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Lymphology 37 (2004) 98-106

Children's Hospital, Pediatrics I, Gottingen, Germany

ABSTRACT function has become overruled by the nutritive function in blood vessels of larger animals. It has generally been accepted that the The circular movement of cells is driven by a blood vascular system is primary and the blood heart, which, however, is not an unique lymphatic vascular system secondary. Diseases organ. Lymph hearts are present in lower of the blood vascular system are the leading vertebrates, still develop transiently in birds, cause for mortality and morbidity in developed and are vestigial in the contractile lymphangion nations. In contrast, lymphedema is seldom which "circulates" immune cells. We conclude life-threatening and can generally be well­ that the definitive lymphatics are perhaps managed by combined physiotherapy. During secondary in mammals, but the blood vascular ontogeny, the blood vessels and the heart system seems to develop on the basis of an develop much earlier than the lymphatic ancestral lymphatic system with lymph hearts. vessels. However, there is growing evidence that the first vascular system occurring during DEVELOPMENT OF THE VASCULAR ontogeny and phylogeny has lymphatic SYSTEM: TRADITIONAL VIEW functions. Defense mechanisms are crucial for all organisms irrespective of their size. The blood vascular system and Macrophages precede the emergence of circulation has been in the focus of scientific erythrocytes during ontogeny, and their research ever since its discovery by William circulation in the hemolymphatic (more Harvey (1). On the other hand, the lymphatic accurately, lymphohematic) system of insects, system has mostly been neglected although which do not possess erythrocytes, shows the lymphatic vessels were discovered by that the lymphatic function is primary Gasparo Aselli (2) at almost the same time. whereas the nutritive function is secondary, The predominant interest in the blood needed only in larger organisms. In molluscs circulation compared to the lymph circulation and arthropods, which have an open vascular is obviously due to its anatomic prominence system, hemocyanin has both oxygen trans­ and accessibility, critical nature of its porting and defense functions. In vertebrates, functions, and the dramatic manifestations of the early blood vessels have structural its disorders as the leading cause for death in characteristics of lymphatics and express the industrialized nations. Diseases of the lymphendothelial receptor flt-4 (Vascular lymphatic vessels are disfiguring; produce Endothelial Growth Factor Receptor-3). swelling, scarring and immune dysregulation, Later, flt-4 becomes restricted to the definitive but are rarely lethal. The dense vasculari­ lymphatics, which are either formed from the zation of tumors has been recognized even by primary vessels or from mesodermal Rudolf Virchow (3), and the intense tumor lymphangioblasts. The primary lymphatic angiogenesis research of recent years is

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 99 mainly based on studies by Judah Folkman well as cell-cell and cell-matrix interactions. (4). Accordingly, the terms vasculogenesis Among them are endothelial and endocardial and angiogenesis have almost without growth factors and receptors such as exception been used in the sense of hemangio­ Vascular Endothelial Growth Factors genesis (and blood vessel development and (VEGFs), their receptors (VEGFR-1,-2,-3; growth). Tumor-induced lymphangiogenesis called flt-1, flk-lIKDR and flt-4, respectively) has been neglected, even denied, but clear and co-receptors, the neuropilins (13, 14). experimental evidence for this phenomenon The angiopoietins and their receptor, Tie2, as has been published recently (5,6). The proper well as the orphan receptor Tiel are of identification of lymphatic endothelial cells critical importance for embryonic hemangio­ (LEes) in histological sections has been genesis (15), and, additionally, members of problematic, and only in the last few years the ephrin ligand/Eph receptor family are have molecules been identified, which allow needed for blood vessel formation (16). Mice for more specific immunohistochemical with a deletion for vascular endothelial (VE)­ staining of LEes (review: 7). There is not only cadherin die of vascular abnormalities (17), a predominance of hemangiogenesis research and the same is true for mice that lack over lymphangiogenesis research, there extracellular matrix molecules that are also is the generally accepted view that blood critical for angiogenesis, such as fibronectin vessels are primary and lymphatic vessels (review: 18). Abnormalities of the development secondary or an offshoot of blood vessels. of the lymphatic vessels are not embryonic According to the biogenetic law of Ernst lethal. Aplasia of lymphatic vessels is only Haeckel, ontogeny is a recapitulation of found in severe genetic aberrations. phylogeny. During ontogeny, the circulatory Hypoplasia of lymphatics is found in children system develops much earlier than the suffering from the N onne-Milroy disease, and lymphatic vascular system. In the chick, the in some of the families, the inherited defect is first blood vessels can be seen after one day of a monoallelic mutation of the VEGFR-3 gene incubation (8), whereas morphological (review: 19). Mice lacking the transcription evidence for LEes is present around day 5 factor Proxl have normal blood vessels but (9). In the mouse, blood vessel development lack lymphatics (11). These mice die around starts at day 7.5 (to); the anlagen of the embryonic day 14.5, but this seems to be due lymphatics can be seen in the jugular region to the abnormal liver development. Therefore, at day 10 (11). In the human, jugular lymph there are many essential genes that control sacs have been found in 6- to 7-week-old cardiovascular development, whereas embryos of 10-14 mm total length (12). This development of lymphatics seems to be rather is 3 to 4 weeks after the development of the unproblematic and disturbed only in severe first blood vessels. In sum, these findings genetic aberrations. seem to show that the blood vascular system Has nature created the highly problematic is primary. Lymphatic vessels occur much blood vascular system first, and only later later and are therefore secondary. developed a lymphatic system, which usually The nutritive function of the circulatory does not produce any serious developmental system is crucial for the development of problems? Is it compatible with the genesis of vertebrates. Therefore, any mutation that higher organisms to produce a highly critical affects the functionality of the cardiovascular organ system out of zero, when almost every system causes early embryonic death. failure will be lethal? Wouldn't it be, from a Numerous genes with essential functions for teleological point of view, much more the development of the circulatory system appropriate to make a critical developmental have been identified. These genes code for step on the basis of an already existing molecules that regulate cell proliferation as foundation?

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NUTRITIVE OR IMMUNE FUNCTION: adipose body (liver-homologue) in the WHAT COMES FIRST? production of anti-microbial proteins. This shows that Drosophila has a well-elaborated It is generally assumed that the need for innate immune system. The cardiovascular nutrition of all parts of the body has been the system of Drosophila ensures rapid driving force for the evolution of the cardio­ circulation of immune cells through the body. vascular system. However, there is an By this means the cells are able to infiltrate a excellent reason for its development even in diseased body compartment much faster than small animals where nutrition is still by would be the case by migration through the diffusion: this reason is immune defense. interstitium only. As in vertebrates, where In fact, both phylogenetically and ontogene­ lymph is composed of interstitial fluid and tically, defense mechanisms develop earlier immune cells, the circulatory system of than the nutritive elements of the blood. In Drosophila and other insects has all the Drosophila, supply with oxygen is typical characteristics of a lymphatic system. accomplished by an elaborate system of fine In some invertebrates, this lymphatic system channels, the tracheal system. Red blood cells takes over an additional function, a nutritive are not present, and the open hemolymphatic one. Oxygen transporting molecules system fulfils functions which are typical comparable to hemoglobin (arylphorin, lymphatic functions. The heart is connected hemocyanin) are present in various insects, to open-ended vessels and pumps interstitial arthropods and molluscs (22), but these fluid and immune cells through the body. molecules still have an additional defense It is therefore more appropriate to call this function. In crustaceans, infection induces the system a lymphohematic or lymphatic cleavage of hemocyanin into antibiotic system. Three types of immune cells are peptides (23). In cephalopods, hemocyanin found in Drosophila: plasmatocytes, crystal binds to various particles and seems to act cells and lamellocytes (20). Plasmatocytes, like an opsonizing agent, which increases the which are capable of phagocytosis, are activity of phagocytes (24). This shows that comparable to the monocyte/macrophage the development of oxygen transporting lineage of the human. Crystal cells seem to molecules is closely linked to immunological have natural killer functions by producing mechanisms. free radicals via melanin synthesis. It is generally described that the first Lamellocytes differentiate in larvae, when blood cells developing in vertebrates are of these are attacked by parasites. They the erythropoietic lineage. In the chick, blood encapsulate the parasites and kill them by the islands, which contain erythroblasts lined by activity of crystal cells. In adult flies, only an outer layer of endothelial cells, are seen in plasmatocytes (phagocytes) are present. Like the yolk sac already after one day of the other two cell types, they are produced incubation. The cells are derived from the during the larval period in so-called lymph posterior (extra-embryonic) mesoderm. This glands, which are located pairwise along the cell population is a transient one, and dorsal vessel. Lymph glands originate from definitive hematopoiesis then takes place at the lateral embryonic mesoderm. They intra-embryonic sites in the so-called AGM differentiate in an anterior to posterior region (!lorta, gonads, mesonephros) (review: gradient and involute during aging. In older 2S). In mammalians including the human, the animals they cannot be found (21). This situation is a very similar one. Extra­ seems to be highly comparable to the involu­ embryonic blood islands can be seen in 13 to tion of the lymphatic organs (bone marrow, 1S-day-old human embryos. The erythrocytes thymus, lymph nodes) in aged humans. In derived therefrom still contain a nucleus. The Drosophila, plasmatocytes interact with the definitive hematopoietic cells develop from

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B

Fig. 1: A) In situ hybridization of a day 2 quail embryo with a VEGFR-3 anti-sense probe (kindly provided by Dr. Anne Eichmann, Paris). Ventral view. The developing aorta and intersomitic vessels (arrows) are positive. In the center, the notochord (N) also expresses VEGFR-3. B) In situ hybridization of a day 10 quail embryo with a VEGFR-3 anti-sense probe. The basilic vein (V) is negative, the accompanying lymphatic trunks (LT) are positive. the splanchnic mesoderm. Mice that lack during development. They can be isolated the transcription factor AMLl (Runx1) do from the epiblast before gastrulation, which is not develop definitive blood cells (26). clearly before the development of blood Successively, all blood cells are formed from islands (28). This situation is reminiscent of pluripotent intra-embryonic stem cells. After an innate immune function and seems to be the erythrocytes, the platelets can be found; fully comparable to the situation described then the neutrophilic granulocytes, thereafter for Drosophila. The fact that the transcrip­ the eosinophils followed by basophils and tion factor Runxl is already active during the lymphocytes; and at last, monocytes are development of immune cells in Drosophila present in the peripheral blood (27). (20) suggests that the intra-embryonic Therefore, it should be stressed that from hematopoiesis of vertebrates is based on these so-called blood cells only erythrocytes ancestral mechanisms, whereas extra­ and platelets are by definition blood cells. embryonic hemangiogenesis is phylogene­ The other cell types, (granulocytes, tically new. Therefore, as in insects, the first lymphocytes and monocytes/macrophages) function of the intra-embryonic vascular are the free cells of the connective tissue, and system during ontogenesis of vertebrates may only a very small minority of them is be defense rather than nutrition, the vessels transiently found in the blood. Furthermore, being lymphatic, rather than blood vessels. it is important to recognize that the first "blood cells" in vertebrate embryos are not LYMPHATIC CHARACTER OF EARLY the erythrocytes. It is generally accepted that, EMBRYONIC VESSELS in adults, monocytes are the precursors of macrophages, but in the embryo the situation The suggestion that the first intra­ is very different. Monocytes are the latest embryonic vessels in vertebrate embryos are cells in the peripheral blood, but, in contrast, lymphatic vessels is also supported by their macrophages are the first immune cells found structure and by the expression of a typical

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 102 lymphendothelial receptor, VEGFR-3 (flt-4). when the definitive lymphatics develop, and Like lymphatic capillaries, embryonic vessels the cardiovascular system then becomes less are not invested by a continuous basal lamina. permeable and focuses on its nutritive They are extremely leaky and become tight at functions. Thus, the definitive lymphatics a time when definitive lymphatics develop. may be secondary but the early embryonic In chick embryos this occurs around day 5 vessels have a primary lymphatic function. (29). Uptake of interstitial fluid seems to be The change from a primary lymphatic to an important function of early embryonic a nutritive function of blood vessels may vessels. Furthermore, the expression of explain the difficulties in characterizing the VEGFR-3 (flt-4) in embryonic blood vessels is different types of vessels in lower vertebrates, a strong indicator of their lymphendothelial especially in fish, where many contradictory function. In Drosophila, there is a single findings have been published. Mayer (38) ancestor for the various VEGF and PDGF tried to explain the different views about the (Platelet-derived Growth Factor) receptors piscine lymphatics by the existence of a found in vertebrates. This receptor, called secondary vascular system, a finding PVR, has three known ligands and directs supported by Vogel and Claviez (39). The embryonic immune cell migration (30-32). secondary vascular system constitutes a Probably only recently did these molecules separate, parallel circulatory system and assume their roles in vessel development, and includes vessels earlier assumed to be it is conceivable that endothelial cells evolved lymphatics (40). It starts from the systemic from immune cells; again suggesting a arteries, forms its own capillary networks, primary defense function of the vascular which supply mainly the oral mucous system. In vertebrates, VEGFs and their membranes and the skin, and then returns to receptors are essential regulators of blood and the systemic venous system. It is thought to lymphatic vessel development. VEGF-A function in skin respiration, osmotic binds VEGFR-1 and -2, and is a key regulation and immune defense. Based on its regulator of physiologic and pathologic blood anatomical and functional characteristics, the vessel development (33). VEGF-C and-D hypothesis has been proposed that the bind VEGFR-2 and -3 and have been shown secondary circulation might be an evolution­ to induce lymphangiogenesis in various ary predecessor of the lymphatic system (41). experimental settings (34). Therefore, In contrast, one might suggest that this part VEGFR-3 is the receptor which is almost of the vascular system has retained its exclusively expressed on LECs, but not on original lymphatic character, and that the BECs. However, this expression pattern holds new, secondary lymphatics have not evolved true only for differentiated tissues. In mouse in these fish. Ligands and receptors of the and chick embryos, VEGFR-3 is expressed in VEGF family that regulate blood vascular all endothelial cells during early development and lymphatic growth and development in (Fig. lA, B), and becomes restricted to the higher vertebrates are also present in fish (42) lymphatics during the organogenic period but their relevance for the secondary vascular (35, 36). VEGFR-3 knock out mice die from system has not been analyzed. cardiovascular malformations before lympha­ tic vessels develop (37). This developmental BLOOD HEART AND LYMPH HEART regulation of VEGFR-3 expression has been interpreted to signify that lymphatics are One may argue that the driving force derived from blood vessels. In contrast, we (vis a tergo) behind the perfusion of the view this expression pattern as a reflection of vascular system is the heart, and that the the lymphatic functions of early embryonic heart is a typical feature of the blood vascular vessels. These functions seem to become lost system. However, lymph hearts are

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Fig. 2: A) Injection of Merkox-blue into an umbilical lymphatic trunk of a day 16 chick embryo. The lymph hearts (arrow) show up in blue. They are located superficially at each side of the vertebral column at the sacro-coccygeal transition. B) Hematoxylin and eosin staining of a lymph heart (LH) ofa day 14 quail embryo. It consists of an inner endothelial lining and a layer of myocytes. A trabeculum transverses the lumen. C) Consecutive section of the lymph heart (LH) shown in B. The endothelial cells are stained with the QHI antibody (8).

characteristic organs of lower vertebrates the venous system in all of these areas, and (43). Similarly to the blood heart, which is lymph hearts range in number from four or located at the interface between veins and six in frogs to more than two hundred in arteries, the lymph hearts are situated at the caecilians. The important function of lymph transition from lymphatic vessels to the veins. hearts is to maintain the directed lymph flow Lymph hearts can generally be found in and entry of lymph into the blood vertebrates in three different areas: jugular, circulation. In reptiles, lympho-venous lumbar and caudal. The amphibian anastomoses exist in the caudal and the lymphatic system mostly communicates with jugular regions but lymph hearts are found

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 104 only in the caudal region (40). Mammals recapitulation of phylogeny during ontogeny and most birds do not possess lymph hearts may still be applicable to the vascular system, but in birds these are still found as transient but the starting point for vessel development embryonic organs (Fig. 2). The structure of may be different from what has been the lymph hearts of birds is very similar to previously suggested: residing in small that of the blood heart. They have an inner animals with primary defense functions of the endocardial lining and a contractile myocar­ vascular system. dium composed of striated muscle cells. Trabeculae can be found as in the blood ACKNOWLEDGMENTS heart as well as a valvular system (44). This finding shows that a heart is not restricted to This study was supported by the the blood vascular system, and it cannot be Deutsche Forschungsgemeinschaft (grant Wi excluded that the blood heart has evolved on 1452/8-1) the basis of a lymph heart. In fact, in Drosophila, the heart and the immune cells develop from a pool of mesodermal REFERENCES precursors, which are characterized by the 1. Harvey, W: Exercitatio anatomica de motu expression of the gene odd (45). A subpopula­ cordis et sanguinis in animalibus. Wilhelm tion of cardiac cells seems to be closely Fitzer, Frankfurt/Main, 1628. related to the lymph glands, suggesting, 2. Asellius, G: De Lacteibus sive lacteis venis again, a primary lymphatic function of the Quarto Vasorum Mesaroicum genere novo invente Gasp. Asellii Cremonesis Antomici circulatory system. In the human, the Ticiensis Qua Sententiae Anatomicae mu/tae, spontaneous, peristaltic contractions in the nel perperam receptae illustrantur. Mediolani, tunica media of lymphatic trunks, generated apud Jo. Baptistam Bidellium. 1627. by pacemaker cells, is highly reminiscent of 3. Virchow, R: Die Cellularpathologie in ihrer the cardiac physiology. Usually, 8 to 10 Begriindung aufphysiologische und pathologische Gewebelehre. Hirschwald, contractions per minute can be observed (46) Berlin, 1858. and respond to stretch and filling volume. 4. Folkman, J: Tumor angiogenesis factor. Cancer Res. 34 (1974), 2109-2113. CONCLUSION 5. Papoutsi, M, G Siemeister, K Weindel, et al: Active interaction of human A375 melanoma cells with the lymphatics in vivo. Histochem. We conclude, that the separation of the Cell Bio!. 114 (2000),373-385. human vascular system into a primary blood 6. Skobe, M, T Hawighorst , DG Jackson, et al: vascular system and a secondary lymphatic Induction of tumor Iymphangiogenesis by vascular system is not appropriate (based on VEGF-C promotes breast cancer metastasis. the evidence). The situation is complicated by Nat. Med. 7 (2001),192-198. 7. Sleeman, JP, J Krishnan, V Kirkin, et al: the fact that the blood vessels and the blood Markers for the lymphatic endothelium: In heart have developed on the basis of a search of the holy grail? Microsc. Res. Tech. primary lymphatic function of the system. 55 (2001), 61-69. The nutritive function seems to be secondary 8. Pardanaud, L, C Altmann, P Kitos, et al: Vasculogenesis in the early quail blastodisc as and has eclipsed the lymphatic function studied with a monoclonal antibody during phylogeny. Embryonic blood vessels recognizing endothelial cells. Development still possess lymphatic functions, and only the 100 (1987), 339-349. definitive lymphatics may be secondary. They 9. Clark, ER, EL Clark: On the origin and early are produced by the activity of transcription development of the lymphatic system of the chick. Contr. Embryo!. 9 (1920), 447-482. factors such as Proxl, which is expressed in 10. Breier, G, F Breviario, L Caveda, et al: specific sites of veins and in mesodermal Molecular cloning and expression of murine precursor cells (11,47). Hackel's view of the vascular endothelial-cadherin in early stage

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