Role Of Ang-2 Research Articles

A Potential Role for Angiopoietin 2 in the Regulation of the Blood–Retinal Barrier in Diabetic Retinopathy

Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.

Angiopoietin-4 Promotes Glioblastoma Progression by Enhancing Tumor Cell Viability and Angiogenesis

Glioblastoma multiforme (GBM) is a highly invasive and vascularized aggressive brain tumor. Less than 10% of GBM patients survive >5 years after diagnosis. Angiogenesis plays an important role in GBM growth, and antiangiogenesis-based therapies have shown clinical efficacy for GBM patients. Unfortunately, therapeutic resistance often develops in these patients, suggesting that GBM cells are capable of switching their dependency on one proangiogenic signaling pathway to an alternative one. Therefore, it is important to identify novel angiogenic factors that play essential roles in tumor angiogenesis and GBM progression. Angiopoietins (Ang-1, Ang-2, and Ang-4) are the ligands of the Tie-2 receptor tyrosine kinase (RTK). The roles of Ang-1 and Ang-2 in tumor angiogenesis have been established. However, little is known about how Ang-4 affects tumor angiogenesis and GBM progression and the mechanism underlying its effects. In our current study, we establish that Ang-4 is upregulated in human GBM tissues and cells. We show that, like endothelial cells, human GBM cells express Tie-2 RTK. We first establish that Ang-4 promotes in vivo growth of human GBM cells by promoting tumor angiogenesis and directly activating extracellular signal-regulated kinase 1/2 (Erk1/2) in GBM cells. Our results establish the novel effects of Ang-4 on tumor angiogenesis and GBM progression and suggest that this pro-GBM effect of Ang-4 is mediated by promoting tumor angiogenesis and activating Erk1/2 kinase in GBM cells. Together, our results suggest that the Ang-4-Tie-2 functional axis is an attractive therapeutic target for GBM.

Abstract 2354: Angiopoietin-2 role in breast cancer metastasis

Abstract Background: Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. The pro-angiogenic effect of Ang-2 is mediated through activation of the Tie-2 receptor that leads to vessel destabilization and dismantling. The latter is an essential step for both angiogenesis and tumor entry into the lumen. We assessed the effects of Ang-2 on tumor vasculature in orthotopic breast carcinoma model MDA-MB-231 that either lacked or overexpressed this factor. Methods: Luciferase-tagged MDA-MB-231 cells were engineered to overexpress human Ang-2. The control line expressed Ang-2 in reverse orientation. Stable production of Ang-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Ang-2 functionality was assessed by migration and proliferation assays using tumor and endothelial cells. Ang2-overexpressing and control MDA-MB-231 cells were orthotopically implanted into mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry using antibodies against MECA-32, smooth muscle actin (SMA-α), VEGFR-3 and Notch-1. Results: Ang-2-overexpressing tumor line produced 11±0.4 ng/ml of Ang-2 while expression in control line was undetectable. Tumor-produced Ang2 was functional as demonstrated by a dose-dependent migration of lymphatic endothelial cells. Soluble Tie-2 inhibited Ang-2 induced migration by 70%. Ang-2 had not effect on proliferation of MDA-MB-231 cells, tumor growth or blood vessel density. However, Ang-2 overexpressing and control tumors substantially differed in vascular morphology. The differences included 170% increase in blood vascular area, 280% increase in number of vessels with open lumen, and 600% increase in lumens’ cross-sectional area. Pericyte coverage shown by SMA-α staining decreased >10-fold (p>0.001) in Ang-2 tumors, demonstrating vessel destabilization. Tumor vascular invasion and pulmonary metastasis increased by 500% and 1100% in Ang-2 tumors compared with controls. Also, blood vessels in Ang-2 tumors displayed 2.0-2.2 fold upregulated Notch-1 and VEGFR-3 expression. Conclusions: These data suggest that Ang-2 increases metastasis via suppression of pericytes’ recruitment, which destabilizes tumor vessels and facilitates entry of tumor cells into the lumen. The increased expression of Notch-1 and VEGFR-3 on blood vessels in Ang-2 overexpressing tumors suggests that signaling of these proteins underlie the vascular morphologic changes in Ang-2-rich environment. This is consistent with prior data demonstrating up-regulation of VEGFR-3 on tumor blood vessels and association of both proteins with increased metastasis. This study demonstrates a key role for Ang-2 in hematogenous metastasis and suggests that suppression of Ang-2 and VEGFR-3 could inhibit breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2354.

Angiopoietin‐1 alters tumor growth by stabilizing blood vessels or by promoting angiogenesis

The maturation of blood vessels requires mural cell adhesion to endothelial cells. Angiopoietin-1 (Ang1), a ligand for Tie2 receptor expressed on endothelial cells, plays a critical role in cell adhesion between mural cells and endothelial cells and in endothelial cell sprouting from preexisting vessels in the absence of mural cells. Much information has been amassed on the Tie2-Ang1 system in physiological blood vessel formation during embryogenesis; however, the role of Ang1 in the tumor environment and its interaction with mural cells has not been well documented. Here we studied how Ang1 regulates maturation of blood vessels using the human colon cancer cell line HT29 and the human prostate cancer cell line PC3, and studied how Ang1 affects tumor growth. In a xenograft tumor model using female nude mice, we found that Ang1 enhanced angiogenesis and resulted in tumor growth in the case of PC3 tumors but suppressed tumor growth in the case of HT29 tumors. In PC3 tumors, the number of mural cells adhering to endothelial cells was less than that in HT29 tumors. Ang1 induced sprouting angiogenesis in PC3 tumors although there was little maturation of blood vessels. On the other hand, there was abundant mural cell adhesion to endothelial cells in HT29 tumors and Ang1 did not induce angiogenesis. These results suggest that Ang1 alters tumor growth in a manner that is dependent on the adhesion of mural cells and their localization in the tumor environment.

Functional Difference of Two Tie2 Ligands, Angiopoietin-1 and -2, in the Regulation of the Adult Bone Marrow Hematopoietic Stem Cells

Majority of the adult HSCs is in quiescent state in cell cycle, and it is likely that maintenance of the HSC quiescence and slow cell-cycle progression are critical for sustaining the self-renewing HSC pool. Indeed, sub-populations of long-term HSC (LT-HSC) are in the quiescent state and have robust reconstitution activity of hematopoiesis. Therefore, it is suggested that quiescence of HSCs is directly linked to the ability for the efficient reconstitution. We previously reported that the side-population (SP) in Lineage− Sca-1+Kit+ (LSK) cells resided in the BM niche is the quiescent HSC population, and is enriched in the Tie2 receptor-positive fraction in LSK. So far, four types of ligands, angiopoietin (Ang)-1~4, have been identified as the ligands of Tie2. We found that Ang-1/Tie2 signaling induces the quiescence or slow cell cycling of HSCs and contributes to the maintenance of long-term repopulating ability of LT-HSCs (Arai et al. 2004). Ang-2 is dispensable for embryonic angiogenesis but is specifically required for postnatal vascular remodeling and lymphatic patterning. In the hematopoietic system, however, the role of Ang-2 in the regulation of LT-HSCs is still unclear. Here, we investigated the functional difference between Ang-1 and Ang-2 in the maintenance of LT-HSCs. First, we examined the expression of CDK inhibitors, cell adhesion molecules, cytokine receptors, transcription factors, and anti-apoptotic factors induced by the Ang-1 and/or Ang2. LSK-SP cells were cultured with stem cell factor (SCF) in the presence or absence of angiopoietins (Ang-1 and/or Ang-2), and gene expressions were analyzed by high-throughput real-time PCR assays using a novel nanofluidic platform (BioMark™ Dynamic Array and Digital Array, Fluidigm). We found that Ang-1 upregulated the expression of p57, p18, α4-integrin, β1-integrin, N-cadherin, Tie2, Hoxb4, Bmi-1 and Bax, compared to control. Although Ang-2 also upregulated p57 and Tie2, its effect was smaller than that of Ang-1. In addition, Ang-1-induced upregulations of p57 and p18 were suppressed by the simultaneous addition of Ang-2. Next we examined the effects of Ang-1 and Ang-2 on the long-term engraftment capacity of LSK-SP cells. LSK-SP cells (Ly5.1+) were cultured in the serum-free medium containing SCF and thrombopoietin (THPO) with or without angiopoietins (Ang-1 and/or Ang-2). After 7 days of culture, cells were transplanted into the recipient mice (Ly5.2+). Percentage of Ly5.1+ cells in the mice transplanted with the HSCs cultured with SCF and THPO was 9.8 ± 4.0 % after 3 months of BM transplantation (BMT). Consistent with our previous report, the LSK-SP cells cultured with Ang-1 efficiently maintained long-term repopulation (LTR), and the percentage of Ly5.1+ cells in the recipient's peripheral blood was 40.8 ± 7.7 % after 3 months of BMT. Although Ang-2 also maintained LTR activity of HSCs (14.5 ± 4.7 %), the frequency of donor-derived cells was significantly lower than the case of Ang-1. Furthermore, the effect of Ang-1 was dramatically interfered by the addition of Ang-2 (10.0 ± 4.5 %). These observations suggest that in the BM niche, especially in the endosteal niche, Ang-1 might be the dominant ligand for Tie2 in LT-HSCs and contribute to the maintenance of the stem cell activities. In addition, we isolated osteoblasts (OB) and mesenchymal progenitor cells (MPCs), the major osteoblastic niche components, from endosteum by collagenase treatment of bone fragments and found that osteoblastic niche cells expressed Ang-1 but not Ang-2, which is consistent with the results of qPCR and LTR-assay. We hypothesized that Ang-2 is involved in the release of HSCs from the niche for the mobilization of HSCs. Collectively, Ang-1 plays a role as the key factor for LTR-activity maintenance of LT-HSCs, and Ang-2 has an antagonistic effect against Ang-1/Tie2 signaling.

Osteoblast-specific Angiopoietin 1 overexpression increases bone mass

Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis.

Angiopoietin-2 Impairs Revascularization After Limb Ischemia

Angiopoietins play important roles in the formation of neovessels and complex vascular networks. Angiopoietin (Ang)-1 and Ang-2 belong to a family of growth factors that display opposing effects on the activation of Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2). Endothelial Ang-2 expression is associated with vessel destabilization and regulates a balance between vascular regression and growth. To elucidate, in particular, the role of Ang-2 after arterial artery occlusion in the mouse limb, we applied a transgenic animal model with targeted Ang-2 expression in endothelial cells. We show here that restoration of blood flow in Ang-2:Tie1 transgenic mice is dramatically impaired when Ang-2 expression is induced in the vasculature. The defective restoration of perfusion in Ang-2 transgenic mice is evidenced by reduced collateral artery growth, which typically occurs to compensate for flow deficits after occlusion of the large conductance artery. Furthermore, reduced movement capacities and higher incidents of necrosis are consequently observed in the transgenic limbs as compared with controls. Mechanistically, the observed effects are attributed to defective smooth muscle cell recruitment in Ang-2 transgenic mice. Moreover, distinct Ang-2 levels in the genetically modified animals clearly correlated with the magnitude of reduced perfusion. In conclusion, our studies define Ang-2 as an important molecule for the progression of collateral artery growth and angiogenesis during ischemia and suggest precise Ang-2 dosage activities to accomplish blood vessel growth.

Angiopoietin-1, Angiopoietin-2 and Tie-2 in the Coronary Circulation of Patients With and Without Coronary Collateral Vessels

The role of the angiopoietin (Ang)/Tie-2 system in coronary collateral growth is not well understood, so the purpose of this study was to investigate and elucidate the relationship of this system to coronary collateral formation in patients with coronary artery disease (CAD). Fifty-nine patients with CAD were recruited. Blood samples from the left ventricle (LV) and coronary sinus (CS) were obtained during cardiac catheterization, and serum concentrations of Ang-1, Ang-2, and Tie-2 were measured by enzyme-linked immunosorbent assay. Patients were then classified as mild CAD (n=30), defined as </=90% stenosis of the coronary arterial luminal diameter, or severe CAD (n=29), which was total (or near total) coronary occlusion requiring coronary collateral growth. Ang-1, Ang-2, and Tie-2 in the LV and CS sera were not significantly different between groups. In the severe CAD group, spillover of Tie-2 (CS-LV value) from the coronary circulation was found in comparison with the mild CAD group (3.43+/-2.22 vs -3.29+/-1.54 ng/ml, p=0.01), whereas the CS-LV values of Ang-1 and Ang-2 did not differ between groups. Tie-2 production was markedly increased in patients with well-developed collaterals. A positive and significant correlation was found between coronary Ang-2 and Tie-2 levels (r=0.44, p<0.001). Tie-2 is probably produced in the coronary circulation and may induce the development or maintenance of coronary collaterals in CAD patients. Furthermore, the role of Ang-2 in the formation of coronary collaterals may be more important than that of Ang-1.

Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.

Enhancement of new vessel formation by Angiopoietin-2/Tie2 signaling in endothelial progenitor cells: A new hope for future therapy?

See article by Kim et al. [6] (pages 394−402) in this issue. The Tie2/angiopoietin pathway is crucial for angiogenesis, blood vessel maturation, and vascular endothelial integrity. The ligands for Tie2 are the angiopoietins, of which angiopoietin-1 (Ang1) and Ang2 have been the most studied. Suppression of plasma leakage, inhibition of vascular inflammation, and prevention of endothelial death are the three widely accepted vascular protective functions for Ang1 [1], the first ligand identified for the Tie2 receptor. However, the role of Ang2, a relative of Ang1 identified by homology screening, remains less clear, because Ang2 exhibits context-dependent behavior. Ang2 can inhibit or stimulate Tie2 receptor phosphorylation under different experimental conditions [2]. Ang2 in vivo acts as a Tie2 antagonist on vascular endothelial cells (ECs), whereas it acts as a Tie2 agonist on lymphatic vessels [3]. Recently, increased Ang2 expression was demonstrated in myocardial ischemia and infarction [4,5], indicating the role of Ang2 in the angiogenic response to tissue ischemia. In this issue of Cardiovascular Research , Kim et al. [6] report a proangiogenic activity of Ang2 in endothelial progenitor cells (EPCs) directly through the Tie2 signaling pathway. Ang2 increased tube formation, migration, and cell survival of EPCs derived from human umbilical cord … *Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. Tel.: +886 2 2833 2211; fax: +886 2 2836 5775. Email address: shyukg{at}ms12.hinet.net

Role of Ang1 and its interaction with VEGF-A in astrocytomas.

Angiopoietins (Ang1 and Ang2) modulate the activity of the endothelial cell (EC)-specific receptor tyrosine kinase Tie2, which together with vascular endothelial growth factor (VEGF-A) and its EC-specific receptors, VEGFR1 and VEGFR2, regulate normal physiological vessel development. The functional role of angiopoietins in tumor angiogenesis, in particular astrocytoma angiogenesis, remains unclear. In this study, we focus on the specific contribution of Ang1 to the vascular growth of glioblastoma multiforme (GBM) and its interactive role with VEGF-A. Subcutaneous and intracranial GBM xenografts were generated using 3 established astrocytoma cell lines (U87, U373, and U343) that were transfected to stably over-express Ang1. GBM xenografts were also generated to express low levels of VEGF-A and high Angl. We found that Ang1 increases the vascular growth of both subcutaneous and intracranial xenografts of GBM by approximately 3-fold. However, the increased vascular growth was only seen in xenografts with concurrent VEGF-A elevation, since decreasing VEGF-A expression resulted in a loss of the pro-angiogenic growth advantage seen with Ang1. Collectively, our data suggest that Ang1 regulates GBM vascularity in a VEGF-A dependent manner, synergizing the initial pro-angiogenic response that is triggered by VEGF-A and promoting the vascular growth of GBM.

Temporal and spatial localization of the angiopoietin-TIE-2 system after myocardial infarction in the rat: Evidence of a role for Ang-2 in angiogenesis Michael J.B. Kutryk, Reena Sandhu, Krystyna Teichert-Kuliszewska, Michael A. Kuliszewski, Sue Nag, Andrew I.M. Campbell, Duncan J. Stewart. Terrence Donnelly Heart Center, St. Michael's Hospital, Toronto, ON, Canada, Dept. of Neuropathology, University of Toronto Health Network, Toronto, ON,

Temporal and spatial localization of the angiopoietin-TIE-2 system after myocardial infarction in the rat: Evidence of a role for Ang-2 in angiogenesis Michael J.B. Kutryk, Reena Sandhu, Krystyna Teichert-Kuliszewska, Michael A. Kuliszewski, Sue Nag, Andrew I.M. Campbell, Duncan J. Stewart. Terrence Donnelly Heart Center, St. Michael's Hospital, Toronto, ON, Canada, Dept. of Neuropathology, University of Toronto Health Network, Toronto, ON,

Direct intramuscular injection of plasmid DNA encoding angiopoietin-1 but not angiopoietin-2 augments revascularization in the rabbit ischemic hindlimb.

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have recently been identified as ligands for the endothelial cell-specific Tie2 receptor. Little is known regarding the impact of these Tie2 ligands on postnatal neovascularization. Accordingly, we tested the hypothesis that gene transfer of plasmid DNA encoding Ang1 and Ang2 could modulate collateral vessel development in a rabbit model of hindlimb ischemia. pAng1* (n=15), pJFE control (no Ang1* insert) (n=9), pAng2 (n=9), pcDNA3 control (no Ang2 insert) (n=10), or saline (n=5) was injected intramuscularly into the rabbit ischemic hindlimb. Collateral vessel development and limb perfusion were assessed before and 30 days after treatment. Calf blood pressure ratio (ischemic to normal hindlimb) was increased 30 days after Ang1* gene transfer versus controls (Ang1*, 0.90+/-0.02; pJFE, 0.76+/-0.05; saline, 0.77+/-0. 03; P<0.05). Angiographic score was higher (P<0.05) in the pAng1* group (0.63+/-0.02) than in the pJFE (0.51+/-0.03) or saline (0. 52+/-0.02) group. Maximal (postpapaverine) blood flow in the ischemic limb was higher (P<0.05) after pAng1* (67.8+/-4.9 mL/min) than pJFE (51.2+/-4.4 mL/min) or saline (52.9+/-4.9 mL/min). Capillary density and capillary/muscle fiber ratio (242+/-12/mm2 and 0.89+/-0.06, respectively) were higher (P<0.01) with pAng1* than pJFE (172+/-11/mm2 and 0.64+/-0.05) or saline (166+/-10/mm2 and 0. 67+/-0.05). Neovascularization was not enhanced with pAng2. Ang1 but not Ang2 gene transfer produces anatomic and physiological evidence of enhanced collateral vessel formation. Ang1 may modulate neovascularization in adult animals and thus represents a feasible therapeutic strategy for patients with tissue ischemia. The role of Ang2 in postnatal neovascularization remains to be clarified.