Human Lymphatic Endothelial Cells Research Articles

Evaluation of the Effect of Botulinum Toxin A on the Lymphatic Endothelial Cells.

Botulinum toxin A (BoTA) is a neurotoxin formed by Clostridium botulinum, with a broad medical application spectrum. While the primary effect of BoTA is on the muscles, the effects of BoTA in other systems including the blood vasculature have already been examined, revealing unexpected actions. However, no studies exist to the best of our knowledge regarding the potential effects of BoTA on the lymphatic vascular system, possessing a critical role in health and disease. Isolated human lymphatic endothelial cells (LECs) were cultured in dedicated in vitro culture systems. The analysis including imaging and cell culture approaches as well as molecular biology techniques is performed to examine the LEC alterations occurring upon exposure to different concentrations of BoTA. Human LECs were cultured and expanded on collagen-coated petri dishes using endothelial basal medium and the commercial product Botox from Allergan as used for all our experiments. Harvested cells were used in various in vitro functional tests to assess the morphologic and functional properties of the BoTA-treated LECs. Gene expression analysis was performed to assess the most important lymphatic system-related genes and pathways. Concentrations of 1, 5 or 10 U of BoTA did not demonstrate a significant effect regarding the proliferation and migration capacity of the LECs versus untreated controls. Interestingly, even the smallest BoTA dose was found to significantly decrease the cord-like-structure formation capacity of the seeded LECs. Gene expression analysis was used to underpin possible molecular alterations, suggesting no significant effect of BoTA in the modification of gene expression versus the starvation medium control. LECs appear largely unaffected to BoTA treatment, with an isolated effect on the cord-like-structure formation capacity. Further work needs to assess the effect of BoTA on the smooth-muscle-cell-covered collecting lymphatic vessels and the possible aesthetic implications of such an effect, due to edema formation. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells.

Recently, a low-level somatic mutation in the NRAS gene (c.182 A > G, Q61R) was identified in various specimens from patients with kaposiform lymphangiomatosis. However, it is unknown how these low-frequency mutated cells can affect the characterization and surrounding environment of their lesions. To understand the pathogenesis and association of these gene abnormalities, we established NRASQ61R mutated lymphatic endothelial cells transfected with lentivirus vector and undertook morphological and functional characterization, protein expression profiling, and metabolome analysis. NRASQ61R human dermal lymphatic endothelial cells showed poor tube formation, a low proliferation rate, and high migration ability, with an increase in the ratio of mutated cells. An analysis of signaling pathways showed inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ERK pathway, which was improved by MAPK kinase (MEK) inhibitor treatment. This study shows the theoretical circumstances induced in vitro by NRASQ61R-mutated cells in the affected lesions of kaposiform lymphangiomatosis patients.

Injectable Thermosensitive Gel CH-BPNs-NBP for Effective Periodontitis Treatment through ROS-Scavenging and Jaw Vascular Unit Protection.

Periodontitis, a prevalent inflammatory condition in the oral cavity, is closely associated with oxidative stress-induced tissue damage mediated by excessive reactive oxygen species (ROS) production. The jaw vascular unit (JVU), encompassing both vascular and lymphatic vessels, plays a crucial role in maintaining tissue fluid homeostasis and contributes to the pathological process in inflammatory diseases of the jaw. This study presents a novel approach for treating periodontitis through the development of an injectable thermosensitive gel (CH-BPNs-NBP). The gel formulation incorporates black phosphorus nanosheets (BPNs), which are notable for their ROS-scavenging properties, and dl-3-n-butylphthalide (NBP), a vasodilator that promotes lymphatic vessel function within the JVU. These results demonstrate that the designed thermosensitive gel serve as a controlled release system, delivering BPNs and NBP to the site of inflammation. CH-BPNs-NBP not only protects macrophages and human lymphatic endothelial cells from ROS attack but also promotes M2 polarization and lymphatic function. In in vivo studies, this work observes a significant reduction in inflammation and tissue damage, accompanied by a notable promotion of alveolar bone regeneration. This research introduces a promising therapeutic strategy for periodontitis, leveraging the unique properties of BPNs and NBP within an injectable thermosensitive gel.

MiR-455–3p regulates lymphangiogenesis in silicosis by regulating VEGF-C/VEGFR3

Silicosis is a disease characterized by lung inflammation and fibrosis caused by long-term inhalation of free silicon dioxide (SiO2). Recent studies have found that a large number of lymphatic hyperplasia occurs during the occurrence and development of silicosis. miRNAs play an important role in lymphangiogenesis. However, the regulation and mechanism of miRNAs on lymphangiogenesis in silicosis remain unclear. In this study, lymphangiogenesis was observed in silicosis rats, and VEGF-C-targeted miRNAs were screened, and the effect of miRNAs on the formation of human lymphatic endothelial cells (HLECs) tubular structure was investigated in vitro. The results showed that SiO2 promoted the expressions of Collagen Ι and α-SMA, TNF-α, IL-6 and VEGF-C increased first and then decreased, and promoted the formation of lymphatic vessels. Bioinformatics methods screened miR-455–3p for targeted binding to VEGF-C, and dual luciferase reporter genes confirmed VEGF-C as the target gene of miR-455–3p, and miR-455–3p was down-regulated in the lung tissue of silicosis rats. Transfection of miR-455–3p Inhibitors down-regulated the expression level of miR-455–3p and up-regulated the expression levels of VEGF-C and VEGFR-3 in HLECs, enhanced migration ability and increased tube formation. Transfection of miR-455–3p Mimics showed an opposite trend. These results suggest that miR-455–3p further regulates the tubular structure formation of HLECs by regulating VEGF-C/VEGFR3. Therefore, targeting miR-455–3p may provide a new therapeutic strategy for SiO2-induced silicosis injury.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Endoplasmic reticulum stress is attenuated by glycolysis in lymphatic malformations.

This study aims to investigate the role of endoplasmic reticulum stress (ER stress) in human dermal lymphatic endothelial cells (HDLECs) and lymphatic malformations (LMs) and its relationship with aerobic glycolysis and inflammation. The proliferation and apoptosis of HDLECs were examined with lipopolysaccharide (LPS) treatment. ER stress-associated proteins and glycolysis-related markers were detected by western blot. Glycolysis indexes were detected by seahorse analysis and lactic acid production assay kits. Immunohistochemistry was used to reveal the ER stress state of lymphatic endothelial cells (LECs) in LMs. LPS induced ER stress in HDLECs but did not trigger detectable apoptosis. Intriguingly, LPS-treated HDLECs also showed increased glycolysis flux. Knockdown of Hexokinase 2, a key enzyme for aerobic glycolysis, significantly inhibited the ability of HDLECs to resist ER stress-induced apoptosis. Moreover, compared to normal skin, glucose-regulated protein 78 (GRP78/BIP), and phosphorylation protein kinase R-like kinase (p-PERK), two key ER stress-associated markers, were upregulated in LECs of LMs, which was correlated with the inflected state. In addition, excessively activated ER stress inhibited the progression of LMs in rat models. These data indicate that glycolysis could rescue activated ER stress in HDLECs, which is required for the accelerated development of LMs. Inflammation enhances both ER stress and glycolysis in LECs while glycolysis is required to attenuate the pro-apoptotic effect of ER stress. Endoplasmic reticulum (ER) stress is activated in lymphatic endothelial cells (LECs) of LMs, especially in inflammatory condition. The expression of ER stress-related proteins is increased in LMs and correlated with Hexokinase 2 expression. Pharmacological activation of ER stress suppresses the formation of LM lesions in the rat model. ER stress may be a promising and effective therapeutic target for the treatment of LMs.

Quantitative proteomics reveals CLR interactome in primary human cells

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine and cancer. To date, the network of proteins interacting with CLR (“CLR interactome”) in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation (IP) utilising anti-human CLR antibody with label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and quantitative in situ proximity ligation assay (PLA). By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4,902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ PLA). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease.

The lymphatic vasculature regulates lung homeostasis through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin after CS exposure that causes endothelial dysfunction characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of tissue factor pathway inhibitor (TFPI) and increased expression of adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.

S100A6 drives lymphatic metastasis of liver cancer via activation of the RAGE/NF-kB/VEGF-D pathway

Patients diagnosed with lymph node (LN) metastatic liver cancer face an exceedingly grim prognosis. In-depth analysis of LN metastatic patients' characteristics and tumor cells’ interactions with human lymphatic endothelial cells (HLECs), can provide important biological and therapeutic insights. Here we identify at the single-cell level that S100A6 expression differs between primary tumor and their LN metastasis. Of particular significance, we uncovered the disparity in S100A6 expression between tumors and normal tissues is greater in intrahepatic cholangiocarcinoma (ICC) patients, frequently accompanied by LN metastases, than that in hepatocellular carcinoma (HCC), with rare occurrence of LN metastasis. Furthermore, in the infrequent instances of LN metastasis in HCC, heightened S100A6 expression was observed, suggesting a critical role of S100A6 in the process of LN metastasis. Subsequent experiments further uncovered that S100A6 secreted from tumor cells promotes lymphangiogenesis by upregulating the expression and secretion of vascular endothelial growth factor-D (VEGF-D) in HLECs through the RAGE/NF-kB/VEGF-D pathway while overexpression of S100A6 in tumor cells also augmented their migration and invasion. Taken together, these data reveal the dual effects of S100A6 in promoting LN metastasis in liver cancer, thus highlighting its potential as a promising therapeutic target.

Single-Cell RNA Sequencing Identifies Response of Renal Lymphatic Endothelial Cells to Acute Kidney Injury.

The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. Lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI. The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells. We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type. In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.

P005 Exosome miR-132-3p derived from Adipose Stem Cells in Creeping fat Regulates RASA1/ERK1/2-dependant Lymphatic Functions and Improve Colitis and Mesenteritis

Abstract Background Creeping fat (CrF) is an important pathological characteristics of Crohn’s disease (CD) and characterized by lymphangiogenesis and abnormal lymphatic functions. Adipose tissue derived stem cells (ADSCs) can regulate surrounding cells function by delivering exosomes (exo). However, whether CrF-ADSC-exo could affect lymphatic endothelium to regulate lymphatic functions, and further influence procession of mesenteritis and colitis remains elusive. Methods Paired CrF and uninvolved mesenteric adipose tissue (uMAT) samples were collected from CD patients during operation, and exosomes were separated by ultra-centrifugation. Human lymphatic endothelial cells (HLECs) were cultured in vitro and evaluated the abilities in proliferation, migration and tube formation. IL-10 KO mice was used for assessing the treatment of CrF-ADSC-exo in experimental colitis and mesenteritis. Results ADSC-derived exosomes from paired CrF and uMAT samples were confirmed to transfer to LEC by immunofluorescence microscope. In vitro, CrF-ADSC-exo effectively stimulated HLECs in proliferation, migration and tube formation. In vivo, CrF-ADSC-exo injection increased lymphangiogenesis in MAT and improved lymphatic drainage, and further alleviated colitis and mesenteritis in IL-10 KO mice. We next adopted high-throughput sequencing of paired CrF-ADSC-exo and uMAT-ADSC-exo, and validated significantly high expression of miR-132-3p in CrF-ADSC-exo. In vitro, upregulation or downregulation of miR-132-3p in HLECs stimulated or inhibited the lymphatic abilities in proliferation, migration and tube formation, respectively. Mechanically, we demonstrated that CrF-ADSC-exo regulated lymphatic function through miR-132-3p-RASA1-ERK1/2 axis by western blot. CrF-ADSC-exo miR-132-3p improved lymphatic drainage function in MAT and reduced inflammatory colon and mesentery in IL-10 KO mice. The higher level of miR-132-3p in CrF from CD patients was tightly correlated with inflammatory mediators expressions when compared with uMAT and healthy MAT. Conclusion Our study discovers that CrF-ADSC-exo improves lymphatic functions and alleviates colitis and mesenteritis through miR-132-3p/RASA1/ERK1/2 axis, which discloses new role of CrF involved in CD procession and provides novel insight for CD treatment.

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Hyaluronic Acid Hydrogels with Phototunable Supramolecular Cross-Linking for Spatially Controlled Lymphatic Tube Formation.

The dynamics of the extracellular matrix (ECM) influences stem cell differentiation and morphogenesis into complex lymphatic networks. While dynamic hydrogels with stress relaxation properties have been developed, many require detailed chemical processing to tune viscoelasticity, offering a limited opportunity for in situ and spatiotemporal control. Here, a hyaluronic acid (HA) hydrogel is reported with viscoelasticity that is controlled and spatially tunable using UV light to direct the extent of supramolecular and covalent cross-linking interactions. This is achieved using UV-mediated photodimerization of a supramolecular ternary complex of pendant trans-Brooker's Merocyanine (BM) guests and a cucurbit[8]uril (CB[8]) macrocycle. The UV-mediated conversion of this supramolecular complex to its covalent photodimerized form is catalyzed by CB[8], offering a user-directed route to spatially control hydrogel dynamics in combination with orthogonal photopatterning by UV irradiation through photomasks. This material thus achieves spatial heterogeneity of substrate dynamics, recreating features of native ECM without the need for additional chemical reagents. Moreover, these dynamic hydrogels afford spatial control of substrate mechanics to direct human lymphatic endothelial cells (LECs) to form lymphatic cord-like structures (CLS). Specifically, cells cultured on viscoelastic supramolecular hydrogels have enhanced formation of CLS, arising from increased expression of key lymphatic markers, such as LYVE-1, Podoplanin, and Prox1, compared to static elastic hydrogels prepared from fully covalent cross-linking. Viscoelastic hydrogels promote lymphatic CLS formation through the expression of Nrp2, VEGFR2, and VEGFR3 to enhance the VEGF-C stimulation. Overall, viscoelastic supramolecular hydrogels offer a facile route to spatially control lymphatic CLS formation, providing a tool for future studies of basic lymphatic biology and tissue engineering applications.

Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway

BackgroundObesity has emerged as a global epidemic. Recent research has indicated that diet-induced obesity can be prevented by promoting lacteal junction zippering. Berberine, which is derived from natural plants, is found to be promising in weight reduction, but the underlying mechanism remains unspecified. PurposeTo determine whether berberine protects against obesity by regulating the lacteal junction and to explore potential molecular mechanisms. MethodsFollowing the induction of the diet-induced obese (DIO) model, mice were administered low and high doses of berberine for 4 weeks. Indicators associated with insulin resistance and lipid metabolism were examined. Various methods, such as Oil Red O staining, transmission electron microscopy imaging, confocal imaging and others were used to observe the effects of berberine on lipid absorption and the lacteal junction. In vitro, human dermal lymphatic endothelial cells (HDLECs) were used to investigate the effect of berberine on LEC junctions. Western Blot and immunostaining were applied to determine the expression levels of relevant molecules. ResultsBoth low and high doses of berberine reduced body weight in DIO mice without appetite suppression and ameliorated glucolipid metabolism disorders. We also found that the weight loss effect of berberine might contribute to the inhibition of small intestinal lipid absorption. The possible mechanism was related to the promotion of lacteal junction zippering via suppressing the ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signaling pathway. In vitro, berberine also promoted the formation of stable mature junctions in HDLECs, involving the same signaling pathway. ConclusionBerberine could promote lacteal junction zippering and ameliorate diet-induced obesity through the RhoA/ROCK signaling pathway.

Trametinib inhibits lymphatic vessel invasion of bone in a mouse model of Gorham-Stout disease.

Gorham-Stout disease (GSD) is a rare lymphatic anomaly that can be caused by somatic activating mutations in KRAS. This discovery has led investigators to suggest that MEK inhibitors could be a novel treatment for GSD. However, the effect of MEK inhibitors on bone disease in animal models of GSD has not been investigated. We recently reported that Osx-tTA;TetO-Vegfc mice exhibit a phenotype that resembles GSD. Osx-tTA;TetO-Vegfc mice overexpress VEGF-C in bone, which stimulates the development of lymphatic vessels in bone and the gradual loss of cortical bone. The objective of this study was to characterize the effect of trametinib, an FDA-approved MEK1/2 inhibitor, on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Immunoblotting was performed to assess the effect of trametinib on VEGF-C-induced phosphorylation of ERK1/2, AKT, and S6 in primary human lymphatic endothelial cells (LECs). Prevention and intervention experiments were performed to determine the effect of trametinib on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. We found that trametinib blocked VEGF-C-induced phosphorylation of ERK1/2 in primary human LECs. We also found that trametinib prevented VEGF-C-induced lymphatic invasion of bone and cortical bone loss in Osx-tTA;TetO-Vegfc mice. Additionally, trametinib slowed the progression of disease in Osx-tTA;TetO-Vegfc mice with established disease. However, it did not reverse disease in Osx-tTA;TetO-Vegfc mice. Our results show trametinib impacts bone disease in Osx-tTA;TetO-Vegfc mice. These findings further support the testing of MEK inhibitors in patients with GSD and other RAS pathway-driven complex lymphatic anomalies with bone involvement.

Age‐related alterations in meningeal immunity drive impaired central nervous system waste removal

AbstractBackgroundThe meningeal lymphatic network enables the drainage of cerebrospinal fluid (CSF) and facilitates the removal of central nervous system (CNS) waste. During aging and in Alzheimer’s disease, impaired meningeal lymphatic drainage promotes the buildup of toxic misfolded proteins in the CNS. Reversing this age‐related dysfunction represents a promising strategy to augment CNS waste clearance, however, the mechanisms underlying this decline remain elusive.MethodYoung (2‐3 month old) and aged (20‐24 month old) C57BL/6J mice were used to study age‐related meningeal phenotypes by single‐cell RNA‐sequencing (scRNA‐seq), high dimensional flow cytometry, and immunohistochemistry. Lymphatic drainage to deep cervical lymph nodes was determined by CSF‐delivery of ovalbumin‐A594. CSF‐delivery of AAVs or antibodies were used to model age‐related deficits in young mice. Peripheral chronic neutralizing antibody administration was utilized to attenuate age‐related alterations in aged mice. 3D in vitro microfluidic devices of human lymphatic endothelial cells were used to model physiological flow parameters.ResultscRNA‐seq of meningeal lymphatic endothelial cells from aged mice revealed a response signature to the inflammatory cytokine IFNγ, which was increased in the aged meninges due to T cell accumulation. Chronic elevation of meningeal IFNγ in young mice via AAV‐mediated over‐expression attenuated CSF drainage—comparable to the deficits observed in aged mice. Mechanistically, this occurred through disruption of lymphatic adherans junctions which was confirmed in 3D in vitro microfluidic systems, and through antibody‐mediated VE‐Cadherin disruption. Therapeutically, IFNγ neutralization alleviated age‐related impairments in meningeal lymphatic function.ConclusionHere we demonstrate that alterations in meningeal immunity underlie the age‐related lymphatic impairment and impaired CNS waste removal. These data suggest manipulation of meningeal immunity as a viable approach to normalize CSF drainage and alleviate the neurological deficits associated with impaired waste removal in neurodegenerative disease.