Angiopoietin-2 Overexpression Research Articles

ADAMTS13 controls vascular remodeling by modifying VWF reactivity during stroke recovery

AbstractAngiogenic response is essential for ischemic brain repair. The von Willebrand factor (VWF)–cleaving protease disintegrin and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) is required for endothelial tube formation in vitro, but there is currently no in vivo evidence supporting a function of ADAMTS13 in angiogenesis. Here we show that mice deficient in ADAMTS13 exhibited reduced neovascularization, brain capillary perfusion, pericyte and smooth muscle cell coverage on microvessels, expression of the tight junction and basement membrane proteins, and accelerated blood-brain barrier (BBB) breakdown and extravascular deposits of serum proteins in the peri-infarct cortex at 14 days after stroke. Deficiency of VWF or anti-VWF antibody treatment significantly increased microvessels, perfused capillary length, and reversed pericyte loss and BBB changes in Adamts13−/− mice. Furthermore, we observed that ADAMTS13 deficiency decreased angiopoietin-2 and galectin-3 levels in the isolated brain microvessels, whereas VWF deficiency had the opposite effect. Correlating with this, overexpression of angiopoietin-2 by adenoviruses treatment or administration of recombinant galectin-3 normalized microvascular reductions, pericyte loss, and BBB breakdown in Adamts13−/− mice. The vascular changes induced by angiopoietin-2 overexpression and recombinant galectin-3 treatment in Adamts13−/− mice were abolished by the vascular endothelial growth factor receptor-2 antagonist SU1498. Importantly, treating wild-type mice with recombinant ADAMTS13 at 7 days after stroke markedly increased neovascularization and vascular repair and improved functional recovery at 14 days. Our results suggest that ADAMTS13 controls key steps of ischemic vascular remodeling and that recombinant ADAMTS13 is a putative therapeutic avenue for promoting stroke recovery.

Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis.

Despite many angiogenic factors playing crucial roles in metabolic homeostasis, effects of angiopoietin-2 (ANG-2) in adipose tissue (AT) remain unclear. Utilizing a doxycycline-inducible AT-specific ANG-2 overexpression mouse model, we assessed the effects of ANG-2 in AT expansion upon a high-fat diet (HFD) challenge. ANG-2 is significantly induced, with subcutaneous white AT (sWAT) displaying the highest ANG-2 expression. ANG-2 overexpressing mice show increased sWAT vascularization and are resistant to HFD-induced obesity. In addition, improved glucose and lipid metabolism are observed. Mechanistically, the sWAT displays a healthier expansion pattern with increased anti-inflammatory macrophage infiltration. Conversely, ANG-2 neutralization in HFD-challenged wild-type mice shows reduced vascularization in sWAT, associated with impaired glucose tolerance and lipid clearance. Blocking ANG-2 causes significant pro-inflammatory and pro-fibrotic changes, hallmarks of an unhealthy AT expansion. In contrast to other pro-angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A), this is achieved without any enhanced beiging of white AT.

Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation.

Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.

Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial-mesenchymal transition-induced angiogenesis.

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial–mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further investigations suggested that overexpression of ANG2 might increase OSCC metastasis by promoting angiogenesis in nude mice. This stimulatory effect could be achieved by inducing abnormal EMT and by reducing apoptosis and increasing proliferation of cells.

Abstract 18963: Over-expression of Angiopoietin-2 in Human Patients With Continuous-flow Left Ventricular Assist Devices Induces Abnormal Angiogenesis and Predicts Gastrointestinal Bleeding Events

Arteriovenous malformations are common in patients with Continuous-Flow Left Ventricular Assist Devices (LVAD) and may reflect deregulation of angiogenesis. Angiopoietin-2 (Ang-2) is a stimulator of angiogenesis produced by endothelial cells in response to thrombin. In this study, we evaluate Ang-2 expression in patients with LVAD compared with stable heart failure (HF) or orthotopic heart transplant (OHT) and test the hypothesis that elevated Ang-2 in patients with LVAD leads to increased angiogenesis. Blood samples were taken from 79 patients (n= 21 HF, 41 LVAD, 17 OHT) during routine heart catheterization. Ang-2 was measured by ELISA and thrombin was measured by Western Blot. Cultured human umbilical vein endothelial cells (HUVECs) were incubated with plasma from each patient and Ang-2 expression was measured by RT-PCR. HUVECs were then incubated on Matrigel with serum from each patient in the presence or absence of an Ang-2 blocking antibody and tube formation was measured by microscopy. Also, vena caval endothelial cells were isolated from discarded guide wires used during catheterization and Ang-2 expression was measured by quantitative immunofluorescence. In patients with LVAD vs. HF or OHT, Ang-2 was elevated in both serum (12.3±9.6, 4.5±2.1, and 4.9±2.0 ng/mL respectively, p<0.01) and endothelial cells (56.3±14.1, 31.3±4.2, 35.0±11.2 AU respectively, p<0.05). On Matrigel, serum from patients with LVAD vs. HF or OHT induced increased tube formation (57.1±3.2, 39.2±7.1, 34.7±5.1 tubes per low power field respectively, p<0.05) and this elevation normalized with Ang-2 blocking antibody. Plasma from patients with LVAD vs. HF or OHT induced Ang-2 gene over-expression in HUVECs (1.6±0.4, 1.0±0.3, 0.6±0.2 RQ respectively, p<0.01) and was associated with higher plasma thrombin. In patients with LVAD, serum Ang-2 >10 ng/mL was associated with a higher 3 month risk of GI bleeding vs. Ang-2 <10 ng/mL (30% vs. 0%, p<0.01). Therefore, we conclude that over-expression of Ang-2 in patients with LVAD induces increased angiogenesis and predicts GI bleeding events. Thrombin is increased in patients with LVAD and their plasma induces Ang-2 over-expression in vitro . Therefore, Ang-2 overexpression may contribute to AVM formation in patients with LVAD.

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis.

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.

Hydrogen Sulfide Prevents Hyperoxia-induced Lung Injury by Downregulating Reactive Oxygen Species Formation and Angiopoietin-2 Release

Oxygen therapy is a life-sustaining treatment for patients with respiratory failure. However, prolonged exposure to high oxygen concentrations often results in hyperoxia-induced acute lung injury (HALI). At present, no effective therapeutic intervention can attenuate the development of HALI. In the present study, we investigated whether hydrogen sulfide (H2S) can confer lung protection in a mouse model of HALI. C57BL/6 mice were either exposed to room air or 90 vol% oxygen and received either the H2S donor sodium hydrosulfide (NaHS, 10 mg/kg) or vehicle. Lung injury was assessed by an HALI score in tissue sections. Bronchoalveolar lavage fluid was analyzed for protein content and cellular infiltration. Reactive oxygen species (ROS) were detected by dihydroethidium staining. Angiopoietin- 2 was detected by Western Blotting. Pulmonary epithelial, endothelial, and macrophage cells were stimulated to produce ROS either in the absence or presence of NaHS. Mice exposed to hyperoxia developed substantial lung injury, characterized by an elevated HALI score, cellular infiltration, protein leakage, ROS production, and overexpression of angiopoietin-2. NaHS treatment abolished morphological indices of HALI. Angiopoietin-2 expression was significantly reduced by NaHS in vivo. In endothelial cells and macrophages, angiopoietin-2 was released due to ROS formation and decreased in the presence of NaHS. In conclusion, H2S protects from HALI by preventing ROS production and angiopoietin-2 release.

Somatic PTPRT and ANGPT2 Mutations in Large Granulocyte Leukemia

AbstractAbstract 1302 Introduction:T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T-cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene (Koskela et al, NEJM, 2012). In the remaining T-LGL patients, the pathogenetic mutations are not known. Methods:To identify additional somatic mutations, we chose two STAT3 mutation negative T-LGL leukemia patients for exome sequencing. CD8+ T-cells were used as test cells and matched CD4+ T-cells as control. The exome was captured with the Nimblegen SeqCap EZ Exome Library v2.0 and the sequencing was performed with the Illumina HiSeq2000 sequencing platform. Candidate somatic mutations were identified with a bioinformatics pipeline consisting of BWA for sequence alignment, Samtools for alignment filtering and Varscan for somatic mutation calling. Results:Index patient 1 was diagnosed with T-LGL leukemia at the age of 70 and a TCR repertoire assay revealed one minor T- cell clone in the leukemic sample (Vβ7.1: 28 %). Exome sequencing revealed 10 nonsynonymous nucleotide variants with p-values lower than 0.01, of which the tumor suppressor gene Protein tyrosine phosphatase (PTP) receptor T (PTPRT) had a variant frequency of 14%. PTPRT has previously been found to reverse Tyr705 phosphorylation on STAT3, a modification associated with STAT3 deactivation. In this novel mutation, a highly conserved hydrophobic valine residue is converted into methionine (V995M). The mutation occurs in the cytoplasmic part of the protein, within the tyrosine-protein phosphatase 1 domain. The PTPRT V995M mutation may thereby affect STAT3 activity by reducing dephosphorylation of Tyr705, thus increasing the expression of STAT3 target genes.Index patient 2 was a 40 year-old male with untreated T-LGL leukemia. A TCR repertoire assay showed one predominant T-cell clone in the leukemic T-cells (Vβ13.2: 70%). Exome sequencing revealed 8 nonsynonymous nucleotide variants with p-values lower than 0.01. The missense mutation K436E in Angiopoietin-2 (ANGPT2), presenting with the lowest somatic p-value (1,06−09) and highest variant frequency (34%), was the most relevant candidate involved in the pathogenesis of leukemia. The mutation occurs on the surface of ANGPT2 within the well-conserved fibrinogen C-terminal domain. This domain binds the receptor TIE2 and the change in the polarity induced by K436E mutation is likely to affect the binding of TIE2 by ANGPT2. Overexpression of ANGPT2 has previously been shown to confer an adverse prognostic factor in other forms of leukemia. While these mutations appear biologically relevant and exciting, we have not yet seen them in other LGL patients screened so far (n=80). Conclusions:Somatic mutations in the PTPRT and ANGPT2 genes may represent rare genetic causes for T-LGL leukemia. Screening for these mutations in a larger cohort of patients is warranted. The mutation in the PTPRT gene is particularly exciting as it may directly impact the STAT3 pathway, which is a common pathogenetic event in T-LGL leukemia. Inactivating mutations of the PTPRT gene may have the same functional consequence as activating mutations of STAT3 in LGL patients. Disclosures:Koskela:Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Kallioniemi:TEKES-FiDiPro: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Inhibition of endothelial/smooth muscle cell contact loss by the investigational angiopoietin-2 antibody MEDI3617

A tumor's dependence on angiogenesis for survival and growth has led to the advancement of a variety of blood vessel directed anticancer treatment strategies. Overexpression of angiopoietin-2 (Ang-2) in tumor vasculature and its crucial role in angiogenesis, i.e. the destabilization of endothelial/peri-endothelial cell interactions, now raises the possibility of additional novel anti-angiogenic therapeutics. The present study utilized a co-culture sphere model to (i) demonstrate the destabilizing effect of Ang-2 on endothelial/smooth muscle cell contact and (ii) evaluate the impact of the investigational Ang-2 antibody MEDI3617 on endothelial/smooth muscle cell dissociation. Real time imaging of spheres showed both exogenous Ang-2 and PMA induced endogenous Ang-2 secretion resulted in sphere destabilization (loss of endothelial cells from smooth muscle cell core). The presence of MEDI3617 inhibited this process. To assess the anti-angiogenic potential of MEDI3617 in vivo, nude mice were injected intradermally with human renal cell carcinoma cells (Caki-1, Caki-2) and the number of blood vessels induced over a 3day period was scored. MEDI3617 (2, 10, 20mg/kg) significantly reduced the initiation of blood vessels for both tumor models at all doses investigated. These data indicate that MEDI3617 treatment significantly impairs the initiation of angiogenesis by inhibiting the Ang-2 mediated disruption of endothelial/muscle cell interaction associated with blood vessel destabilization and thereby reduces tumor cell induced angiogenesis. The results support the notion that targeting the angiopoietin/Tie2 axis may offer novel anti-angiogenic strategies for cancer treatment.

Expression of angiopoietin‐1 and ‐2, and its clinical significance in human bladder cancer

To investigate the relationship between angiopoietin-1 and -2 expression and the clinicopathological variables and clinical outcome in patients with bladder cancer treated by surgical resection, as both have been recently identified as antagonistic angiogenic factors which regulate tumour growth. The expression of angiopoietin-1 and -2 were assessed by immunohistochemistry in tissue sections from 52 transitional cell carcinomas of the bladder (33 grade 1, 15 grade 2, four grade 3, including two associated with carcinoma in situ; 22 were stage Ta, 19 T1 and 11 T2 tumours). Normal bladder specimens were also resected during each operation as controls. The expression angiopoietins were related to the clinicopathological variables of the tumours. Positive immunostaining was detected in 18 samples (35%) for angiopoietin-1 and in 23 (44) for angiopoietin-2. There was no significant difference in survival according to tumour angiopoietin-1 status in the patients, but in contrast the overall survival of patients with angiopoietin-2-positive tumours was significantly lower than for those with angiopoietin-2-negative tumours (P < 0.05). Positive angiopoietin-2 expression was significantly correlated with histological grade (P = 0.026), histological stage (P = 0.009) and poor prognosis (P < 0.05). On multivariate analysis, positive angiopoietin-2 expression was an independent negative predictor for survival (P = 0.042). These results suggest that angiopoietin-2 overexpression is associated with tumour progression, thereby indicating a poor prognosis for some patients treated by surgical resection for bladder carcinoma.

Angiopoietin-2 Is Implicated in the Regulation of Tumor Angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.