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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.

Background and Purpose Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these diseases. Experimental Approach In vitro and in vivo angiogenesis and/or lymphangiogenesis were assessed in VEGF- or hypoxia-stimulated endothelial and retinal cells and in animal models of oxygen-induced retinopathy ( OIR), streptozotocin-induced diabetic retinopathy ( SIDR), suture-induced inflammatory corneal neovascularization ( SICNV) and silver nitrate-induced corneal neovascularization. HUVECs and retinal cells were cultured under hypoxic conditions or incubated with VEGF to identify the molecular mechanisms involved. Key Results The imidazole-based alkaloid derivative LCB54-0009 inhibited capillary-like tube formation in VEGF-induced HUVECs without inducing cytotoxic effects. Intravitreal injection of LCB54-0009 into retinas suppressed the formation of the pathological neovascular tufts and increased vascular permeability in both OIR of mice and SIDR of rats. Furthermore, subconjunctival injection of LCB54-0009 into the cornea suppressed corneal inflammation and inflammation-associated angiogenesis and lymphangiogenesis in SICNV of mice and silver nitrate cauterization of rats. These pharmacological activities were associated with effects on HIF-1α protein stability and HIF-1α/ NF- κB redox sensitivity through its antioxidant activities. LCB54-0009 also inhibited the hypoxia-induced expression of angiopoietin-2, and VEGF-induced VEGFR-2 activation and downstream signalling, resulting in the down-regulation of the expression of pro-angiogenic factors and pro-inflammatory mediators and an up-regulation of the expression of anti-angiogenic factors. Conclusions and Implications LCB54-0009 is a potential candidate molecule for blocking pathological angiogenesis and lymphangiogenesis mediated by HIF-1α- angiopoietin-2 expression and VEGFR-2 activation. [ABSTRACT FROM AUTHOR]

Summary: Vascular endothelial growth factor A (VEGF) is highly expressed in adipose tissue. Its role, however, has not been fully elucidated. Here, we reveal the metabolic role of adipose-VEGF by studying mice with deletion (VEGFAdΔ) or doxycycline-inducible overexpression of a VEGF transgene (VEGFAdTg) in the adipose tissue. VEGFAdΔ mice have reduced adipose vascular density and show adipose hypoxia, apoptosis, inflammation, and metabolic defects on a high-fat diet. In contrast, induction of VEGF expression in VEGFAdTg mice leads to increased adipose vasculature and reduced hypoxia. The latter changes are sufficient to counteract an established compromising effect of high-fat diet on the metabolism, indicating that metabolic misbalance is reversible by adipose vessel density increase. Our data clearly show the essential role of VEGF signaling for adequate adipose function. Besides revealing insights into the molecular mechanisms of obesity-related metabolic diseases, this study points to the therapeutic potential of increased adipose angiogenesis. [Copyright &y& Elsevier]

Among many proangiogenic growth factors, angiopoietin-1 (Ang1, or ANGPT1) is unique because it can induce distinctive vascular remodeling through highly organized angiogenesis and tightening of endothelial cell (EC) junctions. These effects are mediated by synchronous activation of both vascular Tie2 and nonvascular integrin signaling, making Ang1 a viable candidate for therapeutic neovascularization and vascular protection. Ang1 helps delay diabetic complications by restoring microvascular function and can maintain the quiescence of some adult stem cells. Interestingly, Ang1 is dispensable for maintaining normal vasculature throughout adulthood, challenging the original concept of its functions in cell survival and stabilization in quiescent vasculature. This review summarizes recent advances in understanding the biomedical implications of Ang1 and discusses its multifaceted roles in vascular diseases, the mechanisms underlying its effects, and potential therapeutic applications. [Copyright &y& Elsevier]

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. [ABSTRACT FROM PUBLISHER]

Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis. Its principal pharmacologic use is in dental products where it has antibacterial, antifungal, and anti-inflammatory activities that reduce gingival inflammation and supragingival plaque formation. Angiogenesis is indispensable for inflammation, and most angiogenesis is dependent on vascular endothelial growth factor (VEGF). However, the effect of sanguinarine on angiogenesis is not known. In the present study, we examined the effect of sanguinarine on VEGF-induced angiogenesis in vitro and in vivo. Interestingly, sanguinarine markedly suppressed VEGF-induced endothelial cell migration, sprouting, and survival in vitro in a dose-dependent manner at nanomolar concentrations. Furthermore, sanguinarine potently suppressed blood vessel formation in vivo in mouse Matrigel plugs and the chorioallantoic membrane of chick embryos. Our biochemical assays indicated that sanguinarine strongly suppressed basal and VEGF-induced Akt phosphorylation, while it did not produce any changes in VEGF-induced activation of ERK1/2 and PLCγ1. Therefore, we conclude that sanguinarine is a potent antiangiogenic natural product, and its mode of action could involve the blocking of VEGF-induced Akt activation. Thus, in addition to antibacterial, antifungal, and anti-inflammatory activities, sanguinarine has a novel antiangiogenic role. [Copyright &y& Elsevier]

Despite the advent of oral phosphodiesterase-5 inhibitors, curative treatment for erectile dysfunction (ED) remains unavailable. Recently, the link between ED and cardiovascular disease was unveiled and the main etiology of ED was found to be vasculogenic. Therefore, neovascularization is a promising strategy for curing ED. Angiopoietin-1 (Ang1) is an angiogenic growth factor that promotes the generation of stable and functional vasculature. Here, we demonstrate that local delivery of the soluble, stable, and potent Ang1 variant, COMP-Ang1 gene or protein, into the penises of hypercholesterolemic mice increases cavernous angiogenesis, eNOS phosphorylation, and cGMP expression, resulting in full recovery of erectile function and cavernous blood flow up to 8 weeks after treatment. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished in Nos3-/- mice and in the presence of the NOS inhibitor, L-NAME. COMP-Ang1 also restored the integrity of endothelial cell-cell junction by down-regulating the expression of histone deacetylase 2 in the penis of hypercholesterolemic mice and in primary cultured mouse cavernous endothelial cells. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy and ED. [ABSTRACT FROM AUTHOR]

Controlled angiogenesis and lymphangiogenesis are essential for tissue development, function, and repair. However, aberrant neovascularization is an essential pathogenic mechanism in many human diseases, including diseases involving tumor growth and survival. Here, we have demonstrated that mice deficient in C-type lectin family 14 member A (CLEC14A) display enhanced angiogenic sprouting and hemorrhage as well as enlarged jugular lymph sacs and lymphatic vessels. CLEC14A formed a complex with VEGFR-3 in endothelial cells (ECs), and CLEC14A KO resulted in a marked reduction in VEGFR-3 that was concomitant with increases in VEGFR-2 expression and downstream signaling. Implanted tumor growth was profoundly reduced in CLEC14A-KO mice compared with that seen in WT littermates, but tumor-bearing CLEC14A-KO mice died sooner. Tumors in CLEC14A-KO mice had increased numbers of nonfunctional blood vessels and severe hemorrhaging. Blockade of VEGFR-2 signaling suppressed these vascular abnormalities and enhanced the survival of tumor-bearing CLEC14A-KO mice. We conclude that CLEC14A acts in vascular homeostasis by fine-tuning VEGFR-2 and VEGFR-3 signaling in ECs, suggesting its relevance in the pathogenesis of angiogenesis-related human disorders. [ABSTRACT FROM AUTHOR]

Introduction Erectile dysfunction ( ED) is a major complication of radical prostatectomy. Men with radical prostatectomy-induced ED respond less positively to oral phosphodiesterase-5 inhibitors. Aim The study aims to examine whether and how stromal vascular fraction ( SVF) restores erectile function in mice with cavernous nerve injury ( CNI). Methods Twelve-week-old male C57BL/6J mice were used and the animals were distributed into five groups: sham operation group and CNI group receiving a single intracavernous injection of phosphate-buffered saline ( PBS) or SVF (1 × 104, 1 × 105, or 3 × 105 cells/20 μL, respectively). SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. Main Outcome Measures Two weeks after injection, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to platelet/endothelial cell adhesion molecule-1, phosphohistone H3, and phosphorylated endothelial nitric oxide synthase (phospho- eNOS). We also performed Western blot for angiopoietin-1 ( Ang-1), vascular endothelial growth factor- A, hepatocyte growth factor, phospho- eNOS, and eNOS in the corpus cavernosum tissue. Results Local delivery of SVF restored erectile function in a dose-dependent manner in CNI mice. The highest erectile response was noted at a dose of 3 × 105 cells, for which the response was comparable with that in the sham operation group. Local delivery of SVF significantly increased the expression of angiogenic factor proteins and induced cavernous endothelial cell proliferation and eNOS phosphorylation compared with that in the PBS-treated CNI group. SVF-induced promotion of cavernous angiogenesis and erectile function was diminished in the presence of soluble antibody to Tie2, a receptor tyrosine kinase of Ang-1. Conclusion Secretion of angiogenic factors from SVF is an important mechanism by which SVF induces cavernous endothelial regeneration and restores erectile function. These findings suggest that cavernous endothelial regeneration by using SVF may represent a promising treatment strategy for radical prostatectomy-induced ED. Song K-M, Jin H-R, Park J-M, Choi MJ, Kwon M-H, Kwon K-D, Batbold D, Yin GN, Kim WJ, Koh GY, Ryu J-K, and Suh J-K. Intracavernous delivery of stromal vascular fraction restores erectile function through production of angiogenic factors in a mouse model of cavernous nerve injury. J Sex Med 2014;11:1962-1973. [ABSTRACT FROM AUTHOR]

Summary: Current antiangiogenic therapy is limited by its cytostatic nature and systemic side effects. To address these limitations, we have unveiled the role of RhoJ, an endothelial-enriched Rho GTPase, during tumor progression. RhoJ blockade provides a double assault on tumor vessels by both inhibiting tumor angiogenesis and disrupting the preformed tumor vessels through the activation of the RhoA-ROCK (Rho kinase) signaling pathway in tumor endothelial cells, consequently resulting in a functional failure of tumor vasculatures. Moreover, enhanced anticancer effects were observed when RhoJ blockade was employed in concert with a cytotoxic chemotherapeutic agent, angiogenesis-inhibiting agent, or vascular-disrupting agent. These results identify RhoJ blockade as a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects. [Copyright &y& Elsevier]

Background--There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/ VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. Methods and Results--The VEGF-angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. Conclusions--The VEGF-angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool. [ABSTRACT FROM AUTHOR]

Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)-derived Flk1+ mesoder-mal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyz-ing VEGF-AA/EGFR2 downstream signal-ing pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-assoclated protein kinase (ROCK) inhibi-tor Y27632 profoundly promoted the dif-ferentiation and expansion of ECs from Flk1+ MPCs while reducing the differentia-tion and expansion of mural cells. The ROCK suppression-Induced expansion of ECs appears to have resulted from promo-tion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs ob-tained by the combination of ROCK sup-pression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotyplc analyses re-vealed that they were differentiated to-ward arterial EC. Further incubation of the ICAM2+ ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-Induced ECs displayed neo-vasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, effi-cient, and versatile method for obtaining ample amount of ESC-derlved ECs at high purity suitable for use in therapeutic neovascularization. (Blood. 2012;120(13): 2733-2744). [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Angiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation. Methods: Expression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and β-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody. Results: Expression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity. Conclusions: These results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases. [Copyright &y& Elsevier]

Abstract: Despite that angiopoietin-2 (Ang2) produces more versatile and dynamic functions than angiopoietin-1 (Ang1) in angiogenesis and inflammation, the molecular mechanism that underlies this difference is still unknown. To define the role of oligomerization of Ang2 in activation of its receptor, Tie2, we designed and generated different oligomeric forms of Ang2 by replacement of the amino-terminal domains of Ang2 with dimeric, tetrameric, and pentameric short coiled-coil domains derived from GCN4, matrillin-1, and COMP. COMP-Ang2 strongly binds and activates Tie2, whereas GCN4-Ang2 and MAT-Ang2 weakly to moderately bind and activate Tie2. Although native Ang2 strongly binds to Tie2, it does not activate Tie2. Accordingly, COMP-Ang2 strongly promotes endothelial cell survival, migration, and tube formation in a Tie2-dependent manner, and the potency of COMP-Ang2 is almost identical to that of COMP-Ang1. Furthermore, the potency of COMP-Ang2-induced enhanced angiogenesis in the wound healing region is almost identical to the potency of COMP-Ang1-induced enhanced angiogenesis. Overall, there is no obvious difference between COMP-Ang2 and COMP-Ang1 in in vitro and in vivo angiogenesis. Our results provide compelling evidence that proper oligomerization of Ang2 is a critical determinant of its binding and activation of Tie2. [Copyright &y& Elsevier]

Abstract: Introduction: Ischemic necrosis of the femoral head (INFH) can lead to loss of femoral head architecture and deformity. Moreover, the process of bone healing is intimately associated with angiogenesis. We considered that COMP-Ang1 (an angiogenic factor) might preserve femoral head structure and facilitate bone repair. Methods: INFH was induced in the femoral head of rats by dissecting the cervical periosteum and placing a ligature tightly around the femoral neck. Two weeks later, COMP-Ang1 was injected directly into infarcted areas. Rats were divided into the following groups; 1) the sham-operated group (the sham group), 2) the bovine serum albumin-injected group (the BSA group), and 3) the COMP-Ang1-injected group (the COMP-Ang1 group) (n =20/group). At 8 weeks post-surgery animals were sacrificed and radiologic and histomorphometric assessments were performed. Results: Radiographs obtained at 8 weeks post-surgery showed better preservation of femoral head architecture in the COMP-Ang1 group than in the BSA group. Histological findings and immunostainings of endothelial cells for factor VIII revealed that COMP-Ang1 group animals showed higher levels of vascularity in the secondary ossification center of infarcted femoral heads. Conclusions: When INFH was surgically induced in rats, an intraosseous injection of COMP-Ang1 preserved the trabecular framework of the osseous epiphysis and prevented femoral head deformities by promoting angiogenesis and bone remodeling. [Copyright &y& Elsevier]

Abstract: Angiopoietin (Ang) signaling through the Tie2 receptor regulates vasculature. The role of Ang signaling in pulmonary hypertension is well investigated, but its role in lung development is not elucidated. Here, we show that the Tie2 agonist ligand, Ang1, was detected in lung tissue at birth and its expression gradually increased in mice, whereas its antagonist Ang2 was abundant at birth and decreased inversely with Ang1. Mice expressing the potent chimeric Ang1 protein COMP-Ang1 in surfactant protein C (SPC)-positive lung epithelial cells, showed 50% lethality at birth due to respiratory failure. Surviving mice displayed impaired adaptive responsive respiratory function. Histological analysis revealed that pulmonary artery and alveolar structure were significantly dilated, and alveolar density was decreased to approximately a third of controls. Thus, the precise regulation of Tie2 signaling through an Ang1/Ang2 expression switch is important to construct a mature lung vascular network system required for normal lung development. [Copyright &y& Elsevier]

Angiopoietin-1 (Angi) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Angi and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Kruppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Angl/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Angl/Tie2 signal-mediated vascular quiescence. Angi stimulated KLF2 promoter- driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Angi-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Angi. Constitutive active phosphoinositide 3-kinase (P13K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either P13K or AKT and depletion of AKT abrogated Angi-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Angi-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Angi on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEG F-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Angl/Tie2 signal stimulates transcriptional activity of MEF2 through a P13K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses. [ABSTRACT FROM AUTHOR]

Angiopoietin-1 (Ang1) and its receptor, Tie2, play critical roles in blood vessel formation. Ang1 triggers a variety of signaling events in endothelial cells leading to vasculogenic and angiogenic processes. However, the underlying mechanism for Ang1/Tie2 signaling is not fully understood. Here, we show that Tie2 and phospholipase D (PLD) are localized in the caveolae, specialized subdomains of the endothelial cell plasma membrane enriched with signaling molecules. Interestingly, Ang1 increased PLD activities in a dose- and time-dependent manner. Ang1-induced MEK/ERK activation was abrogated when PLD was inhibited, suggesting that PLD mediates Ang1-induced MEK/ERK activation. Moreover, PLD inhibitor, 1-butanol, inhibited Ang1-induced endothelial cell migration. Our results indicate that: (1) caveolae may be the platform for Tie2/PLD association in endothelial cells; (2) PLD is a new mediator of Ang1/Tie2-induced signaling pathway, and it participates in MAPK activation and endothelial cell migration. [Copyright &y& Elsevier]

Angiopoietins have been increasingly implicated to play important roles in blood vessel formation, remodeling, maturation, and maintenance. However, their roles in tumor angiogenesis and hence tumor growth and metastasis still remain uncertain. In this work, angiopoietin 1 expression was amplified in human cervical cancer HeLa cells by stable transfection or recombinant human adenovirus-mediated gene transfer. We show that increased angiopoietin 1 expression promoted in vivo growth of human cervical cancers in mice by promoting tumor angiogenesis and inhibiting tumor cell apoptosis. Furthermore, we also show for the first time that overexpression of angiopoietin 1 also leads to increased tumor vessel plasticity with a large number of vessels lacking periendothelial supporting cells. These results indicate that angiopoietin 1 promotes tumor angiogenesis and tumor vessel plasticity of human cervical cancer in mice. [Copyright &y& Elsevier]