A novel pro-lymphangiogenic function for Th17/IL-17

Abstract Th17 cells, in addition to their proinflammatory functions, have been recognized as potent inducers of angiogenesis in autoimmune diseases and malignancies. In the present study, we demonstrate distinct mechanisms by which IL-17 induces lymphangiogenesis. Using the mouse cornea micropocket and cell culture assays, our data demonstrate that IL-17 directly promotes growth of lymphatic vessels by inducing increased expression of prolymphangiogenic VEGF-D and proliferation of lymphatic endothelial cells. However, IL-17–induced growth of blood vessels is primarily mediated through IL-1β secretion by IL-17–responsive cells. Furthermore, in vivo blockade of IL-17 in a preclinical model of Th17-dominant autoimmune ocular disease demonstrates a significant reduction in the corneal lymphangiogenesis and in the progression of clinical disease. Taken together, our findings demonstrate a novel prolymphangiogenic function for Th17/IL-17, indicating that IL-17 can promote the progression and amplification of immunity in part through its induction of lymphangiogenesis.

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Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101931118 ◽

2021 ◽

Vol 118 (31) ◽

pp. e2101931118

Author(s):

Shira Landau ◽

Abigail Newman ◽

Shlomit Edri ◽

Inbal Michael ◽

Shahar Ben-Shaul ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Vessel ◽

Blood Vessels ◽

Lymphatic Vessel ◽

Lymphatic Vessels ◽

Lymphatic Endothelial Cells ◽

Supporting Cells ◽

Vessel Formation ◽

Vessel Networks

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-β signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

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Loss of Primary Cilia Protein IFT20 Dysregulates Lymphatic Vessel Patterning in Development and Inflammation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.672625 ◽

2021 ◽

Vol 9 ◽

Author(s):

Delayna Paulson ◽

Rebecca Harms ◽

Cody Ward ◽

Mackenzie Latterell ◽

Gregory J. Pazour ◽

...

Keyword(s):

Endothelial Cells ◽

Lymphatic Vessel ◽

Primary Cilia ◽

Fluid Shear Stress ◽

Lymphatic Vessels ◽

Intraflagellar Transport ◽

Membrane Receptors ◽

Lymphatic Endothelial Cells ◽

Corneal Lymphangiogenesis

Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.

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Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-κB and Prox1

Blood ◽

10.1182/blood-2008-12-196840 ◽

2010 ◽

Vol 115 (2) ◽

pp. 418-429 ◽

Cited By ~ 124

Author(s):

Michael J. Flister ◽

Andrew Wilber ◽

Kelly L. Hall ◽

Caname Iwata ◽

Kohei Miyazono ◽

...

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Molecular Mechanisms ◽

Lymphatic Vessels ◽

Growth Factor Receptor ◽

Receptor Expression ◽

Lymphatic Endothelial Cells ◽

Inflammatory Stimuli ◽

And Migration

Abstract The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-κB) family; however, the role of NF-κB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-κB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-κB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-κB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

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Aspirin suppresses components of lymphangiogenesis and lymphatic vessel remodeling by inhibiting the NF-κB/VCAM-1 pathway in human lymphatic endothelial cells

Vascular Medicine ◽

10.1177/1358863x18760718 ◽

2018 ◽

Vol 23 (3) ◽

pp. 201-211 ◽

Cited By ~ 1

Author(s):

Orawin Prangsaengtong ◽

Phatcharida Jantaree ◽

Kriengsak Lirdprapamongkol ◽

Lukana Ngiwsara ◽

Jisnuson Svasti ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Cancer Metastasis ◽

Lymphatic Vessels ◽

Mrna Levels ◽

Lymphatic Endothelial Cells ◽

Tubule Formation ◽

Toxic Dose

Lymphangiogenesis is the process of new vessel formation from pre-existing lymphatic vessels. The process mainly involves cell adhesion, migration, and tubule formation of lymphatic endothelial cells. Tumor-induced lymphangiogenesis is an important factor contributing to promotion of tumor growth and cancer metastasis via the lymphatic system. Finding the non-toxic agents that can prevent or inhibit lymphangiogenesis may lead to blocking of lymphatic metastasis. Recently, aspirin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit in vivo lymphangiogenesis in tumor and incision wound models, but the mechanisms of actions of aspirin on anti-lymphangiogenesis have been less explored. In this study, we aim to explore the mechanism underlying the anti-lymphangiogenic effects of aspirin in primary human dermal lymphatic microvascular endothelial (HMVEC-dLy) cells in vitro. Pretreatment of aspirin at non-toxic dose 0.3 mM significantly suppressed in vitro cord formation, adhesion, and the migration abilities of the HMVEC-dLy cells. Western blotting analysis indicated that aspirin decreased expression of vascular cell adhesion molecule-1 (VCAM-1), at both protein and mRNA levels, and these correlated with the reduction of NF-κB p65 phosphorylation. By using NF-κB inhibitor (BAY-11-7085) and VCAM-1 siRNA, we showed that VCAM-1 expression is downstream of NF-κB activation, and this NF-κB/VCAM-1 signaling pathway controls cord formation, adhesion, and the migration abilities of the HMVEC-dLy cells. In summary, we demonstrate the potential of aspirin as an anti-lymphangiogenic agent, and elucidate its mechanism of action.

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HIV-1 Matrix Protein p17 Promotes Lymphangiogenesis and Activates the Endothelin-1/Endothelin B Receptor Axis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.113.302478 ◽

2014 ◽

Vol 34 (4) ◽

pp. 846-856 ◽

Cited By ~ 21

Author(s):

Francesca Caccuri ◽

Christine Rueckert ◽

Cinzia Giagulli ◽

Kai Schulze ◽

Daniele Basta ◽

...

Keyword(s):

Endothelial Cells ◽

Lymph Node ◽

Structure Formation ◽

Highly Active Antiretroviral Therapy ◽

Matrix Protein ◽

Lymphatic Endothelial Cells ◽

Endothelin 1 ◽

Tumor Dissemination ◽

Hiv 1

Objective— AIDS-related lymphomas are high grade and aggressively metastatic with poor prognosis. Lymphangiogenesis is essential in supporting proliferation and survival of lymphoma, as well as tumor dissemination. Data suggest that aberrant lymphangiogenesis relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. HIV-1 matrix protein p17 was found to accumulate and persist in lymph nodes of patients even under highly active antiretroviral therapy. Because p17 was recently found to exert a potent proangiogenic activity by interacting with chemokine (C-X-C motif) receptors 1 and 2, we tested the prolymphangiogenic activity of the viral protein. Approach and Results— Human primary lymph node–derived lymphatic endothelial cells were used to perform capillary-like structure formation, wound healing, spheroids, and Western blot assays after stimulation with or without p17. Here, we show that p17 promotes lymphangiogenesis by binding to chemokine (C-X-C motif) receptor-1 and chemokine (C-X-C motif) receptor-2 expressed on lymph node–derived lymphatic endothelial cells and activating the Akt/extracellular signal–regulated kinase signaling pathway. In particular, it was found to induce capillary-like structure formation, sprout formation from spheroids, and increase lymph node–derived lymphatic endothelial cells motility. The p17 lymphangiogenic activity was, in part, sustained by activation of the endothelin-1/endothelin receptor B axis. A Matrigel plug assay showed that p17 was able to promote the outgrowth of lymphatic vessels in vivo, demonstrating that p17 directly regulates lymphatic vessel formation. Conclusions— Our results suggest that p17 may generate a prolymphangiogenic microenvironment and plays a role in predisposing the lymph node to lymphoma growth and metastasis. This finding offers new opportunities to identify treatment strategies in combating AIDS-related lymphomas.

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Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment

Expert Reviews in Molecular Medicine ◽

10.1017/erm.2015.2 ◽

2015 ◽

Vol 17 ◽

Cited By ~ 41

Author(s):

Esak Lee ◽

Niranjan B. Pandey ◽

Aleksander S. Popel

Keyword(s):

Endothelial Cells ◽

Cancer Cells ◽

Cancer Progression ◽

Tumour Growth ◽

Tumour Progression ◽

Lymphatic Vessels ◽

Cell Types ◽

Malignant Cell ◽

Lymphatic Endothelial Cells ◽

Tumour Blood

Tumour and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments and cancer cells promotes tumour growth and metastasis. Blood and lymphatic endothelial cells (BEC and LEC) are two of the components in the microenvironments. Tumour blood vessels (BV), comprising BEC, serve as conduits for blood supply into the tumour, and are important for tumour growth as well as haematogenous tumour dissemination. Lymphatic vessels (LV), comprising LEC, which are relatively leaky compared with BV, are essential for lymphogenous tumour dissemination. In addition to describing the conventional roles of the BV and LV, we also discuss newly emerging roles of these endothelial cells: their crosstalk with cancer cells via molecules secreted by the BEC and LEC (also called angiocrine and lymphangiocrine factors). This review suggests that BEC and LEC in various microenvironments can be orchestrators of tumour progression and proposes new mechanism-based strategies to discover new therapies to supplement conventional anti-angiogenic and anti-lymphangiogenic therapies.

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Endothelin-1 Stimulates Lymphatic Endothelial Cells and Lymphatic Vessels to Grow and Invade

Cancer Research ◽

10.1158/0008-5472.can-08-1879 ◽

2009 ◽

Vol 69 (6) ◽

pp. 2669-2676 ◽

Cited By ~ 67

Author(s):

Francesca Spinella ◽

Emirena Garrafa ◽

Valeriana Di Castro ◽

Laura Rosanò ◽

Maria Rita Nicotra ◽

...

Keyword(s):

Endothelial Cells ◽

Lymphatic Vessels ◽

Lymphatic Endothelial Cells ◽

Endothelin 1

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Abstract 546: L-NAME Attenuates the Cardiotonic Steroids-induced Monolayer Permeability in Lymphatic Endothelial Cells

Hypertension ◽

10.1161/hyp.62.suppl_1.a546 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Juan Castor ◽

Darijana Horvat ◽

Walter E Cromer ◽

Thomas J Kuehl ◽

David C Zawieja ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Tight Junctions ◽

Cell Monolayer ◽

Hypertensive Disorder ◽

Lymphatic Endothelial Cells ◽

Vascular Leak Syndrome ◽

Vascular Leak ◽

Monolayer Permeability

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na + /K + ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture. Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of β-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure β-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The β-catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not. Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the β-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.

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Organ-specific lymphatic vasculature: From development to pathophysiology

Journal of Experimental Medicine ◽

10.1084/jem.20171868 ◽

2017 ◽

Vol 215 (1) ◽

pp. 35-49 ◽

Cited By ~ 88

Author(s):

Tatiana V. Petrova ◽

Gou Young Koh

Keyword(s):

Endothelial Cells ◽

Dietary Fat ◽

Lymphatic Vessels ◽

Developmental Origins ◽

Lymphatic Endothelial Cells ◽

Tissue Microenvironment ◽

Organ Specific ◽

Intestinal Lymphatics ◽

Lymphatic Vasculature ◽

Fat Uptake

Recent discoveries of novel functions and diverse origins of lymphatic vessels have drastically changed our view of lymphatic vasculature. Traditionally regarded as passive conduits for fluid and immune cells, lymphatic vessels now emerge as active, tissue-specific players in major physiological and pathophysiological processes. Lymphatic vessels show remarkable plasticity and heterogeneity, reflecting their functional specialization to control the tissue microenvironment. Moreover, alternative developmental origins of lymphatic endothelial cells in some organs may contribute to the diversity of their functions in adult tissues. This review aims to summarize the most recent findings of organotypic differentiation of lymphatic endothelial cells in terms of their distinct (patho)physiological functions in skin, lymph nodes, small intestine, brain, and eye. We discuss recent advances in our understanding of the heterogeneity of lymphatic vessels with respect to the organ-specific functional and molecular specialization of lymphatic endothelium, such as the hybrid blood-lymphatic identity of Schlemm’s canal, functions of intestinal lymphatics in dietary fat uptake, and discovery of meningeal lymphatic vasculature and perivascular brain lymphatic endothelial cells.

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Vascular Growth Factors and Lymphangiogenesis

Physiological Reviews ◽

10.1152/physrev.00005.2002 ◽

2002 ◽

Vol 82 (3) ◽

pp. 673-700 ◽

Cited By ~ 260

Author(s):

Lotta Jussila ◽

Kari Alitalo

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Lymphatic System ◽

Lymphatic Vessels ◽

The Body ◽

Vascular Tumors ◽

Vascular Endothelial ◽

Lymphatic Endothelial Cells ◽

Independent Manner ◽

Tumor Spread

Blood and lymphatic vessels develop in a parallel, but independent manner, and together form the circulatory system allowing the passage of fluid and delivering molecules within the body. Although the lymphatic vessels were discovered already 300 years ago, at the same time as the blood circulation was described, the lymphatic system has remained relatively neglected until recently. This is in part due to the difficulties in recognizing these vessels in tissues because of a lack of specific markers. Over the past few years, several molecules expressed specifically in the lymphatic endothelial cells have been characterized, and knowledge about the lymphatic system has started to accumulate again. The vascular endothelial growth factor (VEGF) family of growth factors and receptors is involved in the development and growth of the vascular endothelial system. Two of its family members, VEGF-C and VEGF-D, regulate the lymphatic endothelial cells via their receptor VEGFR-3. With the aid of these molecules, lymphatic endothelial cells can be isolated and cultured, allowing detailed studies of the molecular properties of these cells. Also the role of the lymphatic endothelium in immune responses and certain pathological conditions can be studied in more detail, as the blood and lymphatic vessels seem to be involved in many diseases in a coordinated manner. Discoveries made so far will be helpful in the diagnosis of certain vascular tumors, in the design of specific treatments for lymphedema, and in the prevention of metastatic tumor spread via the lymphatic system.

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