Endothelial Cells Exhibit Two Waves of P-selectin Surface Aggregation Under Endotoxic and Oxidative Conditions

  • Nathaly Barrionuevo
  • Sebastian Gatica
  • Pedro Olivares
  • Claudio Cabello-Verrugio
  • Felipe SimonEmail author


Sepsis is a clinical syndrome characterized by the presence of circulating microbial endotoxins and oxidative stress. Endotoxin and oxidative stress activate endothelial cells via a convergent signaling pathway (TLR4/MyD88/PI3 K/PLCɣ/NF-B) that stimulates both the transcription of SELP gene (which encodes for human P-selectin) and the release of P-selectin from Weibel–Palade bodies (WPBs). However, time course pattern of P-selectin surface aggregation has not been established in endothelial cells under 24 h of endotoxic or oxidative stress. Our study shows that P-selectin has at least two waves of aggregation at the cell surface: one 10 min and the other 12 h after endotoxic or oxidative stress. The first wave depends exclusively on WPB delivery to the cell membrane, while the second depends on P-selectin translation machinery, ER–Golgi sorting, and WPB surface delivery. Understanding adhesion molecule dynamics in endothelial cells could provide further molecular insights to develop diagnostic or therapeutic tools to aid in the management of sepsis.


Adhesion molecules Endothelial cells Oxidative stress Endotoxemia Sepsis 



Endoplasmic reticulum


Human umbilical vein endothelial cell






Nuclear factor–kappa B


Phospholipase C gamma


Reactive oxygen species


Tissue factor


Toll-like receptor 4


Tumor necrosis factor–⍺


Von Willebrand Factor


Weibel–Palade bodies



This work was supported by research grants from Fondo Nacional de Desarrollo Científico y Tecnológico—FONDECYT 1161288, and 1161646, Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) PhD Scholarship 21171566, Millennium Institute on Immunology and Immunotherapy P09-016-F. Programa de Cooperación Científica ECOS-CONICYT C16S02. BASAL Grant CEDENNA FB0807. The Millennium Nucleus of Ion Channels-Associated Diseases (MiNICAD) is a Millennium Nucleus supported by the Iniciativa Científica Milenio of the Ministry of Economy, Development and Tourism (Chile).


  1. 1.
    Singer M, Deutschman CS, Seymour CW et al (2016) The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA 315:801–810. CrossRefGoogle Scholar
  2. 2.
    Paoli CJ, Reynolds MA, Sinha M et al (2018) Epidemiology and costs of sepsis in the United States—an analysis based on timing of diagnosis and severity level. Crit Care Med 46:1889–1897. CrossRefGoogle Scholar
  3. 3.
    Angus DC, van der Poll T (2013) Severe sepsis and septic shock. N Engl J Med 369:840–851. CrossRefGoogle Scholar
  4. 4.
    Dauphinee SM, Karsan A (2006) Lipopolysaccharide signaling in endothelial cells. Lab Invest 86:9–22. CrossRefGoogle Scholar
  5. 5.
    Lewis AJ, Seymour CW, Rosengart MR (2016) Current murine models of sepsis. Surg Infect 17:385–393. CrossRefGoogle Scholar
  6. 6.
    Echeverría C, Montorfano I, Tapia P et al (2014) Endotoxin-induced endothelial fibrosis is dependent on expression of transforming growth factors β1 and β2. Infect Immun 82:3678–3686. CrossRefGoogle Scholar
  7. 7.
    Pérez L, Muñoz-Durango N, Riedel CA et al (2017) Endothelial-to-mesenchymal transition: cytokine-mediated pathways that determine endothelial fibrosis under inflammatory conditions. Cytokine Growth Factor Rev 33:41–54. CrossRefGoogle Scholar
  8. 8.
    Simon F, Fernández R (2009) Early lipopolysaccharide-induced reactive oxygen species production evokes necrotic cell death in human umbilical vein endothelial cells. J Hypertens 27:1202–1216. CrossRefGoogle Scholar
  9. 9.
    Nuñez-Villena F, Becerra A, Echeverría C et al (2011) Increased expression of the transient receptor potential melastatin 7 channel is critically involved in lipopolysaccharide-induced reactive oxygen species-mediated neuronal death. Antioxid Redox Signal 15:2425–2438. CrossRefGoogle Scholar
  10. 10.
    Galley HF (2011) Oxidative stress and mitochondrial dysfunction in sepsis. Br J Anaesth 107:57–64. CrossRefGoogle Scholar
  11. 11.
    Pan J, McEver RP (1995) Regulation of the human P-selectin promoter by Bcl-3 and specific homodimeric members of the NF-kappa B/Rel family. J Biol Chem 270:23077–23083CrossRefGoogle Scholar
  12. 12.
    Gotsch U, Jäger U, Dominis M, Vestweber D (1994) Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo. Cell Adhes Commun 2:7–14. CrossRefGoogle Scholar
  13. 13.
    McEver RP, Beckstead JH, Moore KL et al (1989) GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 84:92–99. CrossRefGoogle Scholar
  14. 14.
    Rollin S, Lemieux C, Maliba R et al (2004) VEGF-mediated endothelial P-selectin translocation: role of VEGF receptors and endogenous PAF synthesis. Blood 103:3789–3797. CrossRefGoogle Scholar
  15. 15.
    Kiskin NI, Babich V, Knipe L et al (2014) Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane. PLoS ONE 9:e108093. CrossRefGoogle Scholar
  16. 16.
    Babich V, Meli A, Knipe L et al (2008) Selective release of molecules from Weibel-Palade bodies during a lingering kiss. Blood 111:5282–5290. CrossRefGoogle Scholar
  17. 17.
    Nightingale TD, McCormack JJ, Grimes W et al (2018) Tuning the endothelial response: differential release of exocytic cargos from Weibel-Palade bodies. J Thromb Haemost 16:1873–1886. CrossRefGoogle Scholar
  18. 18.
    Lorenzon P, Vecile E, Nardon E et al (1998) Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors. J Cell Biol 142:1381–1391CrossRefGoogle Scholar
  19. 19.
    Chen X, Cheng Z, Werling D et al (2016) Bovine P-selectin mediates leukocyte adhesion and is highly polymorphic in dairy breeds. Res Vet Sci 108:85–92. CrossRefGoogle Scholar
  20. 20.
    Grooby WL, Krishnan R, Russ GR (1997) Characterization of ovine umbilical vein endothelial cells and their expression of cell adhesion molecules: comparative study with human endothelial cells. Immunol Cell Biol 75:21–28. CrossRefGoogle Scholar
  21. 21.
    Doré M, Sirois J (1996) Regulation of P-selectin expression by inflammatory mediators in canine jugular endothelial cells. Vet Pathol 33:662–671. CrossRefGoogle Scholar
  22. 22.
    Stocker CJ, Sugars KL, Harari OA et al (2000) TNF-alpha, IL-4, and IFN-gamma regulate differential expression of P- and E-selectin expression by porcine aortic endothelial cells. J Immunol 164:3309–3315CrossRefGoogle Scholar
  23. 23.
    Yao L, Setiadi H, Xia L et al (1999) Divergent inducible expression of P-selectin and E-selectin in mice and primates. Blood 94:3820–3828Google Scholar
  24. 24.
    Vischer UM, Wagner DD (1993) CD63 is a component of Weibel-Palade bodies of human endothelial cells. Blood 82:1184–1191Google Scholar
  25. 25.
    Metcalf DJ, Nightingale TD, Zenner HL et al (2008) Formation and function of Weibel-Palade bodies. J Cell Sci 121:19–27. CrossRefGoogle Scholar
  26. 26.
    Cerny J, Feng Y, Yu A et al (2004) The small chemical vacuolin-1 inhibits Ca(2 +)-dependent lysosomal exocytosis but not cell resealing. EMBO Rep 5:883–888. CrossRefGoogle Scholar
  27. 27.
    Zhang K, Wang P, Huang S et al (2014) Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS. Mol Biol Rep 41:4253–4259. CrossRefGoogle Scholar
  28. 28.
    González-Pacheco FR, Caramelo C, Castilla MA et al (2002) Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma. Nephrol Dial Transplant 17:392–398CrossRefGoogle Scholar
  29. 29.
    Echeverría C, Montorfano I, Hermosilla T et al (2014) Endotoxin induces fibrosis in vascular endothelial cells through a mechanism dependent on transient receptor protein melastatin 7 activity. PLoS ONE 9:e94146. CrossRefGoogle Scholar
  30. 30.
    Sarmiento D, Montorfano I, Cáceres M et al (2014) Endotoxin-induced vascular endothelial cell migration is dependent on TLR4/NF-κB pathway, NAD(P)H oxidase activation, and transient receptor potential melastatin 7 calcium channel activity. Int J Biochem Cell Biol 55:11–23. CrossRefGoogle Scholar
  31. 31.
    Parthasarathi K, Ichimura H, Monma E et al (2006) Connexin 43 mediates spread of Ca2 + -dependent proinflammatory responses in lung capillaries. J Clin Invest 116:2193–2200. CrossRefGoogle Scholar
  32. 32.
    Burgazli KM, Venker CJ, Mericliler M et al (2014) Importance of large conductance calcium-activated potassium channels (BKCa) in interleukin-1b-induced adhesion of monocytes to endothelial cells. Eur Rev Med Pharmacol Sci 18:646–656Google Scholar
  33. 33.
    Neri T, Pergoli L, Petrini S et al (2016) Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells. Toxicol In Vitro 32:333–338. CrossRefGoogle Scholar
  34. 34.
    Birch KA, Pober JS, Zavoico GB et al (1992) Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. J Cell Biol 118:1501–1510CrossRefGoogle Scholar
  35. 35.
    Veal EA, Day AM, Morgan BA (2007) Hydrogen peroxide sensing and signaling. Mol Cell 26:1–14. CrossRefGoogle Scholar
  36. 36.
    Lu Y-C, Yeh W-C, Ohashi PS (2008) LPS/TLR4 signal transduction pathway. Cytokine 42:145–151. CrossRefGoogle Scholar
  37. 37.
    Echeverría C, Montorfano I, Sarmiento D et al (2013) Lipopolysaccharide induces a fibrotic-like phenotype in endothelial cells. J Cell Mol Med 17:800–814. CrossRefGoogle Scholar
  38. 38.
    Into T, Kanno Y, Dohkan J-I et al (2007) Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells. J Biol Chem 282:8134–8141. CrossRefGoogle Scholar
  39. 39.
    Stottmeier B, Dick TP (2016) Redox sensitivity of the MyD88 immune signaling adapter. Free Radic Biol Med 101:93–101. CrossRefGoogle Scholar
  40. 40.
    Montorfano I, Becerra A, Cerro R et al (2014) Oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a TGF-β1 and TGF-β2-dependent pathway. Lab Invest 94:1068–1082. CrossRefGoogle Scholar
  41. 41.
    Sarmiento D, Montorfano I, Cerda O et al (2015) Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel. Microvas Res 98:187–196. CrossRefGoogle Scholar
  42. 42.
    Zhang K, Wang P, Huang S et al (2014) Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS. Mol Biol Rep 41:4253–4259. CrossRefGoogle Scholar
  43. 43.
    Hickey MJ, Kanwar S, McCafferty DM et al (1999) Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen. J Immunol 162:1137–1143Google Scholar
  44. 44.
    Harari OA, McHale JF, Marshall D et al (1999) Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence. J Immunol 163:6860–6866Google Scholar
  45. 45.
    Eppihimer MJ, Wolitzky B, Anderson DC et al (1996) Heterogeneity of expression of E- and P-selectins in vivo. Circ Res 79:560–569. CrossRefGoogle Scholar
  46. 46.
    Ishiwata N, Takio K, Katayama M et al (1994) Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. J Biol Chem 269:23708–23715Google Scholar
  47. 47.
    Chen AY, Ha JN, DeLano FA, Schmid-Schönbein GW (2012) Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat. J Leukoc Biol 92:183–194. CrossRefGoogle Scholar
  48. 48.
    Tchernychev B, Furie B, Furie BC (2003) Peritoneal macrophages express both P-selectin and PSGL-1. J Cell Biol 163:1145–1155. CrossRefGoogle Scholar
  49. 49.
    Jilma B, Blann A, Pernerstorfer T et al (1999) Regulation of adhesion molecules during human endotoxemia. No acute effects of aspirin. Am J Respir Crit Care Med 159:857–863. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Facultad de Ciencias de la Vida, Universidad Andres BelloSantiagoChile
  2. 2.Millennium Institute on Immunology and ImmunotherapySantiagoChile
  3. 3.Center for the Development of Nanoscience and Nanotechnology (CEDENNA), Universidad de Santiago de ChileSantiagoChile
  4. 4.Millennium Nucleus of Ion Channels-Associated Diseases (MiNICAD), Universidad de ChileSantiagoChile

Personalised recommendations