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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.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell. 1996;87:161–1169.

2. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996;87:1171–1180.[Medline] [Order article via Infotrieve]

3. Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995;376:70–74.[Medline] [Order article via Infotrieve]

4. Thurston G, Suri G, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM. Leakage resistant blood vessels in mice transgenically over-expressing angiopoietin-1. Science. 1999;286:2511–2514.[Abstract/Free Full Text]

5. Thurston G, Rudge JS, Ioffe E, Zhou H, Mahon S, Glazer N, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460–463.[Medline] [Order article via Infotrieve]

6. Dejana E. Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis. J Clin Invest. 1996;98:1949–1953.[Medline] [Order article via Infotrieve]

7. Hirase T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, Rubin LL. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci. 1997;110:1603–1613.[Abstract]

8. Paraskevi A, Navarro P, Zanetti A, Lampugnani GM, Dejana E. Histamine induces tyrosine phosphorylation of endothelial cell-to-cell adherens junctions. Arterioscler Thromb Vasc Biol. 1999;7180:2286–2297.

9. Esser S, Lampugnani MG, Corada M, Dejana E, Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci. 1998;111:1853–1865.[Abstract]

10. Kevil CG, Payne DK, Mire E, Alexander JS. Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins. J Biol Chem. 1998;273:15099–15103.[Abstract/Free Full Text]

11. Allport JR, Ding H, Collins T, Gerritsen ME, Luscinkas FW. Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteosome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions. J Exp Med. 1997;186:517–527.[Abstract/Free Full Text]

12. Allport JR, Muller WA, Luscinskas FW. Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow. J Cell Biol. 1999;148:203–216.[Abstract/Free Full Text]

13. Corada M, Mariotti M, Thurston G, Smith K, Kunkel R, Brockhaus M, Lampugnani MG, Martin-Padura I, Stoppacciaro A, Ruco L, McDonald DM, Ward PA, Dejana E. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc Natl Acad Sci U S A. 1999;96:9815–9820.[Abstract/Free Full Text]

14. Ayalon O, Sabanai H, Lampugnani MG, Dejana E, Geiger B. Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells. J Cell Biol. 1994;126:247–258.[Abstract/Free Full Text]

15. Litwin M, Clark K, Noack L, Furze J, Berndt M, Albelda S, Vadas M, Gamble J. Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule CD31. J Cell Biol. 1997;139:219–228.[Abstract/Free Full Text]

16. Bird IN, Taylor V, Newton JP, Spragg JH, Simmons DL, Salmon M, Buckley CD. Homophilic PECAM-1 CD31 interactions prevent endothelial cell apoptosis but do not support cell spreading or migration. J Cell Sci. 1999;112:1989–1997.[Abstract]

17. Underwood PA, Bean PA, Whitelock JM. Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen. Atherosclerosis. 1998;141:141–152.[Medline] [Order article via Infotrieve]

18. Papapetropoulos A, García-Cardeña G, Dengler TJ, Maisonpierre PC, Yancopoulos GD, Sessa WC. Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest. 1999;79:213–223.[Medline] [Order article via Infotrieve]

19. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani M, Dejana E. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol.. 1996;16:488–496.[Abstract/Free Full Text]

20. Smith WB, Noack L, Khew-Goodall Y, Isenmann S, Vadas MA, Gamble JR. Transforming growth factor-ß1 inhibits the production of IL-8 and the transmigration of neutrophils through activated endothelium. J Immunol. 1996;157:360–368.[Abstract]

21. Xia P, Wang L, Gamble JR, Vadas MA. Activation of sphingosine kinase by tumor necrosis factor-ß inhibits apoptosis in human endothelial cells. J Biol Chem. 1999;274:33143–33147.[Abstract/Free Full Text]

22. Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest. 1999;103:5–9.[Medline] [Order article via Infotrieve]

23. Newman PJ. Cell adhesion in vascular biology: the biology of PECAM-1. J Clin Invest. 1997;99:3–8.[Medline] [Order article via Infotrieve]

24. Fawcett J, Buckley C D, Holness CL, Bird IN, Spragg JH, Saunders J, Harris A, Simmons DL. Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts. J Cell Biol. 1995;128:1229–1241.[Abstract/Free Full Text]

25. Kim CS, Wang T, Madri JA. Platelet endothelial cell adhesion molecule-1 expression modulates endothelial cell migration in vitro. Lab Invest. 1998;78:583–590.[Medline] [Order article via Infotrieve]

26. Luo J, Paranya G, Bischoff J. Non-inflammatory expression of E-selectin is regulated by cell growth. Blood. 1999;93:3785–3791.[Abstract/Free Full Text]

27. Koch AE, Halloran MM, Haskell CJ, Shah MR, Polverini PJ. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature. 1995;376:517–519.[Medline] [Order article via Infotrieve]

28. Fujikawa K, de Aos Scherpenseel I, Jain SK, Presman E, Christensen RA, Varticovski L. Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells. Exp Cell Res. 1999;253:663–672.[Medline] [Order article via Infotrieve]

29. Kim I, Kim HG, So JN, Kim JH, Kwak HJ, Koh GY. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3-kinase/Akt signal transduction pathway. Circ Res. 2000;86:24–29.[Abstract/Free Full Text]

30. Moser R, Schleiffenbaum B, Groscurth P, Fehr J. Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J Clin Invest. 1989;83:444–55.

31. Furie MB, McHugh DD. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor. J Immunol. 1989;143:3309–3317.[Abstract]

32. Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med. 1993;178:449–460.[Abstract/Free Full Text]

33. Vaporciyan AA, DeLisser HM, Yan HC, Mendiguren II, Thom SR, Jones ML, Ward PA, Albelda SM. Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science. 1993;262:1580–1582.[Abstract/Free Full Text]

34. Zehnder JL, Hirai K, Shatsky M, McGregor JL, Levitt LJ, Leung LLK. The cell adhesion molecule CD31 is phosphorylated after cell activation. J Biol Chem. 1992;267:5243–5249.[Abstract/Free Full Text]

35. Caveda L, Martin-Padura I, Navarro P, Breviario F, Corada M, Gulino D, Lampugnani MG, Dejana E. Inhibition of cultured cell growth by vascular endothelial cadherin cadherin-5/VE-cadherin. J Clin Invest. 1996;98:886–893.[Medline] [Order article via Infotrieve]

36. Sheibani N, Frazier WA. Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype. Mol Biol Cell. 1998;9:701–713.[Abstract/Free Full Text]

37. Stromblad S, Cheresh DA. Integrins, angiogenesis and vascular cell survival. Chem Biol. 1996;3:881–885.[Medline] [Order article via Infotrieve]

38. Ingber DE. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc Natl Acad Sci U S A. 1990;87:3579–3583.[Abstract/Free Full Text]

39. Re F, Zanetti A, Sironia M, Polentarutti N, Lanfrancone L, Dejana E, Colotta F. Inhibition of anchorage dependent cell spreading triggers apoptosis in cultured endothelial cells. J Cell Biol. 1994;127:537–546.[Abstract/Free Full Text]

40. Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G, Defilippi P. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 1998;16:6622–6632.

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				X. Li, M. Stankovic, C. S. Bonder, C. N. Hahn, M. Parsons, S. M. Pitson, P. Xia, R. L. Proia, M. A. Vadas, and J. R. Gamble Basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1 Blood, April 1, 2008; 111(7): 3489 - 3497. [Abstract] [Full Text] [PDF]

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				T. Mammoto, S. M. Parikh, A. Mammoto, D. Gallagher, B. Chan, G. Mostoslavsky, D. E. Ingber, and V. P. Sukhatme Angiopoietin-1 Requires p190 RhoGAP to Protect against Vascular Leakage in Vivo J. Biol. Chem., August 17, 2007; 282(33): 23910 - 23918. [Abstract] [Full Text] [PDF]

				A. S. Williams, R. Issa, S. Y. Leung, P. Nath, G. D. Ferguson, B. L. Bennett, I. M. Adcock, and K. F. Chung Attenuation of Ozone-Induced Airway Inflammation and Hyper-Responsiveness by c-Jun NH2 Terminal Kinase Inhibitor SP600125 J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 351 - 359. [Abstract] [Full Text] [PDF]

				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]

				S. Hughes, T. Gardiner, L. Baxter, and T. Chan-Ling Changes in Pericytes and Smooth Muscle Cells in the Kitten Model of Retinopathy of Prematurity: Implications for Plus Disease Invest. Ophthalmol. Vis. Sci., March 1, 2007; 48(3): 1368 - 1379. [Abstract] [Full Text] [PDF]

				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. Ahmmed, X.-P. Gao, C. Tiruppathi, M. Broman, and A. B. Malik Angiopoietin-1 Opposes VEGF-Induced Increase in Endothelial Permeability by Inhibiting TRPC1-Dependent Ca2 Influx Circ. Res., June 24, 2005; 96(12): 1282 - 1290. [Abstract] [Full Text] [PDF]

				C. C. Weber, H. Cai, M. Ehrbar, H. Kubota, G. Martiny-Baron, W. Weber, V. Djonov, E. Weber, A. S. Mallik, M. Fussenegger, et al. Effects of Protein and Gene Transfer of the Angiopoietin-1 Fibrinogen-like Receptor-binding Domain on Endothelial and Vessel Organization J. Biol. Chem., June 10, 2005; 280(23): 22445 - 22453. [Abstract] [Full Text] [PDF]

				V. Limaye, X. Li, C. Hahn, P. Xia, M. C. Berndt, M. A. Vadas, and J. R. Gamble Sphingosine kinase-1 enhances endothelial cell survival through a PECAM-1-dependent activation of PI-3K/Akt and regulation of Bcl-2 family members Blood, April 15, 2005; 105(8): 3169 - 3177. [Abstract] [Full Text] [PDF]

				J I Patel, P G Hykin, Z J Gregor, M Boulton, and I A Cree Angiopoietin concentrations in diabetic retinopathy Br. J. Ophthalmol., April 1, 2005; 89(4): 480 - 483. [Abstract] [Full Text] [PDF]

				M. Scharpfenecker, U. Fiedler, Y. Reiss, and H. G. Augustin The Tie-2 ligand Angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism J. Cell Sci., February 15, 2005; 118(4): 771 - 780. [Abstract] [Full Text] [PDF]

				J. E. Markkanen, T. T. Rissanen, A. Kivela, and S. Yla-Herttuala Growth factor-induced therapeutic angiogenesis and arteriogenesis in the heart-gene therapy Cardiovasc Res, February 15, 2005; 65(3): 656 - 664. [Abstract] [Full Text] [PDF]

				C. Lemieux, R. Maliba, J. Favier, J.-F. Theoret, Y. Merhi, and M. G. Sirois Angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses Blood, February 15, 2005; 105(4): 1523 - 1530. [Abstract] [Full Text] [PDF]

				D. W. Gilroy and P. Vallance Resolution for Sepsis? Circulation, January 4, 2005; 111(1): 2 - 4. [Full Text] [PDF]

				B. Witzenbichler, D. Westermann, S. Knueppel, H.-P. Schultheiss, and C. Tschope Protective Role of Angiopoietin-1 in Endotoxic Shock Circulation, January 4, 2005; 111(1): 97 - 105. [Abstract] [Full Text] [PDF]

				S. Fukuhara, A. Sakurai, H. Sano, A. Yamagishi, S. Somekawa, N. Takakura, Y. Saito, K. Kangawa, and N. Mochizuki Cyclic AMP Potentiates Vascular Endothelial Cadherin-Mediated Cell-Cell Contact To Enhance Endothelial Barrier Function through an Epac-Rap1 Signaling Pathway Mol. Cell. Biol., January 1, 2005; 25(1): 136 - 146. [Abstract] [Full Text] [PDF]

				M. Mura, C. C. dos Santos, D. Stewart, and M. Liu Vascular endothelial growth factor and related molecules in acute lung injury J Appl Physiol, November 1, 2004; 97(5): 1605 - 1617. [Abstract] [Full Text] [PDF]

				Y. Xu, Y.-j. Liu, and Q. Yu Angiopoietin-3 Is Tethered on the Cell Surface via Heparan Sulfate Proteoglycans J. Biol. Chem., September 24, 2004; 279(39): 41179 - 41188. [Abstract] [Full Text] [PDF]

				X. Li, C. N. Hahn, M. Parsons, J. Drew, M. A. Vadas, and J. R. Gamble Role of protein kinase C{zeta} in thrombin-induced endothelial permeability changes: inhibition by angiopoietin-1 Blood, September 15, 2004; 104(6): 1716 - 1724. [Abstract] [Full Text] [PDF]

				U. Fiedler, M. Scharpfenecker, S. Koidl, A. Hegen, V. Grunow, J. M. Schmidt, W. Kriz, G. Thurston, and H. G. Augustin The Tie-2 ligand Angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies Blood, June 1, 2004; 103(11): 4150 - 4156. [Abstract] [Full Text] [PDF]

				S. C. Satchell, K. L. Anderson, and P. W. Mathieson Angiopoietin 1 and Vascular Endothelial Growth Factor Modulate Human Glomerular Endothelial Cell Barrier Properties J. Am. Soc. Nephrol., March 1, 2004; 15(3): 566 - 574. [Abstract] [Full Text] [PDF]

				P. Kouklis, M. Konstantoulaki, S. Vogel, M. Broman, and A. B. Malik Cdc42 Regulates the Restoration of Endothelial Barrier Function Circ. Res., February 6, 2004; 94(2): 159 - 166. [Abstract] [Full Text] [PDF]

				K. G. Peters, C. D. Kontos, P. C. Lin, A. L. Wong, P. Rao, L. Huang, M. W. Dewhirst, and S. Sankar Functional Significance of Tie2 Signaling in the Adult Vasculature Recent Prog. Horm. Res., January 1, 2004; 59(1): 51 - 71. [Abstract] [Full Text]

				P. Dandona, A. Aljada, P. Mohanty, H. Ghanim, A. Bandyopadhyay, and A. Chaudhuri Insulin Suppresses Plasma Concentration of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 Diabetes Care, December 1, 2003; 26(12): 3310 - 3314. [Abstract] [Full Text] [PDF]

				O. Stoeltzing, S. A. Ahmad, W. Liu, M. F. McCarty, J. S. Wey, A. A. Parikh, F. Fan, N. Reinmuth, M. Kawaguchi, C. D. Bucana, et al. Angiopoietin-1 Inhibits Vascular Permeability, Angiogenesis, and Growth of Hepatic Colon Cancer Tumors Cancer Res., June 15, 2003; 63(12): 3370 - 3377. [Abstract] [Full Text] [PDF]

				B. H. Jeon, F. Khanday, S. Deshpande, A. Haile, M. Ozaki, and K. Irani Tie-ing the Antiinflammatory Effect of Angiopoietin-1 to Inhibition of NF-{kappa}B Circ. Res., April 4, 2003; 92(6): 586 - 588. [Full Text] [PDF]

				D. P. Hughes, M. B. Marron, and N. P.J. Brindle The Antiinflammatory Endothelial Tyrosine Kinase Tie2 Interacts With a Novel Nuclear Factor-{kappa}B Inhibitor ABIN-2 Circ. Res., April 4, 2003; 92(6): 630 - 636. [Abstract] [Full Text] [PDF]

				A. I. Nykanen, R. Krebs, A. Saaristo, P. Turunen, K. Alitalo, S. Yla-Herttuala, P. K. Koskinen, and K. B. Lemstrom Angiopoietin-1 Protects Against the Development of Cardiac Allograft Arteriosclerosis Circulation, March 11, 2003; 107(9): 1308 - 1314. [Abstract] [Full Text] [PDF]

				A. Siflinger-Birnboim and A. Johnson Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L435 - L451. [Abstract] [Full Text] [PDF]

				D. Karmpaliotis, I. Kosmidou, E. P. Ingenito, K. Hong, A. Malhotra, M. E. Sunday, and K. J. Haley Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L585 - L595. [Abstract] [Full Text] [PDF]

				S. C. Satchell, S. J. Harper, J. E. Tooke, D. Kerjaschki, M. A. Saleem, and P. W. Mathieson Human Podocytes Express Angiopoietin 1, a Potential Regulator of Glomerular Vascular Endothelial Growth Factor J. Am. Soc. Nephrol., February 1, 2002; 13(2): 544 - 550. [Abstract] [Full Text] [PDF]

				D. A. Long, A. S. Woolf, T. Suda, and H. T. Yuan Increased Renal Angiopoietin-1 Expression in Folic Acid-Induced Nephrotoxicity in Mice J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2721 - 2731. [Abstract] [Full Text] [PDF]

				S. M. Dudek and J. G. N. Garcia Cytoskeletal regulation of pulmonary vascular permeability J Appl Physiol, October 1, 2001; 91(4): 1487 - 1500. [Abstract] [Full Text] [PDF]

				A. Tedgui and Z. Mallat Anti-Inflammatory Mechanisms in the Vascular Wall Circ. Res., May 11, 2001; 88(9): 877 - 887. [Abstract] [Full Text] [PDF]

				I. Kim, S.-O. Moon, S. K. Park, S. W. Chae, and G. Y. Koh Angiopoietin-1 Reduces VEGF-Stimulated Leukocyte Adhesion to Endothelial Cells by Reducing ICAM-1, VCAM-1, and E-Selectin Expression Circ. Res., September 14, 2001; 89(6): 477 - 479. [Abstract] [Full Text] [PDF]

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