2. EMBRYONIC DEVELOPMENT OF THE LYMPHOVASCULAR SYSTEM AND TUMOR LYMPHANGIOGENESIS

AND TUMOR LYMPHANGIOGENESIS

JÖRG WILTING, MARIA PAPOUTSI, KERSTIN BUTTLER, AND JÜRGEN BECKER

Children’s Hospital, Pediatrics I, University of Goettingen, Robert-Koch-Strasse, Goettingen, Germany

INTRODUCTION

The embryonic development of the lymphatic vascular system starts considerably later than the blood vascular system. In chick embryos, the first blood vessels can be seen after 1 day of incubation, whereas morphological evidence for lymphatic endothelial cells (LECs) is present around day 5. However, with specific marker molecules, such as the transcription factor Prox1, LEC precursors can be identified in day-3.5 embryos.

In the mouse, blood vessel development starts at embryonic day (ED) 7.5, whereas the

anlagen of lymph vessels can be seen in the jugular region at ED 10. In human

embryos there is a period of 3–4 weeks between the appearance of the first blood vas-

cular endothelial cells (BECs) and LECs. There is good evidence that LECs develop

from specialized parts of the venous system; however, there is growing evidence that

scattered mesenchymal cells integrate into the growing fetal lymphatics. Similarly,

lymphatics induced by tumors are derived mainly from local vessels, but, to some

extent, pathologic lymphatics seem to develop by integration of circulating cells with

lymphendothelial characteristics. In embryos, like in tumors, the most potent inducers

of lymphangiogenesis are vascular endothelial growth factor (VEGF)-C and -D, which

act mainly via VEGF receptor-3 (flt-4) on LECs.We have shown that blocking of this

interaction prevents lymphangiogenesis in experimental A375 melanomas, while

blocking of VEGF-A greatly inhibits blood vessel development (hemangiogenesis) in

such tumors. However, local invasive growth of A375 melanoma cells is not significantly inhibited, showing that this tumor cell line induces hemangiogenesis, lymphangiogenesis, and invasiveness by distinct mechanisms.

EMBRYONIC LYMPHANGIOGENESIS Development of Embryonic Lymph Sacs

The first obvious morphological criteria of the developing lymphatics are the lymph sacs, which are located in close vicinity to deep embryonic veins. Studies on mammalian embryos have shown that there are eight lymph sacs: three paired and two unpaired (30).The first lymph sacs develop in the jugular region, which is the area where the cranial and caudal jugular veins fuse into the common cardinal vein.

The LECs of the jugular lymph sacs develop considerably later than the first BECs.

In the chick, the first blood vessels can be seen after 1 day of incubation (26), whereas jugular lymph sacs are present around day 5 (5). However, with specific markers, such as the transcription factor Prox1, their precursors can be identified in day-3.5 embryos (40). In the mouse, blood vessel development starts at ED 7.5 (2).

The anlagen of the lymph vessels can be seen in the jugular region at ED 10 (34).

The consecutive development of BECs and LECs has led to the hypothesis that LECs are derived from BECs, specifically from neighboring veins (29). Recently, Oliver and Harvey (22) have supported this hypothesis on the basis of the expres- sion pattern of the homeobox transcription factor Prox1. Prox1-deficient mice die at ED 14.5. They possess a normal blood vascular system, but the development of the lymphatics is arrested at ED 10.5 (34).The first Prox1-positive endothelial cells (ECs) are located in the jugular segment of the cardinal veins, and it has been pos- tulated that these venous ECs are the precursors for LECs in the embryo (22). In avian embryos, the expression pattern of Prox1 is identical with that of mice. The jugular segment of the cardinal veins is Prox1-positive, and labeling of the early (day 4) blood vessels results in a signal in day 6.5 jugular lymph sacs (40), suggesting a blood vascular origin of lymph sacs. The lymphangiogenic protein vascular endothelial growth factor-C (VEGF-C) is essential for the development of lymph sacs. Mice deficient for VEGF-C, which is the ligand of VEGF receptor-3 (VEGFR-3), do not form lymph sacs. This deficiency can be rescued by the application of VEGF-C (14).The studies are in line with the traditional view of the development of LECs from specific parts of the venous system, also called “cen- trifugal theory.”The main representative of this theory was Sabin (29, 30), who had performed India ink injections into the JLS of pig embryos. At about the same time, an opposing theory, the “centripetal theory,” had been set up by Huntington and McClure (10) and Kampmeier (13), who had studied cat and pig embryos.

According to them, the lymphatics develop by confluence of mesenchymal cells,

and only the lymph sacs might be of venous origin (13). By means of grafting

experiments we have recently shown that LECs of quail embryos are able to

integrate into the lymph sacs of chick embryos (38), suggesting an additional,

mesenchymal source for lymph sac ECs.

Transdifferentiation of Venous into Lymphatic Endothelial Cells

The lymph vessels of the body wall in the head, neck, and thoracic region are derived from the jugular segment of the cardinal veins. ECs in the cardinal veins obviously have the potency to give rise to two subtypes of ECs: venous and lymphatic.There are two likely mechanisms behind this phenomenon. Either bipotential ECs develop into the two lineages after asymmetric cell division, or venous ECs transdifferentiate into lym- phatic ECs. Both mechanisms could be regulated by the transcription factor Prox1, which was originally cloned in mice due to its homology to the Drosophila homeobox protein prospero (23).Asymmetric distribution of prospero in the cytoplasm of ganglion mother cells of Drosophila regulates asymmetric division into prospero-negative neu- roblasts and prospero-positive neurons or glia cells (11).Alternatively, Prox1 may induce transdifferentiation of venous ECs into lymphatic ECs. Transcriptional profiling of BECs that were transfected with Prox1 cDNA has provided evidence for a shift toward a lymphatic phenotype (9, 28). In the jugular region, a subpopulation of ECs in the cardinal veins start to express Prox1 in ED 10 mouse and ED 4 chick embryos (34, 40). VEGFR-3 expression becomes restricted to the Prox1-positive LECs. Before, VEGFR-3 is expressed in all blood vessels of the embryo and VEGFR-3 knockout (ko) mice die of cardiovascular failure before lymph vessels develop (6).The ECs of the lymph sacs downregulate BEC markers such as type IV collagen and laminin, and upregulate LEC markers such as secondary lymphoid chemokine (SLC/CCL21) and LYVE-1, a sialoglycoprotein receptor for hyaluronan (1, 35).

Mesenchymal Lymphangioblasts

In the early days of lymphangiogenesis research employing serial sectioning of embryos, it was suggested that lymph vessels develop from “mesenchymal clefts” (10).

Using modern terminology, this means the authors assumed that lymph vessels

develop from scattered lymphangioblasts in the embryonic mesenchyme.These cells

aggregate into tubes and form a communicating vascular system. It is well established

that the blood vascular system of higher vertebrates develops in such a way.The pre-

cursor cells of BECs are called angioblasts and hemangioblasts, according to their poten-

tial to form endothelial cells or both endothelial and blood cells (36). Experimental

studies on chick and quail embryos have provided evidence that the paraxial and

splanchnic mesoderm contain cells with lymphangiogenic potential. Cells derived

from these compartments are capable of integrating into embryonic lymphatics, and

lymphangiogenic potential of mesodermal cells is present even before Prox1 is

expressed in any of these cells (25, 31, 40). During subsequent development, coex-

pression of Prox1 and the endothelial marker QH1 in scattered mesodermal cells of

quail embryos seems to be a marker of lymphangioblasts (40). Like in avian embryos,

expression of LEC markers can also be observed in scattered mesodermal cells of

murine embryos (Fig. 1). These cells are located in various mesodermal compart-

ments of the embryo, e.g., in the dermatomes along the body axis (3). Scattered cells

in this region are positive for Prox1 and LYVE-1 (Fig. 1a,b). We assume that these

cells integrate into the lymph vessels in the dermis of the back.

Evidence for the existence of embryonic lymphangioblasts in other vertebrate species has been provided recently. In Xenopus tadpoles, lymph vessels are obviously derived from both the venous system and from scattered mesenchymal cells (20).

Like in other species, Prox1 and VEGF-C are essential for the development of lymph vessels in tadpoles. However, in the zebrafish, the primary source for lymph vessels seems to be the venous system (41).

TUMOR LYMPHANGIOGENESIS

Growth of tumors is largely dependent on active interactions of tumor cells with the blood vessels of the neighboring healthy tissue (7), and a high density of tumor microvessels is a high risk for hematogenic metastasis (33). However, in human solid cancer, the lymph node status is the most important prognostic indicator for the clin- ical outcome of patients (15, 16, 18, 19), but it has long been denied that tumors induce lymphangiogenesis (8, 32, 42), and it is still under debate if there is significant lymphangiogenesis in human tumors. An elaborated system of lymphatics has been observed in close proximity to invasive areas in breast cancer (4, 16), and lymphangiosis carcinomatosa, the destruction of the lymphatic endothelial lining by tumor cells, is an unfavorable prognostic finding (12). Although increased proliferation of LECs in the vicinity of human tumors can hardly be detected, numerous studies have reported on a positive correlation between expression of lymphangiogenic factors (VEGF-C and -D) and lymphatic vessel density or lymph node metastases (review: (27)). In small laboratory animals, tumors grow much faster and lymphangiogenesis can be meas- ured. We have developed a model of tumor-induced lymphangiogenesis employing the chorioallantoic membrane (CAM) of avian embryos (24).

Tumor-Induced Lymphangiogenesis in the Avian Chorioallantoic Membrane The CAM of reptiles and birds is an extraembryonic organ with respiratory func- tions. Furthermore, it serves as embryonic urinary bladder and mobilizes calcium

Figure 1. Staining of ED 11 mouse embryos with antibodies against markers of lymphendothelial cells.

(a) Prox1 demarcates scattered cells (arrows) in the dermatomes and lymphatics close to the jugular vein (v).

(b) LYVE-1 demarcates scattered cells in the dermatomes close to the neural tube (nt)

from the eggshell. In chick embryos, the CAM develops on day 4, matures on day 12, and involutes on day 18–19. Hatching of chicks takes places around day 20–21. The CAM contains an almost two-dimensional blood vascular system of interdigitating arteries and veins connected to an intraepithelial capillary plexus.

Additionally, there is a network of lymphatic capillaries, mostly along the arteries, but also surrounding larger veins. We have shown that VEGF-A applied on the CAM induces hemangiogenesis but not lymphangiogenesis, whereas VEGF-C induces lymphangiogenesis with a mild hemangiogenic side effect in higher doses (21, 37).VEGF-C-expressing human A375 melanoma cells induce development of lymphatics both at the tumor margins and within the tumor, with a BrdU labeling index of LECs of 11.6% (24). Huge dilated lymphatics can be seen at the tumor margins. These lymphatic capillaries are characterized by their extremely thin lin- ing, which clearly distinguishes them from the blood capillaries.Within the tumors morphological criteria cannot be used to differentiate blood from lymphatic capillaries; however, with specific markers such as Prox1, LECs can be identified.

Within the experimental melanomas, while the density of tumor cells increases, the lumina of the lymphatic vessels are lost. This is due to compression or destruction of the vessels. Also, preexisting lymphatic vessels become compressed or obstructed by the growing tumor mass, as shown by fluorescence microlymphangiography in the mouse (17). Proliferation of LECs is markedly reduced to 3.9% BrdU labeling, when A375 melanoma cells are stably transfected with cDNA for the soluble form of VEGFR-3 (sFlt4). Soluble VEGFR-3 markedly inhibits lymphangiogenesis in experimental A375 melanomas, but it does not inhibit hemangiogenesis, and the cells form huge vascularized tumors on the surface of the CAM (24, 39).This behav- ior completely changes after transfection of A375 melanoma cells with cDNA for the soluble form of VEGFR-1 (sFlt1), which binds VEGF-A with high affinity.The cells do not form vascularized solid tumors on the surface of the CAM.They remain avascular and become necrotic (Fig. 2a,b). However, the cells retain their high inva- sive potential. They invade the tumor stroma, interact with local vessels, and form tumor nodules (Fig. 2a,c–e). The nodules become surrounded by dilatated lymph capillaries, and finally the melanoma cells penetrate the lymphatics (Fig. 2e).

The data strongly suggest that:

1. VEGF-C is a lymphangiogenic factor that can be secreted by tumor cells.

Inhibition of VEGFR-3 signaling strongly reduces tumor lymphangiogenesis, but not hemangiogenesis and tumor growth, and the high invasive potential of A375 melanoma cells also remains unaffected.

2. VEGF-A is a hemangiogenic factor secreted by A375 melanoma cells. Its inhibi- tion greatly reduces formation of solid, vascularized tumors, but does not pre- vent interaction of the cells with the vascularized stroma of the host and formation of metastases.

In sum, tumor-hemangiogenesis, -lymphangiogenesis, and -invasiveness are distinct,

although partially overlapping, mechanisms.

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