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Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro . These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have opposing effects on vascular stability through their receptor Tie2, but there is evidence for Tie2-independent functions of Ang2. Here, Hakanpaa et al. show that Ang2 directly activates β1-integrin, leading to rearrangement of the actin cytoskeleton and decreased VE-cadherin in cell–cell junctions.

Vascular endothelial growth factor C (VEGF‐C) binding to its tyrosine kinase receptor VEGFR‐3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF‐C/VEGFR‐3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF‐C in adult vasculature are unknown. We show here that VEGF‐C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF‐C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The  Vegfc‐ deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high‐fat diet, the Vegfc ‐deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism. Synopsis A new mouse model allows effective, timed and long lasting deletion of vascular endothelial growth factor C (VEGF‐C) by the Cre‐Lox system and highlights its relevance for intestinal lymphatic vessel maintenance and lipid absorption. Vegfc deletion halted the growth of lymphatic vessels at all stages of development. In adults with a normally developed lymphatic system, Vegfc deficiency specifically induced intestinal lymphatic vessel atrophy. Lymphatic vessel atrophy in the adult intestine had no impact on animal welfare but reduced dietary lipid uptake and high fat diet‐induced obesity. Graphical Abstract A new mouse model allows effective, timed and long lasting deletion of vascular endothelial growth factor C (VEGF‐C) by the Cre‐Lox system and highlights its relevance for intestinal lymphatic vessel maintenance and lipid absorption.

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO + -clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues. The lymphatic vasculature is essential to maintain fluid homeostasis and immune surveillance, including in the brain where lymphatic vessels were only recently identified. Here, Jacob et al. provide an anatomical map of lymphatic vessels in the vertebral column, where they find these contribute to fluid drainage and immune responses.

In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development. Ultramicroscopy of wholemount mouse embryos uncovers the first, previously unknown lymphatic structures in mammals: the dorsal longitudinal lymphatic vessel and the ventral primordial thoracic duct, which eventually connect with the cardinal vein as previously described.

Branching morphogenesis is a ubiquitous process that gives rise to high exchange surfaces in the vasculature and epithelial organs. Lymphatic capillaries form branched networks, which play a key role in the circulation of tissue fluid and immune cells. Although mouse models and correlative patient data indicate that the lymphatic capillary density directly correlates with functional output, i.e., tissue fluid drainage and trafficking efficiency of dendritic cells, the mechanisms ensuring efficient tissue coverage remain poorly understood. Here, we use the mouse ear pinna lymphatic vessel network as a model system and combine lineage-tracing, genetic perturbations, whole-organ reconstructions and theoretical modeling to show that the dermal lymphatic capillaries tile space in an optimal, space-filling manner. This coverage is achieved by two complementary mechanisms: initial tissue invasion provides a non-optimal global scaffold via self-organized branching morphogenesis, while VEGF-C dependent side-branching from existing capillaries rapidly optimizes local coverage by directionally targeting low-density regions. With these two ingredients, we show that a minimal biophysical model can reproduce quantitatively whole-network reconstructions, across development and perturbations. Our results show that lymphatic capillary networks can exploit local self-organizing mechanisms to achieve tissue-scale optimization. The lymphatic system is a transport network that controls immune response and tissue fluid circulation in the body. Here the authors combine experiment and theory to reveal that developing lymphatic capillary networks exploit complementary branching strategies to optimize tissue coverage.

Poor data transfer and interoperability between electronic health record (EHR) systems has been a challenge hindering availability and usability of patient information in clinical practice and evidence-based decision-making. To improve data transfer and interoperability, patient information should be documented in a structured format. This also applies to medication-related patient information and results of the interventions, such as medication reviews (MRs), to individually optimize medication regimens, especially in older adults. This study aimed to identify what information obtained from MRs should be documented in a structured form in EHRs at a national and organizational level. The study was conducted as a 3-round Delphi consensus survey in 2020. The electronic survey was based on a comprehensive inventory of international and national MR procedures in various settings. Expert panelists (N=41) independently assessed which topics should be documented in a structured form in EHRs. The interprofessional panel (N=41) consisted of 12 physicians, 13 pharmacists, 10 nurses, and 6 information management professionals (participation rate 66%-76% in rounds 1-3; consensus limit set at 80%). The responses were analyzed quantitatively and qualitatively. Consensus was reached on 97.3% (108/111) of predetermined topics to be documented in a structured form in EHRs. Of these, 39 concerned the MR process, 25 related to potentially drug-induced symptoms, 11 related to burden of risks for adverse drug effects, 12 related to laboratory tests and other test results, 12 related to medication adherence, and 9 related to the use of intoxicants. The patient's blood pressure (mean 4.85, SD 0.53; on a Likert scale 1-5), kidney function (mean 4.81, SD 0.56), and risk of bleeding (mean 4.81, SD 0.56) were ranked as the 3 most important topics to be documented in a structured form. The panel reached a consensus that the information obtained from MRs should be made available to all health care professionals in the national digital repository for patient data and to patients to some extent. The interprofessional expert panel strongly agreed on the results of the MRs that should be documented in a structured form in EHRs and made available to both health professionals involved in care teams and patients themselves.

Notch cell interaction mechanism governs cell fate decisions in many different cell contexts throughout the lifetime of all Metazoan species. It links the fate of one cell to that of its neighbors through cell-to-cell contacts, and binding of Notch receptors expressed on one cell to their membrane bound ligands on an adjacent cell. Environmental cues, such as growth factors and extracellular matrix molecules, superimpose a dynamic regulation on this canonical Notch signaling pathway. In this review, we will focus on Notch signaling in the vertebrate vascular and nervous systems and examine its role in angiogenesis, neurogenesis, and neurovascular interactions. We will also highlight the molecular relationships of the Notch pathway with vascular endothelial growth factors (VEGFs) and their high-affinity tyrosine kinase VEGF receptors, key regulators of both angiogenesis and neurogenesis.

In allergic airway inflammation, VEGFR-3-mediated lymphangiogenesis occurs in humans and mouse models, yet its immunological roles, particularly in adaptive immunity, are poorly understood. Here, we explored how pro-lymphangiogenic signaling affects the allergic response to house dust mite (HDM). In the acute inflammatory phase, the lungs of mice treated with blocking antibodies against VEGFR-3 (mF4-31C1) displayed less inflammation overall, with dramatically reduced innate and T-cell numbers and reduced inflammatory chemokine levels. However, when inflammation was allowed to resolve and memory recall was induced 2 months later, mice treated with mF4-31C1 as well as VEGF-C/-D knockout models showed exacerbated type 2 memory response to HDM, with increased Th2 cells, eosinophils, type 2 chemokines, and pathological inflammation scores. This was associated with lower CCL21 and decreased TRegs in the lymph nodes. Together, our data imply that VEGFR-3 activation in allergic airways helps to both initiate the acute inflammatory response and regulate the adaptive (memory) response, possibly in part by shifting the TReg/Th2 balance. This introduces new immunomodulatory roles for pro-lymphangiogenic VEGFR-3 signaling in allergic airway inflammation and suggests that airway lymphatics may be a novel target for treating allergic responses.