VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l -cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS–ER stress-autophagy axis in the vascular endothelial cells.     

VEGF-induced activation of phosphoinositide 3-kinase is dependent on focal adhesion kinase

Vascular endothelial growth factor (VEGF)-A stimulates formation of new blood vessels (angiogenesis). This process includes migration of endothelial cells from the preexisting vessel toward the source of the growth factor. We show that VEGF-A-induced migration of porcine aortic endothelial cells expressing VEGF receptor-2 (VEGFR-2) is dependent on activation of phosphoinositide 3-kinase (PI3-kinase). There is no direct interaction between VEGF receptor-2 and PI3-kinase; instead PI3-kinase is activated downstream of focal adhesion kinase (FAK) in VEGF-A-stimulated cells. Thus, VEGF-A stimulation leads to complex formation between FAK and PI3-kinase and overexpression of dominant-negative FAK decreases VEGF-A-induced PI3-kinase activation. FAK activation by VEGF-A increases with increasing concentration of growth factor, without apparent collapse of the cytoskeleton, in contrast to the effect of platelet-derived growth factor. FAK activation is mediated via the C-terminal tail of VEGFR-2 and loss of VEGF-A-induced FAK activation in cells expressing mutant VEGFR-2 correlates with loss of migration capacity. These data show that VEGF-A-induced FAK and PI3-kinase activation are required for migration of cells expressing VEGFR-2, via a pathway independent of direct interaction with the receptor.     

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.     

Role of Src in angiopoietin 1-induced capillary morphogenesis of endothelial cells: Effect of chronic hypoxia on Src inhibition by PP2

Signal transduction pathways leading to angiopoietin 1 (Ang1)-induced capillary morphogenesis by endothelial cells remain poorly defined. Angiogenic cellular responses by endothelial cells may be modulated in vivo by chronic hypoxia, such as that induced by tumors. Here, we studied Ang1-induced capillary morphogenesis in human umbilical-vein endothelial cells (HUVECs) cultured chronically under normoxic (21% oxygen) or hypoxic (1.5% oxygen) conditions. Downregulation of Src using a small interfering RNA (siRNA) inhibited Ang1-induced capillary morphogenesis of HUVECs cultured under both conditions by blocking cell spreading and protrusion. Ang1 upregulated the Src-dependent secretion of vascular endothelial growth factor-A (VEGF-A). Blockade of endogenous VEGF-A also inhibited Ang1-induced capillary morphogenesis. Addition of exogenous VEGF-A restored cell spreading and protrusion, leading to Ang1-induced capillary morphogenesis of Src siRNA-treated HUVECs, suggesting that Ang1-induced VEGF-A secretion through Src was required for capillary morphogenesis. PP2 inhibited both Ang1-induced capillary morphogenesis and Src activation in HUVECs cultured under normoxic conditions, but the PP2 activity was significantly impaired in HUVECs cultured under hypoxic conditions. Expression of multidrug resistance-associated protein 1 (MRP 1) was upregulated in hypoxic HUVECs, and treatment with MRP 1 siRNA restored the inhibitory action of PP2. Taken together, our results suggest that Ang1 induces capillary morphogenesis in HUVECs through Src-dependent upregulation of endogenous VEGF-A. Conditions of chronic hypoxia impaired the effect of PP2, possibly via MRP 1.     

Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells.

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.     

A Heat-Shock Protein Axis Regulates VEGFR2 Proteolysis,Blood Vessel Development and Repair

Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration in vitro. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair in vivo consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.     

Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.     

Pro-angiogenic properties of orosomucoid (ORM)

The acute phase protein orosomucoid (ORM), also known as alpha1-acid glycoprotein (AGP), is found to be increased in infection, inflammation and cancer. Recently, we demonstrated that ORM is produced by endothelial cells and detectable in urine samples of patients with bladder cancer. However, it was not clarified yet whether ORM plays a role in new vessel formation. To this aim we performed overexpression and gene silencing for ORM in human microvascular endothelial cells (HDMECs). ORM purified from human plasma was used individually or in combination with VEGF-A in endothelial tube formation, migration and proliferation assay. The in vivo effect of ORM in angiogenesis was studied using the chicken chorionallantois membrane (CAM) with subsequent counting of blood vessels on histological sections from the stimulated areas of CAM tissue. Our data show that ORM alone enhances migration but not proliferation of HDMECs. ORM alone does not induce endothelial tubes in vitro but simultaneous application of ORM with VEGF-A increases the number and the network of VEGF-A-induced endothelial tubes. Remarkably, ORM alone induces new vessel formation in vivo using CAM assay and supports the VEGF-A-induced new vessel formation in this assay. Taken together, our results let assume that ORM has pro-angiogenic properties and supports the angiogenic effect of VEGF-A. Thus, ORM seems to be involved in the regulation of angiogenesis.     

Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells

The role of hypoxia on endothelial cell function and response to growth factors is unknown. Here, we tested the hypothesis that hypoxia re-programs endothelial function by modulating vascular endothelial growth factor receptor levels which in turn alter intracellular signaling and cell function. Hypoxia stimulated VEGF-A and VEGFR1 expression but decreased VEGFR2 levels in endothelial cells. During hypoxia, plasma membrane VEGFR1 levels were elevated whereas VEGFR2 levels were depleted. One functional consequence of hypoxia is a reduction in VEGF-A-stimulated and VEGFR2-regulated intracellular signaling including lowered endothelial nitric oxide synthase activation. Venous, arterial and capillary endothelial cells subjected to hypoxia all exhibited reduced cell migration in response to VEGF-A. A mechanistic explanation is that VEGFR1:VEGFR2 ratio is substantially increased during hypoxia to block VEGF-A-stimulated and VEGFR2-regulated endothelial responses to maximize cell viability and recovery.     

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.     

Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers

Vascular endothelial growth factor (VEGF-A) exerts its effects through receptor tyrosine kinases VEGF receptor-1 (VEGFR-1) and VEGFR-2, which are expressed on most endothelial cell types in vitro and in vivo. We have examined VEGF-A-induced signal transduction in porcine aortic endothelial (PAE) cells individually expressing VEGFR-1 or VEGFR-2, and cells co-expressing both receptor types. We show that VEGF-A-stimulated PAE cells co-expressing VEGFR-1 and -2 contain receptor heterodimers. VEGF-A-stimulation of all three cell lines (expressing VEGFR-1, -2 and -1/2) resulted in signal transduction with different efficiencies. Thus, tyrosine phosphorylation of phospholipase Cgamma, and accumulation of inositol polyphosphates were efficiently transduced in the VEGFR-1/2 cells whereas cells expressing VEGFR-1 responded poorly in these assays. In contrast, VEGF-A-induced activation of phosphoinositide 3-kinase and induction of Ca2+ fluxes were transduced well by VEGFR-1 and VEGFR-2 homo- and heterodimers. The pattern of Ca2+ fluxes was unique for each type of VEGF receptor dimer. Our data show that signal transduction induced by VEGF-A is transduced in distinct manners by homo- and heterodimers of VEGF receptors.     

Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells

Integrin alpha x (ITGAX), a member of the integrin family, usually serves as a receptor of the extracellular matrix. Recently, accumulating evidence suggests that ITGAX may be involved in angiogenesis in dendritic cells. Herein, we report a direct role of ITGAX in angiogenesis during tumor development. Overexpression of ITGAX in human umbilical vein endothelial cells (HUVECs) enhanced their proliferation, migration, and tube formation and promoted xenograft ovarian tumor angiogenesis and growth. Further study showed that overexpression of ITGAX activated the PI3k/Akt pathway, leading to the enhanced expression of c-Myc, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor 2 (VEGFR2), whereas, the treatment of cells with PI3K inhibitor diminished these effects. Besides, c-Myc was observed to bind to the VEGF-A promoter. By Co-Immunoprecipitation (Co-IP) assay, we manifested the interaction between ITGAX and VEGFR2 or the phosphorylated VEGFR2. Immunostaining of human ovarian cancer specimens suggested that endothelial cells of micro–blood vessels displayed strong expression of VEGF-A, c-Myc, VEGFR2, and the PI3K signaling molecules. Also, overexpression of ITGAX in HUVECs could stimulate the spheroid formation of ovarian cancer cells. Our study uncovered that ITGAX stimulates angiogenesis through the PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression during cancer development.     

Regulation of endothelial glutathione by ICAM-1 governs VEGF-A-mediated eNOS activity and angiogenesis

Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.     

Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.     

Proteolytic Cleavage of Versican and Involvement of ADAMTS-1 in VEGF-A/VPF-Induced Pathological Angiogenesis

Malignant tumors and chronic inflammatory diseases induce angiogenesis by overexpressing vascular endothelial growth factor A (VEGF-A/VPF). VEGF-A-induced pathological angiogenesis can be mimicked in immunoincompetent mice with an adenoviral vector expressing VEGF-A¹⁶⁴ (Ad-VEGF-A¹⁶⁴). The initial step is generation of greatly enlarged “mother” vessels (MV) from preexisting normal venules by a process involving degradation of their rigid basement membranes. Immunohistochemical and Western blot analyses revealed that versican, an extracellular matrix component in the basement membranes of venules, is degraded early in the course of MV formation, resulting in the appearance of a versican N-terminal DPEAAE fragment associated with MV endothelial cells. The protease ADAMTS-1, known to cleave versican near its N terminus to generate DPEAAE, is also upregulated by VEGF-A in parallel with MV formation and localizes to the endothelium of the developing MV. The authors also show that MMP-15 (MT-2 MMP), a protease that activates ADAMTS-1, is upregulated by VEGF-A in endothelial cells in vitro and in vivo. These data suggest VEGF-A initiates MV formation, in part, by inducing the expression of endothelial cell proteases such as ADAMTS-1 and MMP-15 that act in concert to degrade venular basement membrane versican. Thus, versican is actively processed during the early course of VEGF-A-induced pathological angiogenesis.     

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.     

Fucoidan extract derived from Undaria pinnatifida inhibits angiogenesis by human umbilical vein endothelial cells

In recent years, anti-angiogenic therapy has become an effective strategy for inhibiting tumor growth. Fucoidan is a class of fucose-enriched sulfated polysaccharides found in brown algae, and it is known to have strong anti-tumor property. Using a human umbilical vein endothelial cells (HUVEC)-based cell culture model, the present study investigated the anti-angiogenic activity of fucoidan extracted from the brown seaweed Undaria pinnatifida. Treatment of HUVECs with various concentrations of fucoidan resulted in significant inhibition of cell proliferation, cell migration, tube formation and vascular network formation. However, significant inhibition of cell proliferation only occurred with longer treatment time (48 h instead of 24h or less). About 40% of cell proliferation and cell migration and 61% of tube formation by HUVECs were inhibited by 400 μg/ml fucoidan, the maximum concentration tested. These results appeared to suggest that modulation of angiogenesis by fucoidan might not occur through growth inhibition and apoptosis. Ex vivo angiogenesis assay demonstrated that at 100 μg/ml, fucoidan caused significant reduction in microvessel outgrowth. Western blot and RT-PCR analyses indicated that at 400 μg/ml, fucoidan significantly reduced the expression of the angiogenesis factor VEGF-A in the suppression of angiogenesis activity. Our results showed that fucoidan isolated from U. pinnatifida may have a new therapeutic potential in the prevention angiogenesis-related diseases.     

Phosphorylation of Akt and ERK1/2 is required for VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.     

SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.