1887

Abstract

A transient increase in trans-endothelial cell permeability in dengue patients leads to vascular leakage and shock syndrome. Here, we analysed the molecular mechanisms that cause permeability changes in human dermal microvascular endothelial cells (HMEC-1) using a direct dengue virus (DENV) infection model or treatment with NS1, a secreted DENV non-structural protein. In HMEC-1 cells, both treatments increase permeability with a concordant increase in the secretion of angiopoietin-2 (Ang-2). There is phosphorylation and loss of the junction protein VE-Cadherin from the inter-endothelial cell junctions and phosphorylation of RhoA. Direct virus infection results in activation of Src by phosphorylation, whereas NS1 treatment alone does not lead to Src activation. Furthermore, treatment with recombinant Ang-1, a physiological antagonist of Ang-2, prevents Ang-2 release, VE-Cadherin phosphorylation and internalization, and phosphorylation of RhoA and Src, resulting in restoration of barrier function. The permeability increase could also be prevented by blocking the Ang1/2 signalling receptor, Tie-2, or using a Rho/ROCK-specific inhibitor. Dasatinib, a Src-family kinase (SFK) inhibitor that inhibits Src phosphorylation, prevents enhanced permeability induced by direct DENV infection whereas in NS1 protein-treated cells its effect is less significant. The results provide important insights on the mechanisms of increased trans-endothelial permeability in DENV infection, and suggest the therapeutic potential of using recombinant Ang-1 or targeting these key molecules to prevent vascular leakage in dengue.

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2018-10-24
2024-12-08
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References

  1. Halstead SB. Dengue. Lancet 2007; 370:1644–1652 [View Article][PubMed]
    [Google Scholar]
  2. Noisakran S, Chokephaibulkit K, Songprakhon P, Onlamoon N, Hsiao HM et al. A re-evaluation of the mechanisms leading to dengue hemorrhagic fever. Ann N Y Acad Sci 2009; 1171:E24E35 [View Article][PubMed]
    [Google Scholar]
  3. Kurane I. Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comp Immunol Microbiol Infect Dis 2007; 30:329–340 [View Article][PubMed]
    [Google Scholar]
  4. Srikiatkhachorn A, Ajariyakhajorn C, Endy TP, Kalayanarooj S, Libraty DH et al. Virus-induced decline in soluble vascular endothelial growth receptor 2 is associated with plasma leakage in dengue hemorrhagic Fever. J Virol 2007; 81:1592–1600 [View Article][PubMed]
    [Google Scholar]
  5. Chuang YC, Lei HY, Liu HS, Lin YS, Fu TF et al. Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability. Cytokine 2011; 54:222–231 [View Article][PubMed]
    [Google Scholar]
  6. Furuta T, Murao LA, Lan NT, Huy NT, Huong VT et al. Association of mast cell-derived VEGF and proteases in Dengue shock syndrome. PLoS Negl Trop Dis 2012; 6:e1505 [View Article][PubMed]
    [Google Scholar]
  7. Michels M, van der Ven AJ, Djamiatun K, Fijnheer R, de Groot PG et al. Imbalance of angiopoietin-1 and angiopoetin-2 in severe dengue and relationship with thrombocytopenia, endothelial activation, and vascular stability. Am J Trop Med Hyg 2012; 87:943–946 [View Article][PubMed]
    [Google Scholar]
  8. Ong SP, Ng ML, Chu JJ. Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability. Med Microbiol Immunol 2013; 202:437–452 [View Article][PubMed]
    [Google Scholar]
  9. Anderson R, Wang S, Osiowy C, Issekutz AC. Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes. J Virol 1997; 71:4226–4232[PubMed]
    [Google Scholar]
  10. Avirutnan P, Zhang L, Punyadee N, Manuyakorn A, Puttikhunt C et al. Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E. PLoS Pathog 2007; 3:e183 [View Article][PubMed]
    [Google Scholar]
  11. Beatty PR, Puerta-Guardo H, Killingbeck SS, Glasner DR, Hopkins K et al. Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med 2015; 304:ra141
    [Google Scholar]
  12. Modhiran N, Watterson D, Muller DA, Panetta AK, Sester DP et al. Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med 2015; 7:304ra142 [View Article][PubMed]
    [Google Scholar]
  13. Glasner DR, Ratnasiri K, Puerta-Guardo H, Espinosa DA, Beatty PR et al. Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components. PLoS Pathog 2017; 13:e1006673 [View Article][PubMed]
    [Google Scholar]
  14. Chen HR, Chuang YC, Lin YS, Liu HS, Liu CC. Dengue virus nonstructural protein 1 induces vascular leakage through macrophage migration inhibitory factor and autophagy. PLoS Negl Trop Dis 2016; 10:e0004828 [View Article][PubMed]
    [Google Scholar]
  15. Milam KE, Parikh SM. The angiopoietin-Tie2 signaling axis in the vascular leakage of systemic inflammation. Tissue Barriers 2015; 3:e957508 [View Article][PubMed]
    [Google Scholar]
  16. Nwariaku FE, Liu Z, Zhu X, Nahari D, Ingle C et al. NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction. Blood 2004; 104:3214–3220 [View Article][PubMed]
    [Google Scholar]
  17. Azzi S, Hebda JK, Gavard J. Vascular permeability and drug delivery in cancers. Front Oncol 2013; 3:211 [View Article][PubMed]
    [Google Scholar]
  18. Gavard J, Patel V, Gutkind JS. Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia. Dev Cell 2008; 14:25–36 [View Article][PubMed]
    [Google Scholar]
  19. Giannotta M, Trani M, Dejana E. VE-cadherin and endothelial adherens junctions: active guardians of vascular integrity. Dev Cell 2013; 26:441–454 [View Article][PubMed]
    [Google Scholar]
  20. Mikelis CM, Simaan M, Ando K, Fukuhara S, Sakurai A et al. RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock. Nat Commun 2015; 6:6725 [View Article][PubMed]
    [Google Scholar]
  21. Brindle NP, Saharinen P, Alitalo K. Signaling and functions of angiopoietin-1 in vascular protection. Circ Res 2006; 98:1014–1023 [View Article][PubMed]
    [Google Scholar]
  22. Parikh SM, Mammoto T, Schultz A, Yuan HT, Christiani D et al. Excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans. PLoS Med 2006; 3:e46 [View Article][PubMed]
    [Google Scholar]
  23. Fiedler U, Scharpfenecker M, Koidl S, Hegen A, Grunow V et al. The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood 2004; 103:4150–4156 [View Article][PubMed]
    [Google Scholar]
  24. Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med 2006; 12:235–239 [View Article][PubMed]
    [Google Scholar]
  25. Gavrilovskaya IN, Gorbunova EE, Mackow NA, Mackow ER. Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability. J Virol 2008; 82:5797–5806 [View Article][PubMed]
    [Google Scholar]
  26. van de Weg CA, Pannuti CS, van den Ham HJ, de Araújo ES, Boas LS et al. Serum angiopoietin-2 and soluble VEGF receptor 2 are surrogate markers for plasma leakage in patients with acute dengue virus infection. J Clin Virol 2014; 60:328–335 [View Article][PubMed]
    [Google Scholar]
  27. Ades EW, Candal FJ, Swerlick RA, George VG, Summers S et al. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol 1992; 99:683–690 [View Article][PubMed]
    [Google Scholar]
  28. Peyrefitte CN, Pastorino B, Grau GE, Lou J, Tolou H et al. Dengue virus infection of human microvascular endothelial cells from different vascular beds promotes both common and specific functional changes. J Med Virol 2006; 78:229–242 [View Article][PubMed]
    [Google Scholar]
  29. Singh S, Anupriya MG, Sreekumar E. Comparative whole genome analysis of dengue virus serotype-2 strains differing in trans-endothelial cell leakage induction in vitro. Infect Genet Evol 2017; 52:34–43 [View Article][PubMed]
    [Google Scholar]
  30. Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelial permeability. Vascul Pharmacol 2002; 39:187–199 [View Article][PubMed]
    [Google Scholar]
  31. Wallez Y, Cand F, Cruzalegui F, Wernstedt C, Souchelnytskyi S et al. Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site. Oncogene 2007; 26:1067–1077 [View Article][PubMed]
    [Google Scholar]
  32. Wessel F, Winderlich M, Holm M, Frye M, Rivera-Galdos R et al. Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin. Nat Immunol 2014; 15:223–230 [View Article][PubMed]
    [Google Scholar]
  33. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 2000; 57:976–983[PubMed]
    [Google Scholar]
  34. Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC et al. Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem 2004; 47:6658–6661 [View Article][PubMed]
    [Google Scholar]
  35. Gorbunova EE, Gavrilovskaya IN, Pepini T, Mackow ER. VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability. J Virol 2011; 85:2296–2303 [View Article][PubMed]
    [Google Scholar]
  36. Krishnamurti C, Kalayanarooj S, Cutting MA, Peat RA, Rothwell SW et al. Mechanisms of hemorrhage in dengue without circulatory collapse. Am J Trop Med Hyg 2001; 65:840–847 [View Article][PubMed]
    [Google Scholar]
  37. Jacobs M, Levin M. An improved endothelial barrier model to investigate dengue haemorrhagic fever. J Virol Methods 2002; 104:173–185 [View Article][PubMed]
    [Google Scholar]
  38. Dewi BE, Takasaki T, Kurane I. In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection. J Virol Methods 2004; 121:171–180 [View Article][PubMed]
    [Google Scholar]
  39. Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002; 186:1165–1168 [View Article][PubMed]
    [Google Scholar]
  40. Komarova Y, Malik AB. Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu Rev Physiol 2010; 72:463–493 [View Article][PubMed]
    [Google Scholar]
  41. Kanlaya R, Pattanakitsakul SN, Sinchaikul S, Chen ST, Thongboonkerd V. Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration. J Proteome Res 2009; 8:2551–2562 [View Article][PubMed]
    [Google Scholar]
  42. Gavard J. Endothelial permeability and VE-cadherin: a wacky comradeship. Cell Adh Migr 2013; 7:455–461 [View Article][PubMed]
    [Google Scholar]
  43. Turowski P, Martinelli R, Crawford R, Wateridge D, Papageorgiou AP et al. Phosphorylation of vascular endothelial cadherin controls lymphocyte emigration. J Cell Sci 2008; 121:29–37 [View Article][PubMed]
    [Google Scholar]
  44. Lambeng N, Wallez Y, Rampon C, Cand F, Christé G et al. Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues. Circ Res 2005; 96:384–391 [View Article][PubMed]
    [Google Scholar]
  45. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005; 437:497–504 [View Article][PubMed]
    [Google Scholar]
  46. Baumeister U, Funke R, Ebnet K, Vorschmitt H, Koch S et al. Association of Csk to VE-cadherin and inhibition of cell proliferation. EMBO J 2005; 24:1686–1695 [View Article][PubMed]
    [Google Scholar]
  47. Braga VM, Machesky LM, Hall A, Hotchin NA. The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J Cell Biol 1997; 137:1421–1431 [View Article][PubMed]
    [Google Scholar]
  48. van Hinsbergh VW, van Nieuw Amerongen GP. Intracellular signalling involved in modulating human endothelial barrier function. J Anat 2002; 200:549–560 [View Article][PubMed]
    [Google Scholar]
  49. Nelson CM, Pirone DM, Tan JL, Chen CS. Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA. Mol Biol Cell 2004; 15:2943–2953 [View Article][PubMed]
    [Google Scholar]
  50. Adam AP, Sharenko AL, Pumiglia K, Vincent PA. Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to decrease barrier function of endothelial monolayers. J Biol Chem 2010; 285:7045–7055 [View Article][PubMed]
    [Google Scholar]
  51. Saharinen P, Eklund L, Alitalo K. Therapeutic targeting of the angiopoietin-TIE pathway. Nat Rev Drug Discov 2017; 16:635–661 [View Article][PubMed]
    [Google Scholar]
  52. van der Heijden M, van Nieuw Amerongen GP, Chedamni S, van Hinsbergh VW, Johan Groeneveld AB. The angiopoietin-Tie2 system as a therapeutic target in sepsis and acute lung injury. Expert Opin Ther Targets 2009; 13:39–53 [View Article][PubMed]
    [Google Scholar]
  53. Manakkadan A, Joseph I, Prasanna RR, Kunju RI, Kailas L et al. Lineage shift in Indian strains of Dengue virus serotype-3 (Genotype III), evidenced by detection of lineage IV strains in clinical cases from Kerala. Virol J 2013; 10:37 [View Article][PubMed]
    [Google Scholar]
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