Proliferating mesodermal cells in murine embryos exhibiting macrophage and lymphendothelial characteristics

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: Our 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

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

<p>Abstract</p> <p>Background</p> <p>Postnatal 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.</p> <p>Results</p> <p>In 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 <it>de novo </it>vasculogenesis for blood and lymph vessels.</p> <p>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 <it>in vitro </it>and <it>in vivo </it>spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels.</p> <p>Conclusion</p> <p>The 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.</p

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Precursor cells of the lymphatic endothelium

Seit mehr als einhundert Jahren wird der Ursprung des Lymphgefäßsystems kontrovers diskutiert. Die ersten morphologisch erfassbaren Lymphgefäßanlagen bei Vögeln, Säugetieren und dem Menschen sind die sogenannten Lymphsäcke. Die tradierte Lehrmeinung über die Lymphgefäßentwicklung beschreibt die Entstehung der Lymphsäcke aus spezifischen Segmenten tiefer embryonaler Venen. Aus den Lymphsäcken gehen später alle anderen Lymphgefäße hervor. Eine gegensätzliche Theorie postuliert die Entwicklung der Lymphsäcke und Lymphgefäße aus mesenchymalen Vorläuferzellen (Lymphangioblasten).In dieser Arbeit wurde die Existenz von Lymphangioblasten bei Mäusen und in humanem Blut auf zellulärer und molekularer Ebene untersucht. Als erste Anlagen des Lymphgefäßsystems in frühen Mausembryonen waren lymphendotheliale Segmente der Kardinal- und Dottersackvenen nachweisbar. Ähnliche Expressionsprofile von Lymphendothelzellen (LEC) der jugulären Lymphsäcke und verstreuten Einzelzellen im Mesenchym der Dermatome und des Mediastinums deuten auf eine Beteiligung dieser Lymphangioblasten an der Entstehung und somit auf einen dualen Ursprung der Lymphgefäße hin. Zum Teil lagen die Einzelzellen in großem Abstand von den sich entwickelnden Lymphsäcken und waren zeitlich auch schon vor deren Entstehung vorhanden. Die Entwicklung der Lymphgefäße wird durch den Wachstumsfaktor VEGF-C gefördert. Durch Applikation von VEGF-C an Schnittkulturen von Mausembryonen konnte lymphangiogenes Potential in weiten Bereichen der Embryonen nachgewiesen werden.Zur Identifizierung und Charakterisierung von zirkulierenden lymphendothelialen Vorläuferzellen beim Menschen wurden mononukleäre Zellen aus peripherem Blut (PBMC) von Kindern untersucht. In Analogie zu den Lymphangioblasten in Mausembryonen, wiesen Subpopulationen der PBMC charakteristische Marker von Lymphendothelzellen auf. RNA-Expressionsstudien bestätigten diese Resultate. Molekulargenetische Untersuchungen von primären humanen Blut- und Lymphendothelzellen haben eine signigikant erhöhte Expression von PPAR? in LEC gezeigt. Die Bedeutung dieses Moleküls wurde daraufhin an knock-out-Mäusen untersucht. Weiterführende Analysen zur Charakterisierung und Genexpression von LEC bei Mäusen und des in vitro Verhaltens von kindlichen PBMC könnte zum besseren Verständnis von Regulationsmechanismen bei der Lymphangiogenese beitragen

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 lymphangiogenesis

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

Immunofluorescence studies of initial lymphatics and epifascial lymphatic collectors.

<p><b>A-C)</b> Staining of initial lymphatics with the antibodies Prox-1 (<b>A, B</b>) and vimentin (<b>A, C</b>). Vimentin is expressed in initial lymphatics. L, lumen of the lymphatics. Arrowheads point to the nuclei of LECs. Bar = 25μm. <b>D)</b> Staining of a lymphatic collector with antibodies against vimentin (green). Note expression in LECs, in numerous cells of the adventitia and some scattered cells in the media. Bar = 200μm. <b>E</b>) Focal expression of β-catenin in lymphatic collectors (L) and in larger vessels of the adventitia. Bar = 100μm. <b>F</b>) Focal expression of ESAM-1 in initial lymphatics (L) and strong expression in dermal capillaries. Dapi (blue) marks all nuclei. Bar = 35μm.</p

Immunofluorescence studies of lymphatic collectors and dermis.

<p><b>A, B</b>) Staining with anti-cKIT (CD117) antibodies of lymphatic collectors. Granulated, round cells that express c-KIT (arrows) are located in the adventitia of the collectors. Bar = 250μm in A, and 25μm in B. <b>C-E</b>) Staining of a lymphatic collectors with anti-CD34 (red) and anti-αSMA (green) antibodies. <b>C,D</b>) In large collectors, CD34+ cells (arrow) are found in the adventitia, and in the outer parts of the media between the αSMA-positive SMCs. Bar = 150μm. <b>D</b>) Higher magnification of C showing nucleated CD34+ cells (arrow). No double-positive cells are visible. Bar = 60μm. <b>E</b>) Smaller caliber collector stained with anti-CD34 (red) and anti-αSMA (green) antibodies. CD34+ cells are found in all parts of the media. Bar = 200μm. <b>F</b>) Staining of foreskin with anti-CD34 (green) and anti-Lyve-1 (red) antibodies. Blood capillaries beneath the epidermis express CD34. The Lyve-1-positive lymphatics are CD34-negative. Dapi (blue) marks all nuclei. Bar = 150μm.</p

Semi- and ultra-thin sections of human epifascial lymphatic collectors.

<p><b>A)</b> Semithin cross-section showing the collapsed lumen (L) of the collector. The media (M) is densely packed with smooth muscle cells (SMCs). Note the thin circular layer of SMCs (arrow) at the border to the adventitia. <b>B-F</b>) Ultrathin sections. <b>B</b>) TEM picture showing a capillary in the media of a collector, consisting of endothelial cells and pericytes. E, erythrocyte. <b>C</b>) TEM picture showing the lumen (L) of a lymphatic collector. The lymphatic endothelial cell possesses slender processes directed towards the SMCs in the media. Note the existence of dark (arrow) and light (asterisk) SMCs. <b>D</b>) TEM picture of the collector shown in A). The collapsed lumen is lined by lymphatic endothelial cells (LEC). Note the rivet-like junction (arrow) between a SMC and a LEC. <b>E)</b> TEM picture of the media showing SMCs. The majority of the SMCs has a dark cytoplasm, some are light (asterisk). <b>F</b>) TEM picture of a dark SMC with ‘starfish morphology’. The lumen (L) of the collector is lined by LECs. Bars = 150 μm in A, 2μm in B,C,E,F, and 1μm in D.</p