Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration.
Vascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI=10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS). These altered proteins were involved in several biological processes, for example, mRNA stability/processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS.
Kanlaya
Rattiyaporn
R
Medical Proteomics Unit, Office for Research and Development, Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Pattanakitsakul
Sa-nga
SN
Sinchaikul
Supachok
S
Chen
Shui-Tein
ST
Thongboonkerd
Visith
V
D000199:Actins / Q000378:metabolism*; D000704:Analysis of Variance; D015703:Antigens, CD / Q000032:analysis; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D003716:Dengue Virus / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D005434:Flow Cytometry; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000032:analysis; D054884:Host-Pathogen Interactions; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D036904:Leukocyte Rolling; D008565:Membrane Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D020652:Peptide Elongation Factor 2 / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D062826:Zonula Occludens-1 Protein; D000099152:Cadherin 5
D000199:Actins; D015703:Antigens, CD; D015820:Cadherins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D008565:Membrane Proteins; D020652:Peptide Elongation Factor 2; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D020543:Proteome; D062826:Zonula Occludens-1 Protein; D000099152:Cadherin 5; C569042:TJP1 protein, human