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1.  Lymphangiogenic factors, mechanisms, and applications 
Lymphangiogenesis, the growth of lymphatic vessels, is essential in embryonic development. In adults, it is involved in many pathological processes such as lymphedema, inflammatory diseases, and tumor metastasis. Advances during the past decade have dramatically increased the knowledge of the mechanisms of lymphangiogenesis, including the roles of transcription factors, lymphangiogenic growth factors and their receptors, and intercellular and intracellular signaling cascades. Strategies based on these mechanisms are being tested in the treatment of various human diseases such as cancer, lymphedema, and tissue allograft rejection. This Review summarizes the recent progress on lymphangiogenic mechanisms and their applications in disease treatment.
doi:10.1172/JCI71603
PMCID: PMC3934166  PMID: 24590272
2.  Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet 
Molecular Metabolism  2014;4(2):93-105.
Objective
Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown.
Methods
K14–VEGFR-3–Ig (sR3) mice that constitutively express soluble-VEGFR-3–Ig in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments.
Results
We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity.
Conclusions
These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance.
doi:10.1016/j.molmet.2014.11.006
PMCID: PMC4314545
Vegfc; Obesity; Adipose tissue; Inflammation; Macrophages; sR3, soluble-VEGFR-3; HFD, high-fat diet; SWAT, subcutaneous white adipose tissue; EWAT, epididymal white adipose tissue; CM, conditioned medium
3.  Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity 
The Journal of Clinical Investigation  2014;124(9):3960-3974.
Schlemm’s canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α9 and exhibited continuous vascular endothelial–cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.
doi:10.1172/JCI75392
PMCID: PMC4153702  PMID: 25061877
4.  The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel 
The Journal of Clinical Investigation  2014;124(9):3975-3986.
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.
doi:10.1172/JCI75395
PMCID: PMC4153703  PMID: 25061878
5.  Interactions between VEGFR and Notch signaling pathways in endothelial and neural cells 
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.
doi:10.1007/s00018-013-1312-6
PMCID: PMC3648205  PMID: 23479133
Angiogenesis; Neurogenesis; Neurovascular interactions; Notch; VEGFs; VEGFRs
6.  VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart 
EMBO Molecular Medicine  2014;6(3):307-321.
Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.
Subject Categories Cardiovascular System; Metabolism
See also: C Kupatt and R Hinkel (March 2014)
doi:10.1002/emmm.201303147
PMCID: PMC3958306  PMID: 24448490
angiogenesis; endothelial cell; ischemia; metabolism; VEGF-B
7.  Activation of Hypoxia Response in Endothelial Cells Contributes to Ischemic Cardioprotection 
Molecular and Cellular Biology  2013;33(16):3321-3329.
Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2gt/gt mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2gt/gt hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2gt/gt hearts than in wild-type hearts. Hif-p4h-2gt/gt mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2gt/gt hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.
doi:10.1128/MCB.00432-13
PMCID: PMC3753900  PMID: 23775121
8.  Tie1 deletion inhibits tumor growth and improves angiopoietin antagonist therapy 
The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.
doi:10.1172/JCI68897
PMCID: PMC3904604  PMID: 24430181
9.  The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish 
Development (Cambridge, England)  2013;140(19):4081-4090.
Blood vessels and neurons grow often side by side. However, the molecular and cellular mechanisms underlying their parallel development remain unclear. Here, we report that a subpopulation of secondary motoneurons extends axons ventrally outside of the neural tubes and rostrocaudally as a fascicle beneath the dorsal aorta (DA) in zebrafish. We tried to clarify the mechanism by which these motoneuron axons grow beneath the DA and found that Vegfc in the DA and Vegfr3 in the motoneurons were essential for the axon growth. Forced expression of either Vegfc in arteries or Vegfr3 in motoneurons resulted in enhanced axon growth of motoneurons over the DA. Both vegfr3 morphants and vegfc morphants lost the alignment of motoneuron axons with DA. In addition, forced expression of two mutant forms of Vegfr3 in motoneurons, potentially trapping endogenous Vegfc, resulted in failure of growth of motoneuron axons beneath the DA. Finally, a vegfr3 mutant fish lacked the motoneuron axons beneath the DA. Collectively, Vegfc from the preformed DA guides the axon growth of secondary motoneurons.
doi:10.1242/dev.091702
PMCID: PMC3913045  PMID: 24046321
Vegfc; Vegfr3; Motoneuron; Wiring; Guidance
10.  Impaired humoral immunity and tolerance in K14-VEGFR-3-Ig mice that lack dermal lymphatic drainage 
Lymphatic vessels transport interstitial fluid, soluble antigen, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower antibody titers. In contrast, although delayed, T cell responses were robust after 21 days, including high levels of antigen-specific CD8+ T cells and production of IFN-γ, IL-4 and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. Additionally, one-year-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.
doi:10.4049/jimmunol.1103545
PMCID: PMC3424306  PMID: 22844119
lymphedema; VEGF-C; lymph node; adaptive immunity; contact hypersensitivity
11.  VEGF-A regulated by progesterone governs uterine angiogenesis and vascular remodelling during pregnancy 
EMBO Molecular Medicine  2013;5(9):1415-1430.
The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of ‘vascular sinus folding’ (VSF) and mural cell drop-out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy — just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF-A secreted from the progesterone receptor (PR)-expressing decidual stromal cells which are largely distributed in the anti-mesometrial region (AMR). In comparison, P4-PR-regulated VEGF-A-VEGFR2 signalling, ligand-independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand-independent manner, with marked reduction of VEGF-A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors — VEGFR2 and Tie2 — strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner.
doi:10.1002/emmm.201302618
PMCID: PMC3799495  PMID: 23853117
decidual angiogenesis; pregnant uterus; vascular sinus folding; vascular regression; VEGF-A
12.  Immune cells control skin lymphatic electrolyte homeostasis and blood pressure 
The Journal of Clinical Investigation  2013;123(7):2803-2815.
The skin interstitium sequesters excess Na+ and Cl– in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl– accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl–, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure–regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3–mediated modification of cutaneous lymphatic capillary function.
doi:10.1172/JCI60113
PMCID: PMC3696542  PMID: 23722907
13.  PHD2 regulates arteriogenic macrophages through TIE2 signalling 
EMBO Molecular Medicine  2013;5(6):843-857.
Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.
doi:10.1002/emmm.201302695
PMCID: PMC3779447  PMID: 23616286
arteriogenesis; ischaemia; macrophages; PHD2; TIE2
14.  A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy 
The EMBO Journal  2013;32(5):629-644.
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.
doi:10.1038/emboj.2012.340
PMCID: PMC3590982  PMID: 23299940
lymph vessel development; ultramicroscopy; VEGFR-3; CCBE1; VEGF-C
15.  Angiopoietin-Like 4 Mediates PPAR Delta Effect on Lipoprotein Lipase-Dependent Fatty Acid Uptake but Not on Beta-Oxidation in Myotubes 
PLoS ONE  2012;7(10):e46212.
Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.
doi:10.1371/journal.pone.0046212
PMCID: PMC3464237  PMID: 23056264
16.  Blocking Fibroblast Growth Factor Receptor Signaling Inhibits Tumor Growth, Lymphangiogenesis, and Metastasis 
PLoS ONE  2012;7(6):e39540.
Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread.
doi:10.1371/journal.pone.0039540
PMCID: PMC3382584  PMID: 22761819
17.  Essential Role of the Coxsackie - and Adenovirus Receptor (CAR) in Development of the Lymphatic System in Mice 
PLoS ONE  2012;7(5):e37523.
The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.
doi:10.1371/journal.pone.0037523
PMCID: PMC3356332  PMID: 22624044
18.  Effects of Angiopoietin-2-Blocking Antibody on Endothelial Cell–Cell Junctions and Lung Metastasis 
Background
Angiopoietin-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.
Methods
We 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.
Results
Adenoviral 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.
Conclusion
Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions.
doi:10.1093/jnci/djs009
PMCID: PMC3309130  PMID: 22343031
19.  VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling 
Nature Cell Biology  2011;13(10):1202-1213.
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−; Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
doi:10.1038/ncb2331
PMCID: PMC3261765  PMID: 21909098
20.  The yin, the yang, and the Angiopoietin-1 
The Journal of Clinical Investigation  2011;121(6):2157-2159.
Twenty years after the discovery of the vascular endothelial Tie receptor tyrosine kinases and 15 years after the discovery of the Tie2 ligand, angiopoietin-1 (Angpt1, also known as Ang1), a study published in the current issue of the JCI reveals an unexpected loss-of-function phenotype of mice conditionally deleted of the Angpt1 gene. The results suggest that Angpt1 is needed as a vascular stabilizing factor that organizes and limits the angiogenesis response and protects from pathological consequences, such as tissue fibrosis.
doi:10.1172/JCI58196
PMCID: PMC3104784  PMID: 21606600
21.  Use of Cancer-Specific Genomic Rearrangements to Quantify Disease Burden in Plasma from Patients with Solid Tumors 
Genes, chromosomes & cancer  2010;49(11):1062-1069.
Detection of recurrent somatic rearrangements routinely allows monitoring of residual disease burden in leukemias, but is not used for most solid tumors. However, next-generation sequencing now allows rapid identification of patient-specific rearrangements in solid tumors. We mapped genomic rearrangements in three cancers and showed that PCR assays for rearrangements could detect a single copy of the tumor genome in plasma without false positives. Disease status, drug responsiveness, and incipient relapse could be serially assessed. In future, this strategy could be readily established in diagnostic laboratories, with major impact on monitoring of disease status and personalizing treatment of solid tumors.
doi:10.1002/gcc.20815
PMCID: PMC3145117  PMID: 20725990
22.  Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer 
Molecular Cancer  2011;10:36.
Background
Most bladder cancer patients experience lymphatic metastasis in the course of disease progression, yet the relationship between lymphangiogenesis and lymphatic metastasis is not well known. The aim of this study is to elucidate underlying mechanisms of how expanded lymphatic vessels and tumor microenvironment interacts each other and to find effective therapeutic options to inhibit lymphatic metastasis.
Results
The orthotopic urinary bladder cancer (OUBC) model was generated by intravesical injection of MBT-2 cell lines. We investigated the angiogenesis, lymphangiogenesis, and CD11b+/CD68+ tumor-associated macrophages (TAM) by using immunofluorescence staining. OUBC displayed a profound lymphangiogenesis and massive infiltration of TAM in primary tumor and lymphatic metastasis in lymph nodes. TAM flocked near lymphatic vessels and express higher levels of VEGF-C/D than CD11b- cells. Because VEGFR-3 was highly expressed in lymphatic vascular endothelial cells, TAM could assist lymphangiogenesis by paracrine manner in bladder tumor. VEGFR-3 expressing adenovirus was administered to block VEGF-C/D signaling pathway and clodronate liposome was used to deplete TAM. The blockade of VEGF-C/D with soluble VEGF receptor-3 markedly inhibited lymphangiogenesis and lymphatic metastasis in OUBC. In addition, the depletion of TAM with clodronate liposome exerted similar effects on OUBC.
Conclusion
VEGF-C/D are the main factors of lymphangiogenesis and lymphatic metastasis in bladder cancer. Moreover, TAM plays an important role in these processes by producing VEGF-C/D. The inhibition of lymphangiogenesis could provide another therapeutic target to inhibit lymphatic metastasis and recurrence in patients with invasive bladder cancer.
doi:10.1186/1476-4598-10-36
PMCID: PMC3080348  PMID: 21481239
23.  Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse 
The Journal of Clinical Investigation  2011;121(5):2000-2012.
In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.
doi:10.1172/JCI44751
PMCID: PMC3083794  PMID: 21540548
24.  Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation 
The Journal of Experimental Medicine  2010;207(10):2255-2269.
The role of lymphangiogenesis in inflammation has remained unclear. To investigate the role of lymphatic versus blood vasculature in chronic skin inflammation, we inhibited vascular endothelial growth factor (VEGF) receptor (VEGFR) signaling by function-blocking antibodies in the established keratin 14 (K14)–VEGF-A transgenic (Tg) mouse model of chronic cutaneous inflammation. Although treatment with an anti–VEGFR-2 antibody inhibited skin inflammation, epidermal hyperplasia, inflammatory infiltration, and angiogenesis, systemic inhibition of VEGFR-3, surprisingly, increased inflammatory edema formation and inflammatory cell accumulation despite inhibition of lymphangiogenesis. Importantly, chronic Tg delivery of the lymphangiogenic factor VEGF-C to the skin of K14-VEGF-A mice completely inhibited development of chronic skin inflammation, epidermal hyperplasia and abnormal differentiation, and accumulation of CD8 T cells. Similar results were found after Tg delivery of mouse VEGF-D that only activates VEGFR-3 but not VEGFR-2. Moreover, intracutaneous injection of recombinant VEGF-C156S, which only activates VEGFR-3, significantly reduced inflammation. Although lymphatic drainage was inhibited in chronic skin inflammation, it was enhanced by Tg VEGF-C delivery. Together, these results reveal an unanticipated active role of lymphatic vessels in controlling chronic inflammation. Stimulation of functional lymphangiogenesis via VEGFR-3, in addition to antiangiogenic therapy, might therefore serve as a novel strategy to treat chronic inflammatory disorders of the skin and possibly also other organs.
doi:10.1084/jem.20100559
PMCID: PMC2947063  PMID: 20837699
25.  Zebrafish Tie-2 shares a redundant role with Tie-1 in heart development and regulates vessel integrity 
Disease Models & Mechanisms  2010;4(1):57-66.
SUMMARY
Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease.
doi:10.1242/dmm.005033
PMCID: PMC3014345  PMID: 21045210

Results 1-25 (59)