Kerstin Buttler

Mesenchymal cells with leukocyte and lymphendothelial characteristics in murine embryos

The development of lymphatic endothelial cells (LECs) from deep embryonic veins or mesenchymal lymphangioblasts is controversially discussed. Studies employing quail‐chick grafting experiments have shown that various mesodermal compartments of the embryo possess lymphangiogenic potential, whereas studies on murine embryos have been in favor of a venous origin of LECs. We have investigated NMRI mice from embryonic day (ED) 9.5 to 13.5 with antibodies against the leukocyte marker CD45, the pan‐endothelial marker CD31, and the lymphendothelial markers Prox1 and Lyve‐1. Early signs of the development of lymphatics are the Lyve‐1‐ and Prox1‐positive segments of the jugular and vitelline veins. Then, lymph sacs, which are found in the jugular region of ED 11.5 mice, express Prox1, Lyve‐1, and CD31. Furthermore, scattered cells positive for all of the four markers are present in the mesenchyme of the dermatomes and the mediastinum before lymphatic vessels are present in these regions. Their number increases during development. A gradient of increasing CD31 expression can be seen the closer the cells are located to the lymph sacs. Our studies provide evidence for the existence of scattered mesenchymal cells, which up‐regulate lymphendothelial and down‐regulate leukocyte characteristics when they integrate into growing murine lymphatics. Such stem cells may also be present in the human and may be the cell of origin in post‐transplantation Kaposi sarcoma. Developmental Dynamics 235:1554–1562, 2006.

Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels

BackgroundPostnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated.ResultsIn an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels.Mouse lung microvascular endothelial cells (MLMVECs) were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony). These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs) in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels.ConclusionThe lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

Proliferating mesodermal cells in murine embryos exhibiting macrophage and lymphendothelial characteristics

BackgroundThe data on the embryonic origin of lymphatic endothelial cells (LECs) from either deep embryonic veins or mesenchymal (or circulating) lymphangioblasts presently available remain inconsistent. In various vertebrates, markers for LECs are first expressed in specific segments of embryonic veins arguing for a venous origin of lymph vessels. Very recently, studies on the mouse have strongly supported this view. However, in the chick, we have observed a dual origin of LECs from veins and from mesodermal lymphangioblasts. Additionally, in murine embryos we have detected mesenchymal cells that co-express LEC markers and the pan-leukocyte marker CD45. Here, we have characterized the mesoderm of murine embryos with LEC markers Prox1, Lyve-1 and LA102 in combination with macrophage markers CD11b and F4/80.ResultsWe observed cells co-expressing both types of markers (e.g. Prox1 – Lyve-1 – F4/80 triple-positive) located in the mesoderm, immediately adjacent to, and within lymph vessels. Our proliferation studies with Ki-67 antibodies showed high proliferative capacities of both the Lyve-1-positive LECs of lymph sacs/lymphatic sprouts and the Lyve-1-positive mesenchymal cells.ConclusionOur data argue for a dual origin of LECs in the mouse, although the primary source of embryonic LECs may reside in specific embryonic veins and mesenchymal lymphangioblasts integrated secondarily into lymph vessels. The impact of a dual source of LECs for ontogenetic, phylogenetic and pathological lymphangiogenesis is discussed.

Lymphatics and Inflammation

Inflammation is a local or systemic tissue reaction caused by external or internal stimuli with the objective to remove the noxa, inhibit its further dissemination and eventually repair damaged tissue. Blood vessels and perivascular connective tissue are important regulators of the inflammatory process. After a short initial ischemic phase, inflamed tissue is characterized by hyperaemia and increased permeability of capillaries. Therefore, blood vessels have been in the focus of inflammation research for quite some time, whereas lymphatic vessels have been neglected. Their reactivity is not immediately obvious, and, their identification within the tissue has hardly been possible until lymphatic endothelial cell (LEC)-specific molecules have been identified a few years ago. This has opened up the possibility to study lymphatics in normal and diseased tissues, and to isolate LECs for transcriptome and proteome analyses. Initial studies now provide evidence that lymphatics are not just a passive route for circulating lymphocytes, but seem to be directly involved in both the induction and the resolution of inflammation. This review provides a summary on the basics of inflammation, the structure of lymphatics and their molecular markers, human inflammation-associated diseases and their relation to lymphatics, animal models to study the interaction of lymphatics and inflammation, and finally inflammation-associated molecules expressed in LECs. The integration of lymphatics into inflammation research opens up an exciting new field with great clinical potential.

Similarities and Differences of Human and Experimental Mouse Lymphangiomas

Lymphangioma is a disfiguring malformation of early childhood. A mouse lymphangioma model has been established by injecting Freunds incomplete adjuvant (FIA) intraperitoneally, but has not been compared with the human disease. We show that, in accordance with studies from the 1960s, the mouse model represents an oil‐granuloma, made up of CD45‐positive leukocytes and invaded by blood and lymph vessels. Several markers of lymphatic endothelial cells are expressed in both mouse and human, like CD31, Prox1, podoplanin, and Lyve‐1. However, the human disease affects all parts of the lymphovascular tree. We observed convolutes of lymphatic capillaries, irregularly formed collectors with signs of disintegration, and large lymph cysts. We observed VEGFR‐2 and ‐3 expression in both blood vessels and lymphatics of the patients, whereas in mouse VEGFR‐2 was confined to activated blood vessels. The experimental mouse FIA model represents a vascularized oil‐granuloma rather than a lymphangioma and reflects the complexity of human lymphangioma only partially. Developmental Dynamics 236:2952–2961, 2007.

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice.

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC–MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogeneis.

Homeobox transcription factor Prox1 in sympathetic ganglia of vertebrate embryos: correlation with human stage 4s neuroblastoma.

Previously, we observed expression of the homeobox transcription factor Prox1 in neuroectodermal embryonic tissues. Besides essential functions during embryonic development, Prox1 has been implicated in both progression and suppression of malignancies. Here, we show that Prox1 is expressed in embryonic sympathetic trunk ganglia of avian and murine embryos. Prox1 protein is localized in the nucleus of neurofilament-positive sympathetic neurons. Sympathetic progenitors represent the cell of origin of neuroblastoma (NB), the most frequent solid extracranial malignancy of children. NB may progress to life-threatening stage 4, or regress spontaneously in the special stage 4s. By qRT-PCR, we show that Prox1 is expressed at low levels in 24 human NB cell lines compared with human lymphatic endothelial cells (LECs), whereas equal immunostaining of nuclei can be seen in embryonic LECs and sympathetic neurons. In NB stages 1, 2, 3, and 4, we observed almost equal expression levels, but significantly higher amounts in stage 4s NB. By immunohistochemistry, we found variable amounts of Prox1 protein in nuclei of NB cells, showing intra and interindividual differences. Because stage 4s NB are susceptible to postnatal apoptosis, we assume that high Prox1 levels are critical for their behavior.

Morphological and Molecular Characterization of Human Dermal Lymphatic Collectors

Millions of patients suffer from lymphedema worldwide. Supporting the contractility of lymphatic collectors is an attractive target for pharmacological therapy of lymphedema. However, lymphatics have mostly been studied in animals, while the cellular and molecular characteristics of human lymphatic collectors are largely unknown. We studied epifascial lymphatic collectors of the thigh, which were isolated for autologous transplantations. Our immunohistological studies identify additional markers for LECs (vimentin, CCBE1). We show and confirm differences between initial and collecting lymphatics concerning the markers ESAM1, D2-40 and LYVE-1. Our transmission electron microscopic studies reveal two types of smooth muscle cells (SMCs) in the media of the collectors with dark and light cytoplasm. We observed vasa vasorum in the media of the largest collectors, as well as interstitial Cajal-like cells, which are highly ramified cells with long processes, caveolae, and lacking a basal lamina. They are in close contact with SMCs, which possess multiple caveolae at the contact sites. Immunohistologically we identified such cells with antibodies against vimentin and PDGFRα, but not CD34 and cKIT. With Next Generation Sequencing we searched for highly expressed genes in the media of lymphatic collectors, and found therapeutic targets, suitable for acceleration of lymphatic contractility, such as neuropeptide Y receptors 1, and 5; tachykinin receptors 1, and 2; purinergic receptors P2RX1, and 6, P2RY12, 13, and 14; 5-hydroxytryptamine receptors HTR2B, and 3C; and adrenoceptors α2A,B,C. Our studies represent the first comprehensive characterization of human epifascial lymphatic collectors, as a prerequisite for diagnosis and therapy.

Integration of CD45-positive leukocytes into newly forming lymphatics of adult mice

The embryonic origin of lymphatic endothelial cells (LECs) has been a matter of controversy since more than a century. However, recent studies in mice have supported the concept that embryonic lymphangiogenesis is a complex process consisting of growth of lymphatics from specific venous segments as well as the integration of lymphangioblasts into the lymphatic networks. Similarly, the mechanisms of adult lymphangiogenesis are poorly understood and have rarely been studied. We have recently shown that endothelial progenitor cells isolated from the lung of adult mice have the capacity to form both blood vessels and lymphatics when grafted with Matrigel plugs into the skin of syngeneic mice. Here, we followed up on these experiments and studied the behavior of host leukocytes during lymphangiogenesis in the Matrigel plugs. We observed a striking co-localization of CD45+ leukocytes with the developing lymphatics. Numerous CD45+ cells expressed the LEC marker podoplanin and were obviously integrated into the lining of lymphatic capillaries. This indicates that, similar to inflammation-induced lymphangiogenesis in man, circulating CD45+ cells of adult mice are capable of initiating lymphangiogenesis and of adopting a lymphvasculogenic cellular differentiation program. The data are discussed in the context of embryonic and inflammation-induced lymphangiogenesis.