Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor-3 (VEGFR3), 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 antibodies, 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) is 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 Ang2 blocking antibody decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of 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 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.

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.

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. ANGPT2 443 is a naturally occurring, lower-oligomeric protein isoform whose expression is increased in cancer. Here we use a knock-in mouse line (mice expressing Angpt2 443 ), 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 Angpt2 443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2 443 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, Angpt2 443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2 443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2 DAP ) that inhibited ANGPT1-or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor 5 1 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. STATEMENT OF SIGNIFICANCEThis study identifies the role of the N-terminal oligomerization domain of Angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of Angiopoietin-2 in cancer.Research.

No 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.

<p>Supplementary Materials and Methods</p>

<p>Supplementary Figure S1: Amino acid sequence of ANGPT2, ANGPT2443 and ANGTPT2DAP. Supplementary Figure S2: Competitive inhibition of ANGPT4-induced TIE2 phosphorylation by ANGPT2 isoforms. Supplementary Figure S3. TIE2 phosphorylation by ANGPT2 isoforms at the retracting cell edges. Supplementary Figure S4. Generation of Angpt2443 allele and the effects of Angpt2443 expression on the retina. Supplementary Figure S5. MMTV-PyMT mammary gland tumor model. Supplementary Figure S6. ANGPT2 at the Weibel-Palade bodies and the subendothelial space. Supplementary Figure S7. B16F10 melanoma lung colonization model. Supplementary Figure S8. E0771 breast cancer cell transplantation model.</p>

<p>Supplementary Tables I-III</p>