Dengue and Rho family proteins. Earlier studies have


Dengue virus infection
was found to cause increased trans-endothelial cell permeability in HMEC-1
cells between 24h to 48h post-infection, with a peak at 36h.  We carried out a proteomics analysis using
LC-MS/MS approach in DENV-infected HMEC-1 cells at 24h and 48h post-infection
to decipher the molecules involved in the observed transient vascular leakage. We
could identify modulation of proteins involved in viral processes, platelet
aggregation and immunity. Functional analysis of one of the modulated proteins,
Nucleophosmin (NPM1), elucidated its role in regulating DENV replication. The
differential proteome in virus infected cells also contained a number of
proteins involved in cell adhesion such as catenin, and Rac and Rho family
proteins. Earlier studies have shown that these molecules are major downstream mediators
in Angiopoietin/Tie2 pathway and Sphingosine-1-phosphate
(S1P) signaling pathway which regulate vascular barrier integrity during
inflammation.

Our study further
focused on the role of Angiopoietin/Tie2 signaling
pathway in DENV-induced vascular permeability. We could observe an up-regulation
in the level of Ang-2 transcripts in
HMEC-1 cells and also an increased secretion of the protein in the culture
supernatants upon infection with DENV. 
There was a corresponding decrease in the level of Ang-2 immunostaining
in the Weibel-palade bodies in infected cells. There was activation of Tie2
receptor and Src molecules, and increased levels of Rho family of GTPases upon
infection. A significant increase in p-VE-Cadherin along with a loss of VE-Cadherin
staining from the cell-cell junction in the infected cells as well as the
neighboring uninfected cells was observed indicating
de-stabilization of inter-endothelial cell junctions. Inhibition of the Rho
family proteins using a Rho kinase (ROCK) inhibitor decreased the FITC Dextran
permeability in the infected cells, supporting the functional significance of
this family of proteins in inducing leakage. Addition of Ang-1, a physiological
antagonist of Ang-2 activity, re-stabilized the barrier integrity by inhibiting
the phosphorylation of VE-Cadherin upon DENV infection.  Our findings suggest the involvement of Src
and Rho family proteins in the Ang/Tie-2 pathway in DENV-induced vascular
leakage.

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Apart from identifying
the role of specific cellular proteins in restricting DENV replication, the study
elucidates the mechanism of Ang-1/Tie2 pathway in regulating vascular
permeability in DENV-infected endothelial cells. Our data suggest that
therapeutic use of Ang-1 could help in alleviating vascular leakage in dengue
hemorrhagic fever and shock syndrome. 

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