Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Publisher Copyright: © 2022 American Society for Clinical Investigation. All rights reserved.Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with smallmolecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.Peer reviewe

Tehostettu perhetyö lasten kokemana

Opinnäytetyö on laadullinen tutkimus, jossa tutkittiin lasten kokemuksia tehostetusta perhetyöstä sekä sitä, miten lapset voivat perhetyössä vaikuttaa. Tavoitteena oli avata tehostetun perhetyön sisältöjä lapsen näkökulmasta käsin. Opinnäytetyö toteutettiin yhteistyössä Kouvolan kaupungin tehostetun perhetyön kanssa. Tutkimuksen kohteena oli tehostetun perhetyön asiakkaat, joilla oli tutkimuksen tekohetkellä lastensuojelun asiakkuus. Tutkimukseen osallistui kahdeksan 5–12-vuotiasta lasta. Aineisto kerättiin tapaamalla lapsia ryhmänä yhteensä neljä kertaa marraskuussa 2017. Tapaamiset olivat toiminnallisia tuokioita, joiden lomassa keskustelimme perhetyön sisällöistä. Keskustelut noudattivat teemahaastattelun mukaisesti tehtyä runkoa. Aineisto litteroitiin ja analysoitiin sisällönanalyysia käyttäen. Tutkimuskysymykset olivat, miten lapset kokevat tehostetun perhetyön ja miten lapset voivat vaikuttaa tehostettuun perhetyöhön. Tutkimuksen tuloksissa lapset kuvaavat perhetyötä pääsääntöisesti perheohjaajien ja heidän kanssaan tehdyn toiminnan kautta. Lasten kokemusten mukaan perheohjaajat toimivat paljon lasten kanssa. Suurin osa lapsista koki, että perheohjaajat antavat heille aikaa ja tekevät asioita heidän toiveidensa mukaisesti. Perheohjaajien käynnit tuntuvat lasten mielestä pääosin mukavilta, mutta lapset nostivat esiin myös muunlaisia tunteita. Perhetyö on lapsilähtöistä työtä ja se käy ilmi tämän tutkimuksen tuloksissa. Tutkimukseen osallistuneet lapset kokevat saavansa vaikuttaa ja osallistua. Lapsen osallisuus näyttää toteutuvan aikuisen läsnäolon kautta. Tutkimustulokset korostavat lasten kanssa vietetyn yhteisen ajan merkitystä perheohjaajan työn sisällöissä. Tutkimustuloksissa nousseita lasten kokemuksia voidaan hyödyntää tehostetun perhetyön kehittämisessä yhä lapsilähtöisempään suuntaan.The thesis is a qualitative study whose purpose was to explore children’s experiences of child welfare family work and how the children are able to affect family work. The aim was to describe the contents of the family work from children’s perspective. The thesis was carried out in co-operation with the intensified child welfare family work unit in the city of Kouvola. The subjects of the study were children who were customers of the intensified family work at the time of the study. There were eight children at the age of 5–12 years who took part in the study. The material for the study was collected by meeting the children as a group four times in November 2017. The meetings were interactive by nature and contained discussions about the contents of the family work along with the activities. The discussions followed a structure of a theme interview. The material was transcribed and analysed by using a content analysis. Research questions were how children experience intensified family work and how children can affect intensified family work. In the study material, children describe the family work with references to the family workers and the activity that they had done together. In children’s experiences, the family workers act much with the children. Most of the children thought that the family workers had time for them and did things according to children’s wishes. The visits by the family workers seemed to be mostly pleasant, but children also had experiences of other kind. Family work is child-oriented work and that shows in the results of this study. The children who responded to this study think that they are able to influence and participate in the family work. Children’s participation appears to be realized through the presence of an adult. In the content of the family work, the results of the study emphasize the significance of the time spent together. Children’s experiences that are presented in the results of the study can be used when intensified family work is developed in a more child-oriented way

Cooperation of angiopoietin-2 and angiopoietin-4 in Schlemm’s canal maintenance

Abstract Purpose: Defects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm’s canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively. Methods: Angpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes. Results: Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2−/−;Angpt4−/− mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity. Conclusions: Our results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina

Abstract The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found Angpt4 expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of Angpt4 resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function

The amino-terminal oligomerization domain of angiopoietin-2 affects vascular remodeling, mammary for gland tumor growth, and lung metastasis in mice

Abstract Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2₄₄₃ is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2₄₄₃), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2₄₄₃ caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2₄₄₃ differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2₄₄₃ promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2₄₄₃ was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Abstract Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling