Number Of Lymphatic Endothelial Cells Research Articles

Lymphatic endothelial cell-specific NRAS p.Q61R mutant embryos show abnormal lymphatic vessel morphogenesis.

Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.

Prokineticin-1 induces normal lymphangiogenic activity and is involved in lymphangiogenesis and lymph node metastasis in colorectal cancer.

Background: Prokineticin family correlates with important roles in several biological processes, including homeostasis. We discovered novel functions of prokineticin1 (PROK1) in lymphangiogenesis and lymphnode metastasis in colorectal cancer.Materials and Methods: We examined changes in the number of lymphatic endothelial cells after PROK1 stimulation. PROK1 protein was stimulated with subcutaneously implanted in mice. Also a high-PROK1-expressing colorectal cancer cell line and anti-PROK1 antibody(Ab) were subcutaneously implanted in mice, and then examine lymphangiogenesis. PROK1 expression and the number of lymph vessels were examined in the primary lesion of 391 patients whose colorectal tumors had been resected.Results: When PROK1 was used as a stimulus, the number of lymphatic cells increased compared to unstimulated cells. And the number of lymph vessels in the skin of mice increased compared to mice implanted without PROK1. The number of lymph vessels in the primary tumor tissue increased when PROK1 was highly expressed compared to cases with non-detectable PROK1 expression. When PROK1 was expressed in human colorectal tumors, the rate of lymphnode metastasis was significantly higher than that in cases with non-detectable PROK1 expression.Conclusions: PROK1 is a lymphangiogenic factor involved in the formation of new lymph vessels and lymphnode metastasis in human colorectal cancer.

Abstract A48: Interactions between lymphatic vessels and cancer-associated fibroblasts in tumor immunity

Abstract Aggressive tumors often hijack the lymphatic vasculature in order to invade and metastasize to their draining lymph nodes (LNs) and ultimately to distant organs. The clinical relevance of lymphatic metastasis has been demonstrated for many cancers, most notably breast cancer, which has been observed to spread preferentially via the lymphogenous route, with the initial sites of metastasis frequently being the tumor-draining LN. Indeed, expression of vascular endothelial growth factor (VEGF)-C, the principal lymphangiogenic growth factor, by the tumor or recruited ancillary cells has been shown to correlate both with LN and distant metastasis, and shorter overall survival, and LN status represents an important prognostic factor for patients diagnosed with breast cancer. Although traditionally considered as passive conduits for tumor cell escape, it is becoming increasingly clear that lymphatic vessels play more active roles in facilitating tumor progression and metastasis. In addition to directly promoting tolerance upon VEGF-C-induced remodeling of the tumor-associated lymphatic vasculature, increased lymphatic density also leads to increased lymph flow from the tumor to the draining LN, causing mechanical stress-induced changes in the stroma surrounding the tumor that may synergize to further suppress anti-tumor immunity. We previously reported that heightened interstitial flow can drive myofibroblast differentiation, commonly identified by the expression of α-smooth muscle actin (α-SMA), and that such flow drives them to align and contract the extracellular matrix in a TGF-β1-dependent manner, leading to stromal stiffening, another feature that is strongly correlated with tumor progression, invasion, and poor prognosis in breast cancer. Here we show that lymphatic endothelial cells (LECs) may contribute directly to stromal stiffening through production of TGF-β1 upon stimulation with VEGF-C. To explore this hypothesis, we developed an orthotopic model of cells isolated from the spontaneous MMTV-PyMT model of breast cancer and stably transfected these cells to over-express VEGF-C, and found that increased VEGF-C secretion correlated with lymphangiogenesis as well as an increase in overall TGF-β1 within these tumors. Furthermore, VEGF-C induces TGF-β1 secretion by LECs in vitro, and in the spontaneous MMTV-PyMT model, inhibition of VEGF-C signaling upon administration of a VEGFR-3 blocking antibody yielded a reduction in overall TGF-β1 in the tumor, as well as specifically in the number of LECs producing TGF-β1 in vivo. Taken together, these results suggest a direct involvement of tumor-associated lymphatic vessels in stromal stiffening during tumor progression, linking tumor mechanobiology with tumor immunology. Citation Format: Ingrid M. van Mier, Jennifer M. Munson, Maria A.S. Broggi, Michele De Palma, Melody A.S. Swartz. Interactions between lymphatic vessels and cancer-associated fibroblasts in tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A48.

Essential Role of Apelin Signaling During Lymphatic Development in Zebrafish

Apelin and its cognate receptor Aplnr/Apj are essential for diverse biological processes. However, the function of Apelin signaling in lymphatic development remains to be identified, despite the preferential expression of Apelin and Aplnr within developing blood and lymphatic endothelial cells in vertebrates. In this report, we aim to delineate the functions of Apelin signaling during lymphatic development. We investigated the functions of Apelin signaling during lymphatic development using zebrafish embryos and found that attenuation of Apelin signaling substantially decreased the formation of the parachordal vessel and the number of lymphatic endothelial cells within the developing thoracic duct, indicating an essential role of Apelin signaling during the early phase of lymphatic development. Mechanistically, we found that abrogation of Apelin signaling selectively attenuates lymphatic endothelial serine-threonine kinase Akt 1/2 phosphorylation without affecting the phosphorylation status of extracellular signal-regulated kinase 1/2. Moreover, lymphatic abnormalities caused by the reduction of Apelin signaling were significantly exacerbated by the concomitant partial inhibition of serine-threonine kinase Akt/protein kinase B signaling. Apelin and vascular endothelial growth factor-C (VEGF-C) signaling provide a nonredundant activation of serine-threonine kinase Akt/protein kinase B during lymphatic development because overexpression of VEGF-C or apelin was unable to rescue the lymphatic defects caused by the lack of Apelin or VEGF-C, respectively. Taken together, our data present compelling evidence suggesting that Apelin signaling regulates lymphatic development by promoting serine-threonine kinase Akt/protein kinase B activity in a VEGF-C/VEGF receptor 3-independent manner during zebrafish embryogenesis.

THU0035 Lymphotoxin Beta Regulates Lymphatic Vessel Expansion in Ectopic Lymphoneogenesis

BackgroundSecondary lymphoid organs are characterized by concordance in time, function and response to stimuli between the lymphatic and vascular system that allows tight regulation of cellular recruitment and exit from...

Notch1 functions as a negative regulator of lymphatic endothelial cell differentiation in the venous endothelium

In development, lymphatic endothelial cells originate within veins and differentiate via a process requiring Prox1. Notch signaling regulates cell-fate decisions, and expression studies suggested that Jag1/Notch1 signaling functions in veins during lymphatic endothelial specification. Using an inducible lymphatic endothelial Prox1CreER(T2) driver, Notch signaling was suppressed by deleting Notch1 or expressing dominant-negative Mastermind-like in Prox1+ endothelial cells. Either loss of Notch1 or reduced Notch signaling increased Prox1+ lymphatic endothelial progenitor cell numbers in the veins, leading to incomplete separation of venous and lymphatic vessels. Notch loss of function resulted in excessive Prox1+ lymphatic cells emerging from the cardinal vein and significant lymphatic overgrowth. Moreover, loss of one allele of Notch1 in Prox1 heterozygous mice rescued embryonic lethality due to Prox1 haploinsufficiency and significantly increased Prox1+ lymphatic endothelial progenitor cell numbers. Expression of a constitutively active Notch1 protein in Prox1+ cells suppressed endothelial Prox1 from E9.75 to E13.5, resulting in misspecified lymphatic endothelial cells based upon reduced expression of podoplanin, LYVE1 and VEGFR3. Notch activation resulted in the appearance of blood endothelial cells in peripheral lymphatic vessels. Activation of Notch signaling in the venous endothelium at E10.5 did not arterialize the cardinal vein, suggesting that Notch can no longer promote arterialization in the cardinal vein during this developmental stage. We report a novel role for Notch1 in limiting the number of lymphatic endothelial cells that differentiate from the veins to assure proper lymphatic specification.

Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Fibrosis Is a Key Inhibitor of Lymphatic Regeneration

Lymphedema is a common debilitating sequela of lymph node dissection. Although numerous clinical studies suggest that factors that lead to fibrosis are associated with the development of lymphedema, this relationship has not been proven. The purpose of these experiments was therefore to evaluate lymphatic regeneration in the setting of variable soft-tissue fibrosis. A section of mouse tail skin including the capillary and collecting lymphatics was excised. Experimental animals (n = 20) were treated with topical collagen type I gel and a moist dressing, whereas control animals (n = 20) underwent excision followed by moist dressing alone. Fibrosis, acute lymphedema, lymphatic function, gene expression, lymphatic endothelial cell proliferation, and lymphatic fibrosis were evaluated at various time points. Collagen gel treatment significantly decreased fibrosis, with an attendant decrease in acute lymphedema and improved lymphatic function. Tails treated with collagen gel demonstrated greater numbers of lymphatic vessels, more normal lymphatic architecture, and more proliferating lymphatic endothelial cells. These findings appeared to be independent of vascular endothelial growth factor C expression. Decreased fibrosis was associated with a significant decrease in the expression of extracellular matrix components. Finally, decreased soft-tissue fibrosis was associated with a significant decrease in lymphatic fibrosis as evidenced by the number of lymphatic endothelial cells that coexpressed lymphatic and fibroblast markers. Soft-tissue fibrosis is associated with impairment in lymphatic regeneration and lymphatic function. These defects occur as a consequence of impaired lymphatic endothelial cell proliferation, abnormal lymphatic microarchitecture, and lymphatic fibrosis. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of normal capillary lymphatics.

VEGF-A produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes

AbstractLymphangiogenesis is involved in tumor cell metastasis and plays a major role in chronic inflammatory disorders. To investigate the role of lymphangiogenesis in inflammation, we induced and maintained delayed-type hypersensitivity (DTH) reactions in the ears of mice and then analyzed the resulting lymphangiogenesis in the inflamed tissue and draining lymph nodes (LNs) by quantitative fluorescence-activated cell sorting (FACS) and by immunofluorescence. Long-lasting inflammation induced a significant increase in the number of lymphatic endothelial cells, not only in the inflamed ears but also in the ear-draining auricular LNs. Inflammation-induced lymphangiogenesis was potently blocked by systemic administration of a vascular endothelial growth factor (VEGF)-A neutralizing antibody. Surprisingly, tissue inflammation specifically induced LN lymphangiogenesis but not LN angiogenesis. These findings were explained by analysis of both VEGF-A protein and mRNA levels, which revealed that VEGF-A was expressed at high mRNA and protein levels in inflamed ears but that expression was increased only at the protein level in activated LNs. Inflammation-induced lymphangiogenesis in LNs was independent of the presence of nodal B lymphocytes, as shown in B cell-deficient mice. Our data reveal that chronic inflammation actively induces lymphangiogenesis in LNs, which is controlled remotely, by lymphangiogenic factors produced at the site of inflammation.

Histomorphometrical study in cavernous lymphangioma of the tongue

To study the histomorphometrical characteristics of lymphatic vessels in cavernous lymphangiomas of the tongue. Immunohistochemical stainings were prepared in the 20 specimens with three antibodies [D2-40, CD31 and proliferating cell nuclear antigen (PCNA)]. Three-dimensional (3D) reconstruction and histometrical analysis of the lymphatic vessels was also examined. Distinctly positive staining for D2-40 was found in dilated lymphatic vessels located in the lamina propria beneath the thinned covering epithelium. Small blood vessels stained positively for CD31 were present in the lamina propria. PCNA-positive lymphatic endothelial cells were scattered in both control and lymphangioma. The 3D architecture of lymphatic vessels was characterized by a complex network with irregular branches in the lamina propria. Histometrical analysis showed that the number of lymphatic endothelial cells per lymphatic vessel perimeter in cavernous lymphangioma was significantly higher than that in control. There were no significant differences in the lymphatic density and the ratio of PCNA-positive lymphatic endothelial cell nuclei to the total number of lymphatic endothelial cell nuclei between control and lymphangioma. These results indicate the absence of excessive endothelial cell proliferation in dilated lymphatic vessels in cavernous lymphangioma. Cavernous lymphangioma may be attributed to the enlargement of lymphatic vessels without the tumorous proliferation of lymphatic endothelial cells.