Lymphatic Endothelial Cell Markers Research Articles

The prognostic influence of lymphatic endothelium-specific hyaluronan receptor 1 in cancer: A systematic review.

Lymphangiogenesis is a key process in cancer development and metastasis. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) is a widely used marker for lymphatic endothelial cells (LEC), which also mediates immune and cancer cell migration. Recently, LYVE‐1–positive tumor cells were shown to acquire LEC‐like phenotype and exploit this receptor for lymphatic dissemination. Furthermore, selective targeting of LYVE‐1 impaired the growth of cancer‐related vasculature and reduced metastasis in vivo, signifying its role in therapeutic and prognostic applications. Although numerous studies have investigated the role of LYVE‐1 in cancer, a unifying detailed review of its prognostic utility is lacking to date. Thus, we compiled and critically appraised evidence from clinical studies comprising a total of 2352 patients diagnosed with different types of cancer and using a variety of experimental approaches. Collectively, most studies revealed a significant association between LYVE‐1 overexpression and dismal outcome of at least one survival estimate. Furthermore, the importance of vasculature location, intra‐ or peritumoral, and the influence of various lymphangiogenesis‐related parameters, such as lymphatic vessel density and invasion, were discussed. However, the specificity of LYVE‐1 staining is challenged by its expression in non‐LEC cells, implying the need for double labelling to better estimate its prognostic significance. In conclusion, this is to our knowledge the first comprehensive systematic review on the prognostic value of LYVE‐1 in cancer. More well‐designed studies across different populations and the development of standardized protocols would be paramount for the consistency of LYVE‐1 findings and for its potential transferability to clinical practice in future.

Immunohistochemical and immunofluorescence expression profile of lymphatic endothelial cell markers in oral cancer.

Lymphangiogenesis makes an important contribution to the tumour microenvironment (TME), but little is known about this in oral squamous cell carcinoma (OSCC). Archival formalin-fixed paraffin-embedded specimens (28 OSCC, 10 inflamed and 6 normal oral mucosa controls) were processed using immunohistochemistry (IHC) with antibodies against lymphatic markers D2-40 (podoplanin), LYVE-1, VEGFR3 and Prox1. After the endothelial cells had been highlighted by the various markers for lymphatic endothelium, the positive stained cells and vessels were identified and counted in a systematic manner to determine microvessel density. Double-labelling immunofluorescence (DLIF) was used to investigate the specificity of D2-40 and LYVE-1 to lymphatic endothelial cells (LECs) as opposed to blood ECs. There was higher D2-40 and Prox1 lymphatic vessel density (P=.001) in the OSCC group when compared with both control groups. Some malignant keratinocytes expressed lymphatic markers, as did a much smaller number of epithelial cells in the control groups. DLIF showed that no vessels co-expressed D2-40/CD34 or LYVE/CD34. Some D2/40+ LVs were LYVE- . D2-40 was the most specific LEC marker in OSCC tissues. These results establish that the OSCC TME contains significantly more lymphatic vessels expressing D2-40 and Prox1 than the control groups, which may play a role in facilitating lymphatic invasion and metastases.

Sema4C modulates the migration of primary tumor-associated lymphatic endothelial cells via an ERK-mediated pathway.

Although lymphatic endothelial cells (LECs) serve a positive role in tumor lymphatic metastasis, the regulation of LECs undergoing migration similar to that of tumor cells remains poorly understood. A previous study revealed that semaphorin 4C (Sema4C) could be a marker of LECs in cervical cancer. Thus, the present study aimed to understand the mechanism via which Sema4C could promote the development of tumor-associated characteristics in LECs in cervical cancer. Primary tumor-associated LECs (TLECs) were distinguished from cervical cancer by flow cytometry. The promigratory ability was assessed using the Transwell assay. Lentivirus infection was used to alter the expression of Sema4C in TLECs. Confocal laser scanning was used to determine the infection efficiency of lentivirus infection. Sema4C/ERK/E-cadherin pathway was measured by reverse transcription-quantitative PCR and western blotting. The co-localization of Sema4C and the lymphatic marker lymphatic vessel endothelial hyaluronan receptor 1 was verified. Primary tumor-associated LECs (TLECs) were isolated from a mouse xenograft cervical tumor model. It was revealed that overexpressing Sema4C stimulated the migratory ability of TLECs, downregulated E-cadherin expression and stimulated ERK phosphorylation, whereas knocking down Sema4C had the opposite effects. The treatment of PD98059 (ERK inhibitor) blocked the pro-migratory ability of TLECs, which indicated a dependence on the ERK signaling pathway. It was identified that the Sema4C/ERK/E-cadherin pathway may be critical for the migration of TLECs, which may promote lymph node metastasis. Therefore, Sema4C could be a promising target for the treatment of cervical cancer with lymphatic metastasis.

Epitope Mapping of the Anti-California Sea Lion Podoplanin Monoclonal Antibody PMab-269 Using Alanine-Scanning Mutagenesis and ELISA.

Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN (seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63-82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alanine-substituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope might contribute to the pathophysiological investigations of seaPDPN.

Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model.

The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel-tumor cell coculture assay.

Development of Monoclonal Antibody PMab-269 Against California Sea Lion Podoplanin.

The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.

A new approach to lymphedema following breast cancer treatment with lymphatic endothelial cell markers

Amaç: Lenfödem (LE), aksiller lenf nodu diseksiyonu ve radyoterapi ile tedavi edilen meme kanserli olgularda yaygın gelişen iyatrojenik bir komplikasyondur. Bu çalışmada lenfödemli hastalarda meme kanseri tedavisi sonrası, lenfatik sistem endotelinden salınan, homeobox transkripsiyon faktörü (Prox-1), lenfatik damar endotel reseptörü-1 (LVYE-1) ve podoplanin (PDPN) belirteçlerinin serum düzeylerini araştırmayı amaçladık.
 Metod: Çalışmanın LE grubunu meme kanseri tedavisi sonrası üst ekstremitede LE gelişen toplam 44 kadın hasta, kontrol grubunu ise demografik açıdan LE grubu ile benzer özellikler gösteren 44 sağlıklı kadın oluşturdu. Hastaların kol çevreleri ölçüldü ve her iki kol çevresi toplamları arasındaki fark (DSOAC) 2 cm veya ≥ %10 ise LE olarak kabul edildi. Serum Prox-1, LVYE-1 ve PDPN konsantrasyonları ticari ELISA (Enzyme-linked immunosorbent assay) ile ölçüldü.
 Bulgular: LE grubunda Prox-1, LVYE-1 ve PDPN seviyeleri kontrol grubuna göre anlamlı olarak düşük bulundu (sırasıyla; p < 0.01, p = 0.02, p = 0.04). Prox-1 seviyeleri Evre 1 LE'li hastalarda Evre 2 LE’li hastalara göre anlamlı yüksekti (p

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.

Characterization of kaposiform lymphangiomatosis tissue-derived cells.

Kaposiform lymphangiomatosis (KLA) is a recently characterized systemic lymphatic anomaly. Activation of RAS/MAPK and PI3K/AKT/mTOR pathways may affect KLA pathogenesis, but the cellular basis of KLA is unclear. Abnormal-spindle endothelial cells that express lymphatic endothelial cell (LEC) markers are characteristic of KLA histopathology. This study evaluated patient-derived KLA cells to establish their morphological and biological characteristics. We established cell lines from primary KLA tissues of two patients with KLA and examined their morphological and functional characteristics, messenger RNA and protein expression profiles, gene mutations, and responses to inhibitors of the RAS/MAPK and PI3K/AKT/mTOR pathways. Both KLA cell lines showed spindle-shaped morphology, stained positive for podoplanin (PDPN), and exhibited impaired tube-formation properties. They expressed LEC marker PDPN and mesenchymal stem cell markers (CD90, CD105) in the absence of endothelial cell markers (CD34, CD31, VWF), per real-time polymerase chain reaction. Both mTOR inhibitor rapamycin and MEK inhibitor trametinib inhibited growth of the two cell lines. A NRAS p.Q61R variant was found in one of two independent KLA tissue samples, but not in the KLA cells (per targeted next-generation sequencing); and KLA cells with this variant had elevated AKT phosphorylation levels. ERK phosphorylation levels were undetectable in both KLA cell lines. Inhibition of the RAS/MAPK and PI3K/AKT/mTOR pathways may represent potential therapeutic targets in KLA. These patient-derived KLA cell lines will be useful research tools to elucidate KLA etiology, and could pave the way for basic, translational, and preclinical studies of this disease.

Expression of Lymphatic Markers in the Berger's Space and Bursa Premacularis.

We previously reported that the bursa premacularis (BPM), a peculiar vitreous structure located above the macula, contains numerous cells expressing markers of lymphatic endothelial cells, such as podoplanin and LYVE-1. Herein, we examined the expression of lymphatic markers in the Berger’s space (BS), BPM, and vitreous core (VC). BS, BPM, and VC specimens were selectively collected in macular hole and epiretinal membrane patients during vitrectomy and were then immunostained with antibodies for podoplanin, LYVE-1, and fibrillin-1 and -2. By visualization using triamcinolone acetonide, the BS was recognized as a sac-like structure with a septum located behind the lens as well as BPM. Those tissues adhered to the lens or retina in a circular manner by means of a ligament-like structure. Immunostaining showed intense expression of podoplanin and LYVE-1 in the BS. Both BS and BPM stained strongly positive for fibrillin-1 and -2. The VC was faintly stained with antibodies for those lymph-node markers. Our findings indicate that both BS and BPM possibly belong to the lymphatic system, such as lymph nodes, draining excess fluid and waste products into lymphatic vessels in the dura mater of the optic nerve and the ciliary body, respectively, via intravitreal canals.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

New reporter zebrafish line unveils heterogeneity among lymphatic endothelial cells during development.

In zebrafish, lymphatic endothelial cells (LECs) originate from multiple/several distinct progenitor populations and generate organ-specific lymphatic vasculatures. Cell fate and tissue specificities were determined using a combination of genetically engineered transgenic lines in which the promoter of a LEC-specific gene drives expression of a fluorescent reporter protein. We established a novel zebrafish transgenic line expressing eGFP under the control of part of the zebrafish batf3 promoter (Basic Leucine Zipper ATF-Like Transcription Factor 3). Spatiotemporal examination of Tg(batf3MIN:eGFP) transgenic fish revealed a typical lymphatic expression pattern, which does not perfectly recapitulate the expression pattern of existing LEC transgenic lines. eGFP+ cells constitute a heterogeneous endothelial cell population, which expressed LEC and/or blood endothelial cells (BEC) markers in different tissues. In addition, we characterize the renal eGFP+ cell as a population of interest to study kidney diseases and regeneration. Our Tg(batf3MIN:eGFP) reporter zebrafish line provides a useful system to study LEC populations, of which heterogeneity depends on origin of progenitors, tissue environment and physiological conditions. We further developed a novel fish-adapted tissue clearing method, which allows deep imaging and 3D-visualization of vascular and lymphatic networks in the whole organism.

The landscape of ‘podoplanin’ in dermatology: a narrative review

Podoplanin (PDPN) is a specific marker for lymphatic endothelial cells; however, it can be expressed by many other cells including renal podocytes. In dermatology, PDPN has been involved in diverse disorders, such as tumor lymphangiogenesis, as well as in immune-mediated disorders. Herein, we illustrate and discuss the landscape of PDPN in dermatology.

Vanishing bone disease: Decoding the rare entity of Gorham–Stout disease

Vanishing bone disease is a rare entity with an obscure cause and undetermined pathophysiology and characterized by spontaneous and progressive resorption of bones. To date, the etiology of Gorham–Stout syndrome is still controversial. However, LYVE-1, a receptor, and podoplanin, a transmembrane glycoprotein identified by the antibody D2-40, are two extensively used molecular markers of lymphatic endothelial cells. These markers do exist in the medullary and cortical portions of Gorham–Stout disease (GSD) patients' bones. The following case report describes a case of 32-year-old male with GSD involving the maxilla with a brief discussion on etiopathogenesis, clinical features, radiological and histological findings, and diagnosis with treatment options.

Peri-tumoral Lymphovascular density by antipodoplanin antibody D2-40, as a predictor of nodal metastasis in oral squamous cell carcinoma

BACKGROUND:
 The involvement of regional lymph nodes by cancer is a major determinant of prognosis in oral squamous cell carcinoma (OSCC) and is a significant predictor of patient survival. Peri-tumoral lymphangiogenesis is being studied as an important tool in predicting neck lymph node metastasis. Podoplanin, a transmembrane glycoprotein is expressed on lymphatic vessel endothelium and considered as a marker of lymphangiogenesis. The high podoplanin expression has been associated with invasion, progression, and metastasis of oral cancer. D2-40, an anti-podoplanin monoclonal antibody, is a specific marker of lymphatic endothelial cells. The present study was designed to investigate the peritumoral lymphovascular density (PT-LVD) by evaluating D2-40 expression and its potential utility in predicting lymph node metastasis in OSCC.
 METHODS: Seventy-two patients with OSCC were enrolled (36 with early and 36 with advanced-stage tumors). Differential expression of peritumoral lymphovascular density (PT-LVD) was evaluated using D2-40 expression by immunohistochemistry (IHC). Expression was categorized as low and high, derived from the mean of a number of vessels per 10 high power fields in hot-spots. Statistical methods were employed to correlate expression with age, gender, tumor site, stage, and neck lymph node involvement. 
 RESULTS: We observed a significant association between peritumoral lymphovascular density (PT-LVD) with clinical neck lymph node status (p=0.001) and tumor stage (p=0.033). There was no significant correlation of D2-40 expression with age, gender, tumor site, T stage, and tumor differentiation.
 CONCLUSION: PT-LVD is a positive predictor of neck lymph node metastasis. It has translational potential, along with other clinical parameters for decision making in the treatment of the neck in oral cancer.

Lymphatic Remodeling Associated with Hemophilic Joint Bleeding in FVIII-Deficient Mice

Introduction Hemophilic joint bleeding leads to toxic synovial iron accumulation, contributing to synovitis. Mechanisms of blood and iron clearance from the joint remain largely unknown. This study aimed to explore synovial lymphatic vessel remodeling in association with hemarthrosis and iron accumulation in Factor VIII (FVIII)-deficient mice as a potential mechanism of iron clearance. Methods Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and the hematocrit determined on day 2 as a measure of joint bleeding. Synovial tissue was harvested at baseline and 2 weeks after knee injury for gene expression analyses by RNA sequencing. Briefly, RNA was purified from synovial tissue and sequenced (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses. Whole knee joints were harvested at baseline and 2 or 4 weeks after knee injury for histological analysis of iron accumulation, as well as blood and lymphatic vessels. Ferric iron (Fe3+) was detected by Prussian blue staining during joint decalcification. Remodeling blood and lymphatic vessels were detected using antibodies for α-smooth muscle actin (αSMA) and lymphatic vascular endothelial hyaluronan receptor 1 (LYVE1), respectively. Total soft tissue and stained areas in histology images were quantified using ImageJ. Co-localization of αSMA and LYVE1 was assessed by laser scanning confocal microscopy with a Zeiss LSM 800 microscope. Results Knee injury induced hemarthrosis in FVIII-deficient mice (mean day 2 hematocrit: 27 %) and significant soft tissue proliferation, indicative of synovitis, at 2 weeks (2.9-fold increase in mean tissue area, p=0.007). Gene expression analyses by RNA sequencing revealed differential expression of lymphatic markers, including very high up-regulation of chemokine (C-C motif) ligand 21 (194-fold, p=0.001) and moderate up-regulation of semaphorin 7a (4.9-fold, p=0.02). Up-regulation of these genes may promote homing and adherence of macrophages and dendritic cells to lymphatic endothelial cells (LECs), facilitating lymphangiogenesis. Up-regulation of FMS-like tyrosine kinase 4 (receptor for vascular endothelial growth factor (VEGF)-C and -D) (5.7-fold, p=0.0007) and collagen and calcium-binding EGF domain-1 (5.6-fold, p=0.03) may also favor lymphangiogenesis. However, VEGF-C expression was unchanged and VEGF-D was 2.5-fold down-regulated (p=0.0002). Likewise, expression of LEC marker LYVE1 was down-regulated (2.6-fold, p=0.05). These findings stimulated histological evaluations of lymphatic vessels in relation to iron and blood vessels in synovial tissue. Prussian blue staining showed significant increases in ferric iron accumulation at 2 weeks (38-fold, p=0.005) and 4 weeks relative to baseline (23-fold, p=0.03). This coincided with blood vessel remodeling, evidenced by increased αSMA expression (r=0.6, p=0.0008). Unlike αSMA, an abundance of LYVE1 was detected in baseline synovial tissue. LYVE1-positive vessels appeared as small, flat structures, in contrast to larger, more rounded αSMA-positive vessels. Confocal microscopy confirmed staining of distinct blood and lymphatic vascular structures. When normalized for tissue area, LYVE1 expression was 1.8-fold decreased at 2 weeks (p=0.04) after knee injury compared to baseline and returned to near-baseline levels at 4 weeks. There was a significant negative correlation between iron accumulation (Prussian blue) and lymphatic vessel staining normalized to tissue area (r=-0.6223, p=0.006). The morphology of lymphatic vessels after joint bleeding was different to baseline, with many vessels appearing as larger, more confluent structures with visible lumens, sometimes wrapping around αSMA-positive blood vessels. Conclusions Synovial tissue in FVIII-deficient mice contains a rich network of lymphatics. Gene expression changes and altered lymphatic vessel morphology after joint bleeding point toward profound changes in lymphatic homeostasis in inflamed synovium. The reduction in lymphatic vessel density suggests an inadequate lymphangiogenic response, which may hamper iron clearance. The role of the lymphatic network after hemophilic joint bleeding warrants further investigation and may represent a novel target for improving bleed resolution and reducing synovitis. Disclosures von Drygalski: Bioverativ/Sanofi: Honoraria, Research Funding; Takeda: Honoraria; Novo-Nordisk: Honoraria; Biomarin: Honoraria; Uniqure: Honoraria; Pfizer: Research Funding; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees, Other: co-founder.

Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260.

Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.

Absence of lymphatic vessels in term placenta

BackgroundThere has been debate about the existence of lymphatic vessels in placenta. Lymphatic endothelial cell (LEC) markers such as LYVE-1 and podoplanin/D2–40 have been found, although PROX1 has not been detected. The most reliable marker for LECs is the double staining for CD31 and PROX1, which has not been performed yet.MethodsWe studied three term placentas and dissected them into three areas: i.) basal plate area, ii.) intermediate area, and iii.) chorionic plate area. We used immunofluorescence single and double staining with antibodies against CD31, PROX1, LYVE-1, VEGFR-3, D2–40/PDPN, CD34, CCBE-1, and vimentin, as well as nested PCR, qPCR, Western blot and transmission electron microscopy (TEM).ResultsAt TEM level we observed structures that have previously mistakenly been interpreted as lymphatics, however, we did not find any CD31/PROX1 double-positive cells in placenta. Absence of PROX1 was also noted by nested PCR, qPCR and Western blot. Also, LEC marker VEGFR-3 was expressed only in a small number of scattered leukocytes but was absent from vessels. The LEC marker D2–40/PDPN was expressed in most stromal cells, and the LEC marker LYVE-1 was found in a considerable number of stromal cells, but not in endothelial cells, which were positive for CD31, CD34, CCBE-1 and vimentin. Additionally, vimentin was found in stromal cells.ConclusionsOur studies clearly show absence of lymphatics in term placenta. We also show that the functional area of the mother’s endometrium is not penetrated by lymphatics in term pregnancy.

Development of an Anti-Sheep Podoplanin Monoclonal Antibody PMab-256 for Immunohistochemical Analysis of Lymphatic Endothelial Cells.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the Cell-Based Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1, kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256 stained type I alveolar cells of lung, renal glomerulus and Bowman's capsule, and lymphatic endothelial cells of lung and colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.

Telocytes and lymphatic endothelial cells: Two immunophenotypically distinct and spatially close cell entities

Telocytes (TCs) have recently emerged as a peculiar type of stromal cells located in both perivascular and interstitial compartments of multiple anatomical sites in humans, other mammals and vertebrates. Pioneer electron microscopy studies have ultrastructurally defined TCs as “stromal cells with telopodes” (i.e. very long and thin cell processes with a moniliform morphology conferred by the irregular alternation of slender segments and small, bead-like, dilated portions), whereupon it has become apparent that TCs largely correspond to the CD34+ stromal/interstitial cells detectable by immunohistochemical assays. Besides CD34, TCs are also characterized by the expression of platelet-derived growth factor receptor (PDGFR)α. Interestingly, recent works recommended that lymphatic endothelial cell (LEC) markers should be routinely assessed to discriminate with certainty TCs from LECs, because these two cell types may exhibit similar morphological traits, especially when initial lymphatics are sectioned longitudinally and appear as vascular profiles with no obvious lumen. Furthermore, it has been argued that lymphatic microvessels immunostained for the small mucin-type transmembrane glycoprotein podoplanin (PDPN), which is widely used as lymphatic endothelial marker, can be easily misidentified as TCs. Nevertheless, surprisingly these assumptions were not based on double tissue immunostaining for TC and LEC markers. Therefore, the present morphological study was undertaken to precisely investigate the mutual spatial organization and putative relationships of TCs and lymphatic vessels in tissues from different human organs. For this purpose, we carried out a series of double immunofluorescence analyses simultaneously detecting the CD34 or PDGFRα antigen and a marker of LECs, either PDPN or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the connective tissue compartment of different organs, TCs were CD34+/PDGFRα+/PDPN−/LYVE-1− while LECs were CD34−/PDGFRα−/PDPN+/LYVE-1+, thus representing two definitely distinct, though spatially close, cell entities. The arrangement of telopodes to intimately surround the abluminal side of LECs suggests a possible role of TCs in the regulation of lymphatic capillary functionality, which is worth investigating further.