Role Of Lymphatic Endothelial Cells Research Articles

Associations amongst genes, molecules, cells, and organs in breast cancer metastasis.

This paper is a cross fertilization of ideas about the importance of molecular aspects of breast cancer metastasis by basic scientists, a pathologist, and clinical oncologists at the Henry Ford Health symposium. We address four major topics: (i) the complex roles of lymphatic endothelial cells and the molecules that stimulate them to enhance lymph node and systemic metastasis and influence the anti-tumor immunity that might inhibit metastasis; (ii) the interaction of molecules and cells when breast cancer spreads to bone, and how bone metastases may themselves spread to internal viscera; (iii) how molecular expression and morphologic subtypes of breast cancer assist clinicians in determining which patients to treat with more or less aggressive therapies; (iv) how the outcomes of patients with oligometastases in breast cancer are different from those with multiple metastases and how that could justify the aggressive treatment of these patients with the hope of cure.

Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-β1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-β type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

Role of lymphatic endothelial cells as antigen presenting cells in tumor T cell immunity

During tumor development, the tumor microenvironment impacts negatively anti-tumor T cell responses. Indeed tumors develop mechanisms to avoid immunosurveillance by inducing poorly immunogenic tumors or by setting up tolerogenic mechanisms to inhibit effective immune responses. In the context of lymphangiogenic tumors, lymphatic endothelial cells (LECs) substantially expand in the tumor itself and in the tumor draining lymph node, resulting in increased lymphatic drainage, tumor cell dissemination and metastasis. Recently, LECs have been given much attention as putative unconventional antigen presenting cells. Our group and others have studied their immunoregulatory role, pointing out their contribution in adaptive immunity. Previous studies have demonstrated that LECs can directly present tumor-derived antigens to tumor-specific CD8+ T cells leading to their deletion. However, it is unknown whether LECs can directly present tumor antigens to CD4+ T cells. Using a murine tumor cell line that promotes lymphangiogenesis, we show that, when MHCII expression is abrogated in LEC, tumor growth is reduced, effector T cell functions are enhanced, whereas the phenotype of suppressive regulatory T cells seems to be impaired. These results suggest that tumor-associated LECs would function as tolerogenic antigen-presenting cells, a property that could be blocked to improve immunotherapies in cancer. Further experiments will be performed in order to confirm these results, and extend to a mouse model of non-small cell lung cancer which is initiated by transformation of normal cells and is more physiologically relevant to humans.

Tumor Lymphatic Interactions Induce CXCR2-CXCL5 Axis and Alter Cellular Metabolism and Lymphangiogenic Pathways to Promote Cholangiocarcinoma.

Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.

Role of lymphatic endothelial cells in the tumor microenvironment-a narrative review of recent advances.

BackgroundAs lymphatic vessel is a major route for solid tumor metastasis, they are considered an essential part of tumor drainage conduits. Apart from forming the walls of lymphatic vessels, lymphatic endothelial cells (LECs) have been found to play multiple other roles in the tumor microenvironment, calling for a more in-depth review. We hope that this review may help researchers gain a detailed understanding of this fast-developing field and shed some light upon future research.MethodsTo achieve an informative review of recent advance, we carefully searched the Medline database for English literature that are openly published from the January 1995 to December 2020 and covered the topic of LEC or lymphangiogenesis in tumor progression and therapies. Two different authors independently examined the literature abstracts to exclude possible unqualified ones, and 310 papers with full texts were finally retrieved.ResultsIn this paper, we discussed the structural and molecular basis of tumor-associated LECs, together with their roles in tumor metastasis and drug therapy. We then focused on their impacts on tumor cells, tumor stroma, and anti-tumor immunity, and the molecular and cellular mechanisms involved. Special emphasis on lung cancer and possible therapeutic targets based on LECs were also discussed.ConclusionsLECs can play a much more complex role than simply forming conduits for tumor cell dissemination. Therapies targeting tumor-associated lymphatics for lung cancer and other tumors are promising, but more research is needed to clarify the mechanisms involved.

Lymphatics in the liver.

The liver is the largest lymph producing organ. A significant increase in the number of hepatic lymphatic vessels, or lymphangiogenesis, has been reported in various liver diseases, including, but not limited to, cirrhosis, viral hepatitis and hepatocellular carcinoma. Despite its apparent relevance in healthy and diseased livers as these and other observations indicate, the hepatic lymphatic system has been poorly studied. With knowledge of the lymphatic system in other organs and tissues incorporated, this review article addresses the current knowledge of the hepatic lymphatic system and the potential role of lymphatic endothelial cells in the health and the disease of the liver and concludes with a brief description on future directions of the study of the hepatic lymphatic system.

Shaping of Peripheral T Cell Responses by Lymphatic Endothelial Cells.

Lymph node stromal cells (LNSCs) have newly been promoted to the rank of new modulators of T cell responses. The different non-hematopoietic cell subsets in lymph node (LN) were considered for years as a simple scaffold, forming routes and proper environment for antigen (Ag)-lymphocyte encountering. Deeper characterization of those cells has recently clearly shown their impact on both dendritic cell and T cell functions. In particular, lymphatic endothelial cells (LECs) control lymphocyte trafficking and homeostasis in LNs and limit adaptive immune responses. Therefore, the new role of LECs in shaping immune responses has drawn the attention of immunologists. Striking is the discovery that LECs, among other LNSCs, ectopically express a large range of peripheral tissue-restricted Ags (PTAs), and further present PTA-derived peptides through major histocompatibility class I molecules to induce self-reactive CD8+ T cell deletional tolerance. In addition, both steady-state and tumor-associated LECs were described to be capable of exogenous Ag cross-presentation. Whether LECs can similarly impact CD4+ T cell responses through major histocompatibility class II restricted Ag presentation is still a matter of debate. Here, we review and discuss our current knowledge on the contribution of Ag-presenting LECs as regulators of peripheral T cell responses in different immunological contexts, including autoimmunity and cancer.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Steady-state antigen scavenging, cross-presentation, and CD8+ T cell priming: a new role for lymphatic endothelial cells.

Until recently, the known roles of lymphatic endothelial cells (LECs) in immune modulation were limited to directing immune cell trafficking and passively transporting peripheral Ags to lymph nodes. Recent studies demonstrated that LECs can directly suppress dendritic cell maturation and present peripheral tissue and tumor Ags for autoreactive T cell deletion. We asked whether LECs play a constitutive role in T cell deletion under homeostatic conditions. In this study, we demonstrate that murine LECs under noninflamed conditions actively scavenge and cross-present foreign exogenous Ags to cognate CD8(+) T cells. This cross-presentation was sensitive to inhibitors of lysosomal acidification and endoplasmic reticulum-golgi transport and was TAP1 dependent. Furthermore, LECs upregulated MHC class I and the PD-1 ligand PD-L1, but not the costimulatory molecules CD40, CD80, or CD86, upon Ag-specific interactions with CD8(+) T cells. Finally, Ag-specific CD8(+) T cells that were activated by LECs underwent proliferation, with early-generation apoptosis and dysfunctionally activated phenotypes that could not be reversed by exogenous IL-2. These findings help to establish LECs as APCs that are capable of scavenging and cross-presenting exogenous Ags, in turn causing dysfunctional activation of CD8(+) T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph node-resident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags.

Heterogeneous expression of toll‐like receptors in lymphatic endothelial cells derived from different tissues

As lymphatic endothelial cells (LECs) express different lymphatic and vascular markers depending on the organ they are derived from, we analysed whether they also show a heterogeneity of response against pathogens. To this end we analysed, for the presence of mRNA encoding for all human toll-like receptor (TLR), LECs isolated from lymph nodes and thymuses. RNA for TLR1-6 and 9 was identified in thymus-derived cells, whereas cells derived from lymph nodes contained mRNA for TLR1-4, 6 and 9, but failed to express mRNA specific for TLR5. The differential expression of TLRs was confirmed by the phosphorylation of nuclear factor-κB p65 only when the two types of LECs were incubated with the appropriate TLR agonists. The stimulation with specific agonists gives rise to a heterogeneous pattern of cytokine and chemokine secretion: thymus-derived LECs produced preferentially interleukin-6, interferon-inducible protein (IP)-10 and tumour necrosis factor-α, whereas cells prepared from lymph nodes mainly released interleukin-8, monocyte chemotactic protein-1, RANTES and (IP)-10. Finally, cells purified from lymph nodes expressed a higher level of intercellular adhesion molecule-1 than did cells prepared from the thymus when stimulated with several TLR agonists. The expression of a large set of TLRs and the responsiveness to specific agonists suggest that LECs are able to respond to pathogens, and the observed differences reflect specialized functions, redundancy and/or roles of LECs of different origin.

Shear stress-induced ATP-mediated endothelial constitutive nitric oxide synthase expression in human lymphatic endothelial cells

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/cm(2) increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from 10(-9) to 10(-6) M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10(-6)M ATP was significantly reduced by 10(-5) M suramin. Suramin (10(-5) M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, 10(-4) M apamin, 10(-9) M iberiotoxin, 10(-5) M 2-aminoethoxydephenyl borate, or 10(-5)M xestospongin C, but not 10(-5) M glybenclamide or 10(-5) M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small- (SK(Ca)) and big-conductance (BK(Ca)) Ca(2+)-activated K(+) channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphate-mediated release of intracellular Ca(2+) ions and the activation of Ca(2+)-activated K(+) channels in LEC.

VEGF-C Alters Barrier Function of Cultured Lymphatic Endothelial Cells Through a VEGFR-3-Dependent Mechanism

The lymphatic endothelium is an important semi-permeable barrier separating lymph from the interstitial space. However, there is currently a limited understanding of the lymphatic endothelial barrier and the mechanisms of lymph formation. The objectives of this study were to investigate the potential active role of lymphatic endothelial cells in barrier regulation, and to test whether the endothelial cell agonists VEGF-A and VEGF-C can alter lymphatic endothelial barrier function. Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) and human umbilical vein endothelial cells (HUVEC) were respectively used as models of lymphatic and vascular endothelium. Transendothelial electrical resistance (TER) of endothelial monolayers served as an index of barrier function. Cells were treated with VEGF-A, VEGF-C, or the VEGFR-3 selective mutant VEGF-C156S. MAZ51 was used to inhibit VEGFR-3 signaling. The results show that while VEGF-A causes a time-dependent decrease in TER in HUVEC, there is no response in HMLEC-d. In contrast, VEGF-C and VEGF-C156S cause a similar decrease in TER in HMLEC-d that is not observed in HUVEC. These results corresponded to the protein expression of VEGFR-2 and VEGFR-3 in these cell types, determined by Western blotting. In addition, the VEGF-C- and VEGF-C156S-induced TER changes were inhibited by MAZ51. The results indicate differential responses of the lymphatic and vascular endothelial barriers to VEGF-A and VEGF-C. Furthermore, our data suggest that VEGF-C alters lymphatic endothelial function through a mechanism involving VEGFR-3.

Vascular Endothelial Cell Growth Factor Receptor 3–Mediated Activation of Lymphatic Endothelium Is Crucial for Tumor Cell Entry and Spread via Lymphatic Vessels

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.

The physiology of lymphocyte migration through the single lymph node in vivo

Lymphocytes are mobile cells, continually recirculating between the blood and the tissues via the lymph. In order to maintain immune surveillance, the majority of lymphocyte traffic occurs through lymph nodes in vivo. Although a great deal of work has been done to elucidate the molecular mechanisms whereby lymphocytes leave the blood and enter the lymph node, lymphocyte traffic also requires that the lymphocyte successfully transit extravascular tissue and enter the lymph following transendothelial migration. The regulation of cell movement through lymph nodes, specific cellular positioning within the nodes, and eventual entry into the efferent lymphatics are poorly understood. The process of lymphocyte recirculation occurs in a physiological background, and in vivo systems have been particularly useful in uncovering the nuances of the process. This review summarizes available data about the recirculation of lymphocytes through the lymph node and the interaction of recirculating lymphocyte pools in vivo. The importance of factors in afferent lymph, the specific distribution of extracellular matrix proteins, potential soluble regulators of cell traffic, and evidence for an active role of lymphatic endothelial cells in the regulation of lymphocyte traffic are discussed. It seems likely that future work will need to be directed at determining the relative importance of these post-transendothelial migration regulators of lymphocyte traffic.