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The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β 1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1-and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability. The Journal of Clinical Investigation R E S E A R C H A R T I C L E3 4 9 6 jci.org Volume 126 Number 9 September 2016We found that angiopoietins promoted a direct interaction of Tie1 and Tie2 and that this interaction was regulated by integrin β 1 . ANG1-and ANG2-induced Tie2 activation and vascular remodeling were reduced or absent in mice where Tie1 was deleted in vascular endothelial cells. Tie1 deletion also attenuated ANG1-induced Akt activation and nuclear exclusion of FOXO1. We found that Tie1 was suppressed via ectodomain cleavage during acute inflammation and that this was associated with reduced agonistic activity of ANG2 and decreased Tie2 signaling. These results indicate that the agonist action of ANG2 is attenuated in inflammation and that Tie1 is an essential component of the angiopoietin signaling system that has potential for therapeutic targeting in disease. ResultsAngiopoietins induce direct interaction of Tie1 and Tie2. To investigate the dynamics of the Tie receptors in angiopoietin signaling, we transfected HUVECs with retroviral vectors expressing full-length (FL) Tie1 and Tie2, tagged on the C terminus with mCherry and GFP, respectively. Stimulation of FL-Tie2-GFP and FL-Tie1-mCherry expressing endothelial cells with COMP-Ang1 (CAng1) (49) or with recombinant human ANG2 induced colocalization of Tie1 and Tie2 in endothelial cell-cell junctions ( Figure 1A and Supplemental Video 1; supplemental material available online with this article; doi:10.1172/JCI84923DS1) (44,45,50). To investigat...

BackgroundAngiopoietin-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.MethodsWe 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.ResultsAdenoviral 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 mm2, difference = 32.67 mm2, 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.ConclusionOur results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions.

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function. In order to characterize the effects of the angiopoietins on lymphatic vessels, we employed viral vectors for overexpression of Ang1 in adult mouse tissues. We found that Ang1 activated lymphatic vessel endothelial proliferation, vessel enlargement, and generation of long endothelial cell filopodia that eventually fused, leading to new sprouts and vessel development. IntroductionMembers of the vascular endothelial growth factor (VEGF) and angiopoietin families regulate both angiogenesis and lymphangiogenesis by acting on vascular endothelial cells. [1][2][3][4] Angiopoietins (Ang1 and Ang2) bind to the Tie2 receptor tyrosine kinase, expressed almost exclusively on the surface of endothelial cells, and regulate interactions between endothelial and periendothelial cells. Ang1 is an obligate agonist of Tie2, whereas Ang2 can act as either an agonist or an antagonist, depending on the cell type and the surrounding microenvironment. [5][6][7] Ang1 expression in the mouse embryo occurs first in the myocardium and later in a more widespread manner around the developing vessels. 5 Ang2 is expressed in the embryonic dorsal aorta and the major aortic branches and in adults in tissues that are undergoing vascular remodeling. 6,8 Gene-targeting experiments have indicated that Ang1 is necessary for maintaining maximal interactions between endothelial cells, periendothelial cells, and the extracellular matrix. 9 In adult tissues, exogenous Ang1 prevents leakage of plasma components into the interstitium caused by powerful vascular permeability agents, such as VEGF. 10 Furthermore, abundant evidence suggests that members of the angiopoietin and VEGF families collaborate during different stages of angiogenesis. Ang2 is expressed at sites of pericyte detachment and blood vessel remodeling in conjunction with VEGF, whereas in the absence of VEGF, Ang2 activity leads to endothelial cell apoptosis. 6,11,12 In addition, factors that induce angiogenesis in vivo, such as hypoxia and VEGF, have been shown to up-regulate Ang2 in endothelial cells. 13 The role of angiopoietins in lymphangiogenesis has remained unclear although Tie2 mRNA and protein have been detected at least in cultured lymphatic endothelial cells. 14,15 Ang2 knockout mice have lymphatic defects, suggesting that Ang2 is needed for normal lymph vessel formation and stabilization. 8 Replacement of the Ang2 gene with a cDNA encoding Ang1 was sufficient to rescue the lymphatic phenotype but not the blood vascular phenotype. 8 Thus, it is possible that both Ang2 and Ang1 act as agonists of Tie2 in ...

Platelet-derived growth factor-D (PDGF-D IntroductionPlatelet-derived growth factor (PDGF) is a mitogen for various cell types, including fibroblasts and smooth muscle cells. Although originally purified from human platelets, 1 current data indicate that several different cell types can produce PDGF in vitro and in vivo. 2 Until recently, the PDGF family of growth factors was composed of PDGF-A and PDGF-B chain homodimers and heterodimers. Recently, however, 2 novel homologous genes were isolated, PDGF-C 3 and PDGF-D. 4-6 PDGF-C and PDGF-D form disulfide-bonded homodimers, but they do not appear to heterodimerize with PDGF-A or PDGF-B chains, and they differ from the latter by having an N-terminal CUB-domain that is proteolytically cleaved before receptor binding. [3][4][5] PDGFs bind to and activate 2 structurally related protein tyrosine kinase receptors, PDGF receptor-␣ and PDGF receptor-␤. According to published data, the ␣-receptor binds PDGF-AA, PDGF-BB, PDGF-AB, and PDGF-CC, whereas the ␤-receptor binds PDGF-BB and PDGF-DD. [2][3][4] Although the exact biologic functions of PDGF-D are unknown, it has been shown to stimulate tumor growth and angiogenesis, 7-9 and it is implicated in glomerulonephritis. 10 Considerable interest focuses on the potential role of PDGF-D in wound healing. Reepithelialization, extracellular matrix deposition, and angiogenesis are all part of the wound healing process, and PDGFs are involved in various stages of this process (for a review, see Martin 11 ).Although most of the PDGF-B in wounds is produced by cells of hematopoietic origin, 12 wound healing occurs normally in mice that undergo transplantation with bone marrow derived from pdgfb null mice. 13 This result suggests a redundancy of PDGF-B-like growth factors. PDGF-D could perhaps provide a redundant function because it also binds to and activates one of the receptors for PDGF-B.Here we have addressed the effects of PDGF-D overexpression in normal skin and muscle and its effects on wound healing. We overexpressed the human PDGF-D cDNA under the keratin 14 (K14) promoter in the basal skin keratinocytes of transgenic mice. Because this promoter is strongly up-regulated during the woundhealing process, abundant PDGF-D is delivered to the wounds. 14 Furthermore, we cloned full-length PDGF-D and the activated growth factor domain into an adeno-associated virus (AAV) vector, overexpressed it alone or in combination with a known angiogenic factor, vascular endothelial growth factor-E (VEGF-E), and examined its effects on the generated blood vessels. Materials and methods Generation and analysis of K14-PDGF-D transgenic miceHuman PDGF-D cDNA (base pair [bp] 176-1285; GenBank sequence number AF336376) was inserted into the BamHI site of the K14 promoter expression vector 15 ( Figure 1A). The resultant construct was digested with EcoRI and SphI, and the expression cassette was purified. A 5-ng/mL For personal use only. on June 19, 2019. by guest www.bloodjournal.org From solution of the DNA was injected into fertilized eggs of t...

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfunction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium. Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B, VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin alpha 9. VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

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