Negative Regulator Of Angiogenesis Research Articles

AZGP1 deficiency promotes angiogenesis in prostate cancer

BackgroundLoss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive.MethodAZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples.ResultNeither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1−/− mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues.ConclusionAZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

DLL4/Notch blockade disrupts mandibular advancement-induced condylar osteogenesis by inhibiting H-type angiogenesis.

Blocking Delta-like 4 (DLL4)/Notch has emerged as a promising therapeutic target for the treatment of tumours by deregulating angiogenesis. However, DLL4/Notch serves as a negative regulator of angiogenesis in multiple organs while acting as a positive regulator of H-type angiogenesis in postnatal long bones. Therefore, the effect of DLL4/Notch signalling blockade on mandibular condylar osteogenesis attracted our attention. To explore the effect of blocking DLL4/Notch on mandibular advancement (MA)-induced condylar osteogenesis. Six-week-old young male C57BL/6J mice (n = 40) were randomly divided into four groups: control group, MA group, MA + Anti-DLL4 group and MA + IgG group. Of note, IgG served as the isotype control for the anti-DLL4. The femurs, tibias and mandibular condyles were collected after sacrificing mice on Day 31 for morphology, micro-computed tomography, immunofluorescence, histology and immunohistochemistry evaluation. First, DLL4/Notch blockade shortened femoral length and reduced bone mass by inhibiting H-type angiogenesis. Second, DLL4/Notch blockade disrupted MA-induced condylar head volume and quality by inhibiting H-type angiogenesis. Mechanistically, blocking DLL4/Notch reduced the number of runt-related transcription factor 2+ (RUNX2+ ) early osteoprogenitors and the expression of Noggin protein in the condylar subchondral bone by inhibiting H-type angiogenesis. In addition, blockade of DLL4/Notch also destroyed the condylar cartilage layer. DLL4/Notch blockade results in shortened femurs and osteopenia, as well as impaired MA-induced condylar osteogenic volume and quality in growing mice by inhibiting H-type angiogenesis. Therefore, when blocking DLL4/Notch is used as a treatment target for diseases, attention should be paid to its impact on the bone mass of mandibular condyle.

CXCR4 blockade in macrophage promotes angiogenesis in ischemic hindlimb by modulating autophagy

Chemokine receptor CXCR4 plays a crucial role in leukocyte recruitment and inflammation regulation to influence tissue repair in ischemic diseases. Here we assessed the effect of CXCR4 expression in macrophages on angiogenesis in the ischemic hindlimb of a mouse. Inflammatory cells were increased in the ischemic muscles of hindlimb, and CXCR4 was highly expressed in the infiltrated macrophages but not in neutrophils. Myeloid-specific CXCR4 knockout attenuated macrophage infiltration and subsequent reduced inflammatory response in the ischemic hindlimb, accompanied with better blood reperfusion and higher capillary density as compared with that in LysM Cre+/− (Cre) mice. Similar outcomes were also observed in CRE mice whose bone marrow cells were replaced with those from CXCR4-deficient mice. Gene ontology cluster analysis reviewed that Decorin, a negative regulator of angiogenesis, was reduced in CXCR4-deficient macrophages. CXCR4-deficient macrophages were less inducible into M1 phase by lipopolysaccharide and more favorable for M2 polarization under oxygen/glucose deprivation condition. Enhanced autophagy was detected in CXCR4-deficient macrophages, which was associated with less expression of both Decorin and the inflammatory cytokines. In summary, myeloid-specific CXCR4 deficiency reduced monocyte infiltration and the secretion of inflammatory cytokines and Decorin from macrophages, thus blunting inflammation response and promoting angiogenesis in the ischemic hindlimb.

Implication of satellite cell behaviors in capillary growth via VEGF expression-independent mechanism in response to mechanical loading in HeyL-null mice.

Angiogenesis and muscle satellite cell (SC)-mediated myonuclear accretion are considered essential for the robust response of contraction-induced muscle hypertrophy. Moreover, both myonucleus and SCs are physically adjacent to capillaries and are the major sites for the expression of proangiogenic factors, such as VEGF, in the skeletal muscle. Thus, events involving the addition of new myonuclei via activation of SCs may play an important role in angiogenesis during muscle hypertrophy. However, the relevance among myonuclei number, capillary supply, and angiogenesis factor is not demonstrated. The Notch effector HeyL is specifically expressed in SCs in the skeletal muscle and is crucial for SC proliferation by inhibiting MyoD in overload-induced muscle hypertrophy. Here, we tested whether the addition of new myonuclei by SC in overloaded muscle is associated with angiogenic adaptation by reanalyzing skeletal muscle from HeyL-knockout (KO) mice, which show blunted responses of SC proliferation, myonucleus addition, and overload-induced muscle hypertrophy. Reanalysis confirmed blunted SC proliferation and myonuclear accretion in the plantaris muscle of HeyL-KO mice 9 wk after synergist ablation. Interestingly, the increase in capillary-to-fiber ratio observed in wild-type (WT) mice was impaired in HeyL-KO mice. In both WT and HeyL-KO mice, the expression of VEGFA and VEGFB was similarly increased in response to overload. In addition, the expression pattern of TSP-1, a negative regulator of angiogenesis, was also not changed between WT and HeyL-KO mice. Collectively, these results suggest that SCs activation-myonuclear accretion plays a crucial role in angiogenesis during overload-induced muscle hypertrophy via independent of angiogenesis regulators.

Glioma-derived extracellular vesicles promote tumor progression by conveying WT1.

Glioma persists as one of the most aggressive primary tumors of the central nervous system. Glioma cells are known to communicate with tumor-associated macrophages/microglia via various cytokines to establish the tumor microenvironment. However, how extracellular vesicles (EVs), emerging regulators of cell-cell communication networks, function in this process is still elusive. We report here that glioma-derived EVs promote tumor progression by affecting microglial gene expression in an intracranial implantation glioma model mouse. The gene expression of thrombospondin-1 (Thbs1), a negative regulator of angiogenesis, was commonly downregulated in microglia after the addition of EVs isolated from different glioma cell lines, which endogenously expressed Wilms tumor-1 (WT1). Conversely, WT1-deficiency in the glioma-derived EVs significantly attenuated the Thbs1 downregulation and suppressed the tumor progression. WT1 was highly expressed in EVs obtained from the cerebrospinal fluid of human patients with malignant glioma. Our findings establish a novel model of tumor progression via EV-mediated WT1-Thbs1 intercellular regulatory pathway, which may be a future diagnostic or therapeutic target.

The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

ABSTRACT Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvβ6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvβ6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 – HMEC1); we demonstrate that de novo αvβ6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvβ6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from β6 negative PC3 cells, generated by shRNA against β6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of β6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvβ6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.

MicroRNA-210-3p Targets RGMA to Enhance the Angiogenic Functions of Endothelial Progenitor Cells Under Hypoxic Conditions.

Endothelial progenitor cells (EPCs) are multipotential stem cells considered to have immense clinical value for revascularization. However, the clinical application of EPCs has been hampered by their clinical potency in ischemic anoxic environments. This study aimed to explore the effect of microRNA-210 (miR-210) on EPCs under oxygen-glucose deprivation (OGD) conditions. We generated a model of EPCs cultured under OGD conditions to simulate ischemia and explore the expression of miR-210 in vitro. With longer exposure to hypoxia, we found that miR-210-3p expression was highly upregulated in OGD groups compared to that in controls from 4 to 24 h, but not miR-210-5p. We then transfected a miR-210-3p mimic and inhibitor into EPCs, and after 24 h, we exposed them to OGD conditions for 4 h to simulate ischemia. We detected miR-210 by real-time polymerase chain reaction (RT-PCR) and tested the proliferation, migration, and tube formation of normal EPCs and OGD-treated EPCs by CCK-8, transwell chamber, and Matrigel assays, respectively. The direct targets of miR-210-3p were predicted using miRWalk. Compared to that in normal EPCs, higher miR-210-3p expression was found in OGD-treated EPCs (p < 0.05). Moreover, upregulation of miR-210-3p was found to promote proliferation, migration, and tube formation in EPCs under normal and OGD conditions (p < 0.05), whereas down-regulation inhibited these abilities in OGD-treated EPCs (p < 0.05). Repulsive guidance molecule A (RGMA), a negative regulator of angiogenesis, was predicted to be a target of miR-210-3p. Accordingly, upregulation of miR-210-3p was found to inhibit its expression at the protein level in OGD-treated EPCs, whereas downregulation of miR-210-3p inhibited its expression (p < 0.05). A dual-luciferase reporter system confirmed that RGMA is a direct target of miR-210-3p. MicroRNA-210-3p overexpression enhances the angiogenic properties of OGD-treated EPCs by inhibiting RGMA.

The Expanding Role of MT1-MMP in Cancer Progression.

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array of MT1-MMP substrates, and MT1-MMP selective inhibitors have allowed for a more complete mapping of MT1-MMP biological functions. MT1-MMP has extensive sheddase activities, is both a positive and negative regulator of angiogenesis, can act intracellularly and as a transcription factor, and modulates immune responses. We presently examine the multi-faceted role of MT1-MMP in cancer, with a consideration of how the diversity of MT1-MMP behaviors impacts the application of MT1-MMP inhibitors.

Mineralocorticoid receptor negatively regulates angiogenesis through repression of STAT3 activity in endothelial cells.

The mineralocorticoid receptor (MR) plays important roles in cardiovascular pathogenesis. The function of MR in angiogenesis is still controversial. This study aimed to explore the role of endothelial MR in angiogenesis and to delineate the underlying mechanism. Endothelial-hematopoietic MR knockout (EMRKO) mice were generated and subjected to hindlimb ischemia and injection of melanoma cells. Laser Doppler measurements showed that EMRKO mice had improved blood flow recovery and increased vessel density in ischemic limbs. In addition, EMRKO accelerated growth and increased the vessel density of tumors. Matrigel implantation, aortic ring assays, and tube formation assays demonstrated that MRKO endothelial cells (ECs) manifested increased angiogenic potential. MRKO ECs also displayed increased migration ability and proliferation. MRKO and MR knockdown both upregulated gene expression, protein level, and phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stattic, a selective STAT3 inhibitor, attenuated the effects of MRKO on tube formation, migration, and proliferation of ECs. At the molecular level, MR interacted with CCAAT enhancer-binding protein beta (C/EBPβ) to suppress the transcription of STAT3. Furthermore, interactions between MR and STAT3 blocked the phosphorylation of STAT3. Finally, stattic abolished the pro-angiogenic phenotype of EMRKO mice. Taken together, endothelial MR is a negative regulator of angiogenesis, likely in a ligand-independent manner. Mechanistically, MR downregulates STAT3 that mediates the impacts of MR deficiency on the angiogenic activity of ECs and angiogenesis. Targeting endothelial MR may be a potential pro-angiogenic strategy for ischemic diseases. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inhibition of AZIN2-sv induces neovascularization and improves prognosis after myocardial infarction by blocking ubiquitin-dependent talin1 degradation and activating the Akt pathway.

BackgroundWe previously found that loss of lncRNA-AZIN2 splice variant (AZIN2-sv) increases cardiomyocyte (CM) proliferation and attenuates adverse ventricular remodelling post-myocardial infarction (MI). However, whether inhibition of AZIN2-sv can simultaneously induce angiogenesis and thus improve prognosis after MI is unclear.MethodsWe used in situ hybridization and quantitative PCR to determine AZIN2-sv expression in endothelial cells. Knockdown and overexpression were performed to detect the role of AZIN2-sv in endothelial cell function, angiogenesis and prognosis after MI. RNA pulldown, RNA immunoprecipitation and luciferase reporter assays were used to determine the interaction with talin1 (Tln1) protein and miRNA-214 (miR-214). DNA pulldown and chromatin immunoprecipitation (ChIP) assays were used to study AZIN2-sv binding to upstream transcription factors.FindingsAZIN2-sv was enriched in cardiac endothelial cells. The loss of AZIN2-sv reduced endothelial cell apoptosis and promoted endothelial sprouting and capillary network formation in vitro. Moreover, in vivo, the loss of AZIN2-sv induced angiogenesis and improved cardiac function after MI. Mechanistically, AZIN2-sv reduced Tln1 and integrin β1 (ITGB1) protein levels to inhibit neovascularization. AZIN2-sv activated the ubiquitination-dependent degradation of Tln1 mediated by proteasome 26S subunit ATPase 5 (PSMC5). In addition, AZIN2-sv could bind to miR-214 and suppress the phosphatase and tensin homologue (PTEN)/Akt pathway to inhibit angiogenesis. With regard to the upstream mechanism, Bach1, a negative regulator of angiogenesis, bound to the promoter of AZIN2-sv and increased its expression.InterpretationBach1-activated AZIN2-sv could participate in angiogenesis by promoting the PSMC5-mediated ubiquitination-dependent degradation of Tln1 and blocking the miR-214/PTEN/Akt pathway. Inhibition of AZIN2-sv induced angiogenesis and myocardial regeneration simultaneously, thus, AZIN2-sv could be an ideal therapeutic target for improving myocardial repair after MI.FundNational Natural Science Foundations of China.

Abstract 4699: Identification of novel potential biomarkers of response to sunitinib in glioblastoma

Abstract Introduction: Glioblastoma multiforme (GBM) is the most advanced and aggressive form of primary malignant brain tumors in adults. O6-Methylguanine methyltransferase (MGMT) is a DNA repair protein well known for its role in resistance to temozolomide used in standard treatment of patients newly diagnosed with GBM. Sunitinib is an oral multitargeting receptor tyrosine kinase (RTK) inhibitor with antiangiogenic and antiproliferative activities targeting several RTKs. Our group has previously shown the role of MGMT as a negative regulator of angiogenesis and invasion and the differential antiproliferative effect of sunitinib based on expression of MGMT in GBM. Recent gene expression profiling (GEP) studies showed that besides its effects on RTKs, sunitinib affects the expression level of other genes at the transcriptional level in different cancer types. We hypothesized that: (i) the mechanism of action of sunitinib may not be limited to direct inhibition of its known targets (RTKs) in GBM and (ii) expression of MGMT may affect response of GBM tumor cells to sunitinib. We performed GEP analysis to identify genes that could be potentially modulated in response to sunitinib in GBM cell lines isogenic for MGMT. Empty vector (EV)-transfected T98/EV cells with constitutive expression of MGMT and its knockdown counterpart T98/MGMT-shRNA were treated with sunitinib or vehicle control. Treatment of T98/EV and T98/MGMT-shRNA cell lines with sunitinib significantly changed the expression of over 2,000 gene transcripts (analysis performed at Genome Quebec Innovation center, McGill University). Lists of significant genes were fitted into previously known biochemical pathways, available among the canonical pathways from various databases provided by ConsensusPathDB (CPDB). Strikingly, immune system was among the top significantly enriched pathways (p-value&amp;lt;10-3), with some immune pathways not previously reported for response to sunitinib. Conclusion: Our study opens new avenues to further understand the mechanisms of action of sunitinib, investigate its immunomodulatory effects, and identify biomarkers for selection of patients who might benefit from the combination of antiangiogenic and immunebased strategies in GBM. Funding Acknowledgment: Cancer Research Society Operating Grant Competition #22716, and Higher Education Quality Enhancement Project (HEQEP)- Window -4 (Grant Id. CP-4023), University Grant Commission (UGC), Bangladesh. Citation Format: Abu Shadat Mohammod Noman, Bassam Abdulkarim, Siham Sabri. Identification of novel potential biomarkers of response to sunitinib in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4699.

Abnormal angiogenesis in blood outgrowth endothelial cells derived from von Willebrand disease patients.

: Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.

Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro.

Repulsive guidance molecule a (RGMa) is a major neuron guidance factor in central nervous systems. We previously found that inhibition of RGMa could greatly enhance neural function rehabilitation in rats after MCAO/reperfusion. Neuron guidance factors are often regulators of angiogenesis. However, the effect of RGMa on angiogenesis and its mechanisms remain to be determined. Here, we investigated the effect of RGMa on endothelial cell (EC) proliferation, migration, tube formation, and cytoskeleton reassembly. The addition of recombinant RGMa significantly decreased the proliferation, migration, and tube formation of ECs. It also decreased the level of phosphorylated focal adhesion kinase (p-FAK Tyr397). Furthermore, the F-actin of the cytoskeleton assembly was obviously suppressed, with decreased filopodia and lamellipodia after the addition of RGMa. Knockout of neogenin or Unc5b significantly diminished RGMa’s inhibition of EC migration, tube formation, and cytoskeleton reassembly. RGMa-induced p-FAK (Tyr397) decrease was also abolished by knockout of neogenin or Unc5b. These results indicate that RGMa may be a negative regulator of angiogenesis through downregulating VEGF and p-FAK (Tyr397) via neogenin and Unc5b in vitro.

WNT5A regulates adipose tissue angiogenesis via antiangiogenic VEGF-A165b in obese humans.

Experimental studies have suggested that Wingless-related integration site 5A (WNT5A) is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Impaired angiogenesis in fat depots has been implicated in the development of adipose tissue capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction. We have recently demonstrated that impaired adipose tissue angiogenesis is associated with overexpression of antiangiogenic factor VEGF-A165b in human fat and the systemic circulation. In the present study, we postulated that upregulation of WNT5A is associated with angiogenic dysfunction and examined its role in regulating VEGF-A165b expression in human obesity. We biopsied subcutaneous and visceral adipose tissue from 38 obese individuals (body mass index: 44 ± 7 kg/m2, age: 37 ± 11 yr) during planned bariatric surgery and characterized depot-specific protein expression of VEGF-A165b and WNT5A using Western blot analysis. In both subcutaneous and visceral fat, VEGF-A165b expression correlated strongly with WNT5A protein (r = 0.9, P < 0.001). In subcutaneous adipose tissue where angiogenic capacity is greater than in the visceral depot, exogenous human recombinant WNT5A increased VEGF-A165b expression in both whole adipose tissue and isolated vascular endothelial cell fractions (P < 0.01 and P < 0.05, respectively). This was associated with markedly blunted angiogenic capillary sprout formation in human fat pad explants. Moreover, recombinant WNT5A increased secretion of soluble fms-like tyrosine kinase-1, a negative regulator of angiogenesis, in the sprout media (P < 0.01). Both VEGF-A165b-neutralizing antibody and secreted frizzled-related protein 5, which acts as a decoy receptor for WNT5A, significantly improved capillary sprout formation and reduced soluble fms-like tyrosine kinase-1 production (P < 0.05). We demonstrated a significant regulatory nexus between WNT5A and antiangiogenic VEGF-A165b in the adipose tissue of obese subjects that was linked to angiogenic dysfunction. Elevated WNT5A expression in obesity may function as a negative regulator of angiogenesis.NEW & NOTEWORTHY Wingless-related integration site 5a (WNT5A) negatively regulates adipose tissue angiogenesis via VEGF-A165b in human obesity.

GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Von Willebrand factor deficiency leads to impaired blood flow recovery after ischaemia in mice.

Neovascularisation, i. e. arteriogenesis and angiogenesis, is an inflammatory process. Therefore attraction and extravasation of leukocytes is essential for effective blood flow recovery after ischaemia. Previous studies have shown that von Willebrand factor (VWF) is a negative regulator of angiogenesis. However, it has also been shown that VWF facilitates leukocyte attraction and extravasation. We aimed to investigate the role of VWF in arteriogenesis and angiogenesis during post-ischaemic neovascularisation. Wild-type (WT) and VWF deficient (VWF-/-) C57BL/6 mice were subjected to hindlimb ischaemia via double ligation of the left femoral artery, and blood flow recovery was followed over time, using Laser Doppler Perfusion Imaging. Blood flow recovery was impaired in VWF-/- mice. After 10 days, VWF-/- mice showed a 43 ± 5 % recovery versus 68 ± 5 % in WT. Immunohistochemistry revealed that both arteriogenesis in the adductor muscles and angiogenesis in the gastrocnemius muscles were reduced in VWF-/- mice. Furthermore, leukocyte infiltration in the affected adductor muscles was reduced in VWF-/- mice. Residual paw perfusion directly after artery ligation was also reduced in VWF-/- mice, indicating a decrease in pre-existing collateral arteriole density. When we quantified collateral arterioles, we observed a 31 % decrease in the average number of collateral arterioles in the pia mater compared to WT mice (57 ± 3 in WT vs 40 ± 4 pial collaterals in VWF-/-). We conclude that VWF facilitates blood flow recovery in mice. VWF deficiency hampers both arteriogenesis and angiogenesis in a hindlimb ischaemia model. This is associated with impaired leukocytes recruitment and decreased pre-existing collateral density in the absence of VWF.

Nogo-A regulates vascular network architecture in the postnatal brain

Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation μCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.

P5 Pharmacological inhibition of plasma membrane calcium ATPASE 4 improves VEGF-induced angiogenesis

Ischaemic cardiovascular diseases are the leading cause of death worldwide and are often associated with partial or full occlusion of the blood vessel network in the affected organs. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing ones. The pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified as a crucial regulator of angiogenesis through activation of the calcineurin/Nuclear Factor of Activated T-cells (NFAT) signalling pathway. We have previously reported a novel role for the Plasma Membrane Calcium ATPase 4 (PMCA4) as a negative regulator of angiogenesis via interaction with calcineurin. Aurintricarboxylic acid (ATA) has been recently identified as a PMCA4-specific inhibitor. We hypothesise that pharmacological inhibition of PMCA4 with ATA will enhance VEGF-mediated angiogenesis. Here, we show that treatment of endothelial cells with nanomolar concentrations of ATA notably enhances calcineurin/NFAT signalling, and the subsequent expression of the VEGF-induced, NFAT-dependent, pro-angiogenic protein RCAN1.4. Targeting PMCA4 with ATA reduces the level of membrane-associated calcineurin, and the amount of calcineurin co-precipitated with PMCA4 in immunoprecipitation assays, indicating that ATA promotes disruption of the PMCA4/calcineurin interaction. ATA mediated inhibition of PMCA4 also enhances endothelial cell motility, and both in vitro and in vivo blood vessel formation. Low concentrations of ATA do not have any deleterious effects on the viability of endothelial cells or the development of zebra fish embryos, highlighting the potential clinical use of ATA, at low concentrations, to improve blood vessel formation in patients with ischaemic cardiovascular diseases.

Reduced expression of microRNA-497 is associated with greater angiogenesis and poor prognosis in human gliomas

MicroRNA (miR)-497 plays a tumor-suppressive role in several malignancies and is involved in glioma invasiveness and resistance to chemotherapy. To add to the knowledge of the clinical significance of miR-497 in human gliomas, quantitative real-time polymerase chain reaction was performed to detect the expression of miR-497 in 110 pairs of freshly prepared glioma and nonneoplastic brain tissues. Then the associations of miR-497 expression with various clinicopathological characteristics and overall survival of glioma patients were estimated statistically. Gain-of-function assays were also performed to examine the role of miR-497 in glioma angiogenesis. The expression of miR-497 in human glioma tissues was significantly lower than in nonneoplastic brain tissues (P<.001). In addition, low miR-497 expression was significantly associated with advanced World Health Organization grade (P<.001) and low Karnofsky performance scores (P=.02). Moreover, the survival of glioma patients with low miR-497 expression was dramatically shorter than that of patients with high miR-497 expression (P=.001). Forced expression of miR-497 in glioma cells inhibited tube formation by cocultured human brain microvascular endothelial cells. We also found that miR-497 overexpression in glioma cells led to decreased expression of vascular endothelial growth factor. In conclusion, miR-497 may be a favorable prognostic marker in human gliomas, in part by being a negative regulator of angiogenesis, implying its potential as a therapeutic target for this cancer.

Abstract 364: TRPV4 Channel Acts as a Negative Regulator of Angiogenesis by Modulation of Rho/Rho Kinase Pathway

Angiogenesis, the formation new blood vessels from pre-existing ones, is critical for maintenance of normal cardiovascular physiology. However, excessive or insufficient angiogenesis can contribute to various diseases including cancer, atherosclerosis, and retinopathy. While the mechanism by which angiogenesis occurs is well established, little is known about the mechanisms that negatively regulate this process. Therefore, we investigated the role of mechanosensitive ion channel, TRPV4, in the regulation of angiogenesis by employing in vitro, ex vivo, and in vivo techniques. In the present study, we first cultured aortic ring explants isolated from wild-type (WT) and TRPV4KO mice and found a significant increase in the sprouting from TRPV4KO aortic rings after 5 days. Next, we found that endothelial cells (EC) isolated from TRPV4KO mice (TRPV4KO EC) exhibited increased proliferation, migration, as well as abnormal angiogenesis in vitro, compared to their WT counterparts. Further, in vivo Matrigel plug assays revealed abnormal vascular growth in TRPV4KO mice. Mechanistically, we found that absence of TRPV4 results in a significant increase in basal Rho activity and that pharmacological inhibition of the Rho/Rho kinase pathway was able to normalize the abnormal tube formation exhibited by TRPV4KO EC in vitro . To confirm these findings, we examined tumor growth in TRPV4KO mice treated with Rho kinase inhibitor, Y-27632, and anti-cancer drug Cisplatin, alone and in combination. We found that Y-27632 treatment, in conjunction with Cisplatin but not alone, was able to significantly reduce the abnormal tumor growth seen in TRPV4KO mice, suggesting that Rho kinase inhibition may have normalized the tumor vasculature and improved the delivery of Cisplatin. Taken together, these data suggest that TRPV4 is a negative regulator of angiogenesis and potentially a novel target for pathological and/or therapeutic angiogenesis.