LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis.

Liver fibrosis is a very common condition seen in millions of patients with various liver diseases, and yet no effective treatments are available owing to poorly characterized molecular pathogenesis. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2) is a functional ligand of Tie1, a poorly characterized endothelial cell (EC)-specific orphan receptor. Upon binding to Tie1, LECT2 interrupts Tie1/Tie2 heterodimerization, facilitates Tie2/Tie2 homodimerization, activates PPAR signaling, and inhibits the migration and tube formations of EC. In vivo studies showed that LECT2 overexpression inhibits portal angiogenesis, promotes sinusoid capillarization, and worsens fibrosis, whereas these changes were reversed in Lect2-KO mice. Adeno-associated viral vector serotype 9 (AAV9)-LECT2 small hairpin RNA (shRNA) treatment significantly attenuates fibrosis. Upregulation of LECT2 is associated with advanced human liver fibrosis staging. We concluded that targeting LECT2/Tie1 signaling may represent a potential therapeutic target for liver fibrosis, and serum LECT2 level may be a potential biomarker for the screening and diagnosis of liver fibrosis.

Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system.

Angiogenesis, the growth of blood vessels, is a fundamental biological process that controls embryonic development and is also involved in numerous life-threatening human diseases. Much work in the field of angiogenesis research has centred on the vascular endothelial growth factor (VEGF)-VEGF receptor system. The Tie receptors and their angiopoietin (Ang) ligands have been identified as the second vascular tissue-specific receptor Tyr kinase system. Ang-Tie signalling is essential during embryonic vessel assembly and maturation, and functions as a key regulator of adult vascular homeostasis. The structural characteristics and the spatio-temporal regulation of the expression of receptors and ligands provide unique insights into the functions of this vascular signalling system.

Enhanced notch signaling modulates unproductive revascularization in response to nitric oxide-angiopoietin signaling in a mouse model of peripheral ischemia.

INTRODUCTION: Arteriolargenesis can be induced by concomitant stimulation of nitric Oxide (NO)-Angiopoietin receptor (Tie)-Vascular Endothelial Growth Factor (VEGF) signaling in the rat mesentery angiogenesis assay. We hypothesized that the same combination of exogenously added growth factors would also have a positive impact on arteriolargenesis and, consequently, the recovery of blood flow in a model of unilateral hindlimb ischemia. RESULTS AND METHODS: NO-Tie mice had faster blood flow recovery compared to control mice, as assessed by laser speckle imaging. There was no change in capillary density within the ischemic muscles, but arteriole density was higher in NO-Tie mice. Given the previously documented beneficial effect of VEGF signaling, we tested whether NO-Tie-VEGF mice would show further improvement. Surprisingly, these mice recovered no differently from control, arteriole density was similar and capillary density was lower. Dll4 is a driver of arterial specification, so we hypothesized that Notch1 expression would be involved in arteriolargenesis. There was a significant upregulation of Notch1 transcripts in NO-Tie-VEGF compared with NO-Tie mice. Using soluble Dll4 (sDll4), we stimulated Notch signaling in the ischemic muscles of mice. NO-Tie-sDll4 mice had significantly increased capillary and arteriole densities, but impaired blood flow recovery. CONCLUSION: These results suggest that Dll4 activation early on in revascularization can lead to unproductive angiogenesis and arteriolargenesis, despite increased vascular densities. These results suggest spatial and temporal balance of growth factors needs to be perfected for ideal functional and anatomical revascularisation.

The Angiopoietin ligands and Tie receptors: potential diagnostic biomarkers of vascular disease.

The Angiopoietin-1 (Angpt1)/Tie2 signaling pathway is important in regulating vascular function. Angpt1-induced Tie2 activation promotes vascular endothelial cell survival and reduces vascular leakage. Angiopoietin-2 (Angpt2), a weak agonist/antagonist of Tie2, opposes and regulates Angpt1 action. The Tie family of receptor tyrosine kinases, Tie2 and Tie1, exist as either homo-or heterodimers. The molecular complex between the receptors is also crucial in controlling Angpt1 signaling; hence, the molecular balance between Angpt1:Angpt2 and Tie2:Tie1 is important in determining endothelial integrity and vascular stability. This review presents evidence of the change observed in the Angiopoietin/Tie molecules in various pathophysiological conditions and discusses the potential clinical applications of these molecules in vascular complications.

Luteal ANGPT-TIE system during selected stages of pregnancy, and normal and antigestagen-induced luteolysis in the dog.

Abstract: Rapid establishment of a vascular network is essential for normal functionality of the corpus luteum (CL). The early luteal phase is associated with increased expression of the VEGF system in canine CL. Acting in synchrony with angiopoietins (ANGPTs), VEGF system plays major roles in stabilization of blood vessels. However, the expression of the ANGPT system has not yet been investigated in the dog. Therefore, here, we investigated the luteal expression of ANGPT1, -2, and of their receptors TIE1 and -2, in pregnant dogs at selected time points during pregnancy and at normal and antigestagen-induced luteolysis. Additionally, luteal cells from early CL were incubated with PGE2 and its effects on the ANGPT system were assessed. Whereas the luteal ANGPT1 was stable until mid-gestation, TIE1 was elevated post-implantation, their expression decreased toward prepartum luteolysis. The ANGPT2- and TIE2-mRNA did not vary during pregnancy. The ANGPT2/ANGPT1 ratio was elevated during prepartum luteolysis. PGE2 increased ANGPT2, but suppressed ANGPT1 levels. None of the ANGPT-system members was affected by antigestagen treatment in mid-pregnancy. Localization of ANGPT1 was predominantly found in the tunica intima and media of vessels and ANGPT2 stained strongly in luteal cells. Both ANGPTs were localized in macrophages. TIE1 stained in the vascular tunica media, in luteal cells and macrophages, whereas TIE2 was colocalized with ANGPT1 in vascular components. In conclusion, high expression of ANGPT1 during the increased presence of VEGFA in early canine CL implies its contribution to vascular network development. The upregulation of the ANGPT2/ANGPT1 ratio during prepartum luteolysis indicates involvement of the ANGPT system in PGF2α-mediated vascular destabilization.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.

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

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.

Molecular control of angiopoietin signalling.

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.

Signaling pathways governing tumor angiogenesis.

Angiogenesis is regulated by the highly coordinated function of various proteins with pro- and antiangiogenic functions. Proangiogenic factors include vascular endothelial growth factor (VEGF), fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, transforming growth factor, angiopoietins, and several chemokines; antiangiogenic factors include thrombospondin-1, angiostatin, and endostatin. Matrix metalloproteinases display a dual role in vascular development. Notch signaling affects remodeling of the primary vascular network of uniformly sized vessels into functionally and morphologically distinct arteries, veins, and capillaries. Tumors, described as 'wounds that never heal', lose the appropriate balance among these factors. Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we highlight recent advances in our understanding of the regulation of tumor angiogenesis and discuss the potential of molecular targeting as a new therapeutic approach.

How do angiopoietins Tie in with vascular endothelial growth factors?

PURPOSE OF REVIEW: The endothelial cells of the blood and lymphatic vessels are involved in common human diseases. Excess blood and lymphatic vessel growth enhances tumor progression and metastasis, whereas insufficient growth leads to tissue ischemia and lymphedema. Lymphatic and blood vascular endothelial cells are regulated by two endothelial specific receptor tyrosine kinase systems, the vascular endothelial growth factor (VEGF) receptors (VEGFRs) and the Tie receptors, activated by the VEGF and angiopoietin ligands, respectively. Blocking of the VEGF-VEGFR-2 pathway has provided the first antiangiogenic strategy for cancer therapy and here we discuss the other pathways where progress is made for drug development, in particular the angiopoietin (Ang)-Tie receptor pathway. RECENT FINDINGS: VEGF-activated VEGFR-2 is the major transducer of angiogenic signals, but recent results show that the lymphangiogenic VEGFR-3 induces angiogenic sprouting as well. VEGF-B, a member of the VEGF family with low angiogenic activity, has been found to be involved in the regulation of energy metabolism in the heart. Recent reports have implicated Ang2 in tumor angiogenesis and revealed that Tie2 utilizes a unique signaling mechanism at endothelial cell-cell junctions. SUMMARY: The VEGF-VEGFR and Ang-Tie systems regulate different aspects of blood and lymphatic vessel growth. Thus, targeting both systems may be beneficial in maximizing the efficacy of anti/pro-angiogenic therapies.

The complex TIE between macrophages and angiogenesis.

Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre-existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro- and anti-angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2-expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin-TIE pathway.

Insight into the Role of Angiopoietins in Ageing-Associated Diseases.

Angiopoietin (Ang) and its receptor, TIE signaling, contribute to the development and maturation of embryonic vasculature as well as vascular remodeling and permeability in adult tissues. Targeting both this signaling pathway and the major pathway with vascular endothelial growth factor (VEGF) is expected to permit clinical applications, especially in antiangiogenic therapies against tumors. Several drugs targeting the Ang-TIE signaling pathway in cancer patients are under clinical development. Similar to how cancer increases with age, unsuitable angiogenesis or endothelial dysfunction is often seen in other ageing-associated diseases (AADs) such as atherosclerosis, Alzheimer's disease, type 2 diabetes, chronic kidney disease and cardiovascular diseases. Thus, the Ang-TIE pathway is a possible molecular target for AAD therapy. In this review, we focus on the potential role of the Ang-TIE signaling pathway in AADs, especially non-cancer-related AADs. We also suggest translational insights and future clinical applications of this pathway in those AADs.

The role of the Angiopoietins in vascular morphogenesis.

The Angiopoietin/Tie system acts as a vascular specific ligand/receptor system to control endothelial cell survival and vascular maturation. The Angiopoietin family includes four ligands (Angiopoietin-1, Angiopoietin-2 and Angiopoietin-3/4) and two corresponding tyrosine kinase receptors (Tie1 and Tie2). Ang-1 and Ang-2 are specific ligands of Tie2 binding the receptor with similar affinity. Tie2 activation promotes vessel assembly and maturation by mediating survival signals for endothelial cells and regulating the recruitment of mural cells. Ang-1 acts in a paracrine agonistic manner inducing Tie2 phosphorylation and subsequent vessel stabilization. In contrast, Ang-2 is produced by endothelial cells and acts as an autocrine antagonist of Ang-1-mediated Tie2 activation. Ang-2 thereby primes the vascular endothelium to exogenous cytokines and induces vascular destabilization at higher concentrations. Ang-2 is strongly expressed in the vasculature of many tumors and it has been suggested that Ang-2 may act synergistically with other cytokines such as vascular endothelial growth factor to promote tumor-associated angiogenesis and tumor progression. The better mechanistic understanding of the Ang/Tie system is gradually paving the way toward the rationale exploitation of this vascular signaling system as a therapeutic target for neoplastic and non-neoplastic diseases.

Regulation of endometrial vascular remodelling: role of the vascular endothelial growth factor family and the angiopoietin-TIE signalling system.

Angiogenesis, lymphangiogenesis and vascular maturation occur on a regular, physiological basis in human endometrium. These processes form part of a continuum of vascular remodelling involving numerous regulatory factors. Key factors include vascular endothelial growth factor (VEGF)A, VEGFC and VEGFD, and their associated receptors VEGFR1, VEGFR2 and VEGFR3. A second group of vascular regulatory proteins belongs to the angiopoietin (ANG)-TIE system. Although members of the VEGF family and the ANG-TIE system are represented in the endometrium, our understanding of how these different molecules interact to regulate remodelling of the blood and lymphatic vasculature present in the endometrium is still limited. A review of the current information is provided.

Angiopoietin-Tie signaling in kidney diseases: an updated review.

Angiopoietins (Angs) are a family of vascular growth factors that share multiple cellular functions related to cell survival, proliferation, and migration. Angs play physiological and pathological roles through the Tie tyrosine kinase receptors. The Ang-Tie signaling pathway participates in the developmental and tumor-induced angiogenesis and is also involved in many disease settings, such as vascular diseases, systemic inflammation, and cancers. Since Angs are widely expressed in the kidney, an enormous amount of research focuses on their roles in the kidney. In this review, we describe the biological functions of the Ang-Tie signaling pathway and summarize their roles in kidney development and maturation, acute and chronic kidney diseases, diabetic nephropathy, lupus nephropathy, hemolytic uremic syndrome, end-stage renal diseases, and renal cell carcinoma. Understanding the molecular mechanisms of Ang-Tie signaling may reveal potential therapeutic targets for preventing or alleviating kidney diseases.

Angiopoietin: a TIE(d) balance in tumor angiogenesis.

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, alpha(5)beta(1) and alpha(v)beta(5) integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2-dependent and TIE-2-independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway.

Anti-angiogenic activity of para-coumaric acid methyl ester on HUVECs in vitro and zebrafish in vivo.

BACKGROUND: Para-coumaric acid methyl ester (pCAME) is one of the bioactive components of Costus speciosus (Koen) Sm. (Zingiberaceae). This plant is traditionally used in Asia to treat catarrhal fevers, worms, dyspepsia, and skin diseases. PURPOSE: To investigate the anti-angiogenic activity of pCAME and its molecular mechanism of action. STUDY DESIGN: We investigated the anti-angiogenic activity of pCAME on human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish (Danio rerio) in vivo. METHODS: In vitro cell proliferation, would healing, migration and tube formation assays were used, along with in vivo physiological angiogenic vessel formation, tumor-induced angiogenic vessel formation assays on zebrafish model. qRT-PCR and RNA-seq were also used for the target investigation. RESULTS: pCAME could inhibit the proliferation, would healing, migration and tube formation of HUVECs, disrupt the physiological formation of intersegmental vessels (ISVs) and the subintestinal vessels (SIVs) of zebrafish embryos, and inhibit tumor angiogenesis in the zebrafish cell-line derived xenograft (zCDX) model of SGC-7901 in a dose-dependent manner. Mechanistic studies revealed that pCAME inhibited vegf/vegfr2 and ang/tie signaling pathways in zebrafish by quantitative RT-PCR analysis, and regulated multi-signaling pathways involving immune, inflammation and angiogenesis in SGC-7901 zCDX model by RNA-seq analysis. CONCLUSION: pCAME may be a multi-target anti-angiogenic drug candidate and hold great potential for developing novel therapeutic strategy for cancer treatment.

Signal transduction induced in endothelial cells by growth factor receptors involved in angiogenesis.

New vessel formation during development and in the adult is triggered by concerted signals of largely endothelial-specific receptors for ligands of the VEGF, angiopoietin and ephrin families. The signals and genes induced by these receptors operate in the context of additional signals transduced by non-endothelial specific growth factor receptors, inflammatory cytokine receptors as well as adhesion molecules. We summarize here available data on characteristic signaling of the VEGF receptor-2 and the current state of knowledge regarding the additional different receptor tyrosine kinases of the VEGF, Tie and Ephrin receptor families. Furthermore, the potential cross-talk with signals induced by other growth factors and inflammatory cytokines as well as the modulation by VE-cadherin is discussed.

Interaction between Tie receptors modulates angiogenic activity of angiopoietin2 in endothelial progenitor cells.

OBJECTIVE: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. METHODS AND RESULTS: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. CONCLUSIONS: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.

Molecular profile and proliferative responses of rat lymphatic endothelial cells in culture.

BACKGROUND: Lymphangiogenesis plays an important role in metastasis of many solid tumors. To study lymphangiogenesis under controlled conditions, an in vitro model is needed. The goal of this work was to establish such an in vitro model by determining a molecular profile of rat mesenteric lymphatic endothelial cells (RMLEC) and characterizing their proliferative responses to angiogenic and lymphangiogenic factors, such as vascular endothelial growth factor A and C (VEGF-A and VEGF-C). METHODS AND RESULTS: RMLEC strongly expressed most lymphatic-specific markers, including Prox-1, LYVE-1, and VEGFR-3. Proliferation of RMLEC was serum and heparin dependent. In the presence of low (2%) serum concentration, exogenously added VEGF-A and VEGFC stimulated RMLEC in a linear and dose-dependent manner. This effect was abrogated by anti-VEGF-A and VEGF-C antibodies, as well as by soluble Tie-2 and Flt-4 fusion proteins. Abrogation was reversed by VEGF-A, suggesting that this factor as an important regulator of lymphangiogenesis. CONCLUSIONS: Cultured RMLEC preserved a molecular profile consistent with the phenotype of lymphatic endothelium in vivo and respond to either VEGF-A or VEGF-C factors. VEGFA was able to rescue RMLEC proliferation inhibited by a neutralizing VEGF-C antibody or soluble Tie-2 fusion protein. These results support the existence of cross-talk among angiogenic and lymphangiogenic factors. This work established experimental conditions that allow in vitro modeling of lymphatic endothelial responses to lymphangiogenic regulators. Preliminary results using this model suggest that VEGF-A, VEGF-C, and angiopoietins work in concert to promote lymphangiogenesis in vivo.