Lymphendothelial Markers Research Articles

Pulmonary and pleural lymphatic endothelial cells from pediatric, but not adult, patients with Gorham-Stout disease and generalized lymphatic anomaly, show a high proliferation rate.

BackgroundGorham-Stout disease (OMIM 123880) and generalized lymphatic anomaly are two rare disorders of lymphendothelial growth in which thoracic involvement with chylothorax is a feared complication. Currently it is believed that both disorders are prenatal malformations that progress slowly after birth. Several pharmaceuticals with antiproliferative properties, including interferon-α-2b, rapamycin and propranolol, have however been shown to affect the disease course in some patients. Deeper knowledge of the growth characteristics of these malformations are therefore needed to guide the clinical approach.MethodsLymphatic vessels in lung and pleural tissue from both children and adult patients with generalized lymphatic anomaly or Gorham-Stout disease were studied using an immunohistochemical approach, targeting lymphendothelial markers (D2-40/Prox-1) and a proliferation marker (Ki-67).ResultsWe found significant proliferation and growth in these lesions in pediatric patients but not in adults. Furthermore, the data may suggest that the disease process is at least partly reversible.ConclusionsThese malformations of the lymphatic system proliferate at a significant rate long after birth, which could suggest that the clinical approach for children should be different from adults.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0449-4) contains supplementary material, which is available to authorized users.

Peritumoral D2-40 Chalkley score independently predicts metastases and survival in patients with cutaneous malignant melanoma.

Many observational studies investigated the prognostic significance of angiogenesis and lymphangiogenesis in patients with melanoma. However, the obtained results are rather contradictory, probably due to the lack of the consensus methodology. To investigate the prognostic significance of angiogenesis and lymphangiogenesis-related parameters in patients with melanoma, we performed a retrospective investigation following the consensus recommendations for angiogenesis and lymphangiogenesis quantification in solid tumors and reporting recommendations for tumor marker (REMARK) criteria for reporting the results. Blood and lymphatic vessel Chalkley scores, endothelial cell proliferation fractions and microvessel densities were quantified using a double immunostaining for endothelial marker CD34 or lymphendothelial marker D240 and the proliferation marker Ki-67 in 196 patients with melanoma. These parameters were evaluated separately for peritumoral (PT) and intratumoral areas and were correlated with outcome. In multivariate analysis PT D240 Chalkley score was identified as a strongest predictor for sentinel lymph node metastases, non-sentinel lymph node metastases, distant metastases, disease free survival and overall survival in patients with melanoma. If additional studies corroborate our findings, we believe that the inclusion of PT D240 Chalkley counts to the routine pathology examination of melanoma samples would provide additional information for identifying high-risk patients.

Visualization of Intrapulmonary Lymph Vessels in Healthy and Inflamed Murine Lung Using CD90/Thy-1 as a Marker

BackgroundLymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions.Methodology/Principal FindingsImmunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located.Conclusions/SignificanceThese results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions.

Multipotent mesenchymal stem cells acquire a lymphendothelial phenotype and enhance lymphatic regeneration in vivo

Background—The importance and therapeutic value of stem cells in lymphangiogenesis are poorly understood. We evaluated the potential of human and murine mesenchymal stem cells (MSCs) to acquire a lymphatic phenotype in vitro and to enhance lymphatic regeneration in vivo. Methods and Results—We assessed the lymphendothelial differentiation of human and murine MSCs after induction with supernatant derived from human dermal microvascular endothelial cells, isolated lymphatic endothelial cells, and purified vascular endothelial growth factor (VEGF)-C in vitro. We used human or murine progenitor MSC lines and then characterized the lymphatic phenotype by morphology, migratory capacity, and the expression of lymphatic markers such as Prox-1, podoplanin, Lyve-1, VEGF receptor-2, and VEGF receptor-3. Using a murine lymphatic edema model, we assessed the potential of these cells to form a functional lymphatic vasculature in vivo after injection of syngeneic MSCs. Incubation with supernatant from lymphatic endothelial cells induced an endothelium-like morphology and the expression of lymphendothelial markers in both human and murine MSCs in vitro. MSCs showed migratory activity along a VEGF-C gradient, which was enhanced by VEGF-C conditioning. In vivo, the local application of MSCs resulted in a significant decrease in edema formation (20.1%; P0.01 versus untreated tails) after 3 weekly cell injections and restored the drainage of intradermally injected methylene blue after 7 weekly injections. Conclusions—MSCs were capable of expressing a lymphatic phenotype when exposed to lymph-inductive media and purified VEGF-C. Migratory activity toward VEGF-C in vitro suggests homing capability in vivo. Restoration of lymphatic drainage after injection of MSCs in a lymphedema model indicates that MSCs play a role in lymphatic regeneration. The potential clinical application of MSC in wound healing and reduction of lymphatic edema warrants further research. (Circulation. 2009;119:281-289.)

Multipotent Mesenchymal Stem Cells Acquire a Lymphendothelial Phenotype and Enhance Lymphatic Regeneration In Vivo

The importance and therapeutic value of stem cells in lymphangiogenesis are poorly understood. We evaluated the potential of human and murine mesenchymal stem cells (MSCs) to acquire a lymphatic phenotype in vitro and to enhance lymphatic regeneration in vivo. We assessed the lymphendothelial differentiation of human and murine MSCs after induction with supernatant derived from human dermal microvascular endothelial cells, isolated lymphatic endothelial cells, and purified vascular endothelial growth factor (VEGF)-C in vitro. We used human or murine progenitor MSC lines and then characterized the lymphatic phenotype by morphology, migratory capacity, and the expression of lymphatic markers such as Prox-1, podoplanin, Lyve-1, VEGF receptor-2, and VEGF receptor-3. Using a murine lymphatic edema model, we assessed the potential of these cells to form a functional lymphatic vasculature in vivo after injection of syngeneic MSCs. Incubation with supernatant from lymphatic endothelial cells induced an endothelium-like morphology and the expression of lymphendothelial markers in both human and murine MSCs in vitro. MSCs showed migratory activity along a VEGF-C gradient, which was enhanced by VEGF-C conditioning. In vivo, the local application of MSCs resulted in a significant decrease in edema formation (-20.1%; P<0.01 versus untreated tails) after 3 weekly cell injections and restored the drainage of intradermally injected methylene blue after 7 weekly injections. MSCs were capable of expressing a lymphatic phenotype when exposed to lymph-inductive media and purified VEGF-C. Migratory activity toward VEGF-C in vitro suggests homing capability in vivo. Restoration of lymphatic drainage after injection of MSCs in a lymphedema model indicates that MSCs play a role in lymphatic regeneration. The potential clinical application of MSC in wound healing and reduction of lymphatic edema warrants further research.

Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6–8 weeks), untreated old (aged 9–15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F ® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice ( p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.

Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas

BackgroundLymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels.MethodsLymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis.ResultsLECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-α1 and -α9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs.ConclusionLECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.

The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a ‘homing’ mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1 +/CD31 +/CD45 + cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.

Determination of Microvessel Density by Quantitative Real-time PCR in Esophageal Cancer: Correlation with Histologic Methods, Angiogenic Growth Factor Expression, and Lymph Node Metastasis

Angiogenesis and lymphangiogenesis are important steps in tumor growth and dissemination and are of prognostic importance in solid tumors. The determination of microvessel density (MVD) by immunohistology is subject to considerable variability between different laboratories and observers. We compared MVD determination by immunohistology and quantitative real-time PCR and correlated the results with clinical variables. The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2, and lymphatic endothelial markers VEGFR-3, Prox, and LYVE was assessed by quantitative PCR (qPCR) in primary surgical samples. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was quantified by PCR and correlated with MVD and clinical variables. The expression of endothelial antigens vascular endothelial cadherin (CD144), P1H12 (CD146), tie-2, and VEGFR-2 correlated with each other in 54 samples of primary esophageal cancer (P < 0.0001 for all comparisons). MVD determined immunohistologically by CD31 staining in a subgroup of 35 patients correlated significantly with the qPCR method. The expression of angiogenetic growth factors VEGF-A, VEGF-C, VEGF-D, angiopoietin-1, and angiopoietin-2 was significantly associated with MVD (P < 0.0001 for all comparisons). Analysis of the expression of lymphendothelial markers VEGFR-3, Prox, and LYVE revealed concordant results, indicating that quantification of lymphendothelial cells is possible by qPCR. The presence of lymph node metastasis on surgical specimens was significantly correlated with MVD (P < 0.003), VEGFR-2 (P < 0.048), and VEGF-C (P < 0.042) expression. These results indicate that quantification of MVD by qPCR in surgical samples of esophageal carcinoma yields similar results with immunohistology. Interestingly, the extent of angiogenesis and lymphangiogenesis was not related in individual tumor samples. Lymph node metastases could be predicted by MVD and VEGF-C expression.

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.

Orbital lymphangioma with positive immunohistochemistry of lymphatic endothelial markers (vascular endothelial growth factor receptor 3 and podoplanin)

Existence of true orbital lymphangiomas has been questioned in recent years. Therefore an orbital lymphangioma was analyzed with two new specific markers of lymphatic endothelium. Case-report with clinicopathological, immunohistochemical, and ultrastructural findings. A 25-year-old man presented with recurrent lower lid "hematomas" and a pea-sized tumor palpable in the left lower lid. Magnetic resonance imaging showed an inferonasally located orbital tumor which extended to the posterior pole of the eye. The highly vascularized tumor was excised by medial orbitotomy. Histopathologically, the mass consisted of large, erythrocyte-filled cavernous vessels without evidence of smooth muscle cells or pericytes surrounding them. Numerous lymph follicles and small arterioles were scattered between them. Immunohistochemically, endothelial cells lining the lumina of the cavernous vessels were partly positive for podoplanin and vascular endothelial growth factor receptor 3 (flt-4), two markers of lymphatic endothelium. These markers did not react with endothelial cells lining the arterioles. Ultrastructurally, cavernous vessels displayed features characteristic of lymphatic vessels, and the smaller vessels demonstrated signs of arterioles. Ultrastructural analysis and immunohistochemistry using two new markers of lymphatic endothelium suggest a lymphatic nature of large vessels in an orbital lymphangioma. A greater series of vascular orbital tumors must be studied with these new lymph endothelial markers to confirm the existence of true orbital lymphangiomas and to analyze different profiles of lymph endothelial marker expression.