies; that is, from HH (Hamburger and Hamilton,
1951) Stage 24 to 40, stages which span the most active phases of morphogenesis and
coronary vessel vasculogenesis. We also analyzed the expression of various lymphatic
markers in the mouse heart, including Prox-1, LYVE-1, VEGFR-3, and podoplanin, at
different developmental stages for comparison, since lymphatic markers are not truly
lymphatic-specific. Our primary objective was to confirm lymphatic vessel identity in the
mouse heart by investigating the expression patterns of two primary lymphatic markers
Prox-1 and LYVE-1 throughout development (ED 9.5 to adult). Two epicardial cell lines
(Eid et al, 1994; Austin et al, 2008) and quail embryonic epicardial cell primary cultures
were also used to augment our in vivo findings.
Materials and Methods
Animals
Chicken (Gallus gallus; Squire Valleevue Farm, Hunting Valley, OH) and quail
(Coturnix coturnix japonica; Boyd’s Bird Company, Pullman, WA) embryos were
57 incubated in an egg incubator (GQF Mfg Co., Savannah, GA) with a rocking apparatus
and dissected out at Hamburger-Hamilton (HH) Stages 24-40 (Hamburger and Hamilton,
1951). Mice were obtained from Dr. Radhika Atit (Case Western Reserve University,
OH). The adult rat epicardial cell line (Eid et al, 1994) was obtained from Dr. David
Bader (Vanderbilt University, TN). The embryonic mouse epicardial cell line (Austin et
al, 2008) consists of immortalized ED 11.5 mouse epicardial cells and was obtained from
Drs. Austin and Barnett (Vanderbilt University, TN).
Antibodies
The antibodies against Prox-1 (polyclonal rabbit anti-human, 5 ug/ml; Research
Diagnostics Inc., Concord, MA), LYVE-1 (polyclonal rabbit anti-mouse, 10 ug/ml;
Angio-Proteomie, Boston, MA), VEGFR-3 (polyclonal rabbit anti-mouse, 4 ug/ml; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), podoplanin (monoclonal hamster anti-mouse,
5 ug/ml, eBioscience, San Diego, CA), CD44 (monoclonal mouse anti-chicken, 125
ug/ml; US Biological Inc., Swampscott, MA), and CD31/PECAM (rat anti-mouse, 10
ug/ml; BP Pharmingen, San Jose, CA) were used according to the manufacturer’s
protocol. The endothelial precursor marker QH-1 (mouse anti-quail, 1:2000) was
obtained from the Developmental Studies Hybridoma Bank developed under the auspices
of the NICHD and maintained by The University of Iowa, Department of Biological
Sciences, Iowa City, IA.
Immunostaining of Whole Hearts and Sections
58 Intact hearts from staged embryonic quail embryos were dissected and fixed in
4% paraformaldehyde. The hearts were incubated in the primary antibodies anti-Prox-1
and anti-QH-1 overnight. The hearts were then incubated with the appropriate fluorescent
secondary antibodies (Molecular Probes, Eugene, OR) and observed under the
stereoscope. Cryosections of quail and chicken embryonic hearts were examined for
Prox-1 immunofluorescence as well. ED 9.5-15 mouse hearts and adult mouse hearts
were also sectioned and stained for Prox-1, LYVE-1, podoplanin, and VEGFR-3. The
cardiac myocyte marker MF20/titin was used for co-localization. Stained sections were
mounted with DAPI for nuclear visualization, and examined under the Nikon DIAPHOT
200 fluorescence microscope. Images were captured with the digital camera and
QCapture Pro software.
Primary Cultures
Quail embryonic hearts at HH Stage 21 were harvested and placed on Matrigel-
coated coverslips to allow the epicardial cells to grow out onto the matrix. The hearts
were removed after 24 hours and the quail epicardial cells (QECs) were maintained in
culture at 37°C and 5% CO2 in M199 media ((Mediatech Inc., Manassas, VA)
supplemented with 10% FBS. The coverslips were then rinsed in PBS, fixed in 4%
paraformaldehyde, and stained for Prox-1. After being mounted with DAPI for nuclear
visualization, the coverslips were examined under the Nikon DIAPHOT 200 fluorescence
microscope. Images were captured with the digital camera and QCapture Pro software.
Cell Culture
59 The adult rat epicardial cells (ARECs) were maintained in culture at 37°C and 5%
CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) media with sodium pyruvate
(Mediatech Inc., Manassas, VA) and supplemented with 10% fetal bovine serum (FBS;
Invitrogen, Carlsbad, CA). The embryonic mouse epicardial cells (EMECs) were
maintained in culture at 33°C and 5% CO2 in DMEM media without sodium pyruvate
(Mediatech Inc., Manassas, VA) and supplemented with 10% FBS. The ARECs and
EMECs were seeded onto poly-L-lysine-coated coverslips and collagen-coated coverslips
respectively and once they were confluent, the coverslips were rinsed in PBS, fixed in 4% paraformaldehyde, and stained for Prox-1, LYVE-1, and VEGFR-3. The coverslips were then mounted with DAPI for nuclear visualization, and examined under the Nikon
DIAPHOT 200 fluorescence microscope. Images were captured with the digital camera and QCapture Pro software.
Western Blotting
Tissue samples were sonicated in RIPA lysis buffer (with protease inhibitors) and
assayed for protein concentration. The sample lysate was then electrophoresed on an 8%
SDS-PAGE gel and transferred onto a PVDF membrane (Millipore, Bedford, MA).
Membranes were blocked in a solution of 5% milk/TBS-T and incubated with the primary antibody Prox-1 overnight. The monoclonal antibody anti-β-actin was used as a
loading control. After washing, the blots were incubated with the appropriate HRP-linked
secondary antibodies (Cell Signaling, Beverly, MA) and signals were then detected using
an enhanced chemiluminescence detection system (Pierce Chemical Co., Rockford, IL).
60 Phosphatase Assay
HH Stage 30 quail heart samples were exposed to a calf intestinal alkaline
phosphatase enzyme (NE Biolabs, Ipswich, MA) for 1½ hours at 37°C in 1X NE
Reaction Buffer. The enzyme was then inactivated with the addition of 50 mM EDTA.
Dephosphorylated samples were run on a Western Blot alongside control samples that
were not enzyme-treated.
Results
The formation of the Prox-1-positive epicardial lymphatic network in quail
In order to analyze lymphatic development within the embryonic heart, we stained
quail hearts ranging from HH Stage 24 to HH Stage 40 for the lymphatic marker Prox-1,
with co-localization for the endothelial precursor marker QH-1 that labels both lymphatic
and blood vessel precursors as has been shown in the chorioallantoic membrane (Parsons-
Wingerter et al, 2006). These stages were chosen because they span a time in
development when vasculogenesis is active in the epicardium, leading to the formation of
the first definitive blood vessels including the coronaries. At HH Stage 24, after the heart
has just looped and developed distinct atrial chambers, QH-1-positive cells were found at
the base of the outflow tract and were also scattered over the atrium and ventricle in an
equidistant pattern, but there were no distinctly detectable Prox-1-positive cells on the
epicardial surface of the embryonic heart (Figure 2-1 A, B). Between HH Stages 26-28, with the septation of the outflow tract into the aorta and pulmonary trunk, large, irregularly shaped regions of Prox-1-positive/QH-1-positive cells started to appear along
61 the great vessels with the greatest concentration appearing at the cranial region (Figure
2-1 C, D).
At HH Stages 29-30, after the heart had completed septation into four chambers,
we observed several Prox-1-positive/QH-1-positive vessels that ran down the length of
the great vessels, including the aorta and pulmonary artery, and formed a widespread, branching network at their base and over the surface of the ventricles (Figure 2-2 A, B).
At HH Stages 38-40, when cardiac morphogenesis is largely complete, the Prox-1- positive vessels encircling the great vessels were larger and well developed, but those covering the ventricles were obscured by the thickness of the epicardium. Clusters of
Prox-1-positive cells were also apparent at regular intervals along the lymphatics of the great vessels, which may be indicative of lymphatic valve formation (Figure 2-2 C). At these later stages, Prox-1-positive/QH-1-positive vessels were also found surrounding the posterior interventricular blood vessel running within the epicardial connective tissue of the interventricular sulcus on the posterior aspect of the embryonic heart (Figure 2-2 D).
Thus, the avian embryonic heart acquired an extensive lymphatic network throughout cardiac morphogenesis (Figure 2-3) during the embryonic stages when coronary vessel development is actively proceeding.
It also appears that Prox-1 may play roles in different cell types during cardiac
development in addition to contributing to the formation of the cardiac lymphatic vessels,
according to the distribution patterns of the staining found in sections of the heart. For example, in HH stage 24 sections, Prox-1-positive cells were found in the atrial and
ventricular myocardium, as well as in the lining and mesenchyme of the forming
endocardial cushions (Figure 2-4 A, B). One or two Prox-1-positive cells were also
62 found in the ventricular epicardium in sections at this stage. At HH stages 29-30, Prox-1-
positive cells were also located in the endocardium and mesenchyme of the aortic and
atrioventricular valves (Figure 2-4 C). In all of the valves, Prox-1 appeared to be expressed specifically in cells of the fibrosa layer, otherwise known as the arterial aspect of the valvular cusp which is composed primarily of collagen fibers (Gross and Kugel,
1931; Schoen, 2005). Prox-1 stained both atrial and ventricular cardiac myocytes as well, but the nuclear staining intensity in the cardiac myocytes was lower than that exhibited by the lymphatic vessels or the cells lining the valves.
Co-localization of Prox-1 with other lymphatic markers in mouse
Mouse heart sections were labeled for additional lymphatic markers such as
LYVE-1 and VEGFR-3 in order to compare with Prox-1 localization, since commercially
available antibodies for LYVE-1 and VEGFR-3 do not work in chicken or quail tissues.
At ED 9.5-10.5, Prox-1 was found only in cardiac myocytes, while LYVE-1 and
VEGFR-3 were expressed in cells/vessels within the myocardium (Figure 2-5 A-D). The
LYVE-1-positive cells did not stain for the cardiac myocyte marker MF20 or the
lymphatic marker podoplanin (Figure 2-5 E-H) but they did express the endothelial cell
marker CD31/PECAM (Figure 2-5 I-K).
At ED 13.5, Prox-1 labeled the nuclei of cells organized into vessels running
along the aorta and pulmonary trunk (Figure 2-6 A), but there was no co-localization
with LYVE-1 which was expressed in myocardial vessels as well as individual cells
found in the epicardium of the heart (Figure 2-6 B-D). Some Prox-1-negative/LYVE-1-
positive cells were found scattered in close vicinity of the Prox-1-positive lymphatic
63 vessels (Figure 2-6 A, B). Expression patterns for LYVE-1, which is homologous to the
hyaluronan receptor CD44, in the ED 13.5 mouse embryo heart were similar to staining
patterns observed for CD44 in the HH Stage 30 chick embryo heart. CD44 was present in
cells within the chicken epicardium and myocardium, with certain cells found next to
Prox-1-positive vessels, but no co-localization was seen at this stage (Figure 2-6 E-G).
Examination of semi-thin (0.5-1 micron) resin embedded mouse tissue sections (results
not shown) and confocal microscopy (Figure 2-7) revealed LYVE-1-positive cells to be
within the mesothelial layer of the epicardium and the subepicardial mesenchyme of the
ED 13.5 mouse embryo heart, as well as in the myocardium.
In the ED 15 mouse heart, the Prox-1-positive lymphatic vessels had begun to
extend from the great vessels down towards the ventricles, while the LYVE-1-positive
cells were present in large numbers on the great vessels, atria and ventricles (Figure 2-8
A, B). Several of the LYVE-1-positive cells appeared to be coalescing into a tubular
network and, in the sections, showed co-localization with Prox-1 staining in the
lymphatics encircling the great vessels (Figure 2-8 C-F). The LYVE-1-positive cells
found on the ventricles and within the myocardium remained Prox-1-negative (Figure 2-
8 E, F).
In the adult mouse heart, the epicardial lymphatics, such as those at the
atrioventricular junction and apex of the heart, stained positive for the four lymphatic
markers: Prox-1, LYVE-1, VEGFR-3 and podoplanin (Figure 2-9 A-H). However, the
myocardial network of vessels and cells expressed only LYVE-1 and VEGFR-3 and were
negative for Prox-1 and podoplanin (Figure 2-9 I-L). The epicardial lymphatic vessels
also expressed the endothelial marker CD31, as did a subset of the myocardial LYVE-1-
64 positive vessels, while other LYVE-1-positive cells within the myocardium were
negative for CD31 (Figure 2-10). Our findings from the mouse studies suggest that there
are three different types of cells/vessels in the heart with lymphatic phenotype (Figure 2-
11).
Lmyphatic markers in epicardial cells in vitro
For comparison, two epicardial cell lines and cultured embryonic epicardial cells
were also analyzed for expression of lymphatic markers. Primary cultures of quail
epicardial cells (QECs) were isolated from hearts at stages when there were no apparent
Prox-1-positive cells found in the epicardium in sections. Some of these isolated cells in culture were QH-1-positive but unexpectedly many expressed Prox-1 in both the nucleus and cytoplasm (Figure 2-12 A-C). An adult rat epicardial cell line (ARECs) was found to express three lymphatic markers: Prox-1, LYVE-1, and VEGFR-3, but not in the subcellular localization reported for adult lymphatic endothelial cells (Figure 2-12 D-F).
Prox-1 was found at low levels homogeneously throughout the cytoplasm of all the cells,
while LYVE-1 staining was present both in the cytoplasm and in a pattern suggesting its
presence in the plasma membranes in the majority of the cells. VEGFR-3 was present in
the perinuclear region in most of the cells. In the embryonic mouse epicardial cells
(EMECs), a subset of cells were observed to express all three lymphatic markers, and the
subcellular localization for each marker was the same as for the ARECs (Figure 2-12 G-
I).
Prox-1 Western Blots
65 Western blot analysis of heart tissue extracts from HH Stage 30 quail and chicken
embryos, post-hatched chickens, ED 15 mouse embryos, postnatal mice, and adult mice
revealed 2 Prox-1-positive protein bands (Figure 2-13 A). In fact, for the embryonic
mouse samples, the two bands of the Prox-1 doublet were equally intense but in the postnatal and adult mice, the upper band grew steadily weaker in intensity in comparison to the lower band. Furthermore, only one Prox-1 band was detected in the extracts from
the ARECs and EMECs (Figure 2-13 A). The single Prox-1 band for the two cell lines
had a molecular weight of 83 kDa and was consistent with the upper band of the doublet
that appeared in the other samples. This difference may be due to the fact that the whole
heart extracts consisted primarily of myocardial tissue, while the cell lines were purely
epicardial cells. Anti-Prox-1 also detected 2 bands in Western blots of lens extracts from
HH Stage 30 quail embryos (Figure 2-13 A). In previous studies, Prox-1 appeared as a
single thick band for lens extracts from other species (Del Rio-Tsonis et al, 1999). This
was also the case in our preliminary trials with the quail lens extract but with further
dilution of the protein concentrations, 2 bands were resolved with the upper band being
very faint. Avian embryonic heart tissue was also treated with an alkaline phosphatase
and analyzed using Western Blotting in comparison to control samples that had not been
exposed to the enzyme. In the dephosphorylated samples, the upper band for Prox-1 was
greatly reduced after 1½ hours of phosphatase treatment (Figure 2-13 B). Srinivasan et al
(1998) found that Prospero, the Drosophila homolog of Prox-1, also had several phosphoisoforms. In Western Blots, the upper band for Prospero (220 kDa) was reduced
significantly with phosphatase treatment, analogous to our findings with Prox-1. We
suggest therefore that Prox-1 has a phosphorylated form, and that phosphorylation of this
66 transcription factor may be associated with its activation or a change in its subcellular
localization and/or function as is the case for Prospero.
Discussion
The three sets of cardiac cells with lymphatic phenotype
Lymphatic markers that are currently used are not completely specific to
lymphatic endothelial cells, thereby complicating efforts to map the lymphatics in various
organs and tissues. For example, in the heart, Prox-1 is found in the lymphatics, as well
as in cardiac myocytes and valvular cells (Wilting et al, 2007 and our studies). According
to Kaipainen et al (1995), VEGFR-3 is found in venous and lymphatic endothelia in the
early mouse embryo. From about ED 14.5 onwards however, VEGFR-3 appeared to be
expressed only in the lymphatic endothelium. Nevertheless, in our studies, we have used
several lymphatic markers (Prox-1, LYVE-1, VEGFR-3 and podoplanin) in the developing and adult mouse heart in order to confirm the specificity of the lymphatic staining.
We detected two sets of cells in the epicardium expressing lymphatic markers.
One group was positive for nuclear-localized Prox-1 and appeared as a sheet of cells in
the serosa of the future aorta and pulmonary trunk that migrated down into the
epicardium of the heart. These Prox-1-positive cells expressed QH-1 in the quail, but did not initially express CD44 in the chick embryo or LYVE-1 (homologous to CD44) in the mouse embryo. Therefore, if the Prox-1-positive cells are originating from the cardinal vein and from the lymph sacs, they have lost LYVE-1 expression. A down-regulation of
LYVE-1 expression in lymphatic precursors has not been previously reported. In the
67 mouse embryo, the Prox-1-positive/LYVE-1-negative cells then organized into a network of lymphatic vessels that eventually became positive for LYVE-1 and maintained their
Prox-1 positivity. Thus, LYVE-1 seems to be up-regulated once these Prox-1-positive cells have organized into vessels. The functional significance of this pattern of LYVE-1 expression is not known.
The other set of epicardial cells that we detected were Prox-1-negative/LYVE-1-
positive cells and were present prior to the appearance of the Prox-1-positive cells
migrating over the aorta and pulmonary trunk into the heart. The Prox-1-negative/LYVE-
1-positive cells were equidistantly scattered over the entire heart except for the distal
portion of the OFT and reminiscent in their location to CD44-positive cells in the chick
embryo heart or to QH-1-positive cells in the quail embryo heart prior to septation.
However, despite the similarity in staining patterns of the epicardial cells for each of
these markers, we cannot at this time conclude that these markers are all expressed by the
same set of cells. At later stages in the mouse (ED 15), we saw Prox-1-positive/LYVE-1-
positive cells that appeared to be organized into a network of vessels at the base of the
heart with Prox-1-negative/LYVE-1-positive cells scattered nearby. In the adult, the
epicardium was populated by cells/vessels expressing both LYVE-1 and nuclear-
localized Prox-1. These findings led us to the question “Does the subset of Prox-1-
negative/LYVE-1-positive cells in the embryonic epicardium get incorporated into the
Prox-1-positive/LYVE-1-positive vessels that we later observed in this layer and become
Prox-1-positive themselves or do they have another fate altogether not related to the
lymphatics?” The LYVE-1-positive cells in the epicardium of the heart may undergo a
fate similar to what was observed in a study conducted by Buttler et al (2006). In their
68 murine model, LYVE-1-positive mesenchymal cells were found to also express the
leukocytic marker CD45. However, once some of these LYVE-1-positive cells were
incorporated into the developing dermal lymphatics, they down-regulated CD45
expression, and up-regulated PECAM and Prox-1 expression. The cells therefore down-
regulated their leukocyte phenotype and up-regulated lymphendothelial characteristics.
Similarly, in the quail chorioallantoic membrane, it appeared that scattered Prox-1-
negative/QH-1-positive cells were incorporated into Prox-1-positive/QH-1-positive
lymphatic vessels and became Prox-1-positive once connected to the vessel (Parsons-
Wingerter et al, 2006).
QH-1 is known to detect quail angioblasts, hemangioblasts, and precursors of the
blood vessel endothelial cells (Reese et al, 2002; Wada et al, 2003; Tomanek, 2005;
Tomanek et al, 2006). QH-1 also labels lymphatic precursors in the chorioallantoic
membrane (CAM) more intensely than it labels blood vascular endothelial cells (Parsons-
Wingerter et al, 2006). This raises the question of how many of the QH-1-positive cells in the embryonic epicardium are precursors of blood vessel endothelial cells and whether any could be destined to become lymphatic endothelial cells. In our studies, the overall pattern of LYVE-1-positive cells in the epicardium of the embryonic mouse heart at ED
13.5 is reminiscent of the pattern of QH-1-positive cells found in quail embryo hearts at
HH Stage 24. These findings raise the possibility that the QH-1-positive cells scattered over the epicardium in the quail embryonic heart represent precursor cells that can become blood or lymphatic endothelial cells. Unfortunately, it is not possible to support this idea using immunohistological methods at this time because QH-1 only recognizes quail cells and currently available LYVE-1 antibodies do not recognize quail LYVE-1.
69 LYVE-1 expression by itself may not indicate that the mouse epicardial cells will become
lymphatic precursors because LYVE-1 is also expressed in the endothelial lining of liver
sinusoids and in vessels within the spleen (Mouta Carreira et al, 2001). The significance
of LYVE-1 expression in epicardial cells requires further study.
A third set of cells noted on the endocardial side of the myocardium at an early
stage in the mouse expressed LYVE-1, VEGFR-3 and PECAM, but not Prox-1 or
podoplanin. Furthermore, cells with similar positive protein expression for LYVE-1 and
VEGFR-3 populate the thickness of the adult myocardium. When the trajectory of these
LYVE-1-positive/VEGFR-3-positive myocardial vessels was followed in serial sections
of the adult mouse heart and reconstructed using 3D imaging software, they were found
to contain red blood cells and were continuous with the ventricular lumen. These vessels also stained positive for the venous endothelial marker EphrinB4. We hypothesize that these vessels may be: (1) a population of lymphatic precursor cells, (2) the “deep lymphatic network” which may have a different gene expression profile compared to the epicardial “superficial lymphatic network”, or (3) a network of Thebesian vessels that have been described as arterioluminal shunts in the heart. The identity and function of these subsets of cells in the embryo and the adult require further investigation.
Thus, it appears that there are three types of cells with lymphatic phenotype in the mouse heart which include: (1) Prox-1-positive cells that migrate into the heart within the
epicardium of the great vessels to eventually express other lymphatic markers such as
LYVE-1, VEGFR-3, and podoplanin; (2) LYVE-1-positive cells in the ventricular
epicardium, a subset of which eventually co-localize with Prox-1 expression in the
developing epicardial lymphatics; and (3) LYVE-1-positive/VEGFR-3 positive cells
70 within the myocardium that remain Prox-1-negative throughout development, perhaps
serving as a ‘pool’ of cells that have not reached terminal lymphatic differentiation but
could potentially be recruited for future lymphangiogenesis.
The lymphangiogenic potential of the epicardium
Surprisingly, both the adult rat epicardial cells (ARECs) and embryonic mouse
epicardial cells (EMECs) expressed three lymphatic markers but in a different subcellular
localization than in mature lymphatics. The mature lymphatics express LYVE-1 and
VEGFR-3 on the cell surface and nuclear-localized Prox-1. Most of the ARECs and
EMECs displayed intense immunostaining for LYVE-1 primarily in the cytoplasm and
VEGFR-3 in the perinuclear region, with low levels of Prox-1 in the cytoplasm. A subset
of cells in primary cultures of quail embryo epicardial cells also exhibited nuclear-
localized Prox-1 expression. We confirmed the presence of Prox-1 in the cultured cells by
Western blot analysis. These findings are intriguing for several reasons. First, the diffuse
pattern and low intensity of the Prox-1 staining in the cell lines raises the possibility that
the protein could be expressed in the same manner in the epicardial cells of the
embryonic quail, chicken and mouse heart but was undetected by immunostaining
because of the weak cytoplasmic staining and the thin profile of these squamous
epithelial cells in histological sections. If we extrapolate from the mouse lens system
(Duncan et al, 2002), where Prox-1 is translocated from the cytoplasm to the nucleus of
the lens fiber during terminal differentiation, then the diffuse cytoplasmic staining of the
epicardial cells may reflect their undifferentiated state. The fact that the ARECs and
EMECs expressed three lymphatic markers albeit at low levels and not in the subcellular
71 location exhibited by mature lymphatic endothelial cells raises the possibility that these
cells may be poised to differentiate into lymphatic endothelial cells, when exposed to an
appropriate stimulus.
Summary
Our findings (summarized in Figure 2-11) indicated that there are three types of
cells with lymphatic phenotype in the developing mouse heart which include: (1) Prox-
1-positive cells that appear to migrate into the heart within the epicardium of the great
vessels to eventually express other lymphatic markers such as LYVE-1, VEGFR-3, and podoplanin, (2) LYVE-1-positive cells in the ventricular epicardium, a subset of which eventually co-localized with Prox-1 expression in the developing epicardial lymphatics,
and (3) LYVE-1-positive/VEGFR-3-positive cells within the myocardium that remained
Prox-1-negative throughout development, perhaps serving as a ‘pool’ of cells that have
not reached terminal lymphatic differentiation but could potentially be recruited for
future lymphangiogenesis. In addition, our epicardial cell lines expressed three lymphatic
markers and thus appear to have some lymphatic potential, so this cell culture model
seems worth pursuing to uncover some of the molecular mechanisms that induce early
steps in epicardial cell differentiation.
Acknowledgements
Confocal microscopy was performed in the Pediatric Imaging Center at Rainbow
Babies & Children’s Hospital in Cleveland, Ohio. We are grateful to Dr. Midori Hitomi with the Department of Pathology at University Hospitals in Cleveland, Ohio, for her
72 help in obtaining the semi-thin sections. We are thankful for Dr. Susann Brady-Kalnay’s advice (Department of Molecular Biology and Microbiology, Case Western Reserve
University, Cleveland, Ohio) for the phosphatase experiments. We also thank Robert
Thompson, Edward Clark, and David Sedmera for stimulating discussion and directing us
to as well as providing us with key papers.
73 Figure 2-1. Lymphatic markers in the early quail embryonic heart. No cells with
nuclear-localized Prox-1 immunostaining (green) were detected on the epicardial surface
of the HH stage 24 heart (A), but QH-1-positive (red) cells were present on the atria, ventricles, and the base of the outflow tract (B). Nuclear-localized Prox-1-positive/QH-1- positive cells on the great vessels first appeared in the embryonic heart at HH Stage 26
(C, D). No Prox-1-positive cells were detected on the ventricular and atrial surfaces of the heart at this stage (not shown). At = atrium, OFT = outflow tract, V = ventricle. Bars
= 100 um.
74
75 Figure 2-2. Lymphatic markers in the septated quail embryonic heart. Prox-1- positive (green)/QH-1-positive (red) lymphatic vessels began to form a branching network over the great vessels (A) and ventricles (B) in the HH Stage 29-30 heart. The individual QH-1-positive (red) cells (A, arrow) adjacent to the lymphatic vessels immunostained with a greater intensity than cells within the vessels. Clusters of Prox-1- positive cells (C, arrow) were observed along lymphatic vessels of the HH Stage 38 heart. At this stage, lymphatic vessels (D, white arrow) were also found on the posterior aspect of the heart surrounding major blood vessels (D, red arrow). Bars = 100 um.
76
77 Figure 2-3. Diagram of the progressive development of the Prox-1-positive
lymphatic network in the avian embryonic heart from HH Stage 24 to 38. Green
coloring represents Prox-1-positive cells and vessels. Dotted pink lines indicate presumed
pathways for the development of the ventricular lymphatics at HH Stage 38. The quail
epicardium becomes opaque at later stages and obscures Prox-1 staining. For reference,
septation of the heart is complete by HH Stage 30 and the coronary arteries connect to the aortic lumen by HH Stage 32.
78
79 Figure 2-4. Prox-1 immunofluorescence in quail heart sections. At HH Stage 24, nuclear-localized Prox-1 expression was found in the ventricular myocardium (A) and the endocardial cushions (B). At HH Stage 30, Prox-1 was also found in the cells lining the various cardiac valves, including the aortic valve (C). Bars = 100 um.
80
81 Figure 2-5. Lymphatic markers in the ED 9.5 mouse heart. (A) Prox-1 was found in
cardiac myocytes in the ventricle while LYVE-1 (B) and VEGFR-3 (C) were expressed
in cells/vessels on the endocardial side of the myocardium at the site of the future
interventricular septum. (D) is a high magnification view of the Prox-1-positive cardiac
myocytes in the boxed region in (A). Sections in A-C were 7 microns thick and
immediately adjacent to each other. The LYVE-1-positive cells/vessels did not co-
localize with the cardiac myocyte marker MF20 (E, F) or with the lymphatic marker
podoplanin (G, H), but they did stain for the endothelial cell marker CD31/PECAM (I-
K). The overlay image (K) for the LYVE-1 and CD31 staining revealed that only a
subset of the CD31-positive cells were LYVE-1-positive. Double-positive cells are
yellow. Bars = 100 um.
82
83 Figure 2-6. Prox-1 and LYVE-1/CD44 were initially not co-localized in the mouse or
chick embryo heart. (A) In the ED 13.5 mouse heart, Prox-1 (green) was expressed in
the lymphatic vessels found between the aorta and pulmonary trunk while LYVE-1
(green; homologous to CD44) was found in epicardial cells on the great vessels (B), a
myocardial network of vessels (C), as well as in cells in the ventricular epicardium (D).
Cardiac myocytes were stained for MF20 (red) in (B-D). In the HH Stage 30 chick heart,
CD44 (LYVE-1 homolog) was also expressed in epicardial cells (E, green) close to Prox-
1-positive cells (red) on the great vessels, cells in the myocardium (F), and ventricular epicardial cells (G). In chick sections at this stage, Prox-1 (red) was found in the lymphatics and cardiac myocytes and did not co-localize with CD44 staining (E-G). Ao = aorta, PT = pulmonary trunk. Bars = 100 um.
84
85 Figure 2-7. Confocal microscopy of LYVE-1-positive cells (green) in the ED 13.5 mouse heart (441X magnification). Cardiac myocytes were stained for MF20 (red) and
DAPI (blue) was used for nuclear visualization. LYVE-1 was expressed in (A) mesothelial cells of the epicardium (arrowhead), and cells within the (B) subepicardial mesenchyme and (C) myocardium. Note that the cell indicated in (C) is LYVE-1-positive but MF20-negative. EM = epicardial mesothelium, Myo = myocardium, SM = subepicardial mesenchyme.
86
87 Figure 2-8. Lymphatic markers in the ED 15 mouse heart. (A) In the immunostained whole mount hearts, Prox-1 was expressed in lymphatic vessels along the great vessels and the base of the heart. (B) LYVE-1-positive cells (white arrow) were found on the great vessels, atria and ventricles with some cells arranged in a network (black arrow).
Immunostained sections revealed co-localization of Prox-1 (C) and LYVE-1 (D) staining in the lymphatics on the great vessels but individual LYVE-1-positive cells at the atrioventricular junction (F, white arrowhead) did not stain for Prox-1 (E). (C) and (D) are alternate sections; (E) and (F) are alternate sections. Arrows in (C) and (D) indicate the same region, and arrows in (E) and (F) indicate the same region. At = atrium, PT = pulmonary trunk, V = ventricle. Bars = 100 um.
88
89 Figure 2-9. Lymphatic markers in the adult mouse heart. (A-D) Prox-1, VEGFR-3,
LYVE-1, and podoplanin were expressed in the lymphatic vessels (arrows) at the atrio- ventricular junction of the adult mouse heart (frontal sections). (E-H) All four lymphatic markers were also expressed in the epicardial lymphatic vessels covering the ventricles
(arrows). (I-L) Prox-1 and podoplanin were not expressed in the VEGFR-3- positive/LYVE-1-positive cells and vessels (arrows) found within the ventricular myocardium. V = ventricle. Bars = 100 um.
90
91 Figure 2-10. LYVE-1 and CD31/PECAM co-localization in adult mouse heart sections. In the adult mouse heart, LYVE-1 and CD31 co-stained the epicardial lymphatics (A, arrows) at the apex of the heart and vessels within the ventricular myocardium (B, arrow). There were also cells in the myocardium that were positive for
LYVE-1 expression but did not stain for CD31 (A, arrowhead). Bar = 100 um.
92
93 Figure 2-11. Diagram of the three different types of cells/vessels with lymphatic phenotype in the mouse heart arranged according to location. These include (1) Prox-
1-positive cells migrating into the heart via the great vessels, (2) LYVE-1-positive cells already present in the epicardium of the heart, and (3) LYVE-1-positive cells found within the myocardium.
94
95 Figure 2-12. Lymphatic markers in vitro. (A) Prox-1 expression assessed by immunostaining in quail epicardial cells (QECs) which were also labeled for QH-1 (B)
and mounted with DAPI for nuclear visualization (C). (A-C) are the same field of view.
(D-F) Expression of lymphatic markers (green) in the adult rat epicardial cells (ARECs).
Inserts in (E, F) are confocal images of VEGFR-3 and LYVE-1-expression in the
ARECs. (G-I) Expression of lymphatic markers (red) in the embryonic mouse epicardial
cells (EMECs). DAPI = blue staining. Bar = 100 um.
96
97 Figure 2-13. Prox-1 Western Blots. (A) Samples included HH Stage 30 quail lens extract (lane 1), HH Stage 30 quail embryonic hearts (lane 2), HH Stage 30 chicken embryonic hearts (lane 3), post-hatched chicken hearts (lane 4), postnatal mouse hearts
(lane 5), ED 15 mouse embryo hearts (lane 6), adult mouse heart tissue (lane 7), adult rat epicardial cells (lane 8), and embryonic mouse epicardial cells (lane 9). (B) Control HH
Stage 30 quail heart samples (lane 1) were run alongside dephosphorylated samples (lane
2) for a Western Blot. The intensity of the upper band of the Prox-1 doublet was greatly reduced with phosphatase treatment.
98
99 CHAPTER 3: ACTIVATION OF EPICARDIAL CELLS BY VEGF-C SIGNALING
Ganga Karunamuni1,2, Shi Gu3, Yong Qiu Doughman1, David Bader4, Peter Scacheri5 and Michiko Watanabe1,2,5
Departments of Pediatrics1 and Anatomy2, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106
Department of Biomedical Engineering3, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106
Cell and Developmental Biology4, Vanderbilt University Medical Center, 21st Avenue South and Medical Center Drive, Nashville, TN 37232
Department of Genetics5, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106
Abstract
The proepicardium and epicardium of the developing heart have been shown to
give rise to different cardiac cell types, including fibroblasts, smooth muscle cells, vascular endothelial cells and even cardiomyocytes (reviewed in Cai et al, 2008). Recent
evidence has suggested that the adult epicardium may also contribute cells to these
lineages (Smart et al, 2008). Understanding how these multipotent cells can be activated
to differentiate into a particular cell lineage would be helpful in developing cardiac cell
replacement therapies in cases of heart disease.
We previously demonstrated that adult and embryonic epicardial cell
lines/primary cultures express the lymphatic markers Prox-1, LYVE-1, and VEGFR-3 in
a subset of cells, although not in the same subcellular compartments as mature lymphatic
endothelial cells. In this study, we observed that in WT1-cre mice, epicardial mesothelial
cells and their descendants express lymphatic markers in situ. We also explored the
100 mechanisms that push pluripotent epicardial cells into the lymphatic phenotype. An
understanding of the molecular basis of cardiac lymphangiogenesis could be used to
design therapies aimed at controlling cardiac lymphedema, cancer metastasis and similar
pathological conditions. Explanted embryonic epicardial cells were treated with the
lymphangiogenic growth factor VEGF-C, resulting in an accumulation of Prox-1 in the
nuclei of a subset of cells. The MEK inhibitor UO126 that inhibits the
phosphorylation/activation of ERK reduced Prox-1 expression in nuclei in VEGF-C treated explanted epicardial cells. Similarly, when adult rat epicardial cells (ARECs)
were treated with VEGF-C, a dramatic increase in nuclear Prox-1 and phosphorylated
ERK expression was observed in a subset of cells. This accumulation of nuclear Prox-1
in the ARECs was not induced after FGF or VEGF-A treatment, and was also blocked by
the MEK/ERK inhibitor. Our studies suggest that the lymphangiogenic growth factor
VEGF-C binds to VEGFR-3 and acts via phosphorylated ERK to stimulate the
accumulation of Prox-1, considered to be a “master lymphangiogenic gene”, in the nuclei
of epicardial cells regardless of whether they are adult or embryonic in nature.
101 Introduction
The VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) mediated
signaling pathway regulates lymphangiogenesis at many levels. The growth factors
VEGF-C (Vascular Endothelial Growth Factor C) and VEGF-D (Vascular Endothelial
Growth Factor D) bind to the VEGFR-3/Flt-4 receptor (Joukov et al, 1996; Achen et al,
1998) found on lymphatic endothelial cells (Kaipainen et al, 1995), and stimulate the
growth of new lymphatic vessels from pre-existing ones (Oh et al, 1997; Veikkola et al,
2001). VEGFR-3 activation results in the protein kinase C-dependent activation of Ras,
Raf, and MEK, thereby leading to the phosphorylation and activation of extracellular
signal-related kinases (ERK)-1 and -2, which are implicated in cell proliferation.
VEGFR-3 also mediates the activation of protein kinase B/Akt, which is linked to cell
survival (Makinen et al, 2001). Therefore, VEGFR-3 is considered to transduce signals
resulting in proliferation, migration, and survival of lymphatic endothelial cells (Makinen
et al, 2001).
Lymphedema refers to the accumulation of protein-rich fluid or lymph in the interstitium as a result of lymphatic dysfunction, destruction or blockage, and can be hereditary or acquired. Hereditary lymphedema (Milroy’s disease) is an autosomal dominant disorder (Milroy, 1892; Ferrell et al, 1998) that results primarily from inactivating missense mutations in the kinase domain of the VEGFR-3 receptor
(Karkkainen et al, 2000; Irrthum et al, 2000). In the Chy mouse, a genetic model of
Milroy’s disease involving a heterozygous inactivating VEGFR-3 mutation, cutaneous lymphedema regressed with the adeno-associated delivery of VEGF-C (Karkkainen et al,
2001). The delivery of plasmid DNA coding for human VEGF-C was also effective in
102 treating lymphedema in mouse and rabbit models of acquired lymphedema (Yoon et al,
2003). In VEGF-C knockout mice, the lymphatic system failed to develop; however, this phenotype was rescued through the application of recombinant VEGF-C or VEGF-D, both of which bind to VEGFR-3. Thus, manipulating the VEGFR-3 pathway has obvious therapeutic potential. However, this signaling cascade has also been linked to tumor lymphangiogenesis and metastasis (Karpanen et al, 2001; Skobe et al, 2001; He et al,
2005). Metastatic events have been shown to be reduced after the introduction of blocking antibodies that inhibit VEGFR-3 activation (He et al, 2002; Roberts et al, 2006).
It would appear therefore that exquisite regulation of VEGFR-3 signaling is required for normal lymphatic development. The downstream consequences of VEGF-C activation are not well characterized beyond the identification of signaling cascades in lymphatic endothelial cells.
Prox-1 (Prospero homeobox protein 1), a nuclear-localized transcription factor, has been identified as critical for lymphangiogenesis. Experimentally forced expression of Prox-1 in blood vascular endothelial cells was shown to induce the transcription of lymphatic-specific genes like VEGFR-3 and suppress the expression of blood-specific genes, thereby resulting in a more lymphatic phenotype (Hong et al, 2002; Petrova et al,
2002). Furthermore, Prox-1-null mice lack a lymphatic system and die at ED 14.5 (Wigle et al, 1999), leading many researchers to consider Prox-1 as a candidate for the lymphangiogenic “master gene”. Regulation of Prox-1 expression has been investigated to some extent (Srinivasan et al, 2000). However, the link between the lymphangiogenic
VEGFR-3 signaling pathway and Prox-1 has not yet been elucidated in the lymphatics, including those of the heart.
103 We have previously shown that the adult mouse heart is invested with an
extensive network of epicardial lymphatic vessels which express Prox-1 and are also
labeled with VEGFR-3, LYVE-1 (Lymphatic vessel endothelial hyaluronan receptor),
and podoplanin antibodies (Karunamuni et al, 2010). In addition, Lavine et al (2006) have demonstrated that VEGF-C was expressed in a punctuate pattern in the epicardium of the developing mouse heart, from which the authors proposed that VEGF-C is most
likely secreted by perivascular cells. VEGFR-3 expression was also observed in
epicardial cells from ED 13.5 onwards in mice (Lavine et al, 2006). Therefore, the
VEGFR-3 pathway could potentially regulate lymphatic development in the epicardium.
We used an embryonic epicardial explant model and an adult rat epicardial cell line
(ARECs; Wada et al, 2003), that expresses the lymphatic markers Prox-1, LYVE-1, and
VEGFR-3 (Karunamuni et al, 2010), in order to investigate the interaction between Prox-
1 and VEGFR-3 signaling. A molecular understanding of the lymphangiogenic process
and the lymphangiogenic potential of the epicardium could lead to the development of
therapies to initiate, enhance or inhibit lymphangiogenesis in the heart and elsewhere.
Materials and Methods
Cell line
The adult rat epicardial cell line [34] was obtained from Dr. David Bader (Vanderbilt
University, TN). The adult rat epicardial cells (ARECs) were maintained in culture at
37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) media with sodium
pyruvate (Mediatech Inc., Manassas, VA) and supplemented with 10% fetal bovine
serum (FBS; Invitrogen, Carlsbad, CA).
104
Animals
Quail (Coturnix coturnix japonica; Boyd’s Bird Company, Pullman, WA) embryos were incubated in an egg incubator (GQF Mfg Co., Savannah, GA) with a rocking apparatus and dissected out at Hamburger-Hamilton (HH) Stages 24-30 [46].
Antibodies
The antibodies against Prox-1 (polyclonal rabbit anti-human, 1:200; Angio-Proteomie,
Boston, MA), LYVE-1 (polyclonal rabbit anti-mouse, 1:100; Angio-Proteomie, Boston,
MA), Cy3-conjugated alpha smooth muscle actin (mouse monoclonal, 1:200; Sigma-
Aldrich, St. Louis, MO), and phospho-p44/42 MAPK (Erk1/2) (polyclonal rabbit anti- human, 1:50; Cell Signaling Technology, Danvers, MA) were used according to the manufacturer’s protocol.
Phalloidin
Alexa Fluor 488 phalloidin (1:40; Invitrogen, Carlsbad, CA) is a high-affinity probe for
F-actin that is made from a mushroom toxin conjugated with the bright, photostable, green-fluorescent Alexa Fluor 488 dye. The phalloidin was used according to the manufacturer’s protocol.
WT1-Cre mice
A BAC recombineering strategy was used to insert an IRES/EGFP-CRE cassette into the
3’ UTR of a full length mouse WT1 gene (Wilm et al, 2005). The resultant recombinant
105 BAC clone was used to generate several independent transgenic mouse lines that express
Cre in the epicardial lineage beginning at the proepicardial stage. Embryos were used from timed matings between WT280Cre transgenic males (referred to as WT1-Cre
animals) and Rosa26R (R26R) homozygous females, or ICR wild-type mice, where the
day of plug is E0.5. LacZ-stained sections of ED 16.5 mice were kindly sent to us by Dr.
Andy Wessels (Medical University of South Carolina, SC). The mouse line was
originally generated by Dr. John Burch. Paraffin-embedded sections were dewaxed,
rehydrated, and microwaved for 15 mins in sodium citrate buffer (pH 6) for antigen retrieval. Sections were then washed in phosphate-buffered saline (PBS), blocked with
Triton-X buffer for 30 mins, and incubated with the primary antibodies Prox-1 and
LYVE-1 overnight. After being incubated with the appropriate fluorescent secondary
antibodies (Molecular Probes, Eugene, OR), the slides were mounted with DAPI for
nuclear visualization, and examined under the Nikon DIAPHOT 200 fluorescence microscope. Images were captured with the digital camera and QCapture Pro software.
Growth Factors
The ARECs were seeded onto poly-L-lysine-coated coverslips and left in culture medium
for 24 hrs. Cells were serum-starved for another 24 hrs, and then treated with basic FGF
(40 ng/ml; Peprotech, Rocky Hill, NJ) or VEGF-C (200 ng/ml; GenWay Biotech, San
Diego, CA) or VEGF-A (25 ng/ml; Peprotech, Rocky Hill, NJ) for 30 minutes-24 hrs in serum-free media. Coverslips were rinsed in PBS, fixed in 4% paraformaldehyde, and stained for Prox-1, phalloidin, alpha smooth muscle actin, or phosphorylated ERK. The coverslips were then mounted with DAPI for nuclear visualization, and examined under
106 the Nikon DIAPHOT 200 fluorescence microscope. Images were captured with the
digital camera and QCapture Pro software.
UO126 Inhibitor
The MEK inhibitor UO126 (Cell Signaling Technology, Inc; Danvers, MA) was
dissolved in DMSO and aliquots were stored at -20°C. This inhibitor has been shown to
be a highly selective inhibitor of MEK 1 and MEK 2. For the inhibitor studies, ARECs
were seeded onto poly-L-lysine-coated coverslips and left in culture medium for 24 hrs,
and cells were serum-starved for another 24 hrs. Cells were then pre-treated with DMSO
or UO126 for 2 hrs and subsequently subjected to the 4 following treatments for 30 mins:
(1) DMSO as a negative vehicle-only control, (2) 10 uM UO126, (3) DMSO + 200 ng/ml
VEGF-C, and (4) 10 uM UO126 + 200 ng/ml VEGF-C. Coverslips were rinsed in PBS, fixed in 4% paraformaldehyde, and stained for Prox-1, Cy3-conjugated alpha smooth muscle actin or Alexa 488 phalloidin. The coverslips were then mounted with DAPI for nuclear visualization, and examined under the Nikon DIAPHOT 200 fluorescence microscope. Images were captured with the digital camera and QCapture Pro software.
Quail Heart Explants
Stage 24 quail hearts were chosen since no Prox-1-positive cells were seen in the
epicardium at this stage in either whole mounts or sections. After harvesting, control
hearts were placed in serum-free media while experimental hearts were placed in serum-
free media supplemented with 200 ng/ml of VEGF-C for 30 mins (at 37˚C). Hearts were
then fixed and stained for Prox-1. Stage 24 quail hearts were also placed on collagen-
107 coated coverslips for 1 hr to allow the epicardial cells to grow out onto the matrix. Hearts
were then removed, and cells were treated with media supplemented with 200 ng/ml of
VEGF-C for 30 mins either in the absence or presence of the MEK inhibitor UO126 (in a
37˚C, 5% CO2 incubator). Cells were then fixed and stained for Prox-1. In separate
experiments, the VEGFR-3 inhibitor MAZ51 (EMD Biosciences, NJ) was applied to HH
Stage 27 quail hearts in vivo for 24 hrs. Hearts were then harvested at HH Stage 30 and stained for Prox-1.
Results
Lymphatic progenitors are present in the epicardium
The WT1-Cre transgenic mouse line (Wilm et al, 2005; see Materials and
Methods) has been used previously to lineage label mesothelial cells during development.
The Wilms tumor protein (WT1) has been identified as an expression marker of the
serosal mesothelium, including the epicardial mesothelium of the heart (Armstrong et al,
1993; Moore et al, 1998; Perez-Pomares et al, 1998; Carmona et al, 2001). In other
studies, mesothelial cells have been shown to give rise to vascular smooth muscle cells in the developing lung and gut (Wilm et al, 2005; Que et al, 2008). We obtained sections of
ED 16.5 WT1-Cre mice where cells labeled with LacZ were denoted as descendants of
WT1-expressing cells in the epicardial mesothelium. Certain LacZ-labeled cells co- stained for the lymphatic marker Prox-1 in epicardial lymphatic endothelial cells at the atrio-ventricular junction (Figure 3-1). A subpopulation of LacZ-labeled cells also stained for another lymphatic marker LYVE-1 in individual mesenchymal cells located in the atria, the atrio-ventricular junction, and the pericardium (Figure 3-2). This suggests
108 that at least a subset of endothelial cells within the cardiac lymphatic vessels and mesenchymal lymphangioblasts are derived from mesothelial progenitors in the embryonic epicardium.
VEGF-C induced Prox-1 nuclear accumulation ex vivo via the VEGFR-3 pathway
In a previous study, we did not detect nuclear-localized Prox-1 in the epicardium of the HH (Hamburger and Hamilton) stage 24 quail heart, which is a few stages prior to the appearance of Prox-1-positive cells on the epicardium of the distal outflow tract
(Karunamuni et al, 2010). However, explanted quail hearts at this stage that were treated with the lymphangiogenic growth factor VEGF-C for 30 mins expressed Prox-1 in the nuclei of a subset of cells in the epicardium at the atrio-ventricular junction, while cells in similar locations in control hearts remained Prox-1-negative (Figure 3-3 A-D). Prox-1 nuclear accumulation was also induced by incubation with VEGF-C in primary cultures of epicardial cells grown out from HH stage 24 quail hearts onto collagen-coated coverslips (Figure 3-3 E-F). We then tested whether the effect of VEGF-C in this context is mediated by the VEGFR-3 signaling pathway. The VEGFR-3 inhibitor MAZ51
(EMD Biosciences, NJ) was applied to quail hearts in vivo which were then harvested at
HH Stage 30, when there is typically an extensive branching lymphatic network already covering the heart. The VEGFR-3 inhibitor was found to induce a delay in the migration and development of the Prox-1-positive epicardial lymphatics (Figure 3-4). These findings suggest therefore that the activation of VEGFR-3 via VEGF-C is necessary for
Prox-1 function in epicardial cells.
109 VEGF-C specifically induced Prox-1 nuclear accumulation in the adult epicardium
An adult rat epicardial cell line was utilized for more extensive analysis because it
has characteristics of embryonic epicardial cells and is more accessible to manipulation in vitro. The cell line was derived from a rat epicardial mesothelioma, and has been shown to have properties of embryonic epicardial cells (Eid et al, 1992; Eid et al, 1994; Wada et al, 2003). When left untreated, the adult rat epicardial cells (ARECs) formed a monolayer after 1-2 days and did not express the phenotype of smooth muscle or mesenchymal cells, as demonstrated by staining for alpha smooth muscle actin or fluorescently-labeled phalloidin antibodies respectively (Figure 3-5 A-B). Phosphorylated (activated) ERK was found mainly in the cytoplasm of the cells, which also exhibited a low level of cytoplasmic and nuclear Prox-1 (Figure 3-5 C-D). The cells were first treated with basic
FGF, since FGF signaling has been shown to be important for epicardial development or
differentiation (van Wijk et al, 2009). When treated with FGF for 24 hrs, several smooth
muscle cells were observed (Figure 3-5 E). Some of the cells appeared to detach from
the monolayer and developed stress fibers (Figure 3-5 F), thus adopting a more
migratory phenotype, as was previously shown by Bader and his colleagues (Wada et al,
2003). Phosphorylated ERK expression was observed in the nuclei of the cells, while
Prox-1 expression remained primarily in the cytoplasm (Figure 3-5 G-H).
In contrast, when treated with the lymphangiogenic growth factor VEGF-C for 24
hrs, only a few smooth muscle cells were detected (Figure 3-5 I). The cells developed
stress fibers and short, fine filopodia-like structures at the cell boundaries which were
extended towards each other and the surrounding environment (Figure 3-5 J). In
addition, both phosphorylated ERK and Prox-1 expression were observed in the nuclei of
110 the cells (Figure 3-5 K-L). Further analysis revealed that a subset of the cells became
positive for Prox-1 nuclear expression after just 30 mins of VEGF-C treatment (Figure 3-
6). The frequency of cells with Prox-1 nuclear accumulation was approximately 17% at 5
mins, increasing to approximately 71% by 30 mins and remaining high (approximately
51%) after 24 hrs of VEGF-C treatment (Table 3-1). To test the specificity of this
response, we exposed the cells to VEGF-A, another growth factor that is known to be
expressed in the environment of the embryonic epicardial cells. When treated with the
angiogenic growth factor VEGF-A for 24 hrs, the ARECs developed stress fibers and
filopodia, and several smooth muscle cells were also seen (Figure 3-5 M-N). As with
FGF, phosphorylated ERK expression was found in a subset of cell nuclei, but Prox-1 expression remained primarily cytoplasmic (Figure 3-5 O-P).
In general, with the addition of growth factors, a subset of the adult epicardial cells expressed both cytoplasmic and nuclear phosphorylated ERK and appeared to
differentiate into other lineages, corroborating other studies where ERK activity in both
the nucleus and cytoplasm was associated with cellular differentiation (Marshall, 1995).
These initial findings also suggest that after VEGF-C stimulation, the ERK signaling
pathway may be involved in the nuclear localization of Prox-1 in the adult epicardial cells
by rapidly inducing its concentration in the cell nuclei in a growth factor-specific event.
Our immunohistology findings regarding Prox-1 nuclear accumulation was supported by
Western Blot data for Prox-1 in the absence and presence of VEGF-C (Figure 3-7). Prox-
1 mRNA levels were also similar in cells without and with VEGF-C stimulation
according to real-time PCR analysis, indicating that the nuclear accumulation event is not
likely due to an increase in Prox-1 transcription. Prox-1 nuclear accumulation was also
111 seen in the ARECS after they were treated with Leptomycin B (LC Laboratories;
Woburn, MA), a nuclear export inhibitor that selectively inhibits the Exportin-1 pathway
(Figure 3-8). This is consistent with the findings of Demidenko et al (2001) who reported that Prospero, the Drosophila homolog of Prox-1, was similarly transported out of the nucleus via the Exportin-1 pathway. Nuclear export of Prox-1 is one of several potential pathways that could be regulated by VEGF-C signaling in the epicardial cells.
The MEK inhibitor UO126 blocked Prox-1 nuclear accumulation both in vitro and ex
vivo
The MEK inhibitor UO126 (Cell Signaling Technology, Inc; Danvers, MA)
prevents the phosphorylation of serine residues in MEK 1/2 which normally occurs
downstream of receptor tyrosine kinase stimulation. MEK is hence not available to
phosphorylate and thereby activate ERK. Treating the ARECs with UO126 for 30 mins
caused a reduction in Prox-1 expression in the cell nuclei below baseline (vehicle only),
even in the presence of the lymphangiogenic growth factor VEGF-C (Figure 3-9 A-D).
The UO126 and VEGF-C-treated ARECs also did not display any smooth muscle
staining and although the cells managed to migrate away from the monolayer, their
cytoskeletal organization appeared to have been compromised as indicated by phalloidin
labeling, compared to the cells that were not treated with the MEK inhibitor (Figure 3-
10). Similar results were obtained when explanted quail embryonic epicardial cells were
treated with VEGF-C either in the absence or presence of UO126. In this case, Prox-1
expression was primarily found in specific regions within the explanted epicardial cell
nuclei (Figure 3-9 E) as opposed to the ARECs, where Prox-1 was homogenously
112 localized throughout the cell nuclei after VEGF-C stimulation. With the addition of the
MEK inhibitor UO126, Prox-1 expression was almost completely eliminated from the explanted epicardial cell nuclei (Figure 3-9 F). These findings suggest that phosphorylated ERK, which is activated upon stimulation of receptor tyrosine kinase signaling pathways such as the VEGF-C/VEGFR-3 pathway, is required for the accumulation of Prox-1 in the adult or embryonic epicardial cell nucleus and its inhibition can detrimentally affect cell migration and differentiation.
Discussion
Our studies suggest that the lymphangiogenic growth factor VEGF-C binds to
VEGFR-3 and acts via the ERK pathway to rapidly stimulate the accumulation of Prox-1, considered to be a “master lymphangiogenic gene”, in the nuclei of both adult and embryonic epicardial cells. The inhibition of ERK activation with UO126, even in the presence of VEGF-C, induced a dramatic decrease in Prox-1 expression in the cell nuclei, giving credence to the theory that ERK may directly or indirectly regulate Prox-1 activation and transcriptional function in the epicardium.
The relationship between the ERK pathway and lymphangiogenesis is supported by the finding that human syndromes involving mutations in the Ras/Raf/MEK/ERK pathway are often associated with lymphatic disorders. For example, Noonan Syndrome, characterized by short stature, congenital heart disease, and developmental delay
(Allanson, 1987), has been linked to mutations in the Raf1 gene which can account for 3-
17% of all cases (Pandit et al, 2007). Lymphatic vessel dysplasia, hypoplasia, or aplasia are found in approximately 20% of Noonan Syndrome patients. They lead to generalized
113 lymphedema, peripheral lymphedema, pulmonary lymphangiectasia or intestinal
lymphangiectasia, with the most common manifestation being dorsal limb lymphedema
(Witt et al, 1987). Various degrees of edema can also be present in the fetus, leading to
the disruption of normal tissue migration or organ placement, and resulting in
cryptorchidism, wide-spaced nipples, low-set posteriorly rotated ears, hypertelorism and downward slanting palpebral fissures (Nisbet et al, 1999). Similarly, cardiofaciocutaneous (CFC) syndrome, characterized by a distinctive craniofacial appearance as well as cardiac and cutaneous abnormalities (Roberts et al, 2006), has been linked to MEK mutations which have been reported in about 10-15% of afflicted individuals (Rodriguez-Viciana et al, 2006). Lymphedema and chylothorax (the
accumulation of lymph and emulsified fat in the thoracic cavity) are common findings in
CFC patients. Thus, mutations in Raf and MEK which ultimately overactivate ERK
downstream in the signaling cascade have been linked to lymphatic dysfunction. It has
not been tested whether these problems are due to the misregulation of the lymphatic
gene Prox-1 by the overactivation of ERK.
Our studies show that activated or phosphorylated ERK is required for the nuclear
accumulation of the lymphatic “master regulatory gene” Prox-1 in the epicardium after
stimulation of receptor tyrosine kinase pathways via VEGF-C signaling. One theory is
that Prox-1 may be phosphorylated by ERK, thus inducing its transport into the epicardial
cell nucleus. A serine or threonine followed by a proline (S/TP) has been suggested as the
minimal consensus sequence for ERK (Davis, 1993; Songyang et al, 1996). In the case of
Prox-1, in its C-terminus which consists of the homeodomain and prospero domain,
serine 569 is followed by a proline, a site which has a 99% chance prediction of being
114 phosphorylated according to the phosphorylation prediction site software NetPhos
(Technical University of Denmark). It is likely therefore that this sequence could be the
target of ERK phosphorylation, thereby inducing the nuclear transport of Prox-1 in the
epicardium. Alternatively, the nuclear export of Prox-1 may also be regulated and
perhaps inhibited by VEGF-C signaling and/or ERK activation, since our studies have
demonstrated that inhibiting the Exportin-1 pathway via Leptomycin B can result in the
nuclear accumulation of Prox-1 as well. In addition, there remains the possibility that the
Prox-1 antibody, which has not been well characterized, is more reactive against the
activated form of Prox-1, such as in cases of VEGF-C stimulation, versus its normal
form. This question would warrant further investigation in future studies.
As has been shown in our studies, the adult epicardial cells appeared to retain
characteristics of the embryonic epicardium under conditions of both lymphangiogenic
growth factor stimulation as well as lymphatic signaling pathway inhibition. The embryonic epicardium has been shown to be a multipotent source of stem cells in the heart, giving rise to fibroblasts and smooth muscle cells. It has also been proposed to give rise to endothelial cells and, though controversial, to cardiomyocytes. We have shown that in addition to the above mentioned cell types, embryonic epicardial cells can give rise to a subpopulation of lymphatic endothelial cells as well. The adult epicardium may also potentially induce lymphatic cell differentiation through the activated VEGFR-
3/ERK signaling pathway by altering the subcellular location and the transcriptional activity of the lymphatic regulatory gene Prox-1.
Summary
115 We have made several interesting observations during the course of this study.
First, in a novel finding, lineage tracing experiments revealed that the epicardium gives rise to a subset of lymphatic endothelial cells in the developing heart. Another novel finding was that the lymphangiogenic growth factor VEGF-C stimulated the nuclear accumulation of the lymphatic transcription factor Prox-1 in both adult epicardial cell lines and explanted embryonic epicardial cells in a growth factor-specific event. It was demonstrated previously that Prox-1 is necessary for VEGFR-3 expression but until now, it was not known that VEGFR-3 was also necessary for proper localization of Prox-1.
Finally, the recruitment of phosphorylated ERK is important for Prox-1 nuclear accumulation in epicardial cells, irrespective of whether they are adult or embryonic in nature, and its inhibition can significantly affect epicardial cell migration and differentiation. Thus, we have highlighted certain essential steps in the epicardial cell differentiation pathway towards a more lymphatic lineage. Understanding the mechanisms that control the differentiation of multipotent epicardial cells in situ may be of value in devising therapies for a broad range of diseases.
Acknowledgements
The authors would like to thank Dr. John B. E. Burch (NIH) for creating the mouse lines,
including the one used in this study, and generously providing them to so many
investigators. We also thank Dr. Andy Wessels (Medical University of South Carolina)
for sending us lacZ stained sections of the WT1-cre/ROSA26 mouse embryo. We are
grateful to Dr. Thomas Hering (University of Kentucky) and Dr. Gary Landreth (Case
Western Reserve University) for advising us throughout the study.
116 Figure 3-1. Prox-1 staining in ED 16.5 sections of WT1-Cre/R26R mice. Prox-1-
positive cells (green) in the endothelial lining of lymphatic vessels at the atrioventricular
junction (A-C, arrows) were stained with X-gal by the presence of LacZ (blue in bright
field pictures) (D-F, arrows) in the WT1-Cre/R26R mice. Blue fluorescence staining in images A-C = DAPI. AVJ = Atrioventricular junction. Bars = 100 um.
117
118 Figure 3-2. LYVE-1 staining in ED 16.5 sections of WT1-Cre/R26R mice. Individual
LYVE-1-positive cells (green) that were found in the pericardium, on the atrium, and at the atrioventricular junction (A-D, arrows) also stained for lacZ (E-H, arrows) in the
WT1-Cre/R26R mice. Blue staining in images A-D = DAPI. AVJ = Atrioventricular junction. Bars = 100 um.
119
120 Figure 3-3. VEGF-C treatment in explanted HH Stage 24 quail hearts for 30 mins.
(A, B) Epicardial cells in untreated control hearts were Prox-1-negative. (C, D) Prox-1 nuclear expression was seen in a subset of epicardial cells at the atrio-ventricular junction after VEGF-C exposure. (E) Explanted quail embryonic epicardial cells in controls were
Prox-1-negative. (F) Prox-1 nuclear expression was observed in the explanted epicardial cells after VEGF-C exposure. At = atrium, OFT = outflow tract, V = ventricle. Bars =
100 um.
121
122 Figure 3-4. The effects of inhibiting VEGFR-3 activation in quail hearts in vivo. (A)
Prox-1-positive epicardial lymphatics in HH Stage 30 untreated quail heart. (B)
Reduction in growth and branching (arrow) for Prox-1-positive vessels in quail heart treated with the VEGFR-3 inhibitor MAZ51 (5 uM; EMD Biosciences, NJ) for 24 hrs.
Ao = aorta, LA = left atrium, PT = pulmonary trunk, RA = right atrium. Bar = 100 um.
123
124 Figure 3-5. Adult rat epicardial cells (ARECs) with FGF, VEGF-C and VEGF-A
treatment for 24 hrs. Untreated ARECs did not stain for smooth muscle actin (A). The
actin cytoskeleton was labeled with Alexa Fluor 488 phalloidin (B). Phosphorylated ERK was detected in the cytoplasm (C), and low levels of Prox-1 were found in the cytoplasm and nuclei (D). With the addition of FGF, SMA-positive cells (~ 41%) were observed
(E), and stress fibers were detected with Alexa Fluor 488 phalloidin (F). Phosphorylated
ERK expression was found in the nuclei (G), but Prox-1 remained primarily in the cytoplasm (H). With the addition of VEGF-C, only a few cells (~ 13%) stained for smooth muscle actin (I, arrow), and filopodia were seen at the cell boundaries (J, arrow). Both phosphorylated ERK and high Prox-1 expression were observed in the cell nuclei (K-L, arrows). When treated with VEGF-A, SMA-positive cells (~ 24%) were evident (M) and cells developed filopodia (N). Phosphorylated ERK expression was observed in the nuclei of a subset of cells (O) while Prox-1 expression remained primarily cytoplasmic (P). Blue = DAPI. SMA = smooth muscle actin, pERK = phosphorylated ERK. Bar = 100 um.
125
126 Figure 3-6. Adult rat epicardial cells treated with VEGF-C for 30 mins. (A)
Untreated cells expressed low levels of Prox-1 in the cytoplasm and nuclei. (B) A subset of VEGF-C treated cells showed Prox-1 nuclear accumulation after only 30 mins of treatment. Blue = DAPI, green = Prox-1. Bar = 100 um.
127
128 Figure 3-7. Prox-1 protein levels via Western Blot. Prox-1 protein levels were assessed
in untreated control ARECs in comparison to ARECs treated with VEGF-C for 30 mins.
Total Prox-1 protein levels remained the same with or without VEGF-C. However, with
VEGF-C exposure, cytoplasmic Prox-1 levels decreased while nuclear levels increased.
Beta-actin was used as a loading control. Ctrl = control, cyto = cytoplasmic extract, nuc = nuclear extract, WCE = whole cell extract (total protein).
129
130 Figure 3-8. Adult rat epicardial cells treated with nuclear export inhibitor leptomycin B for 2 hrs. (A) Untreated cells expressed low levels of Prox-1 in the cytoplasm and nuclei. (B) Leptomycin B-treated cells showed accumulation of Prox-1 in the nuclei. Green = Prox-1. Bar = 100 um.
131
132 Figure 3-9. The MEK inhibitor UO126 induced a decrease in Prox-1 nuclear
expression. (A) Control adult rat epicardial cells that had been treated with the vehicle
DMSO displayed low levels of Prox-1 in the nucleus and cytoplasm. (B) Cells treated with VEGF-C and DMSO exhibited an accumulation of Prox-1 in the nuclei. (C) Cells treated with the UO126 inhibitor dissolved in DMSO were observed to have reduced
Prox-1 nuclear expression even below baseline levels. (D) In the presence of VEGF-C,
UO126-treated cells maintained a very low level of punctuate Prox-1 immunostaining in the nuclei. Prox-1 nuclear expression was present in explanted quail epicardial cells treated with VEGF-C (E) but Prox-1 expression was significantly reduced in epicardial cell nuclei after UO126 exposure (F). Blue = DAPI, green = Prox-1. QECs = quail
epicardial cells. Bar = 100 um.
133
134 Figure 3-10. Smooth muscle actin and phalloidin staining in DMSO/UO126 and
VEGF-C-treated ARECs. In the absence of UO126, smooth muscle cells were seen (A, arrow) and cells developed filopodia (B, arrow). In the presence of UO126, no alpha smooth muscle actin positive cells were observed (C), and the actin cytoskeleton appeared disorganized (D) compared to cells not treated with UO126. SMA = smooth muscle actin. Bar = 100 um.
135
136 Table 3-1. Prox-1 nuclear accumulation in the ARECs at different time points of
VEGF-C treatment.
137
Time Period for VEGF-C % of ARECs with Prox-1 Nuclear Treatment Accumulation 0 minutes 0 5 minutes 17.0 15 minutes 31.3 30 minutes 70.6 24 hours 51.2 Percentages are mean values calculated from a sample size of 3. Average standard deviation = 10.8.
138 CHAPTER 4: SUMMARY, SUPPLEMENTARY DATA AND FUTURE
DIRECTIONS
Summary
The purpose of this study was to obtain a better understanding of the lymphangiogenic process in the heart. The lymphatic system serves an important role in the heart by eliminating excess fluid found in the cardiac tissue. In the event of lymphatic dysfunction, destruction or blockage, edema may be observed, as well as inflammation and fibrosis, in the affected regions. The lymphatic system in the adult heart has been relatively well delineated but the embryonic lymphatic network has not been similarly investigated. One of our first goals therefore was to map out the lymphatic system in the developing heart, especially in relation to the epicardium which has been shown to be a multipotent source of stem cells in the heart. The stimulation of the epicardum to form lymphatic precursor cells would be invaluable in enhancing lymphangiogenesis in pathological conditions of lymphatic damage, thus warranting insight into the lymphangiogenic signaling pathway in the heart.
The lymphatic network of the embryonic and adult heart
In our studies (Karunamuni et al, 2010), we tracked the development of the lymphatic vasculature in the epicardium of the developing quail/mouse heart using the lymphatic marker Prox-1 which is also considered to be one of the master lymphatic regulatory genes. At early stages, no Prox-1 expression was seen in the epciardium until individual Prox-1-positive cells migrated into the heart and organized into vessels in the
139 epicardium that covered the aorta and pulmonary trunk, and also extended over the
ventricles. The lymphatics in general follow the pre-existing blood vessel network as they
develop and this remains true for the heart as well, except in a few cases. For example,
when the coronary arteries in the early avian embryonic heart were stained using smooth
muscle actin as a marker, the lymphatic vessels had already begun to spread over the
ventricles in regions where branches of the coronaries had not yet arrived (Figure 4-1).
These Prox-1-positive lymphatic vessels consistently acquired a mature branching pattern
in the adult heart, with valves also forming later in development.
Interestingly enough, in addition to the incoming Prox-1-positive lymphatic cells,
there were two other populations of cells with lymphatic phenotype observed in the heart.
One group of cells was found in the epicardium and expressed another lymphatic marker
LYVE-1 but not Prox-1 initially. A subset of these LYVE-1-positive cells appeared to be incorporated into the epicardial lymphatics later in development and became positive for
Prox-1 expression. We also noted the presence of a myocardial vascular network in the heart which expressed the lymphatic markers LYVE-1 and VEGFR-3 throughout development, but not Prox-1 or podoplanin. When their trajectory was followed in serial sections and reconstructed using 3D imaging software, these myocardial LYVE-1 positive vessels were found to contain blood cells and their lumens were continuous with the ventricular lumen (Figure 4-2). These vessels were also observed to stain for
ephrinB4, a marker for venous endothelial cells (Figure 4-2). We propose that these
LYVE-1 positive vessels within the myocardium could be Thebesian vessels which have been described in the literature as arterioluminal shunts in the heart. Alternatively, these myocardial cells and vessels could be part of the deep lymphatic plexus of the heart
140 which may have different properties from the superficial or epicardial lymphatic plexus, or they could comprise a “pool” of lymphatic precursor cells that could be recruited for cardiac lymphangiogenesis in the future.
Earlier literature reviews of the cardiac lymphatics have usually relied on dye injection experiments to trace the path of a homogeneous, interconnected system of vessels in the heart. The caveats for such experiments are that the dye can leak into vessels other than the lymphatics or that it may not be taken up by all lymphatic vessels themselves especially if they are immature. In contrast, the use and comparison of several lymphatic markers in our studies at different developmental time points allowed for the comprehensive identification of three types of cells/vessels expressing lymphatic markers in the developing mouse heart. The three types of cells/vessels included:
• Prox-1-positive cells that migrated into the heart within the epicardium of the
great vessels and eventually expressed other lymphatic markers such as LYVE-1,
VEGFR-3, and podoplanin once they enter the heart
• LYVE-1-positive cells in the ventricular epicardium, a subset of which appeared
to become incorporated into the epicardial lymphatics by eventually co-
expressing Prox-1
• LYVE-1-positive/VEGFR-3-positive cells and vessels within the myocardium
that appeared to arise from invagination of the ventricular endocardium at the
base of developing trabeculae and remained Prox-1-negative/podoplanin-negative
throughout development
The epicardium as a source of lymphatic cells
141 The embryonic epicardium is currently viewed as a source of stem cells in the
heart by giving rise to various cardiac cell types including fibroblasts, smooth muscle
cells, and though controversial, even cardiac myocytes. Certain lineage studies have also shown that endothelial cells can be derived from this multipotent cell population.
In our studies, lineage tracing experiments using the WT1-cre mice indicate that
both lymphatic endothelial cells and lymphangioblasts may have progenitors in the
epicardium. We also demonstrated that the adult epicardium contains a subset of cells that express lymphatic markers but not in the subcellular localization seen for mature lymphatics. For example, Prox-1 which is nuclear-localized in lymphatic endothelial cells
was primarily cytoplasmic in our adult epicardial in vitro model as well as in the
embryonic epicardial explants. When treated with the lymphangiogenic growth factor
VEGF-C, Prox-1 accumulated in the nucleus in both models in a growth factor-specific
effect, marking a step forward in the differentiation process towards a more lymphatic
phenotype.
The adult epicardium can thus induce a similar response to the embryonic
epicardium when exposed to the appropriate lymphangiogenic stimulus. Since the
embryonic epicardium has been seen to be instrumental in wound healing and regeneration processes in the heart, our findings would suggest that the adult epicardium
may also be able to recapitulate its embryonic developmental phenotype at least in the
realm of lymphatic signaling. In cases where the cardiac lymphatic system has been
damaged due to surgery or heart failure or infection, it may be possible to develop a
gene- or protein-based therapeutic system of epicardial-specific lymphangiogenesis in
order to replace the pre-existing malfunctioning lymphatic vessels.
142
Supplementary Data
Oxygen tension and the epicardial lymphatics
Lymphangiogenic signaling in the heart has been shown to involve a complex pathway involving Prox-1, VEGFR-3, pERK, and possibly a host of other molecules and
genes. We and others have shown that blood vessels are regulated in their development
by microenvironmental hypoxia and hypoxia inducible factors. Because lymphatic
precursors seem to be present during the same time frame as blood vessel development,
they may also be subject to regulation by hypoxia. It would therefore be interesting to
determine whether differential levels of tissue oxygen would impact the formation of the
extensive Prox-1-positive lymphatic network in the developing heart, much like they
affect the forming blood vessels.
Hypoxia, or reduced oxygen tension, has been shown to be important for blood
vessel formation in the embryo (Lee et al, 2001). Several organs, including the heart,
have been shown to exhibit varying levels of tissue hypoxia. Previous studies originating
from our group have shown that when the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-
1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] (Koch, 2002) was injected into the vitelline vein of the chicken embryo in ovo, intensely positive hypoxic regions included
areas around the base of the outflow tract and along where the major coronary vessels
develop (Wikenheiser et al, 2006; Sugishita et al, 2004A; Sugishita et al, 2004B). The
hypoxic response is principally mediated through the transcription factor HIF-1 (Dery et
al, 2005), which was also shown to have an essential role in regulating coronary vessel
143 development (Wikenheiser et al, 2009). Both hypoxia (15% O2 levels) and hyperoxia, or
increased oxygen levels (75-40%), were seen to induce coronary anomalies in the
embryonic heart (Wikenheiser et al, 2009).
The effects of hypoxia or hyperoxia on the lymphatic vasculature have not been
well characterized. Preliminary studies have investigated the link between hypoxia and
the receptor VEGFR-3 which is found on both lymphatic and blood vessel endothelial
cells early in development. Nilsson et al (2004) found that severe hypoxic conditions (1%
O2) stimulated the formation of an extensive vascular network during mouse embryonal
stem cell differentiation. Addtionally, the introduction of neutralizing antibodies against
VEGFR-3 or the sequestering of VEGFR-3 ligands impaired vascularization, suggesting that hypoxia-driven vascular development is dependent on VEGFR-3 activity. However,
lymphatic endothelial cells labeled by Prox-1 were not visibly affected by hypoxic
conditions. It is currently unknown whether the cardiac lymphatics would respond in a
similar fashion under such circumstances.
In our initial studies, we investigated the effect of changing oxygen levels on the cardiac lymphatic vasculature. Fertile quail embryos (Coturnix coturnix japonica; Boyd’s
Bird Company, Pullman, WA) were incubated in an egg incubator (GQF Mfg Co.,
Savannah, GA) with a rocking apparatus until the appropriate stages were reached
(Hamburger and Hamilton, 1951; HH Stage 25-30 [ED 4.5-7]). From HH Stage 25 onwards, embryos were either left at normoxic conditions (21% O2), or subjected to
hyperoxic (75-40% O2) conditions within a modified styrofoam chamber (Hova-Bator,
G.Q.F. Mfg. Co., Savannah, GA) with O2 percentage being measured by an oxygen
regulator (PROOX 360 oxygen regulator; BioSpherix, Redfield, NY) that was connected
144 to an oxygen tank. Ambient oxygen concentrations were chosen for our experiments
based on preliminary and published data from our laboratory and others to allow for
maximal survival during specific stages of coronary vascular development. The survival
rate was approximately 95-98% for all regimens. HH Stage 30 was selected for embryo
harvesting since it has been shown to correspond to a hypoxic peak in the heart
(Wikenheiser et al, 2006) and is still a full day before the coronaries connect to the aorta in the embryo. After hearts were dissected out, they were subjected to whole mount
staining for both the lymphatic marker Prox-1 and the endothelial marker QH-1 which
labels blood and lymphatic vessel endothelial cells, angioblasts, and hemangioblasts.
In normoxic hearts, the Prox-1/QH-1-positive lymphatic vessels encircled the
great vessels such as the aorta and pulmonary trunk and extended downwards in a
branching pattern over the ventricles. With hyperoxia, the Prox-1-positive lymphatic
vasculature appreared to be more disorganized in their development and the small-
diameter capillary-like branches of the lymphatics were either reduced in number or
absent altogether (Figures 4-3, 4-4). Another interesting finding was the clustering or the
accumulation of the Prox-1-positive cells in the peritruncal region at the base of the
outflow tract where typically there is a distinct vessel-free region in normoxic hearts.
These findings suggest that first, increased oxygen levels may be affecting the
distribution or concentration gradients of guidance cues such as growth factors in the
cardiac tissue, thus resulting in endothelial cells/vessels being found in areas normally
devoid of such cells. In addition, hyperoxia could have either directly affected the
development of the lymphatic vessels, for example by regulating Prox-1 through HIF-1
or ROS (reactive oxygen species), which can be induced by hyperoxic levels.
145 Alternatively, this could have been an indirect event where the lymphatics responded to patterning changes of nearby blood vessels that were induced by hyperoxia. In any case,
there appears to be a link between ambient oxygen levels and the lymphatic network in
the embryonic heart, and the investigation of the underlying mechanism warrants further
study.
HIF-1 and Prox-1
Since hypoxia has been found to stimulate angiogenesis or blood vessel formation
(Lee et al, 2001), it plays a key role in the pathophysiology of cancer, heart attack, stroke and other major causes of mortality (Iyer et al, 1998). Highly positive hypoxic regions in the developing heart have included the outflow tract (OFT) myocardium, the atrioventricular junction (AVJ), the interventricular sulci, the base of the OFT and areas where the major coronary vessels developed (Wikenheiser et al, 2006; Sugishita et al,
2004A; Sugishita et al, 2004B).
The transcription factor hypoxia inducible factor 1 (HIF-1) is an important
regulator of the hypoxic response. HIF-1 is a heterodimeric protein that is composed of
HIF-1α and HIF-1β subunits. HIF-1β/ARNT is a nuclear localized protein (Eguchi et al,
1997), and is found in cells under all oxygen conditions, but HIF-1α must be induced.
Under normoxic conditions, HIF-1α is hydroxylated at proline residues within its oxygen-dependent degradation domain, and is subsequently recognized by pVHL, the product of the von Hippel Lindau tumor suppressor gene and a recognition component of the E3 ubiquitin ligase complex (Dery et al, 2005). This promotes the ubiquitylation (or post-translational modification) of HIF-1α which is then targeted for proteasomal
146 degradation. Under hypoxic conditions however, HIF-1α accumulates instantaneously and binds with ARNT in the nucleus to form the HIF-1 transcription complex. This heterodimer then binds to hypoxic response elements (HRE)-containing promoter regions in genes that can promote angiogenesis (such as VEGF), glucose metabolism, cell survival and erythropoietin synthesis among other functions (Minet et al, 2000;
Livingston and Shivdasani, 2001; Semenza, 2003; Dery et al, 2005). HREs contain functionally essential HIF-1 binding sites with the consensus sequence 5’-RCGTG-3’
(Semenza et al, 1996).
Since HIF-1α can induce expression of angiogenic factors such as VEGF, it is
possible that HIF-1α may also be able to regulate expression of lymphatic-associated
factors such as Prox-1, which does contain a HIF-1 binding site with the consensus sequence. The relationship between HIF-1α and Prox-1 has not been determined in any organ as of yet, including the heart.
To this end, Fertile White Leghorn (Gallus gallus) chicken eggs were obtained
from Case Western Reserve University’s Squire Valleevue Farm (Cleveland, OH). Eggs
were incubated in a humidified room air incubator at 37°C until the embryos reached HH
Stage 25. From this stage onwards, embryos were either left at normoxic conditions
(20.8% O2), or subjected to hypoxic (15% O2) and hyperoxic (75-40% O2) conditions within a modified styrofoam chamber (Hova-Bator, G.Q.F. Mfg. Co., Savannah, GA) with O2 percentage measured by an oxygen regulator (PROOX 360 oxygen regulator;
BioSpherix, Redfield, NY) connected to a nitrogen or oxygen tank. Tissues were
harvested at HH Stages 30 and 33, and then analyzed for Prox-1 protein expression using
Western blotting techniques. In brief, hearts from staged chicken embryos were
147 homogenized with a sonicator under ice within an ice-cold lysis buffer [50 mM Tris-HCl
(pH 7.4), 150mM NaCl, 1% NP-40, 1% Triton X-100, 0.25% Na-deoxycholate, 0.1%
SDS, 1 mM EDTA, and a protease inhibitor cocktail (Complete Mini, EDTA-free tablets
(Roche, Mannheim, Germany)] and stored in a -80°C freezer until needed. Protein
concentration was determined by using the DC protein assay (Bio-Rad Laboratories Inc,
Hercules, CA). A total of 100 μg of the whole heart protein lysate was electrophorised on
an 8% SDS-PAGE gel and then transferred onto a polyvinylidene difluoride (PVDF)
membrane (Millipore, Bedford, MA). Membranes were blocked for 1 hour at room
temperature with 5% nonfat milk in Tris-buffered saline with 1% Tween (TBST). The primary antibodies rabbit polyclonal anti-Prox-1 (Research Diagnostics Inc; Concord,
MA) and monoclonal mouse anti-β-tubulin (Sigma, St Louis, MO) were incubated at a dilution of 1:500 and 1:20,000 respectively overnight at 4°C. The monoclonal antibody anti-β-tubulin was used for the loading control. After washing, the blots were incubated with an anti-rabbit IgG (H&L) HRP-linked antibody (Cell Signaling, Beverly, MA) for 1 hour at room temperature at a dilution of 1:1,000. Anti-mouse IgG (H&L) HRP-linked antibody (Cell Signaling, Beverly, MA) was used against β-tubulin at a dilution of
1:10,000 for 1 hr at room temperature. Signals were then detected using an enhanced chemiluminescence detection system (ECL) (Pierce Chemical Co., Rockford, IL).
According to Western Blot analysis, in HH Stage 33 avian whole heart samples,
Prox-1 protein levels decreased under hypoxia (15% O2) and increased under hyperoxia
(75-40% O2) compared to normoxic hearts (Figure 4-5 A). In comparison, HIF-1α levels were shown to have increased under hypoxia and decreased under hyperoxia at this stage according to our previous studies (Wikenheiser et al, 2009). Similarly, in HH Stage 30
148 avian whole heart samples, Prox-1 protein levels decreased under hypoxia (Figure 4-5
B), whereas HIF-1α levels had been seen to increase at this stage (Wikenheiser et al,
2009). From these preliminary findings, it appears therefore that HIF-1α may negatively affect Prox-1 protein levels at least under these circumstances.
TCDD and its effect on Prox-1 protein levels
In further studies, we investigated the effect of the toxin 2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD) on Prox-1 protein expression in the developing heart. TCDD is a halogenated aromatic hydrocarbon that resists chemical and biological degradation and thus persists in the environment, thereby increasing the chances of human exposure. TCDD can accumulate in humans over a lifetime though a diet of meat, poultry, dairy products, mother’s milk, and through exposure from inhalation of smog and cigarette smoke (Ivnitski-Steele I and Walker MK, 2005; Kneer S and Schrenk D,
2006).
Studies using animal models have found that TCDD affects the developing vasculature, and that its toxicity is mediated by activation of the aryl hydro-carbon receptor (AhR). TCDD binds with the AhR transcription factor and stimulates its translocation into the nucleus, where it dimerizes with ARNT. Sustained activation of
AhR through TCDD binding has had adverse effects in the developing heart. For example, TCDD has been shown to inhibit coronary development which was preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis (Ivnitski et al,
2001). TCDD exposure also led to a decrease in myocardial hypoxia and VEGF expression (Ivnitski-Steele et al, 2004), and reduced endothelial cell responsiveness to
149 angiogenic stimuli (Ivnitski-Steele et al, 2005). These findings suggest that TCDD
activated AhR may compete with HIF-1α for binding to ARNT. In the developing heart,
such competitive binding could result in vascular anomalies, especially since previous
studies have demonstrated that changes in HIF-1α expression levels can lead to abnormal
coronary vessel development (Wikenheiser et al, 2006, 2009). We hypothesized therefore that TCDD treatment of the developing heart would reduce HIF-1α levels and thereby affect expression of Prox-1, which we proposed in the previous section to be a candidate for a HIF responsive gene.
For this study, White Leghorn (Gallus gallus) fertile chicken eggs (Privett
Hatchery, Portales, NM) were first weighed to determine the average and range of weight
for preparation of the dosing solutions and then incubated at 37.5°C at 55% humidity in a
forced-draft JamesWay incubator under normoxic conditions (20.8%) until the embryos
reached appropriate stages. The eggs were placed on an automatic rotator such that they
were rocked back-and-forth approximately once per every two hours. Eggs were
incubated undisturbed until day 4.5 (HH Stage 25) when they were removed from the
incubator and laid horizontally. The fat end of the egg was cleaned with an ethanol wipe
and a hole was made in the shell into the air cell with a sharp probe. The dosing solution
(vehicle control - corn oil, TCDD dissolved in corn oil) was administered with a 100 µl
gas tight Hamilton syringe with a 26 g beveled removable needle. The needle was
inserted into the hole in the egg shell to ~ 1 cm and 10 µl of the dosing solution was dispensed into the air cell of the egg. The egg was placed in a vertical position with the fat end upwards for further incubation. Dosing levels were 1.0 and 3.0 pmol of
150 TCDD/gm in 10 μl of corn oil. Stages of experimental exposure were selected based on
results from a previous publication (Wikenheiser et al, 2009).
At HH Stage 30, hearts were harvested and homogenized with a sonicator under
ice within an ice-cold lysis buffer. Protein concentration was determined by using the DC
protein assay (Bio-Rad Laboratories Inc, Hercules, CA). A total of 100 μg of the lysate
was electrophorised on an 8% SDS-PAGE gel and then transferred onto a polyvinylidene
difluoride (PVDF) membrane (Millipore, Bedford, MA). Membranes were blocked for 1 hour at room temperature with 5% nonfat milk in Tris-buffered saline with 1% Tween
(TBST). The primary antibody rabbit polyclonal anti-HIF-1α (Gift of Dr. Faton Agani) and rabbit polyclonal anti-Prox-1 (Research Diagnostics Inc; Concord, MA) were incubated at a dilution of 1:500 overnight at 4°C. The monoclonal antibody anti-β- tubulin was used for the loading control (1:20,000). After washing, the blots were incubated with an anti-rabbit IgG (H&L) HRP-linked antibody (Cell Signaling, Beverly,
MA) for 1 hour at room temperature at a dilution of 1:1,000. Anti-mouse IgG (H&L)
HRP-linked antibody (Cell Signaling, Beverly, MA) was used against β-tubulin at a dilution of 1:10,000 for 1 hr at room temperature. Signals were detected using an enhanced chemiluminescence detection system (ECL) (Pierce Chemical Co., Rockford,
IL).
According to Western Blot analysis, HIF-1α protein levels were seen to decrease
with TCDD exposure while Prox-1 protein expression increased (Figure 4-6). These
results are consistent with our previous findings that HIF-1α expression levels appeared
to have an inverse relationship with Prox-1 levels under conditions of varying ambient
oxygen. We have also observed that different tissue oxygen levels significantly affect the
151 development/migrational patterning and presumably the physiological function of the
Prox-1-positive epicardial lymphatic vasculature. Furthermore, since Prox-1 does have a
HIF binding site with the consensus sequence, it is possible that HIF-1α may bind to
Prox-1 and negatively regulate its expression, thereby regulating its transcriptional function in the developing heart. Further investigation is needed to determine whether components of the HIF signaling cascade can directly affect Prox-1 function, thereby providing us with another method to control the lymphangiogenic process in the epicardium.
Genes regulated by Prox-1 in the epicardium
In the study of DNA-protein interactions, Chromatin ImmunoPrecipitation (ChIP)
has emerged as a powerful technique for the discovery and characterization of protein- mediated transcriptional regulation. ChIP dissects the spatial and temporal dynamics and
interactions of chromatin and its associated factors. This technique allows us to map
minute-by-minute changes at a single promoter, or alternatively, follow a single
transcription factor over the entire human genome. ChIP is very versatile and can give us significant insight into how genes are regulated in their natural context. The principle of
ChIP is simple: the selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a protein or protein modification of interest can be used to determine the relative abundance of that antigen at one or more locations (loci) in the genome. The most commonly ‘chipped’ chromatin is euchromatin. This material
contains active genes and maintains an open and extended structure in order to play an
important role in transcription, DNA repair and gene replication. By contrast
152 heterochromatin, which contains many inactive genes, is difficult to analyze by ChIP, not
least because of its condensed state and generally repetitive DNA sequence. The ChIP
technique involves using in vivo cross-linking to bind chromatin-associated proteins to
DNA and then isolating these complexes by immunoprecipitating them with specific
antibodies. Cross-links (made with formaldehyde) can be removed and the DNA can be
analyzed by PCR or other means to determine whether specific DNA sequences are
associated with the protein of interest.
Unitl recently, ChIP-chip, which combines ChIP with DNA microarrays (Buck et
al, 2005; Mockler et al, 2005), was the most widely used technique to map protein
binding sites on DNA on a genomic scale. However, ChIP-chip suffers from certain
technical limitations: (1) it requires large amounts (several micrograms) of DNA and thus
involves extensive amplification, which introduces bias; (2) it is subject to cross-
hybridization, which hinders the study of repeated sequences and allelic variants; and (3) it is currently expensive to study entire mammalian genomes. ChIP-Seq (Barski et al,
2007; Johnson et al, 2007; Robertson et al, 2007; Mikkelsen et al, 2007), which combines
ChIP with parallel sequencing technology, is on its way to replacing ChIP-chip as the
commonly used approach for genome-wide identification of protein–DNA interactions in
vivo. ChIP-Seq's coverage, high resolution and cost-effectiveness, combined with its
ability to sequence several million bases in a short span of time (1–2 days), allow us to
map and understand protein–DNA interactions on a genome-scale.
In our study, a ChIP-Seq analysis of VEGF-C treated ARECs with nuclear-
localized Prox-1 was performed in order to further investigate the role or function of
Prox-1 in epicardial cell development or differentiation by analyzing the genes to which
153 it binds. In brief, protein-chromatin interactions were first cross-linked in situ, after
which specific DNA fragments were co-immunoprecipitated and sequenced to identify
genome-wide sites associated with Prox-1. DMSO-treated ARECs (with primarily
cytoplasmic Prox-1 expression) and UO126/VEGF-C treated ARECs (with almost no
nuclear levels of Prox-1) were also analyzed for comparison. Analysis of the sequencing
data however revealed a significant amount of background noise, suggesting that there
might be problems with the Prox-1 antibody that was used. The ChIP procedure was also
performed with an RNA pol2 antibody for the DMSO-treated ARECs. Subsequent data from a quantitative PCR using GAPDH primers suggested that the ChIP process wasat least working.
Since the ChIP-Seq procedure did not yield results, another method to determine
which genes are downstream of Prox-1 in VEGF-C activated epicardial cells may be to
transfect the ARECs with Prox-1 siRNA (Dharmacon) and confirm that Prox-1
expression has been knocked down. A combination of electroporation and lipofection
techniques may be needed to ensure that the siRNA is delivered into the cells. An
expression array analysis could then be perfomed on the following cell samples: ARECs
infected with control siRNA, VEGF-C treated ARECs infected with control siRNA,
ARECs infected with the Prox-1 siRNA, and VEGF-C treated ARECs infected with the
Prox-1 siRNA, in order to determine which genes are being regulated by VEGF-C
signaling in the epicardium and/or may be regulated by Prox-1 in these same cells.
Future Directions
154 In future studies, we would like to establish a more definite link between HIF-1α
signaling and Prox-1 function in the developing heart, and thereby investigate the
mechanism for the potential inverse relationship between the two factors. One possibility
would be to decrease HIF-1α activity with the use of a dominant negative version of HIF-
1α (dnHIF-1α) and examine the subsequent effect on the Prox-1-positive lymphatics in
the avian embryonic heart as well as Prox-1 protein expression. One such dnHIF-
1α version consists of HIF-1α that lacks DNA-binding, transactivation, and oxygen-
dependent domains (Chen et al, 2003). Unfortunately, the dominant negative HIF-1α has
not worked satisfactorily enough in the avian model system in our previous studies. An alternative method therefore would be to pharmacologically inhibit HIF-1α in the
developing avian heart through the application of 2-methoxyestradiol (2ME2) (Ricker et
al, 2004). We could then investigate the correlative changes in the development of the
Prox-1-positive epicardial lymphatic vasculature, and the resulting differences in Prox-1
protein levels with Western Blot analysis.
In addition, the adult rat epciardial cells have proved to be an invaluable in vitro
system to dissect out certain underlying mechanisms of epicardial cell development and
differentiation. These cells have been stimulated to migrate over surfaces and follow
different lineages through growth factor stimulation; for example, they have acquired a
more lymphatic phenotype through Prox-1 nuclear accumulation via VEGF-C
stimulation. The next logical step would be to induce lymphatic tubulogenesis or tube
formation in cell cultures, in hopes of ultimately using the epicardial cells in potential
therapies to treat cardiac edema. Such a process may involve two essential components.
First, the correct extracellular matrix (ECM) environment must be in place for the cells to
155 migrate through and form tubes, such as a 3D collagen matrix/hydrogel, perhaps mixed in
with other lymphatic-friendly ECM molecules such as fibronectin, or polyglycolic acid
scaffolds which have had some success in lymph vessel reconstruction (Dai et al, 2010).
Furthermore, fluid flow or some kind of mechanotransductory stress may be needed to
induce lymphatic tubulogenesis in the adult epicardial cells so that these newly formed
vessels may serve a physiological and functional purpose. An environmental chamber
could potentially be desgined to incorporate these elements. Alternatively, the adult
epicardial cells could be fluorescently tagged and injected directly into an in vivo model
where the lymphatics have been compromised and/or cardiac edema has developed, in
order to determine whether they can be incoporated into new lymphatic vessels with
VEGF-C stimulation.
Final Words
In this study, I was able to map out the extensive branching patterns of the
epicardial lymphatic vasculature in both avian and mouse models using an important
lymphatic transcription factor Prox-1 as a lymphatic marker. This Prox-1-positive
epicardial lymphatic network has been found to be extremely sensitive to changes in
environmental conditions, such as inhibiting the lymphangiogenic receptor VEGFR-3 or
altering ambient oxygen levels, thus providing us with potential methods to control cardiac lymphangiogenesis. My work with the epicardial cell line in vitro also highlighted the importance of ERK signaling in Prox-1 nuclear accumulation and thus cellular differentiation towards a more lymphatic phenotype. It must be noted however that lymphatic signaling in the epicardium appears to be an exquisitely regulated process
156 incorporating multiple components and players, much like the formation of the coronary
vasculature. Further investigation of how these lymphatic factors associate with each
other is needed to develop successful therapies aimed at reducing or treating cardiac edema and similar pathological conditions.
157 Figure 4-1. The lymphatics and coronary arteries in the HH stage 30 quail heart. (A)
Prox-1 was used to label the lymphatic vessels which had already extended over the ventricles in a branching pattern. (B) Cy3-alpha conjugated smooth muscle actin was used to label the coronary arteries. Overlay is in (C). Prox-1 = green, smooth muscle actin = red. Bar = 100 um
158
159 Figure 4-2. Myocardial vessels with venous and lymphatic phenotype in the adult mouse heart. (A) Vessels and cells in the myocardium stained with the venous marker
EphB4. (B) Vessels and cells in the myocardium stained with the lymphatic marker
LYVE-1. (C) Overlay of the EphB4 and LYVE-1 staining patterns revealed that a subset of EphB4-positive vessels also expressed LYVE-1. Red blood cells were observed in some of these vessels (arrows). Green = EphrinB4, red = LYVE-1. Bar = 50 um.
160
161 Figure 4-3. Comparison of great vessel lymphatics in normoxic and hyperoxic avian embryonic hearts. In normoxic HH stage 30 quail embryos, an extensive, branching
Prox-1/QH-1-positive lymphatic network was observed on the great vessels of the heart
(A, B). In hyperoxic hearts, the Prox-1/QH-1-positive lymphatic vessels were more disorganized in their patterning (C, D). Green = Prox-1, red = QH-1. Bar = 100 um.
162
163 Figure 4-4. Comparison of ventricular lymphatics in normoxic and hyperoxic avian embryonic hearts. In normoxic HH stage 30 quail embryos, an extensive, branching
Prox-1/QH-1-positive lymphatic network was also observed on the ventricles of the heart
(A, B). In hyperoxic hearts, the Prox-1/QH-1-positive lymphatic vessels appeared to accumulate at the base of the outflow tract and exhibited reduced branching patterns (C,
D). Green = Prox-1, red = QH-1. Bar = 100 um.
164
165 Figure 4-5. Comparison of Prox-1 protein levels under hypoxic and hyperoxic conditions in the developing heart. (A) At HH stage 33 in the avian heart, Prox-1 protein levels decreased with hypoxia and increased with hyperoxia compared to normoxic hearts, according to Western Blot analysis. (B) At HH stage 30, Prox-1 protein levels decreased with hypoxia compared to normoxic hearts. Beta-tubulin was used as a loading control. Norm = normoxic, hyx = hypoxic, hyp = hyperoxic.
166
167 Figure 4-6. Comparison of Prox-1 protein levels with TCDD exposure in the developing heart. With Western Blot analysis, HIF-1α protein levels in avian whole heart samples decreased while Prox-1 protein levels increased. Beta-tubulin was used as a loading control. Ctrl = control hearts injected with corn oil, 1pM = avian hearts injected with 1 pM TCDD, 3pM = avian hearts injected with 3 pM TCDD.
168
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