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Background: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. Methods: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. Results: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. Conclusions: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.

Blood and lymphatic vessels form extensive networks throughout the body, which function in order to deliver oxygen and nutrients to the tissues, to remove extravasated fluid and to absorb dietary lipids. The formation of new blood and lymphatic vessels (termed angiogenesis and lymphangiogenesis) is critical during embryonic development and in the adult, and is regulated by multiple signaling pathways. Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGFRs), as well as the Notch signaling system, are key governors of blood and lymphatic endothelial cell fate, and regulate angiogenesis and lymphangiogenesis in health and disease. Despite the numerous recent advances in the field of vascular biology, many steps in the complex processes of angiogenesis and lymphangiogenesis remain unclear. In this study we investigated the role of VEGFR3 signaling in blood endothelial cells, tip cell specification, as well as the interplay of the receptor with the VEGFR2 and the Notch signaling pathways during angiogenesis. VEGFR3 is a tyrosine kinase receptor that is mainly expressed in lymphatic endothelial cells in the adult. We observed VEGFR3 expression in sprouts that guide the blood vascular endothelium in angiogenic conditions. VEGFR3 blockade with a monoclonal antibody displayed synergistic properties with simultaneous VEGFR2 targeting in reducing angiogenesis and inhibiting tumor growth. Furthermore we found that Notch signaling suppresses VEGFR3 expression in endothelial cells, identifying VEGFR3 as a novel tip cell marker, which is normally repressed by Notch activation. In the next step we employed a combination of genetic and in vitro models to show that loss of VEGFR3 results in a hypervascular phenotype, accompanied by loss of Notch signaling. VEGFR3 could be stimulated by VEGFC and activate Notch in blood endothelial cells. Our results point towards a mechanism where VEGFC produced by macrophages at the vascular front acts via VEGFR3 to activate Notch a, Not available

Background: Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized. Methods: We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided. Results: Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm[superscript 2], difference = 32.67 mm[superscript 2], 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell–cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell–cell junctions in endothelial–tumor cell cocultures. Conclusion: Our results indicate that blocking An

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.