The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

The mammalian lymphatic vasculature is important for returning fluids from the extracellular tissue milieu back to the blood circulation. We showed previously that Prox1 dosage is important for the development of the mammalian lymphatic vasculature. The lack of Prox1 activity results in the complete absence of lymphatic endothelial cells (LECs). In Prox1 heterozygous embryos, the number of LECs is reduced because of a decrease in the progenitor pool in the cardinal vein. This reduction is caused by some progenitor cells being unable to maintain Prox1 expression. In this study, we identified Vegfr3, the cognate receptor of the lymphangiogenic growth factor Vegfc, as a dosage-dependent, direct in vivo target of Prox1. Using various mouse models, we also determined that Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors and that Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. We propose that this feedback loop is the main sensing mechanism controlling the number of LEC progenitors and, as a consequence, the number of budding LECs that will form the embryonic lymphatic vasculature.     

The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells

The homeobox gene Prox1 is crucial for mammalian lymphatic vascular development. In the absence of Prox1, lymphatic endothelial cells (LECs) are not specified. The maintenance of LEC identity also requires the constant expression of Prox1. However, the mechanisms controlling the expression of this gene in LECs remain poorly understood. The SRY-related gene Sox18 is required to induce Prox1 expression in venous LEC progenitors. Although Sox18 is also expressed in embryonic arteries, these vessels do not express Prox1, nor do they give rise to LECs. This finding suggests that some venous endothelial cell-specific factor is required for the activation of Prox1. Here we demonstrate that the nuclear hormone receptor Coup-TFII is necessary for the activation of Prox1 in embryonic veins by directly binding a conserved DNA domain in the regulatory region of Prox1. In addition, we show that the direct interaction between nuclear hormone receptors and Prox1 is also necessary for the maintenance of Prox1 expression during early stages of LEC specification and differentiation.     

Prox1 dosage controls the number of lymphatic endothelial cell progenitors and the formation of the lymphovenous valves

Arteries, veins, and lymphatic vessels are functionally linked, and their physical interaction is tightly regulated. The lymphatic vessels communicate with the blood vessels only at the junction of the jugular and subclavian veins. Here, we characterize the embryonic lymphovenous valves controlling this vital communication and show that they are formed by the intercalation of lymphatic endothelial cells (LECs) with a subpopulation of venous endothelial cells (ECs) at the junction of the jugular and subclavian veins. We found that unlike LEC progenitors, which move out from the veins and differentiate into mature LECs, these Prox1-expressing ECs remain in the veins and do not acquire LEC features. We demonstrate that the development of this Prox1-expressing venous EC population, and therefore of lymphovenous valves, requires two functional copies of Prox1, as the valves are absent in Prox1 heterozygous mice. We show that this is due to a defect in the maintenance of Prox1 expression in venous ECs and LEC progenitors promoted by a reduction in Coup-TFII/Prox1 complex formation. This is the first report describing the molecular mechanism controlling lymphovenous communication.     

Coordinated lymphangiogenesis is critical in lymph node development and maturation

Background: Lymph node (LN) formation requires multiple but coordinated signaling from intrinsic and extrinsic cellular components during embryogenesis. However, the contribution and role of lymphatic vessels (LVs) in LN formation and maturation are poorly defined. Here, using lymphatic‐specific reporters, Prox1‐GFP mice and Vegfc^+/LacZ mice, we analyzed migration, assembly, and ingrowth of lymphatic endothelial cells (LECs) in LNs during pre‐ and postnatal development. Results: Prox1⁺ LECs form string‐like connections rather than lymph sac–like structures until E14.5, but the LEC coverage around LN anlagen completes before birth. Compared to wild‐type littermates, Vegfc^+/LacZ mice had markedly smaller LNs in neonates and adults, presumably due to the decrease in LTi cell clusters and migrating Prox1⁺ LECs during embryogenesis. In addition, Vegfc‐haploinsufficiency or inhibition of VEGFR3 signaling led to an impairment of LN LV ingrowth, resulting in a significant decrease in LN volume. These data indicate that VEGF‐C/VEGFR3 signaling plays a substantial role in normal LN formation through proper migration and organization of LECs. Conclusions: Taken together, our results provide compelling evidence that the contribution of LVs through VEGF‐C/VEGFR3 signaling is critical in LN development and maturation. Developmental Dynamics 245:1189–1197, 2016. © 2016 Wiley Periodicals, Inc.     

The sialomucin CD34 is a marker of lymphatic endothelial cells in human tumors

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.     

Murine Notch1 is required for lymphatic vascular morphogenesis during development

BACKGROUND: The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)‐specific conditional Notch1 knockout mice crossed with an inducible Prox1CreER^T2 driver. RESULTS: LEC‐specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1‐mutant LECs, whereas proliferation was increased. RNA‐seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1‐mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated. CONCLUSIONS: The lymphatic phenotype of LEC‐specific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis. Developmental Dynamics 243:957–964, 2014. © 2014 Wiley Periodicals, Inc.     

Chlamydophila pneumoniae enhances secretion of VEGF, TGF-�� and TIMP-1 from human bronchial epithelial cells under Th2 dominant microenvironment

Purpose

Chlamydophila pneumoniae infection in the airways is thought to be associated with the pathogenesis of asthma, especially in non-atopic severe asthma with irreversible airway obstruction that may be related to airway remodeling. Here, we investigated whether C. pneumoniae infection enhances the secretion of critical chemical mediators for airway remodeling, such as VEGF, TGF-β, and TIMP-1, in human bronchial epithelial cells (BECs) in a Th2-dominant microenvironment.

Methods

Human bronchial epithelial cells (BEAS-2B cells) were infected with C. pneumoniae strain TW183 and cultured in both a Th1-dominant microenvironment with INF-γ and a Th2-dominant microenvironment with IL-4 or IL-13 added to the culture medium. The VEGF, TGF-β, and TIMP-1 levels in the culture supernatants were measured using enzyme-linked immunosorbent assays (ELISA). The activation of NF-κB in each experimental condition was determined using an electrophoretic mobility shift assay.

Results

Chlamydophila pneumoniae-infected BECs showed enhanced secretion of VEGF, TGF-β, and TIMP-1 compared with non-infected BECs. The levels of cytokines secreted from BECs were increased more when IL-13 was added to the culture medium. C. pneumoniae-infected BECs also showed increased NF-κB activation.

Conclusions

These results suggest that C. pneumoniae plays a role in the pathogenesis of airway remodeling in asthma, revealing a Th2-dominant immune response. Further studies are required to clarify the precise mechanism of C. pneumoniae infection in airway remodeling.     

Martentoxin,a large-conductance Ca2+-activated K+ channel inhibitor,attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-α in human umbilical vein endothelial cells (HUVECs). We found that, 1, 10 and 100 µmol/L martentoxin decreased nitric oxide production by HUVECs exposed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-α. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.     

血小板反应素-1对猪胸导管内皮细胞增殖和凋亡的影响

目的探讨血小板反应素-1(thrombospondin-1,TSP-1)对淋巴管内皮细胞(lymphatic endothelial cells,LECs)增殖和凋亡的影响。方法取健康猪的胸导管内皮细胞进行分离、培养;应用凝血因子VIII,血管内皮生长因子受体-3(VEGFR-3)抗体联合对淋巴管内皮细胞进行鉴定;采用MTT法检测TSP-1对LEC的生长抑制作用;采用Hoechst33258荧光染色检测淋巴管内皮细胞凋亡。结果培养的淋巴管内皮细胞呈第VIII因子和VEGFR-3阳性反应,为典型淋巴管内皮细胞。MTT法证实,当浓度为0.8μg/ml￣2.0μg/ml时,TSP-1能明显抑制淋巴管内皮细胞的增殖,且抑制作用呈现剂量依赖关系。Hoechst33258证实,经TSP-1处理后淋巴管内皮细胞,可观察到其核周围有凋亡小体。结论TSP-1对淋巴管内皮细胞增殖有明显的剂量依赖性抑制作用,诱导淋巴管内皮细胞凋亡。     

The KSHV viral IL-6 homolog is sufficient to induce blood to lymphatic endothelial cell differentiation

The predominant tumor cell of Kaposi's Sarcoma (KS) is the spindle cell, a cell of endothelial origin that expresses markers of lymphatic endothelium. In culture, Kaposi's Sarcoma-associated herpesvirus (KSHV) infection of blood endothelial cells drives expression of lymphatic endothelial cell specific markers, in a process that requires activation of the gp130 receptor and the JAK2/STAT3 and PI3K/AKT signaling pathways. While expression of each of the KSHV major latent genes in endothelial cells failed to increase expression of lymphatic markers, the viral homolog of human IL-6 (vIL-6) was sufficient for induction and requires the JAK2/STAT3 and PI3K/AKT pathways. Therefore, activation of gp130 and downstream signaling by vIL-6 is sufficient to drive blood to lymphatic endothelial cell differentiation. While sufficient, vIL-6 is not necessary for lymphatic reprogramming in the context of viral infection. This indicates that multiple viral genes are involved and suggests a central importance of this pathway to KSHV pathogenesis.     

Tumor‐associated lymphatic endothelial cell promotes invasion of cervical cancer cells

The most common way for cervical cancer to spread is through the lymphatic system. Tumor‐associated lymphatic endothelial cell (TLEC) has been considered to play a crucial role in metastasis of certain cancers. The aim of this study was to isolate TLEC from human cervical cancers and explore its involvement in metastasis‐associated behaviors in vitro. Lymphatic vessels in 62 cervix tissue specimens ranging from cervical intraepithelial neoplasia (CIN) to advanced invasive cancer were detected using immunochemical staining with D2‐40 antibody. Relation of lymphatic vessel density (LVD) to clinicopathological characters was analyzed. Primary TLECs were isolated by LYVE‐1 immuno‐magnetic beads from cervical cancer tissues and verified through expression of LEC markers Prox‐1 and D2‐40, and then cultured in vitro. Invasiveness and viability of cells were assessed by transwell assay and typan blue exclusion, respectively. Our results showed that higher LVD was significantly associated with advanced FIGO stage, pelvic lymphatic nodal metastasis (LNM), and poorer cell differentiation. TLECs were successfully primarily isolated and cultured in vitro. Supernatant of TLEC enhanced invasiveness of Hela cell, but did not significantly affect cell viability. In conclusion, TLECs might actively promote lymphatic metastasis of cervical cancer. Further studies are needed to demonstrate the underlying mechanisms.     

自发性高血压大鼠淋巴微循环功能受损

目的:观察自发性高血压大鼠(SHRs)淋巴微循环功能变化.方法:8周龄雄性SHR大鼠(SHR组)和WKY大鼠(WKY组),每组各10只.应用VasT rack测量两组大鼠微淋巴管自律运动;取胸导管分离原代淋巴管内皮细胞(LECs).应用免疫荧光和Western Blot检测LECs血管内皮生长因子受体3(VEGFR3)...     

Tumour necrosis factor superfamily member 15 (Tnfsf15) facilitates lymphangiogenesis via up‐regulation of Vegfr3 gene expression in lymphatic endothelial cells

Lymphangiogenesis is essential in embryonic development but is rare in adults. It occurs, however, in many disease conditions including cancers. Vascular endothelial growth factor‐C/D (VEGF‐C/D) and VEGF receptor‐3 (Vegfr3) play a critical role in the regulation of lymphangiogenesis. We investigated how the VEGF‐C/Vegfr3 signalling system is regulated by tumour necrosis factor superfamily member 15 (Tnfsf15), an endothelium‐derived cytokine. We report here that Tnfsf15, which is known to induce apoptosis in vascular endothelial cells, can promote lymphatic endothelial cell (LEC) growth and migration, stimulate lymphangiogenesis, and facilitate lymphatic circulation. Treatment of mouse LECs with Tnfsf15 results in up‐regulation of Vegfr3 expression; this can be inhibited by gene silencing of death domain‐containing receptor‐3 (DR3; Tnfrsf25), a cell surface receptor for Tnfsf15, with siRNA, or by blocking Tnfsf15–DR3 interaction with a Tnfsf15 neutralizing antibody, 4‐3H. Additionally, Tnfsf15/DR3 signalling pathways in LECs include activation of NF‐κB. Tnfsf15‐overexpressing transgenic mice exhibit a marked enhancement of lymph drainage; this is confirmed by treatment of wild‐type mice with intraperitoneal injection of recombinant Tnfsf15. Moreover, systemic treatment of pregnant Tnfsf15 transgenic mice with 4‐3H leads to inhibition of embryonic lymphangiogenesis. Our data indicate that Tnfsf15, a cytokine produced largely by endothelial cells, facilitates lymphangiogenesis by up‐regulating Vegfr3 gene expression in LECs, contributing to the maintenance of the homeostasis of the circulatory system. This finding also suggests that Tnfsf15 may be of potential value as a therapeutic tool for the treatment of lymphoedema. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Lymph node lymphatic endothelial cells as multifaceted gatekeepers in the immune system

Single-cell technologies have recently allowed the identification of multiple lymphatic endothelial cell (LEC) subsets in subcapsular, paracortical, medullary, and other lymph node (LN) sinus systems in mice and humans. New analyses show that LECs serve key immunological functions in the LN stroma during immune responses. We discuss the roles of different LEC types in guiding leukocyte and cancer cell trafficking to and from the LN parenchyma, in capturing microbes, and in transporting, presenting, and storing lymph-borne antigens in distinct types of lymphatic sinuses. We underscore specific adaptations of human LECs and raise unanswered questions concerning LEC functions in human disease. Despite our limited understanding of human lymphatics – hampering clinical translation in inflammation and metastasis – we support the potential of LN LECs as putative targets for boosting/inhibiting immunoreactivity.     

Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells

OBJECTIVE:

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs).

METHODS:

HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H₂O₂), treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H₂O₂ for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction.

RESULTS:

Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs.

CONCLUSION:

Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.