Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes

OBJECTIVE The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes

In vivo visualization and quantification of collecting lymphatic vessel contractility using near-infrared imaging

Techniques to image lymphatic vessel function in either animal models or in the clinic are limited. In particular, imaging methods that can provide robust outcome measures for collecting lymphatic vessel function are sorely needed. In this study, we aimed to develop a method to visualize and quantify collecting lymphatic vessel function in mice, and to establish an in vivo system for evaluation of contractile agonists and antagonists using near-infrared fluorescence imaging. The flank collecting lymphatic vessel in mice was exposed using a surgical technique and a near-infrared tracer was infused into the inguinal lymph node. Collecting lymphatic vessel contractility and valve function could be easily visualized after the infusion. A diameter tracking method was established and the diameter of the vessel was found to closely correlate to near-infrared fluorescence signal. Phasic contractility measures of frequency and amplitude were established using an automated algorithm. The methods were validated by tracking the vessel response to topical application of a contractile agonist, prostaglandin F2α, and by demonstrating the potential of the technique for non-invasive evaluation of modifiers of lymphatic function. These new methods will enable high-resolution imaging and quantification of collecting lymphatic vessel function in animal models and may have future clinical applications

Differential effects of anaesthesia on the contractility of lymphatic vessels in vivo.

KEY POINTS Contractility of lymphatic collectors is essential for the functionality of the lymphatic system and, thus, for lymph flow. Previously published rates of lymphatic collectors in mice vary from 1.1 to 17 contractions/min with little agreement between investigators. In this study, we focused on the effects of different anaesthesia regimens on lymphatic vessel contractility using in vivo imaging approaches. We show that isoflurane and pentobarbital have an inhibitory effect on lymphatic contractility compared to mice under other anaesthesia regimens and in awake conditions. These results should help to establish a standardization of lymphatic contraction studies in mice and may also have relevance for patients undergoing anaesthesia during surgery. ABSTRACT Contractions of collecting lymphatic vessels are essential for the function of the lymphatic vascular system, due to the lack of a central pump to drive flow. A wide range of physiological contraction frequencies and strengths have been reported in previous in vivo studies in mice. This is probably due to the different types of anaesthesia that have been used and which might have exerted direct influences on lymphatic vessel function. We investigated six commonly used anaesthesia regimens for their influence on lymphatic vessel contractility using near-infrared in vivo imaging approaches. Non-invasive imaging of the lymphatic leg collector revealed distinct effects of the anaesthesia regimens with reduced contraction activity under isoflurane and pentobarbital anaesthesia. Isoflurane also reduced the contractility of near-infrared dye-loaded vessels during invasive imaging of the lymphatic flank collector whereas the combination of ketamine/xylazine/acepromazine had no major effects. The transport time of a lymphatic-specific dye from the skin through the lymphatic vasculature to the systemic bloodstream was also delayed under isoflurane anaesthesia. Based on these results, we recommend use of combinations of ketamine and medetomidine for future non-invasive studies and of ketamine, xylazine and acepromazine for invasive studies. Beyond their importance for facilitating the interpretation and planning of animal studies, our findings might also have relevance for human patients undergoing anaesthesia for surgical procedures

An Important Role of VEGF-C in Promoting Lymphedema Development

Secondary lymphedema is a common complication after cancer treatment, but the pathomechanisms underlying the disease remain unclear. Using a mouse tail lymphedema model, we found an increase in local and systemic levels of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C and identified CD68(+) macrophages as a cellular source. Surprisingly, overexpression of VEGF-C in a transgenic mouse model led to aggravation of lymphedema with increased immune cell infiltration and vascular leakage compared with wild-type littermates. Conversely, blockage of VEGF-C by overexpression of soluble VEGF receptor-3 reduced edema development, diminishing inflammation and blood vascular leakage. Similar findings were obtained in a hind limb lymph node excision lymphedema model. Flow cytometry analyses and immunofluorescence stainings in lymphedematic tissue showed that VEGF receptor-3 expression was restricted to lymphatic endothelial cells. Our data suggest that endogenous VEGF-C causes blood vascular leakage and fluid influx into the tissue, thus actively contributing to edema formation. These data may provide the basis for future clinical therapeutic approaches.Peer reviewe

Transcriptional profiling of breast cancer-associated lymphatic vessels reveals VCAM-1 as regulator of lymphatic invasion and permeability

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin α4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis

DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

BACKGROUND: Prostate cancer (PCa) is one of the leading causes of cancer-related mortality and morbidity in the aging male population and represents the most frequently diagnosed malignancy in men around the world. The Deltex (DTX)-3-like E3 ubiquitin ligase (DTX3L), also known as B-lymphoma and BAL-associated protein (BBAP), was originally identified as a binding partner of the diphtheria-toxin-like macrodomain containing ADP-ribosyltransferase-9 (ARTD9), also known as BAL1 and PARP9. We have previously demonstrated that ARTD9 acts as a novel oncogenic survival factor in high-risk, chemo-resistant, diffuse large B cell lymphoma (DLBCL). The mono-ADP-ribosyltransferase ARTD8, also known as PARP14 functions as a STAT6-specific co-regulator of IL4-mediated proliferation and survival in B cells. METHODS: Co-expression of DTX3L, ARTD8, ARTD9 and STAT1 was analyzed in the metastatic PCa (mPCa) cell lines PC3, DU145, LNCaP and in the normal prostate luminal epithelial cell lines HPE and RWPE1. Effects on cell proliferation, survival and cell migration were determined in PC3, DU145 and/or LNCaP cells depleted of DTX3L, ARTD8, ARTD9, STAT1 and/or IRF1 compared to their proficient control cells, respectively. In further experiments, real-time RT-PCR, Western blot, immunofluorescence and co-immunoprecipitations were conducted to evaluate the physical and functional interactions between DTX3L, ARTD8 and ARTD9. RESULTS: Here we could identify DTX3L, ARTD9 and ARTD8 as novel oncogenic survival factors in mPCa cells. Our studies revealed that DTX3L forms a complex with ARTD8 and mediates together with ARTD8 and ARTD9 proliferation, chemo-resistance and survival of mPCa cells. In addition, DTX3L, ARTD8 and ARTD9 form complexes with each other. Our study provides first evidence that the enzymatic activity of ARTD8 is required for survival of mPCa cells. DTX3L and ARTD9 act together as repressors of the tumor suppressor IRF1 in mPCa cells. Furthermore, the present study shows that DTX3L together with STAT1 and STAT3 is implicated in cell migration of mPCa cells. CONCLUSIONS: Our data strongly indicate that a crosstalk between STAT1, DTX3L and ARTD-like mono-ADP-ribosyltransferases mediates proliferation and survival of mPCa cells. The present study further suggests that the combined targeted inhibition of STAT1, ARTD8, ARTD9 and/or DTX3L could increase the efficacy of chemotherapy or radiation treatment in prostate and other high-risk tumor types with an increased STAT1 signaling

An important role of cutaneous lymphatic vessels in coordinating and promoting anagen hair follicle growth.

The lymphatic vascular system plays important roles in the control of tissue fluid homeostasis and immune responses. While VEGF-A-induced angiogenesis promotes hair follicle (HF) growth, the potential role of lymphatic vessels (LVs) in HF cycling has remained unknown. In this study, we found that LVs are localized in close proximity to the HF bulge area throughout the postnatal and depilation-induced hair cycle in mice and that a network of LVs directly connects the individual HFs. Increased LV density in the skin of K14-VEGF-C transgenic mice was associated with prolongation of anagen HF growth. Conversely, HF entry into the catagen phase was accelerated in K14-sVEGFR3 transgenic mice that lack cutaneous LVs. Importantly, repeated intradermal injections of VEGF-C promoted hair growth in mice. Conditioned media from lymphatic endothelial cells promoted human dermal papilla cell (DPC) growth and expression of IGF-1 and alkaline phosphatase, both activators of DPCs. Our results reveal an unexpected role of LVs in coordinating and promoting HF growth and identify potential new therapeutic strategies for hair loss-associated conditions

Lymphatic outflow of cerebrospinal fluid is reduced in glioma

Glioblastoma is a malignant brain tumor with mean overall survival of less than 15 months. Blood vessel leakage and peritumoral edema lead to increased intracranial pressure and augment neurological deficits which profoundly decrease the quality of life of glioblastoma patients. It is unknown how the dynamics of cerebrospinal fluid (CSF) turnover are affected during this process. By monitoring the transport of CSF tracers to the systemic blood circulation after infusion into the cisterna magna, we demonstrate that the outflow of CSF is dramatically reduced in glioma-bearing mice. Using a combination of magnetic resonance imaging (MRI) and near-infrared (NIR) imaging, we found that the circulation of CSF tracers was hindered after cisterna magna injection with reduced signals along the exiting cranial nerves and downstream lymph nodes, which represent the major CSF outflow route in mice. Due to blockage of the normal routes of CSF bulk flow within and from the cranial cavity, CSF tracers were redirected into the spinal space. In some mice, impaired CSF clearance from the cranium was compensated by a lymphatic outflow from the sacral spine

Lymphatic outflow of cerebrospinal fluid is reduced in glioma.

Glioblastoma is a malignant brain tumor with mean overall survival of less than 15 months. Blood vessel leakage and peritumoral edema lead to increased intracranial pressure and augment neurological deficits which profoundly decrease the quality of life of glioblastoma patients. It is unknown how the dynamics of cerebrospinal fluid (CSF) turnover are affected during this process. By monitoring the transport of CSF tracers to the systemic blood circulation after infusion into the cisterna magna, we demonstrate that the outflow of CSF is dramatically reduced in glioma-bearing mice. Using a combination of magnetic resonance imaging (MRI) and near-infrared (NIR) imaging, we found that the circulation of CSF tracers was hindered after cisterna magna injection with reduced signals along the exiting cranial nerves and downstream lymph nodes, which represent the major CSF outflow route in mice. Due to blockage of the normal routes of CSF bulk flow within and from the cranial cavity, CSF tracers were redirected into the spinal space. In some mice, impaired CSF clearance from the cranium was compensated by a lymphatic outflow from the sacral spine