Culture Of Lymphatic Endothelial Cells Research Articles

Isolation and short-term culturing of primary lymphatic endothelial cells from collecting lymphatics: A techniques study.

To develop an experimental method for routine isolation and short-term culture of primary lymphatic endothelial cells from specific collecting vessels. Lymphatic endothelial cell tubes (LECTs) were isolated from micro-dissected collecting vessels. LECTs were allowed to attach and grow for ~3 weeks before being passaged. Non-purified cultures were partially characterized by immunofluorescence and RT-PCR at passages 1-2. The method was validated in cultures of primary lymphatic endothelial cells (LECs) from male and female mice. After 1 or 2 passages, >60% of the LECs maintained expression of Prox1. Expression of 22 different genes was assessed using RT-PCR. Prox1, Vegfr3, eNos, Cdh5, Pecam1, Cx43, Cx37, and Cx47, among others, were expressed in these short-term cultured LECs, while Myh11, Cnn1, Desmin, and Cd11b were not detected. Prox1 expression, as determined by western blotting, was similar in cultured LECs from age-matched male and female mice. Confocal imaging of intracellular calcium in cultures of primary LECs from Cdh5-GCaMP8 mice demonstrated that a functional phenotype was maintained, similar to lymphatic endothelial cells in freshly isolated vessels. This method provides an innovative tool for routine isolation and study of primary LECs from specific collecting lymphatic vessels from any mouse, and in fact, from other species.

Endothelial‐Cell Modifications in Murine Collecting Lymphatic Vessels in Obesity

The presence of edema within tissues and cavities presented by patients afflicted with obesity suggests that lymphatic dysfunction may be a contributing factor during the onset and/or development of the various ailments associated with this disease. Importantly, obesity has been shown to significantly increase the chances of developing type-2 diabetes, cardiovascular diseases, and lymphedema in breast cancer patients undergoing surgical intervention and radiotherapy. In obesity, vascular endothelial dysfunction is known to result as a consequence of the chronic systemic inflammation. Relevant to this study, the extent of lymphatic endothelial dysfunction in obesity and type-2 diabetes remains to be fully assessed. In order to 1) elucidate the root cause of lymphatic dysfunction and subsequent impaired lymph transport in obesity, and 2) determine if this dysfunction involves an endothelial component; it is essential to isolate the various cell types in the lymphatic wall and compare the specific genetic and proteomic makeup in health and disease. Here we focus on the study and understanding of the disease-driven modifications to lymphatic endothelial cells (LECs). We hypothesize that LECs of collecting lymphatic vessels from obese mice will exhibit differential expression of transcription factors commonly associated with endothelial dysfunction (i.e. LEC specification, valve dysfunction, and barrier disruption). We utilized a diet-induced obesity mouse model. Obesity was induced by feeding C57BL/6J male mice a western diet (ResearchDiets Inc. Cat.No. D12079Bi) for 14 weeks starting at 5 weeks of age. Consistent with previous studies, diet-induced obese animals gained twice as much weight when compared to aged-matched animals fed a control diet (weight gain 18.8 g vs 9.7 g). Non-fasted blood glucose levels were 41.6±10.6% higher in obese animals, which also displayed increased skin moisture content, 45.1±4.7% (obese) vs 32.6±4.8% (control), as determined by a wet-to-dry weight ratio. Our preliminary qRT-PCR results on fluorescence-activated cell sorting (FACS) purified LECs show that diet-induced obesity results in decreased expression of the transcription factor, and LEC marker, Prox1. We have developed a novel protocol for establishing cell cultures of primary LECs from collecting lymphatic vessels isolated from different species. Notably, the same protocol with minor modifications can be used to establish cultures of primary endothelial cells from blood vessels. Preliminary characterization of murine primary LECs using RT-PCR, immunofluorescence, and confocal microscopy suggests that these cultures could be a useful novel tool for lymphatic research. Currently, we are using these cultured primary LECs from db/db mice and a proteomics approach to further assess the effects of obesity and type-2 diabetes in the lymphatic endothelium of collecting lymphatics. We anticipate that these studies will provide critical information to identify novel mechanisms of lymphatic endothelial dysfunction in obesity, type-2 diabetes, and lymphedema, and propose novel therapeutic targets to ameliorate lymphatic dysfunction in these diseases.

Loss of Neuropilin 2 in Adult Lymphatic Endothelium Promotes Lymphedema

The lymphatic system is essential in mediating tissue fluid balance. Lymphatic diseases including lymphedema occur as a result of dysfunctional lymphatic networks and are a significant public health issue with no known cure. Our long‐term goal is to understand the cellular and molecular processes that control lymphatic function in order to better treat lymphatic diseases. Some primary lymphedema patients have mutations in the neuropilin‐2 gene (NRP2). NRP2 is a transmembrane receptor found in lymphatic endothelial cells (LEC) that is unique in its ability to transmit both pro‐lymphangiogenic signals from VEGF‐C/D ligands as well as suppressive signals from Semaphorin‐3F (SEMA3F). Previously, we observed elevated tissue swelling in constitutive Nrp2‐deficient mice compared to wild‐type littermates following acute inflammatory reactions. However, it was unclear whether tissue swelling was due to increased vascular leakage (edema) or decreased lymphatic drainage (lymphedema) or both. We hypothesize that Nrp2 regulates lymphatic drainage function in adult tissues and that loss of Nrp2 in adult lymphatic vessels may aggravate lymphedema and swelling. In order to more carefully dissect the molecular signaling events in adult LEC that may contribute to lymphedema, we created inducible, LEC‐specific Nrp2 knockout mice (prox1‐creERT2;Nrp2f/f) and examined lymphangiogenesis and lymphedema models after tamoxifen treatment in adult mice. Compared to appropriate littermate controls, mice lacking Nrp2 in LEC are poorly lymphangiogenic in VEGFC‐induced corneal lymphangiogenesis models and display massive swelling due to increased interstitial fluid and reduced lymphatic drainage in acute and chronic delayed‐type hypersensitivity models and secondary lymphedema models in vivo. Further, culture of primary dermal mouse LEC (mLEC) isolated from Prox1‐iNrp2‐KO mice following tamoxifen treatment reveal suppressed VEGFR3 activation compared to mLEC isolated from control mice. Taken together, our new data indicate that Nrp2 is a critical mediator of homeostatic lymphatic function and overall fluid balance in adult tissues. Our data suggests that the Nrp2 axis may be a pivotal pathway to stimulate lymphangiogenesis and lymphatic drainage in lymphedema conditions resulting from a paucity of lymphatic vessels.Support or Funding InformationR01 HL141858, R01 HL133216, R01 HL137229

Notch signaling pathway is a potential therapeutic target for extracranial vascular malformations

Notch expression has been shown to be aberrant in brain arteriovenous malformations (AVM), and targeting Notch has been suggested as an approach to their treatment. It is unclear whether extracranial vascular malformations follow the same patterning and Notch pathway defects. In this study, we examined human extracranial venous (VM) (n = 3), lymphatic (LM) (n = 10), and AV (n = 6) malformations, as well as sporadic brain AVMs (n = 3). In addition to showing that extracranial AVMs demonstrate interrupted elastin and that AVMs and LMs demonstrate abnormal α-smooth muscle actin just as brain AVMS do, our results demonstrate that NOTCH1, 2, 3 and 4 proteins are overexpressed to varying degrees in both the endothelial and mural lining of the malformed vessels in all types of malformations. We further show that two gamma secretase inhibitors (GSIs), DAPT (GSI-IX) and RO4929097, cause dose-dependent inhibition of Notch target gene expression (Hey1) and rate of migration of monolayer cultures of lymphatic endothelial cells (hLECs) and blood endothelial cells (HUVEC). GSIs also inhibit HUVEC network formation. hLECs are more sensitive to GSIs compared to HUVEC. GSIs have been found to be safe in clinical trials in patients with Alzheimer’s disease or cancer. Our results provide further rationale to support testing of Notch inhibitors in patients with extracranial vascular malformations.

Isolation of Human Skin Lymphatic Endothelial Cells and 3D Reconstruction of the Lymphatic Vasculature In Vitro.

Studies of lymphangiogenesis and lymphatic endothelial biology in vitro require pure cultures of lymphatic endothelial cells and 3D vascular constructs, which closely resemble native human lymphatic vasculature. We describe a method for the isolation of human dermal microvascular lymphatic endothelial cells and generation of a 3D lymphatic capillary network. The lymphatic vascular construct is generated by coculturing primary lymphatic endothelial cells and fibroblasts in their native matrix, without the use of synthetic scaffolds or exogenous factors. The tissue is stable over many weeks and accurately recapitulates features of human dermal lymphatic microvasculature.

Lymphatic Endothelial Cells under Mechanical Stress: Altered Expression of Inflammatory Cytokines and Fibrosis.

Secondary lymphedema, resulting from damage to lymphatic vessels, is a common sequela following surgical removal of lymph nodes for cancer. Current therapeutics for treating lymphedema are limited and further research on underlying causes is warranted. Published studies on molecular mechanisms of lymphedema primarily focus on lymphatic endothelial cells (LECs), which comprise the innermost lining of lymphatic capillaries and collecting vessels. However, traditional static culture of LECs may not adequately recapitulate the lymphedemous cell phenotype as transcriptomal comparison of human dermal LECs has shown significant differences in ex vivo and in vitro LEC gene expression. In this study, we designed a dynamic culture system, in which LECs were exposed to physiologic and excess mechanical strain to determine if native and lymphedemous phenotypes could be reproduced in vitro. Purified human LECs were cultured in silicon dishes and subjected to 0% (control), 4%, and 8% mechanical strain for 72 hours. Our results indicate that control and stretched LECs maintained a mature phenotype. Extreme stretching at 8% strain significantly increased LEC proliferation and significantly increased Prox1 expression, suggesting a lymphedemous cell phenotype resulting with lymphangiogenesis. Mechanical strain reinforced a mature lymphatic phenotype and excess strain promoted lymphangiogenesis, while altering collagen deposition and cytokine secretion.

Lymph node‐derived lymphatic endothelial cells express functional costimulatory molecules and impair dendritic cell‐induced allogenic T‐cell proliferation

Lymphatic endothelial cells (LECs) interact with different immune cells, including T cells within lymph nodes (LNs). However, direct interactions of LECs with immune cells have yet to be investigated. In vitro studies were performed to characterize primary cultures of human LECs derived from LNs in their capacity of interacting with T cells. The results show that LECs express HLA molecules and functional costimulatory molecules needed for T-cell activation. A direct binding of LECs and T cells was detected in cell cultures connected with a clustering of costimulatory molecules on the contact phase. LECs were also able to take up and process antigens. However, major histocompatibility complex class II(+) LECs fail to induce allogeneic T-cell proliferation. Interestingly, supernatants of IFN-γ activated LECs impair proliferation of T cells cocultured with allogeneic dendritic cells, suggesting an inhibitory role of LECs. Indoleamine 2,3 dioxygenase was identified as one inhibitory molecule, which may be responsible for the impaired CD4(+) T-cell proliferation. Our observations suggest a regulatory function for activated LECs on CD4(+) T cells, which may play a role in vivo in the maintenance of the critical balance between tolerance and recall responses.

Establishment and Characterization of Human Lymphatic Endothelial Cell in Breast Cancer Patients Undergoing Sentinel Lymphadenectomy

Recently remarkable progress has been made in the understanding of lymphangiogenesis due to the availability of specific biomarkers. A sentinel lymph node (SLN) biopsy has already been established as a common surgical procedure, and the clinical usefulness of an SLN biopsy has been confirmed in patients with various types of cancer. In this study, a novel method has been successfully developed for the isolation of anatomically defined lymphatic endothelial cells (LECs) from human sentinel lymphatic channels during an SLN biopsy in breast cancer patients by the use of collagenase II digestion. The isolated cells were cultured in EGM2MV media with 10% fetal bovine serum (FBS) under hypoxic conditions in an atmosphere of 5% O 2, 5% CO2 and 90% N2 at 37°C. The cultured cells exhibited a monolayer with a cobblestone appearance. Immunofluorescence analysis using selective lymphangiogenesis markers showed expression of vascular endothelial growth factor receptor 3 (VEGFR-3), prospero homeobox 1 (Prox-1) and Podoplanin. Newly established LECs showed expressions of vascular endothelial growth factor (VEGF) family members except VEGF-D and corresponding VEGF receptors by the use of conventional RT-PCR. Treatment with VEGF-C156S (500 ng/mL) apparently induced phosphorylation of the protein kinase Akt, MAPK and JNK in human isolated LECs as determined by Western blot analysis. Peak induction of Akt, MAPK and JNK occurred at 10 to 15 minutes after incubation of the isolated LECs with VEGF-C156S. The use of the MTS cell proliferation assay showed a significant increase in the growth of human lymphatic endothelial cells with VEGF-C156S treatment. The effects of VEGF-C156S (500 ng/mL) on proliferation activity was significantly with 3% FBS condition alone (MTS score: 0.54±0.0104, n=3) and a 3% FBS+VEGFC156S (MTS score: 0.572±0.0061, n=3) condition. The isolated and enzymatic digested method adopted for the culture of human LECs is simple and useful for the investigation of the cellular, molecular and genomic properties of LECs.

A Novel Approach for Harvesting Lymphatic Endothelial Cells from Human Foreskin Dermis

The lymphatic system plays an important role on tissue fluid and protein transportation, immune cell trafficking, lipid absorption, and even cancer metastasis, according to recent research. Disorders of the lymphatic system may lead to extremity edema and fibrosis. To investigate the physiological and pathophysiological characteristics of the lymphatic system, isolation and culture of lymphatic endothelial cells (LECs) in vitro are critical, although they remain difficult. The authors introduce a novel approach for harvesting LECs from child foreskin dermis. After dispase digestion to remove epidermis, instead of the scrape procedure as described previously, collagenase was used to digest and harvest all the dermal cells, including vessel endothelial cells. Immunomagnetic beads were then applied to isolate CD34()/CD31(+)cells. These cells were characterized as LECs by immunofluorescent and RT-PCR with LECs specific markers including Prox-1, LYVE-1, and VEGFR-3. Proliferation of isolated cells in response to angiogenic factors and hypoxia were tested. Cells exhibited their typical cobblestone morphology as monolayer cultures under standard growth conditions and were propagated for at least seven passages without their characteristics being altered. LECs specifically responded to various stimuli, especially to VEGFs. Using collagenase digestion procedure followed by immunomagnetic beads sorting, human dermal LECs could be harvested successfully.

Microarray Analysis of VEGF-C Responsive Genes in Human Lymphatic Endothelial Cells

Vascular endothelial growth factor-C (VEGF-C) is considered one of the most important factors influencing lymphatic endothelial cell biology. The goal of this work was to characterize the gene expression response by lymphatic endothelial cells (LECs) to VEGF-C. Primary cultures of human microvascular LECs were exposed to 100 ng/mL VEGF-C for 30 minutes and 6 hours, and their lysates were evaluated by microarray analysis to determine changes in mRNA expression induced by VEGF-C. Characteristic of a response to a growth factor stimulus, the largest number of differentially expressed genes were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and the transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis and neurodegeneration were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C.

Lymphatic endothelium: An important interactive surface for malignant cells

Endothelial cells line the vessels which transport fluid and cells throughout the body. Although much attention has been paid to these cells in the context of the blood vascular system, endothelial cells also line lymphatic vessels. Recent progress in identifying growth factors which drive the development of lymphatic vessels and molecular markers specific for lymphatics has expanded our understanding of the role the lymphatic system plays in human pathology. Techniques for purifying populations of lymphatic endothelial cells also allow the in vitro analysis of this unique surface to explore its role in tumour metastasis, immune cell function and fluid transport. This review provides a synopsis of the recent data pertaining to the purification and culture of lymphatic endothelial cells, and the interaction of tumour cells with lymphatic endothelium.

Basic fibroblast growth factor-mediated lymphangiogenesis of lymphatic endothelial cells isolated from dog thoracic ducts: effects of heparin.

We have attempted to evaluate whether, similar to the angiogenesis of blood vessels, cultures of lymphatic endothelial cells (LEC) isolated from dog thoracic ducts have an ability to induce lymphangiogenesis in response to basic fibroblast growth factor (bFGF), then to examine the effects of heparin on the bFGF-mediated morphogenesis. The effects of bFGF and/or heparin on the proliferation and migration of the LEC were evaluated by changing the number of the subconfluent cells and by wound migration assay, respectively. The effects of the agents on invasion and tube formation of the LEC into a three-dimensional collagen gel and on collagen gel induced tube formation of the LEC were also investigated by a phase-contrast microscope and an electron microscope. The bFGF (10 ng/ml) caused a significant induction of proliferation and migration of the LEC, the induction of which was augmented dose-dependently by an additional treatment with heparin ranging from 1 to 100 microg/ml. The bFGF produced invasion and tube formation of the LEC into a three-dimensional collagen gel. The bFGF also facilitated to form capillary-like tubes of the LEC between two layers of collagen gels. Heparin (10 microg/ml) accelerated both processes of bFGF-mediated lymphangiogenesis of the LEC. These findings suggest that the cultured LEC isolated from dog thoracic ducts have an ability to form lymphatic capillary-like tubes in response to bFGF and that heparin accelerates dose-dependently the process of the bFGF-mediated neovascularization of lymph vessels.

Nitric Oxide Production by Lymphatic Endothelial Cells in Vitro

The present study demonstrates that confluent monolayer cultures of lymphatic endothelial cells produce and secrete NO. Immunofluorescent studies showed that eNOS activity can be stimulated with Ca ionophore to enhance the production of NO. Cells and various cytokines stimulated the production of iNOS which showed the greatest increase in activity at 4 hrs and declined at 18 and 24 hrs. These studies provide evidence that, within the lymphatic vascular lumen, nitric oxide may be produced by the lymphatic endothelium which interact with various vasoactive substances to regulate lymphatic vascular tone. In addition, the production of NO by LEC may be important in the regulation of lymphatic vascular tone in order to more readily accommodate sudden fluctuations in lymph flow and pressure that normally occur during the process of lymph formation and propulsion.

Lymphangiogenesis in vitro: Formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells.

Lymphatic endothelial cells grown long term in culture form lymphatic capillarylike tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.

Lymphatic endothelium isolation, characterization and long term culture

Using a collagenase trypsin-EDTA treatment, we have been able to successfully isolate and grow primary cultures of the lymphatic endothelium (LEC) that were subcultured, frozen for storage, subsequently thawed with good recovery and growth, and serially subcultured. The morphological features of cultured LEC were consistent with that observed for the endothelium of intact lymphatic vessels. A prominent feature of growing cultures was the appearance of large vacuoles in the perinuclear region of the cytoplasm, which became filled with fluid and cell debris engulfed from the culture medium. The basal cell surface lacked a well defined basal lamina and anchoring filaments were observed extending from the basal plasmalemmal surface into the underlying substratum. LEC in cultures were also positive for Factor VIII-related antigen. However, specific granules, characteristic of Weibel-Palade bodies were not observed in ultrathin sections of confluent cultures. F-actin was identified in LEC cultures using fluorescein phalloidin, and in confluent cultures actin filaments were located at the periphery of the cell as a continuous circumferential thin band and short filamentous bundles in the central part of the cell. By using heparin and endothelial cell growth supplement in the culture medium we have been able to grow stable cultures of lymphatic endothelial cells that could be maintained when serially subcultured for over two years. These LEC cultures provide an in vitro model for investigating the function and biochemical properties of the lymphatic endothelium.