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(Circulation Research. 2000;87:603.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Angiopoietin-1 Is an Antipermeability and Anti-Inflammatory Agent In Vitro and Targets Cell Junctions

Jennifer R. Gamble, Jenny Drew, Libby Trezise, Anne Underwood, Michelle Parsons, Lisa Kasminkas, John Rudge, George Yancopoulos, Mathew A. Vadas

From the Vascular Biology Laboratory, Division of Immunology, Hanson Centre for Cancer Research (J.R.G., J.D., L.T., M.P., L.K., M.A.V.), Institute of Medical and Veterinary Science and the University of Adelaide, South Australia; CSIRO Molecular Science (A.U.), North Ryde, New South Wales, Australia; and Regeneron Pharmaceuticals, Inc (J.R., G.Y.), Tarrytown, NY.

Correspondence to J.R. Gamble, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science and the University of Adelaide, Frome Road, Adelaide, South Australia 5000. E-mail jennifer.gamble{at}imvs.sa.gov.au

	Abstract

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Abstract—Inflammation is a basic pathological mechanism that underlies many diseases. An important component of the inflammatory response is the passage of plasma components and leukocytes from the blood vessel into the tissues. The endothelial monolayer lining blood vessels reacts to stimuli such as thrombin or vascular endothelial growth factor by changes in cell-cell junctions, an increase in permeability, and the leakage of plasma components into tissues. Other stimuli, such as tumor necrosis factor- (TNF-), are responsible for stimulating the transmigration of leukocytes. Here we show that angiopoietin-1, a cytokine essential in fetal angiogenesis, not only supports the localization of proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) into junctions between endothelial cells and decreases the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthens these junctions, as evidenced by a decrease in basal permeability and inhibition of permeability responses to thrombin and vascular endothelial growth factor. Furthermore, angiopoietin-1 inhibits TNF-–stimulated leukocyte transmigration. Angiopoietin-1 may thus have a major role in maintaining the integrity of endothelial monolayers.

Key Words: endothelium • inflammation • permeability • angiogenesis • cell junctions

	Introduction

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Angiopoietin-1 (Ang1) is a recently identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2.¹ It is involved in the angiogenic phase of embryonic vascular development with major defects in the interaction of endothelial cells (ECs), with the surrounding mesenchymal cells and extracellular matrix evident in Ang1 knockout mice.² As a result, the vessels are poorly formed, with a lack of branching and remodeling, and are ectatic and leaky. Tie-2 knockout mice have a similar phenotype with additional venous malformation brought about by a disruption of the endothelial-smooth muscle cell interactions.³ Although these observations have shown that, in development, Ang1 is critical for the stabilization of the interaction of ECs with their surrounding matrix, little is known of how the integrity of mature vessels, and in particular that of the endothelial monolayer, is maintained. Two recent publications have suggested that Ang1 is involved in mature blood vessel control. Firstly, mice transgenic for Ang1 have leakage-resistant blood vessels,⁴ and secondly, Ang1, delivered by an adenovirus expression system, inhibited the tissue edema induced by vascular endothelial growth factor (VEGF) and mustard oil, 2 powerful stimulators of vascular leak.⁵

Junctions between ECs give the structural basis for the regulation of the passage of plasma proteins or leukocytes into the tissues.⁶ Two junctional structures involved in this regulation include the tight junctions⁷ and the adherence junctions, which are altered by agents that induce EC permeability or which mediate leukocyte transmigration.^{8 9 10 11 12 13} Junctional proteins also serve to signal neighboring ECs to maintain their quiescent and anti-inflammatory phenotype. In particular, as endothelial junctions are established, platelet EC adhesion molecule-1 (PECAM-1) rapidly moves to these junctions,¹⁴ suppresses the expression of adhesion molecules such as E-selectin,¹⁵ and prevents EC apoptosis.¹⁶

We show here that Ang1 inhibits EC permeability in vitro and suggest that a likely mechanism is through the regulation of the junctional complexes, PECAM-1 and vascular endothelial (VE) cadherin.

	Materials and Methods

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Cells
Human umbilical vein ECs were grown and used as described.¹⁵ For VEGF-induced permeability, human umbilical artery ECs¹⁷ were used because they more consistently responded to VEGF₁₆₅.

Reagents
Ang1 used is as described.¹⁸ Anti-VE cadherin and PECAM-1 antibodies have been described.¹⁵

PECAM-1 Localization
Cells were fixed and stained for PECAM-1 as described.¹⁵ Images were captured using a Bio-Rad laser scanning confocal microscope (MR600). Images under comparison were subjected to equivalent amounts of contrast enhancement. At least 20 fields were analyzed for PECAM localization in each group. These were consecutive fields within the one well and contained 100 to 400 cells in total.

E-selectin expression was analyzed as described.¹⁵

The endothelial permeability assay was performed essentially as described.¹⁹

The neutrophil transendothelial cell migration assay was performed and quantified by an MTT colorimetric assay.^{20 21}

Immunoprecipitation
Human umbilical vein EC monolayers, confluent for at least 24 hours, were treated with control or Ang1. The lysis, extraction, and analysis were performed as described.⁹

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Ang1 Regulates PECAM-1 Localization to Cell Junctions
ECs plated at low density initially attach to the matrix, flatten, and establish contacts with neighboring cells. Pretreatment of cells with Ang1 did not change the number of cells that adhered to the matrix nor the initial single-cell morphology (not shown); however, it resulted in an enhanced PECAM-1 localization to the junctions (Figures 1A and 1B), suggesting that these junctions were forming more rapidly. At 45 minutes after cell plating, Ang1 did not affect the number of cell aggregates, the number of cells in each aggregate, or the expression of cell-surface PECAM-1 or VE cadherin (see online Table 1; online-only data supplement available at http://www.circresaha.org). However, the number of cell contacts that showed PECAM-1 localization increased from 55±5% to 75±5% (n=2 experiments) between control and Ang1 treatment, respectively, and the percentage of cells with the most intense junctional staining of PECAM-1 increased from 1% to 27% (see online Figure 1; online-only data supplement available at http://www.circresaha.org). The changes were specific for Ang1, given that the other angiogenic factors, VEGF and basic fibroblast growth factor, had no such effects (not shown). These data suggest that Ang1 may affect cell junctions, and therefore we extended our analysis to assays that involve junctional regulation.

View larger version (86K): [in this window] [in a new window]   Figure 1. Localization of PECAM-1 after treatment with Ang1. ECs were incubated with control (A) or 0.1 µg/mL Ang (B) for 10 minutes and then allowed to attach to fibronectin-coated slides. Sixty minutes later, the cells were stained for PECAM-1 expression.

Ang1 Inhibits E-Selectin Expression
PECAM-1 has been proposed as an important mechanosensing molecule in ECs,^{22 23} mediating inhibition of proliferation,²⁴ migration,²⁵ apoptosis,¹⁶ and expression of adhesion molecules.¹⁵ As such, the consequence of the enhanced PECAM-1–PECAM-1 engagement mediated by Ang1 should be to mature the cells into a quiescent, nonproliferative, noninflammatory phenotype. E-selectin expression is involved in inflammation,¹⁵ proliferation,²⁶ and angiogenesis,²⁷ and thus its presence can be considered a marker of an activated endothelium. Cells plated at low density express low but significant levels of E-selectin. The induction under this situation is cytokine independent but dependent on integrin attachment and the loss of PECAM-PECAM interaction.¹⁵ Treatment with Ang1 inhibited the E-selectin expression (Figures 2A and 2B) in a dose-dependent manner (see online Figure 2; online-only data supplement available at http://www.circresaha.org). Ang1 had no effect on tumor necrosis factor- (TNF-)–induced E-selectin expression (see online Table 2, groups a through d; online-only data supplement available at http://www.circresaha.org).

View larger version (16K): [in this window] [in a new window]   Figure 2. Ang1 downregulates E-selectin expression. ECs were treated with 0.1 µg/mL Ang1 (ANG) or vehicle (VEH) or left untreated (NIL) for 10 minutes, and then plated onto fibronectin-coated wells and cultured for 16 hours. Cells were then stained for E-selectin. A, Typical FACS profile from 1 individual experiment showing E-selectin expression. CTRL indicates cells stained with an irrelevant antibody. B, Mean fluorescence intensity for E-selectin from 6 experiments. Data are mean±SEM. *P<0.006 compared with vehicle (unpaired t test).

Ang1 Inhibits EC Permeability
Ang1 treatment of EC monolayers inhibited their basal permeability (Figure 3A) in a dose-dependent manner. More strikingly, Ang1 inhibited the permeability induced by 2 classic EC permeability-inducing agents, thrombin and VEGF (Figures 3B and 3C) by 70% and 100%, respectively. The phosphatidylinositol 3-kinase (PI3K) pathway, although implicated in Ang1-mediated EC survival,^{28 29} does not appear to be involved in the regulation of permeability. The PI3K-specific inhibitor LY294002 had no effect on Ang1-induced inhibition of permeability (online Figure 3; online-only data supplement available at http://www.circresaha.org), although it reversed, in parallel experiments, the protective effect of Ang1 on EC survival (not shown). This result suggests that an alternate signaling pathway to PI3K is used by the Tie2 receptor to mediate changes in cell junctions.

View larger version (26K): [in this window] [in a new window]   Figure 3. Ang1 inhibits EC permeability and TNF-–induced transmigration of neutrophils. The EC monolayer was treated for 30 minutes with Ang1 (Ang) or vehicle (Veh) before assay. A, Ang1 inhibits in a dose-dependent manner the basal permeability of ECs in the absence of exogenous stimulation. Data are mean±SEM of 3 to 5 experiments where each group was performed in duplicate or triplicate. *P<0.05 compared with no treatment. B, Ang1 inhibits thrombin-stimulated vascular permeability. Cells were stimulated in the upper well with 2 U/mL thrombin (T) for 15 minutes after 0.1 µg/mL Ang1 treatment. Data are mean±SEM from 2 experiments where each group was performed in duplicate. *P<0.02 compared with vehicle (Veh)+thrombin (paired t test). C, Ang1 inhibits the VEGF-stimulated vascular permeability. This was performed as for Figure 3B, except that stimulation was with 50 ng/mL VEGF (V) for 30 minutes. Data are mean±SEM from 3 experiments where each group was performed singly or in duplicate. *P<0.05 compared with Veh+VEGF (unpaired t test). D, Ang1 inhibits transmigration. EC monolayer was treated for 4 hours with TNF- and then for 30 minutes with 0.1 µg/mL Ang1 (Ang) or Vehicle (Veh). Mean±SEM as a percentage of cells that transmigrated is given for 4 experiments where each group was performed in duplicate or triplicate. *P<0.002 compared with Veh+TNF- (unpaired t test).

The transmigration of leukocytes induced by cytokines such as TNF- is also regulated by endothelial junctions.^{30 31 32 33} Ang1 pretreatment of ECs abolished TNF-–induced transmigration (Figure 3D). The Ang1 effect was not mediated through changes in TNF- signaling, because maximum inhibition of transmigration was seen when Ang1 was added at the end of the stimulation period 15 minutes before polymorphonuclear neutrophil addition. Under these conditions, Ang1 had no effect on the level of TNF-–induced E-selectin expression (see online Table 2, groups e and f; online-only data supplement available at http://www.circresaha.org). Furthermore, Ang1 treatment of the neutrophils did not alter their capacity to transmigrate (not shown).

The inhibitory effect of Ang1 on E-selectin expression, permeability, and transmigration was mediated through the Tie2 receptor, because the Tie2 Fc–soluble protein abolished these responses (see online Figures 4a through 4c; online-only data supplement available at http://www.circresaha.org).

View larger version (34K): [in this window] [in a new window]   Figure 4. Ang1 decreases basal VE cadherin and PECAM-1 phosphorylation. A, Cells were treated with vehicle control (C) or Ang1 (0.1 µg/mL) for 10 (A10) or 30 (A30) minutes. Lysates were immunoprecipitated with anti-VE cadherin–coated beads. Equivalent samples of immunocomplexes were blotted with an antiphosphotyrosine antibody, and the phosphorylation of VE cadherin (VE-C) and associated ß-catenin (B-cat) are shown (upper 2 bands). Gels were stripped and reprobed for VE cadherin and ß-catenin (lower 2 bands, respectively). B, Cells were treated with Ang1 or vehicle control for 30 minutes, immunoprecipitated with anti-PECAM-1–coated beads, blotted for antiphosphotyrosine (upper band), stripped, and reprobed for PECAM-1 (lower band).

Ang1 Alters VE Cadherin and PECAM-1 Phosphorylation
PECAM-1, VE cadherin, and its associated signaling molecules the catenins have been implicated in the regulation of EC junctions. Changes in phosphorylation of PECAM-1 and association of the catenins with VE cadherin are seen during histamine-, thrombin-, and VEGF-induced permeability and during polymorphonuclear neutrophil transmigration.^{8 9 10 11 12} Ang1 treatment for 10 minutes induced a decrease in the basal phosphorylation of VE cadherin, which returned to normal levels by 30 minutes (Figure 4A). In 2 experiments performed, the decrease was 33% and 45% normalized to VE cadherin content. Although no significant change in phosphorylation of ß-catenin was evident, an increase in the amount of ß-catenin associated with VE cadherin was observed. Ang1 also induced a significant decrease in basal PECAM-1 phosphorylation (Figure 4B). In 2 experiments performed, the decrease was 48% and 51% normalized to PECAM-1 content. The changes in phosphorylation of PECAM-1 and VE cadherin and increase in the association of ß-catenin with VE cadherin are consistent with an increase in cell-cell interaction.^{6 8 9 34}

	Discussion

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Enhanced interaction of ECs with their supporting structures has been the chief explanation for the angiogenic effects of Ang1 both in development and in vitro.^{2 18} The results presented here suggest that the additional mechanism of stabilization of cell-cell interactions may also be operative. Indeed, this multilevel effect of Ang1 compounds the antiproliferative and antiapoptotic effects of cell-cell and cell-matrix interactions.^{16 35 36 37 38 39 40}

Our in vitro findings highlight a potentially critical role for Ang1 in maintaining the integrity of endothelial monolayers in mature animals. To our knowledge, this is the first agent that, by an action on ECs, prevents the acute leakiness of blood vessels that is involved in the generation of swelling or edema seen in inflammatory or allergic reactions. Moreover, the degree of inhibition of between 70% and 100% suggests that this is a potentially potent and physiological mechanism. The effects on inhibition of permeability and transmigration, as well as on the prevention of cytokine-independent expression of adhesion proteins, are consistent with its effect on cellular junctions, and this is supported by the alteration in 2 important molecules involved in EC integrity, namely PECAM-1 and VE cadherin. However, other mechanisms of action cannot be ruled out. Nevertheless, these findings suggest a role for Ang1 from embryogenesis to adulthood and open the possibility of its therapeutic use in inflammatory diseases.

	Acknowledgments

 
This work was supported by grants from the National Health and Medical Research Council of Australia and the South Australian Anti-Cancer Foundation. We thank Anna Nitschke and Mari Walker for preparation of the manuscript.

Received June 12, 2000; revision received August 22, 2000; accepted August 23, 2000.

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				S. D. McCarter, S. H. J. Mei, P. F. H. Lai, Q. W. Zhang, C. H. Parker, R. S. Suen, R. D. Hood, Y. D. Zhao, Y. Deng, R. N. N. Han, et al. Cell-based Angiopoietin-1 Gene Therapy for Acute Lung Injury Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 1014 - 1026. [Abstract] [Full Text] [PDF]

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				G. Niu and W. B. Carter Human Epidermal Growth Factor Receptor 2 Regulates Angiopoietin-2 Expression in Breast Cancer via AKT and Mitogen-Activated Protein Kinase Pathways Cancer Res., February 15, 2007; 67(4): 1487 - 1493. [Abstract] [Full Text] [PDF]

				N. Kociok, S. Radetzky, T. U. Krohne, C. Gavranic, and A. M. Joussen Pathological but Not Physiological Retinal Neovascularization Is Altered in TNF-Rp55-Receptor-Deficient Mice Invest. Ophthalmol. Vis. Sci., November 1, 2006; 47(11): 5057 - 5065. [Abstract] [Full Text] [PDF]

				L. Dewachter, S. Adnot, E. Fadel, M. Humbert, B. Maitre, A.-M. Barlier-Mur, G. Simonneau, M. Hamon, R. Naeije, and S. Eddahibi Angiopoietin/Tie2 Pathway Influences Smooth Muscle Hyperplasia in Idiopathic Pulmonary Hypertension Am. J. Respir. Crit. Care Med., November 1, 2006; 174(9): 1025 - 1033. [Abstract] [Full Text] [PDF]

				A. C. Aplin, M. Gelati, E. Fogel, E. Carnevale, and R. F. Nicosia Angiopoietin-1 and vascular endothelial growth factor induce expression of inflammatory cytokines before angiogenesis Physiol Genomics, October 3, 2006; 27(1): 20 - 28. [Abstract] [Full Text] [PDF]

				R. Harfouche and S. N. A. Hussain Signaling and regulation of endothelial cell survival by angiopoietin-2 Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1635 - H1645. [Abstract] [Full Text] [PDF]

				A. I. Nykanen, K. Pajusola, R. Krebs, M. A.I. Keranen, O. Raisky, P. K. Koskinen, K. Alitalo, and K. B. Lemstrom Common Protective and Diverse Smooth Muscle Cell Effects of AAV-Mediated Angiopoietin-1 and -2 Expression in Rat Cardiac Allograft Vasculopathy Circ. Res., June 9, 2006; 98(11): 1373 - 1380. [Abstract] [Full Text] [PDF]

				S. D. McCarter, P. F. H. Lai, R. S. Suen, and D. J. Stewart Regulation of Endothelin-1 by Angiopoietin-1: Implications for Inflammation. Experimental Biology and Medicine, June 1, 2006; 231(6): 985 - 991. [Abstract] [Full Text] [PDF]

				I. Kalomenidis, A. Kollintza, I. Sigala, A. Papapetropoulos, S. Papiris, R. W. Light, and C. Roussos Angiopoietin-2 Levels Are Elevated in Exudative Pleural Effusions Chest, May 1, 2006; 129(5): 1259 - 1266. [Abstract] [Full Text] [PDF]

				N. P.J. Brindle, P. Saharinen, and K. Alitalo Signaling and Functions of Angiopoietin-1 in Vascular Protection Circ. Res., April 28, 2006; 98(8): 1014 - 1023. [Abstract] [Full Text] [PDF]

				E. Ryschich, P. Lizdenis, C. Ittrich, A. Benner, S. Stahl, A. Hamann, J. Schmidt, P. Knolle, B. Arnold, G. J. Hammerling, et al. Molecular Fingerprinting and Autocrine Growth Regulation of Endothelial Cells in a Murine Model of Hepatocellular Carcinoma Cancer Res., January 1, 2006; 66(1): 198 - 211. [Abstract] [Full Text] [PDF]

				D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]

				R. H. Adamson and F. E. Curry Ang-1: Tie-ing up endothelial adhesion? Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H74 - H76. [Full Text] [PDF]

				F. Baffert, T. Le, G. Thurston, and D. M. McDonald Angiopoietin-1 decreases plasma leakage by reducing number and size of endothelial gaps in venules Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H107 - H118. [Abstract] [Full Text] [PDF]

				F. Roviezzo, S. Tsigkos, A. Kotanidou, M. Bucci, V. Brancaleone, G. Cirino, and A. Papapetropoulos Angiopoietin-2 Causes Inflammation in Vivo by Promoting Vascular Leakage J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 738 - 744. [Abstract] [Full Text] [PDF]

				J.-K. Min, Y.-M. Kim, S. W. Kim, M.-C. Kwon, Y.-Y. Kong, I. K. Hwang, M. H. Won, J. Rho, and Y.-G. Kwon TNF-Related Activation-Induced Cytokine Enhances Leukocyte Adhesiveness: Induction of ICAM-1 and VCAM-1 via TNF Receptor-Associated Factor and Protein Kinase C-Dependent NF-{kappa}B Activation in Endothelial Cells J. Immunol., July 1, 2005; 175(1): 531 - 540. [Abstract] [Full Text] [PDF]

D. Jho, D. Mehta, G