Lymphatic Endothelial Cell Markers Research Articles

Role of lymphatic endothelium specific hyaluronan receptor 1 in virus infection and associated diseases.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) serves as a prominent marker for lymphatic endothelial cells (LECs) and is pivotal in the process of lymphangiogenesis, a critical factor in cancer development and metastasis. Overexpression of LYVE-1 has been observed in various cancers, where it is recognized as an adverse prognostic indicator. Targeting LYVE-1 has demonstrated inhibitory effects on tumor cell proliferation, migration, and the formation of lymph node metastases both in vitro and in vivo. While extensive research has focused on the role of LYVE-1 in cancer cells, its involvement in virus infection and associated diseases remains largely unexplored. This review consolidates recent findings regarding the expression of LYVE-1 and its functions in lymphangiogenesis during various viral infections and the development of related diseases, with a particular emphasis on Kaposi's sarcoma herpesvirus. Despite the limited available data, it is evident that further studies are essential to comprehensively understand the contribution of LYVE-1 to viral pathogenesis and oncogenesis.

The discriminative role of PROX-1 immunohistochemistry between venous malformation and lymphatic malformation of the deep type with no visible diagnostic surface skin lesion.

Venous malformations (VMs) are distinguished from lymphatic malformations (LMs) when specific diagnostic skin lesions are present. In the deep type, this is difficult by clinico-radiologic evaluation alone. We aimed to investigate the usefulness of lymphatic vessel endothelial cell (LEC) markers for the differential diagnosis of the deep VMs and LMs. A retrospective study was conducted based on the medical records of patients with VMs and LMs who underwent biopsy with both D2-40 and PROX-1 immunohistochemistry. We compared the initial clinico-radiological diagnosis with the final pathological diagnosis and identified which ones showed a difference. From 261 patients who had VMs and LMs, 111 remained after the exclusion of those who showed definite surface diagnostic features. After pathological diagnosis with the expressions of D2-40 and PROX-1, 38 of 111 (34.2%) patients' final diagnoses were changed. Among these 38 cases, diagnosis was not changed by D2-40 positivity alone, but changed by PROX-1 positivity alone (52.6%) or by both (47.4%). The diagnostic changes were more frequent in the deep category (43.7%) than in the superficial category. Identifying the expression of D2-40, and especially PROX-1, in the differential diagnosis of VMs and LMs may provide important treatment guidelines and understanding their natural course.

Lymphatic vessels accompanying dorsal and basal dural sinuses in the human brain

Recent investigations showed the presence of meningeal lymphatic vessels (mLVs) along the superior sagittal and transverse dural sinuses which drain both fluid and immune cells from the cerebrospinal fluid (CSF) to the deep cervical lymph nodes. This study uses immunohistochemistry (IHC) and the Western Blot technique to show the presence of mLV accompanying the dorsal (superior sagittal, inferior sagittal, transverse, sigmoid, and straight) and basal (cavernous, sphenoparietal, superior, and inferior petrosal) dural sinuses in the human brain. Samples for IHC were obtained from dorsal and basal meningeal dural sinuses of 3 human cadavers and 3 autopsies. Routine histological techniques were carried out for the specimens. Podoplanin (PDPN, lymphatic vessel endothelial cell marker) and CD31 (vascular endothelial cell marker) IHC staining were applied to the 5 µm thick paraffin sections. Furthermore, PDPN and CD31 protein expressions were evaluated using Western Blot to the tissue samples from the same regions of 4 autopsies. Two consecutive sections from each sinus were PDPN, and CD31 was stained to differentiate blood vessels (BV) from mLV. The IHC staining showed the presence of mLVs accompanying both dorsal and basal dural sinuses. The mLVs accompanying the dorsal dural sinuses had a larger dimensions range compared to the basal dural sinuses. However, the number of mLVs along the basal dural sinuses was more than the mLVs along the dorsal ones. Further, fluid channels were closely localized to the mLV, with varying diameters and densities. Western Blotting technique showed the presence of PDPN expression in both dorsal and basal dural sinus samples. The knowledge of the presence of mLV along both dorsal and basal dural sinuses in humans can increase the understanding of how mLV contributes to the brain lymphatic circulation and may help understand the neuropathophysiological processes of various neurological diseases.

Scanning Electron Microscopy Analysis of Lymphatic Regeneration in a Secondary Lymphedema Mouse Model: A Preliminary Study

Under inflammatory conditions including lymphatic disorders, bone marrow-derived myeloid cells often express lymphatic endothelial cell (LEC) markers, and these cells are then called LEC progenitor cells, which extend lymphatic vessels by fusing with existing lymphatic vessels. However, studies on the mechanism of lymphatic regeneration using three-dimensional images of lymphatic structures are limited. In this study, scanning electron microscopy (SEM) was used to observe the three-dimensional structure of lymphangiogenesis in a mouse model of secondary lymphedema. The model was established in C57BL/6J mice via circumferential incision in the inguinal region of the left hind limb. Skin samples were obtained from the lymphedema region on days 2, 5, and 8 after surgery. To determine lymphatic vessel positions using SEM analysis, we detected anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) immunoreactivity in serial sections and overlaid them during SEM observation. On days 2 and 5, spherical cells, probably myeloid cells, were attached and fused to the LYVE-1-positive lymphatic vessel walls. On day 8, spherical cells were converted to string-shaped cells, forming a new lymphatic vessel wall resembling an intraluminal pillar. Our results showed the newly formed lymphatic vessel wall extended into the lumen, suggesting intussusceptive lymphangiogenesis.

Widespread distribution of lymphatic vessels in human dura mater remote from sinus veins.

Background and purpose: Previous experimental studies have shown that meningeal lymphatic vessels are located primarily along the walls of the dural sinus veins. Whether they are more widespread throughout human dura mater has presently not been characterized. The present study explored in humans whether meningeal lymphatic vessels may be identified remote from the sinus veins and whether they differ in the various location of dura mater. Methods: We included 15 patients who underwent neurosurgery, in whom dura mater was removed as part of the planned procedure. Tissue was prepared for immunohistochemistry using the lymphatic endothelial cell markers lymphatic vessel endothelial hyaluronan receptor 1 protein (LYVE-1), podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3). Results: Lymphatic endothelial cell positive cells were found in dura mater at the posterior fossa (n = 8), temporal skull base (n = 5), frontal convexity (n = 1), and cranio-cervical junction (n = 1). They were most commonly seen remote from blood vessels, but also occurred along blood vessels, and seemed to be most abundant at the skull base. Conclusion: The present observations show that human lymphatic vessels are widespread in dura mater, not solely lining the dural sinuses.

Podoplanin-positive cells located in the basal layer of Bowen disease include tumor cells with cancer stem cell properties.

Podoplanin (PDPN) is widely used as a marker of lymphatic endothelial cells. PDPN is also involved in tumor progression, and upregulated PDPN expression is often found in various cancers. In this study, we first immunohistochemically examined PDPN expression in 87 cases of Bowen disease. Positive expression was detected in 64.4% of Bowen disease specimens, and the positive cells were exclusively located in the basal layer and corresponded to palisaded basal cells (PBCs). PBCs have been considered to be residual normal keratinocytes so far, but PDPN expression in cancers is generally associated with poor clinical outcomes. We also examined PDPN expression in 27 cases of Bowen carcinoma. Diffuse and strong PDPN expression was detected in 22.2% of Bowen carcinoma specimens, and another 22.2% showed PDPN expression at the leading edges of tumor nests. These results prompted us to determine whether PDPN-positive cells are more tumorigenic than PDPN-negative cells. We cultured Bowen disease cells using a three-dimensional (3D) cell culture system and examined PDPN expression. In the cultured Bowen disease tissue, PDPN expression was again detected in the basal layer. Then, we isolated 1.2 × 105 PDPN-positive and -negative cells from the 3D organotypic culture of Bowen disease by fluorescence-activated cell sorting analysis and compared their tumorigenicity using 3D culture. The PDPN-positive tumor cells were able to regenerate Bowen disease tissue, but the PDPN-negative tumor cells were not. In addition, the regenerated Bowen disease tissue derived from the PDPN-positive cells exhibited PDPN expression in its basal layer, as the parental Bowen disease tissue did. These results indicate that PDPN-positive cells include tumor cells with cancer stem cell properties. Although the precise mechanism through which PDPN expression is involved in the pathogenesis of Bowen disease needs to be determined, PDPN may be a novel druggable target for Bowen disease.

C61 RAREFACTION OF MYOCARDIAL LYMPHATIC VESSELS IN PATIENTS WITH CARDIAC AMYLOIDOSIS

Abstract Introduction In cardiac amyloidosis (CA), amyloid fibers accumulate in the extracellular spaces of the heart, causing a compression and obliteration of lymphatic vessels, and possibly their disappearance. As lympathic drainage removes proteins from the interstitial spaces, including amyloid precursors, an impairment of lympathic vessels could exacerbate amyloid accumulation, triggering a vicious circle. Methods Lymphatic vessels were visualized by immunohistochemistry using a monoclonal antibody against podoplanin (D2–40, a marker of lymphatic endothelial cells) and the immunopreroxidase technique. We investigated 3 groups of cases: 1) as a reference standard, endomyocardial specimens from the left ventricular (LV) endomyocardium of 10 heart valve donors, deceased for trauma and/or cerebral hemorrhage, with no evidence of cardiac disease on gross and microscopic examination; 2) 15 consecutive LV endomyocardial biopsies (EMBs) from patients with CA, where cardiac amyloid was detected and typed by Congo red staining, immunohistochemistry and proteomics, and 3) 13 surgical endomyocardial biopsies from patients with septal LV hypertrophy, undergoing aortic valve surgery and with CA excluded by histology. Results In donor hearts (n=10), lymphatic vessels were mostly distributed in the subendocardial and subepicardial layers, with some intra–myocardial lymphatic vessels near larger blood vessels. We then examined 15 patients with CA (amyloid transthyretin [ATTR]–CA, n=10; amyloid light–chain [AL]–CA, n=5; 50% men, median age 75 years [interquartile range 71–78]), and 13 patients with CA excluded (55% men, median age 68 years [59–71]). The density of lymphatic vessels in patients with CA was 0.52 vessels/mm2 (0–3.32), vs. 10.90 vessels/mm2 (3.10–26.25) in those without CA (p=0.001). No significant differences were found in LV mass index (p=0.170) or LV ejection fraction (p=0.262). Three patients with ATTR–CA and 2 patients with AL–CA showed no lymphatic vessels on EMB. The density of lymphatic vessels did not differ significantly in patients with ATTR– or AL–CA. Conclusions Patients with CA have significantly decreased endo–myocardial lymphatic vessels as compared to patients without CA, despite no significant difference in the degree of LV wall thickening or LV ejection fraction. These results support the hypothesis that the lymphatic drainage is deranged in patients with CA, possibly promoting further amyloid deposition.

Characteristic Features of Deep Brain Lymphatic Vessels and Their Regulation by Chronic Stress.

Studies have demonstrated that a functional network of meningeal lymphatic vessels exists in the brain. However, it is unknown whether lymphatic vessels could also extend deep into the brain parenchyma and whether the vessels could be regulated by stressful life events. We used tissue clearing techniques, immunostaining, light-sheet whole-brain imaging, confocal imaging in thick brain sections and flow cytometry to demonstrate the existence of lymphatic vessels deep in the brain parenchyma. Chronic unpredictable mild stress or chronic corticosterone treatment was used to examine the regulation of brain lymphatic vessels by stressful events. Western blotting and coimmunoprecipitation were used to provide mechanistic insights. We demonstrated the existence of lymphatic vessels deep in the brain parenchyma and characterized their features in the cortex, cerebellum, hippocampus, midbrain, and brainstem. Furthermore, we showed that deep brain lymphatic vessels can be regulated by stressful life events. Chronic stress reduced the length and areas of lymphatic vessels in the hippocampus and thalamus but increased the diameter of lymphatic vessels in the amygdala. No changes were observed in prefrontal cortex, lateral habenula, or dorsal raphe nucleus. Chronic corticosterone treatment reduced lymphatic endothelial cell markers in the hippocampus. Mechanistically, chronic stress might reduce hippocampal lymphatic vessels by down-regulating vascular endothelial growth factor C receptors and up-regulating vascular endothelial growth factor C neutralization mechanisms. Our results provide new insights into the characteristic features of deep brain lymphatic vessels, as well as their regulation by stressful life events.

Immunohistochemical and Scanning Electron Microscopic Confirmation of the Lymphatic Lacunae in the Uterine Tube Mucosal Folds. What Are the Clinical Implications?

Uterine tubes (UTs) are essential during physiological reproduction. The most intriguing part of its wall is the mucosa. Apart from the epithelial cells vital for its normal function, the connective tissue lamina propria contains wide spaces whose function, morphology and structure are yet to be elucidated. The present study used bioptic samples from 25 premenopausal (mean age 48,33 years, ?=3,56) and 25 postmenopausal women (mean age 57,8 years, ?=7,79). In both study groups, samples were obtained from two anatomically distinct parts of the UT - ampulla and infundibulum with fimbriae. The specimens were processed for scanning electron microscopy (SEM) and immunohistochemical detection of podoplanin (clone D2-40) and VEGFR-3 - two markers of lymphatic endothelial cells. The results showed that specimens from premenopausal and postmenopausal women contain wide lymphatic spaces, also known as lymphatic lacunae. The most probable function of the lacunae in the fimbriae is oocyte pick-up upon ovulation thanks to their ability to get engorged with lymph, thus serving as an erectile-like tissue. The ampullary lacunae are probably responsible for tubal fluid maintenance and recirculation. These results indicate that they are vital for normal reproduction because tubal fluid dynamics are as important as fluid composition. Further research on this topic is highly warranted because more detailed insights into UT function have a great potential to refine the methods of reproductive medicine, e.g. in vitro fertilization (IVF), which are still far from optimal regarding fertility outcomes.

Lymphatic vessels are present in human saccular intracranial aneurysms

Saccular intracranial aneurysm (sIA) rupture leads to subarachnoid haemorrhage and is preceded by chronic inflammation and atherosclerotic changes of the sIA wall. Increased lymphangiogenesis has been detected in atherosclerotic extracranial arteries and in abdominal aortic aneurysms, but the presence of lymphatic vessels in sIAs has remained unexplored. Here we studied the presence of lymphatic vessels in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), using immunohistochemical and immunofluorescence stainings for lymphatic endothelial cell (LEC) markers. Of these LEC-markers, both extracellular and intracellular LYVE-1-, podoplanin-, VEGFR-3-, and Prox1-positive stainings were detected in 83%, 94%, 100%, and 72% of the 36 sIA walls, respectively. Lymphatic vessels were identified as ring-shaped structures positive for one or more of the LEC markers. Of the sIAs, 78% contained lymphatic vessels positive for at least one LEC marker. The presence of LECs and lymphatic vessels were associated with the number of CD68+ and CD163+ cells in the sIA walls, and with the expression of inflammation indicators such as serum amyloid A, myeloperoxidase, and cyclo-oxygenase 2, with the presence of a thrombus, and with the sIA wall rupture. Large areas of VEGFR-3 and α-smooth muscle actin (αSMA) double-positive cells were detected in medial parts of the sIA walls. Also, a few podoplanin and αSMA double-positive cells were discovered. In addition, LYVE-1 and CD68 double-positive cells were detected in the sIA walls and in the thrombus revealing that certain CD68+ macrophages are capable of expressing LEC markers. This study demonstrates for the first time the presence of lymphatic vessels in human sIA walls. Further studies are needed to understand the role of lymphatic vessels in the pathogenesis of sIA.

Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

30. The Effect of Lymphatic Proliferation in Extremity Trauma And Aberrant Wound Healing

Purpose: Traumatic injury results in alterations to tissue organization requiring a highly orchestrated process for proper healing. Following traumatic injury, muscle, tendon, vasculature, nerves, and bone are destroyed resulting in impaired regeneration. There is a paucity of studies investigating the role of lymphatic vasculature network plays in tissue healing following traumatic injury. This is surprising considering the role that the lymphatic system plays in the normal inflammatory response and managing resulting edema which is critical in preventing infection and initiating the healing cascade. Alterations in this process can result in aberrant healing seen following traumatic injury. Utilizing a reliable and reproducible method of traumatic injury, we investigated the role of lymphatic vessels in and aberrant wound healing utilizing novel bioinformatic techniques and 2- and 3-dimensional confocal imaging modalities. Methods: We utilized a murine model of traumatic injury resulting in formation of heterotopic ossification (HO). We performed a 30% total body surface area burn to the dorsum of the mouse and a concurrent Achilles’ tenotomy (Burn/tenotomy; BT) resulting in fully formed HO at the Achilles’ injury site nine weeks following injury. C57B6 and Prox1-eGFP mice labeling lymphatic endothelial cells were injured. To visualize lymphatic endothelium prior to injury, uninjured hindlimbs were harvested and processed utilizing the PEGASOS method for tissue clearing. Hindlimbs were harvested prior to injury, one, three, and nine weeks post-injury for histology. Tissue was also harvested from uninjured mice, one, and six weeks post-injury for single-cell RNA-sequencing (scRNA). Separately, we performed the BT injury in chylous ascites (Chy) mice characterized by dysfunctional lymphatic system due to an inactivating mutation in Vegfr3, a cell-surface receptor critical for the growth and survival of lymphatic endothelium. Mice were harvested nine weeks post-injury to assess the impact on HO formation. Results: Reconstructed 3-D confocal microscopy images of cleared uninjured whole-mount hindlimb tissue stained for lymphatic endothelial cell markers CD31 and LYVE1 show lymphatic vessels at the injury site. Sections from uninjured Achilles’ tendon and one, three, and nine weeks post-injury from Prox1-eGFP mice co-stained with LYVE1 show lymphatics in tendon peritenon, however, no lymphatic vessels within the tendon prior to injury. Following injury, lymphatics endothelium is disrupted, characterized by no positive Prox1-eGFP staining following injury. Interestingly, Prox1-eGFP+ cells are found within the Achilles’ tendon and Prox1-eGFP/LYVE1 double-positive cells forming a lumen are found nine weeks post-injury within the Achilles’ tendon (B; n=3/group, p*<0.5). ScRNA shows expression of lymphatic endothelial growth factor Vegfc following injury by mesenchymal progenitor cells (MPCs), while receptors for the ligand are expressed by the lymphatic endothelial cell cluster. VEGFR3 mutation leads to increased variability of HO formation following injury (E; n=4/6/group). Conclusion: These findings highlight the disruption and regeneration of lymphatic vessels following extremity trauma. For the first time, we propose a mechanism for MPC-lymphatics interaction that drives lymphatic regeneration following extremity injury through VEGFC-VEGFR3 driven lymphangiogenesis.

Quantitative Three-Dimensional Analysis of the Lymphatic Vasculature in the Postnatal Mouse Heart.

The development and maturation of the lymphatic vasculature are essential for organ function with disruption leading to severe phenotypes. For example, malfunction of cardiac lymphatics results in myocardial oedema, persistent inflammation and reduced cardiac output. Thus, it is important to study the process of cardiac lymphatic formation and growth from the early stages of fetal development to adulthood. In the murine heart the lymphatics continue to develop and expand postnatally with extensive growth and patterning occurring up to at least 2 weeks after birth. Here, we describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters, including vessel junction number (i.e., branching density), vessel length, and number of vessel end points in the murine postnatal heart. This protocol is based on the use of reliable antibodies against key markers of lymphatic endothelial cells (LECs), specifically the glycoprotein lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), the vascular endothelial growth factor receptor 3 (VEGFR3; also known as Fms-related receptor tyrosine kinase 4, FLT4), the mucin-type protein podoplanin (PDPN), and the co-receptor neuropilin 2 (NRP2). For imaging and quantitative analysis of the sub-epicardial network in neonatal hearts, VEGFR3 was selected given its exclusive expression in the lymphatic endothelium. In addition to LECs, LYVE1 expression was detected in tissue-resident macrophages, PDPN in the epicardium, and NRP2 in the autonomic nervous system of the heart. Overall, we characterized the expression patterns of commonly used lymphatic markers in the context of the neonatal heart and provide an image analysis pipeline that can be adapted to study other organs and systems (e.g., blood vasculature and nerve system).

Podoplanin expression in salivary gland carcinomas and its clinical significance. An immunohistochemical study.

Podoplanin (D2-40) is alymphatic endothelial marker that is considered as aspecific marker for lymphatic endothelial cells and lymphangiogenesis in salivary gland carcinomas (SGCs). Aim: thepresent study aimed to investigate theimmunohistochemical expression ofpodoplanin in SGCs and to correlate its expression with theclinicopathological parameters and patients' survival. Forty-nine SGC cases were electronically selected. Demographic, clinical, laboratory, and survival data were reviewed and tabulated. Immunohistochemistry was performed using antipodoplanin. Cases were divided into low and high expression based on ascoring system. Ascore of0 and 1 was considered low expression, while > 1 was considered high expression. Podoplanin high expression was seen in 46.9% ofcases, and 53.1% ofcases showed low expression. Significant statistical associations were seen between podoplanin expression and tumour grade ( p ≤ 0.001), tumour-nodal- metastasis (TNM) stage (p ≤ 0.001), tumour size (p ≤ 0.001), nodal metastasis (p ≤ 0.001), tumour type (p = 0.03), prognosis (p ≤ 0.001), and mortality (p ≤ 0.001). The overall survival and progression-free survival differed significantly in cases with high and low expression (p ≤ 0.001). Podoplanin overexpression might be asignificant prognostic indicator for patients with SGCs, implicating that it is apotential therapeutic target to improve survival in these cancer patients.

Evaluation of the degree and distribution of lymphangiectasia in full-thickness canine small intestinal specimens diagnosed with lymphoplasmacytic enteritis and granulomatous lymphangitis.

Intestinal lymphangiectasia (IL) is often observed in dogs with chronic small intestinal diseases. Hypoplasia of the lymphatic vessel due to decreased lymphangiogenesis, which has been suggested in human idiopathic IL, may contribute to the pathogenesis of canine IL. This study aimed to evaluate the diameter and number of lymphatic vessels in full-thickness small intestinal specimens of dogs with IL. Immunohistochemical labeling of lymphatic endothelial cell markers was performed on retrospectively retrieved full-thickness small intestinal specimens. Sixteen dogs with histologically confirmed IL were included, of which 10 had lymphoplasmacytic enteritis (LPE), and six had granulomatous lymphangitis (GL). Nine dogs that died from non-gastrointestinal disorders and with little or no abnormalities in the small intestine were used as controls. Lymphatic vessel diameters in dogs with IL were significantly increased in all layers of the small intestine, including the villus lacteal, lamina propria, submucosa, muscularis, and mesentery, compared with controls (all P<0.01). There was no significant difference in the lymphatic vessel diameters between dogs with LPE and GL (all P>0.05). There was no significant difference in the number of lymphatic vessels between dogs with IL and the controls in all layers of the small intestine (all P>0.05). This study demonstrated that IL was observed in all layers of the small intestine, including the submucosa, muscularis, and mesentery, independent of the underlying disease. Factors other than reduced lymphatic vessels would contribute to the pathogenesis of IL in dogs.

Lymphatic Endothelial Markers and Tumor Lymphangiogenesis Assessment in Human Breast Cancer

Metastasis via lymphatic vessels or blood vessels is the leading cause of death for breast cancer, and lymphangiogenesis and angiogenesis are critical prerequisites for the tumor invasion–metastasis cascade. The research progress for tumor lymphangiogenesis has tended to lag behind that for angiogenesis due to the lack of specific markers. With the discovery of lymphatic endothelial cell (LEC) markers, growing evidence demonstrates that the LEC plays an active role in lymphatic formation and remodeling, tumor cell growth, invasion and intravasation, tumor–microenvironment remodeling, and antitumor immunity. However, some studies have drawn controversial conclusions due to the variation in the LEC markers and lymphangiogenesis assessments used. In this study, we review recent findings on tumor lymphangiogenesis, the most commonly used LEC markers, and parameters for lymphangiogenesis assessments, such as the lymphatic vessel density and lymphatic vessel invasion in human breast cancer. An in-depth understanding of tumor lymphangiogenesis and LEC markers can help to illustrate the mechanisms and distinct roles of lymphangiogenesis in breast cancer progression, which will help in exploring novel potential predictive biomarkers and therapeutic targets for breast cancer.

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.