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Use of the genomic library of short interfering RNA to block vascular collapse from the exposure to diverse pathogenic agents
Author(s) -
Gautam Aarti,
Hammamieh Rasha,
Filippov Andrey,
Jett Marti
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.663.8
Subject(s) - endothelial stem cell , peripheral blood mononuclear cell , endothelium , small interfering rna , biology , vascular permeability , microbiology and biotechnology , in vitro , barrier function , klf2 , immunology , cell culture , gene , transfection , gene expression , genetics , biochemistry , endocrinology
The objective of the project is to find common approaches for diagnosis or treatment of exposure to multiple pathogenic agents that lead to vascular leakage or collapse. Endothelial permeability is associated with dramatic changes in cell shape that occurs upon stimulation with bacterial toxins or inflammatory mediators. For example, Y. pestis , causative agent of plague, rapidly invades from the infection site into the monocytic cells. Upon leukocyte activation, the cascades of mediators stimulate various organ responses which lead to the systemic, often fatal, vascular collapse and end‐organ failure. Concomitantly, endothelial cells are actively involved in host responses to infectious agents, controlling coagulation cascades and regulating leukocytes trafficking. In the current project, we are exploring how peripheral blood mononuclear cells (PBMCs) infected with pathogenic agents , influences endothelial cell barrier function leading to vascular leakage. To identify host factors regulating an established infection, we are performing siRNA screen using siGENOME libraries spanning ~5000 genes characterized as drugable targets. Herein, we report the use of AMAXA nucleofactor 96‐well shuttle system for siRNA screening in human lung microvascular endothelial cell (HMVEC‐L). We have further standardized the 96‐well format using the trans‐well system for assaying vascular permeability with FITC dextran. Cell monolayers are used as in vitro model of vascular endothelium and are incubated with PBMC infected with Y. pestis (CO92 pgm − ). Primary gene list known to affect vascular leakage is being generated. Similar approach is being followed for Staphylococcus aureus enterotoxin B (SEB) that has been known to perturb the endothelial barrier function. These studies will ultimately influence the development of a new avenue of therapy which is currently lacking with these select agents.