Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The Gs-coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

CPT1A regulates breast cancer-associated lymphangiogenesis via VEGF signaling.

BACKGROUND: Lymphangiogenesis is critical for metastasis of a variety of cancers, including breast cancer. CPT1A (carnitine palmitoyltransferase 1a) has been reported to play a critical role in breast cancer progress. However, the molecular mechanism remains elusive. METHODS: In order to investigate the role of CPT1A in HDLEC cells, short hairpin RNA approach was utilized to knock down the CPT1A gene expression. We employed transwell and lymphatic vessel formation assay to examine invasion and lymphangiogenesis of HDLEC (Human dermal lymphatic endothelial cells). RT-qPCR and westernblot analyses were used to determine genes expression in HDLEC and breast cancer cells. Finally, we determined the relative rate of acetyl-CoA/CoA in shNC and shCPT1A HDLEC cells by LC-MS approach. RESULTS: Knockdown of CPT1A in breast cancer cells (MCF-7 and MDA-MB-231) abolished invasion and lymphangiogenesis of HDLEC cells. Mechanistically, CPT1A depletion suppressed the expression of VEGF-C and VEGF-D in MCF-7 and MDA-MB-231 cells. Interestingly, CPT1A knockdown in HDLEC cells exhibited attenuated expression of lymphangiogenic markers (podoplanin, VEGFR-3, VEGF-C, VEGF-D and PROX-1). Consistently, CPT1A -null HDLEC cells displayed compromised invasion and lymphangiogenesis compared with negative control. Further investigation revealed that CPT1A regulated VEGFR3 via acetyl-CoA mediated H3K9ac, which could be abrogated by supplement of acetate. CONCLUSIONS: In present study, we revealed the mechanism by which CPT1A regulates breast cancer-associated invasion and lymphangiogenesis. Our findings provide insights into CPT1A -promoted breast tumor metastasis and provide rationale for understanding breast cancer metastasis.

Phosphorylation of Akt and ERK1/2 is required for VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium.

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.

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.

Inducible capillary formation in lymphatic endothelial cells by blocking lipid phosphate phosphatase-3 activity.

Lymphangiogenesis plays critical roles under normal and/or pathological conditions; however, the molecular contributors to this event were unknown until recently. In the present study, we first employed gene chip analysis and confirmed that lipid phosphate phosphatase-3 (LPP3) expression was increased until capillary formation in the conditionally immortalized rat lymphatic endothelial cell line. Signaling responses occur when several lipids induce acute biological functions; further, lipid phosphate phosphatases (LPPs) control their functions via dephosphorylation; however, there is no report on the association between LPP3 and lymphangiogenesis. siRNA-targeted LPP3 significantly increased capillary formation of human lymphatic endothelial cells; in contrast, it decreased cell adhesion to the basement membrane matrix. Furthermore, the inducible effect of the LPP inhibitor on capillary formation was observed. For the first time, we report that LPP3 abolishes accelerated abnormal lymphangiogenesis. Blocking LPP3 activities may aid in the development of novel therapy for lymph vessel defects.

Conditional gene targeting in mouse high endothelial venules.

High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacterial artificial chromosome recombineering. Crossing these transgenic mice with the ROSA26 reporter strain, which expresses lacZ following Cre-mediated recombination, and staining the resulting progeny with 5-bromo-4-chloro-5-indolyl-beta-D-galactoside indicated that Cre recombinase was specifically expressed in mAb MECA79-reactive HEVs in secondary lymphoid organs but not in any other blood vessels of the transgenic mice. The expression of Cre recombinase correlated with a developmental switch, from immature, mAb MECA367-reactive HEVs to mature, mAb MECA79-reactive HEVs in neonatal lymph nodes. In addition to the HEVs, Cre recombinase was also strongly expressed in the colonic villi, which recapitulated the intrinsic expression of GlcNAc6ST-2 as confirmed in GlcNAc6ST-2(GFP/GFP) knock-in mice and by RT-PCR. Furthermore, treatment with an antimicrobial agent revealed that the colonic expression of Cre recombinase in the transgenic mice was regulated by commensal bacteria in the colon. In addition, Cre recombinase was expressed in a small subset of cells in the brain, testis, stomach, small intestine, and lung. In view of the restricted expression of Cre recombinase, this transgenic mouse line should be useful for elucidating tissue-specific gene functions using the Cre/loxP system.

Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.

The extracellular lysophospholipase D autotaxin (ATX) and its product, lysophosphatidic acid, have diverse functions in development and cancer, but little is known about their functions in the immune system. Here we found that ATX had high expression in the high endothelial venules of lymphoid organs and was secreted. Chemokine-activated lymphocytes expressed receptors with enhanced affinity for ATX, which provides a mechanism for targeting the secreted ATX to lymphocytes undergoing recruitment. Lysophosphatidic acid induced chemokinesis in T cells. Intravenous injection of enzymatically inactive ATX attenuated the homing of T cells to lymphoid tissues, probably through competition with endogenous ATX and exertion of a dominant negative effect. Our results support the idea of a new and general step in the homing cascade in which the ectoenzyme ATX facilitates the entry of lymphocytes into lymphoid organs.

Orbital lymphatics: do they exist?

INTRODUCTION: Although the lymphatic system was first described almost 400 years ago, it is only in very recent years that researchers have been able to identify lymphatic channels with reasonable accuracy. Through advances in molecular biology and the development of endothelial cell markers the long held view that the human orbit is devoid of lymphatics has now been challenged. DISCUSSION: This review discusses the current evidence on this topic, which confirms the presence of orbital lymphatics in lachrymal gland and optic nerve sheath.

A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules.

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules leads to rolling and is critical for recruitment of lymphocytes into peripheral lymph nodes. Peripheral node addressin represents a class of L-selectin ligands recognized by the function-blocking monoclonal antibody MECA-79. Its epitope overlaps with sialyl 6-sulfo Lewis X, an L-selectin recognition determinant. Here, mice lacking two N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6ST-1 and GlcNAc6ST-2) demonstrated elimination of both peripheral node addressin and sialyl 6-sulfo Lewis X in high endothelial venules, considerably reduced lymphocyte homing to peripheral lymph nodes and reduced sticking of lymphocytes along high endothelial venules. Our results establish an essential function for the sulfotransferases in L-selectin ligand synthesis and may have relevance for therapy of inflammatory diseases.

Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span.

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.

Core 2 branching beta1,6-N-acetylglucosaminyltransferase and high endothelial venule-restricted sulfotransferase collaboratively control lymphocyte homing.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to carbohydrate ligands expressed on high endothelial venules (HEV) of the secondary lymphoid organs. Previous studies demonstrated that L-selectin ligand sulfotransferase (LSST) forms 6-sulfo sialyl Lewis x (sLe(x)) on both core 2 branch and MECA-79-positive extended core 1 O-glycans, but the chemical nature and roles of HEV ligands elaborated by LSST and core 2 beta1,6-N-acetylglucosaminyltransferase-1 (Core2GlcNAcT) have been undefined. In the present study, we have generated mutant mice with deficient LSST and show that inactivation of LSST gene alone leads to only partial impairment of lymphocyte homing to peripheral lymph nodes and moderate reduction in lymphocyte counts in the peripheral lymph nodes, despite the fact that L-selectin ligands that contain 6-sulfo sLe(x) are reduced at HEV. By contrast, LSST/Core2GlcNAcT double null mice exhibited a markedly reduced lymphocyte homing and reduced lymphocyte counts as a result of significantly decreased 6-sulfo sLe(x) on HEV L-selectin counterreceptors, relative to LSST- or Core2GlcNAcT-single null mice. Moreover, induction of LSST and Core2GlcNAcT transcripts was observed in HEV-like structure formed in the salivary gland of the non-obese diabetic mouse, which displays chronic inflammation. These results indicate that LSST and Core2GlcNAcT cooperatively synthesize HEV-specific L-selectin ligands required for lymphocyte homing and suggest that LSST and Core2GlcNAcT play a critical role in lymphocyte trafficking during chronic inflammation.

Adhesion of B cell lines to endothelial cells from human lymphoid tissue modulates tyrosine phosphorylation and endothelial cell activation.

Through the production of cytokines and growth factors the endothelium of secondary lymphoid organs plays a crucial role in controlling lymphocyte migration to the lymphoid microenvironment, an essential step in the initiation of the immune response. Here we demonstrate that direct contact of B cell lines with tonsil-derived human endothelial cells resulted in changes in the phosphorylation state of endothelial cells, causing their functional activation. We found a rapid (<15-s) and transient dephosphorylation, followed by a rapid rephosphorylation of tyrosine residues of the focal adhesion kinase, paxillin, and ERK2. Maximal rephosphorylation occurred after 15-30 min of B cell contact. Preincubation of lymphoid B cells with an adhesion-blocking Ab directed against alpha(4)beta(1) integrin abrogated adhesion-mediated changes of endothelial cell tyrosine phosphorylation, suggesting that cell contact was essential. Similar patterns of tyrosine phosphorylation, but with slightly different kinetics were induced after cross-linking of beta(1) integrin or CD40 on endothelial cells. Functional activation of endothelial cells by B cell adhesion was confirmed by the production of IL-6, IL-8, monocyte chemoattractant protein-1, M-CSF, and macrophage inflammatory protein-1beta mRNA. However, direct cross-linking of beta(1) integrin and CD40 failed to accomplish the same functional activation. These data indicate that direct contact of lymphoid B cells with the endothelium from lymphoid tissue induce endothelial cell signaling, resulting in chemokine and cytokine production. This phenomenon may provide a mechanism for the remodeling of the endothelium from lymphoid tissues, thus contributing to the free migration of lymphocytes and other cells into the lymphoid organs.

Histochemical analysis of lymphatic endothelial cells in lymphostasis.

The ultrastructure of endothelial cells of intestinal lymphatics and the thoracic duct (TD) and the relation to lymphostasis were examined in rats and monkeys. Localization of 5'-nucleotidase (5'-Nase) and endothelial nitric oxide synthase (eNOS) was studied. In normal lymphatic endothelial cells, 5'-Nase reaction product was evenly deposited on the cell surface in vivo and on cultured TD endothelial cells (TDECs), whereas eNOS was evenly distributed throughout the nucleus and cytoplasm. TDECs had a long filamentous process extending towards the subendothelial extracellular matrix but became flat and regular within 30-40 minutes after gastric perfusion with olive oil. According to their electron-density, two types of cells were found in the TD endothelial layer. The cells with low electron-density exhibited stronger 5'-Nase activity. Valves were bicuspid formations and the valvular endothelial surface of the convex side showed weaker 5'-Nase activity than the concave side. During TD blockage-induced lymphostasis in rats, the 5'-Nase product was almost not discernible in the TDECs within 2 weeks. Larger vesicles were found in the endothelial cytoplasm of the ligated TD. Their number decreased after 6-12 weeks. The small intestinal lymphatics in the mucosa and submucosa were dilated, with numerous open intercellular junctions. The endothelial lining appeared to have reduced activities for 5'-Nase and eNOS in 9 of 11 experimental animals. The results indicated that the inability of the open intercellular junctions, normally working as one-way endothelial flap valves, may be a key morphological feature after TD blockage. Reduced eNOS and 5'-Nase may functionally influence contractile activity and transport capability of the lymphatic vessels in the lymphostasis.

Differential requirements for core2 glucosaminyltransferase for endothelial L-selectin ligand function in vivo.

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.

Immunohistochemical localisation of ET-1 and eNOS in lymphatic stomata of the porcine broad ligament of the uterus.

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in lymphatic stomata in areas of their special accumulation in the porcine broad ligament during the estrous cycle. The study was performed using polyclonal antibody for ET-1 and monoclonal antibody for eNOS. ET-1 and eNOS immunoreactivities were demonstrated in some thin endothelial lymphatic lacuna walls throughout the estrous cycle. In the mesothelial cell layer, ET-1 and eNOS were detected only in stomata-related cuboidal mesothelial cells, however, the intensity of the immunostaining and distribution of the positive cells varied during the cycle. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the tone of lymphatic stomata during the absorption and passage of fluids, particles and cells from the peritoneal cavity to lymphatic vessels in the porcine broad ligament.

Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase.

Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0-24 h. This migration was inhibited by anti-VCAM-1 or anti-alpha4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.

A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

[Expression of focal adhesion kinase in lymphatic endothelial cells of metastasis].

This study was directed at the expression of focal adhesion kinase (pp125FAK) which is located together with vinculin and talin in the spot of cellular adhesion. The authors' intention was to collect reliable data on this important kinase in the signal pathways so as to provide in-depth materials for exploring the mechanism of tumor metastasis to lymphatic vessels. The immunohistochemical method was used to study the expression of FAK in lymphatic endothelial cells of metastaltci adenocarcinoma of rectum and its adjacent lymph-nodes. The result demonstrated that lymphatic endothelial cells expressed pp125FAK when lymphatic vessels were invaded by cancer cells. Reaction production was located in the cytoplasm. This study provides that there was a strong correlation between the expression of FAK in lymphatic endothelial cells and the metastasis of cancer to lymphatic vessels.