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Screening of siRNA to identify the genes associated with vascular collapse when exposed to Yersinia pestis
Author(s) -
Hoke Allison Violet,
Gautam Aarti,
Filippov Andrey,
Amemiya Kei,
Hammamieh Rasha,
Jett Marti
Publication year - 2012
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.26.1_supplement.1151.10
Subject(s) - yersinia pestis , small interfering rna , vascular permeability , peripheral blood mononuclear cell , biology , gene , in vitro , microbiology and biotechnology , immunology , rna , genetics , virulence , endocrinology
Yersinia pestis , the causative agent of plague, upon leukocyte activation leads to the systemic, often fatal, vascular collapse and end‐organ failure. To investigate genes associated with vascular collapse, we studied the permeability of human lung microvascular endothelial cells (HMVEC‐L) co‐cultured with peripheral blood mononuclear cells (PBMCs) in the presence of bacteria. The monolayer's permeability was measured using fluorescein dextran in a 96‐transwell format. To identify host factors, a primary screen of ~400 siRNAs against drugable target genes was performed and the cells were plated for monolayer formation. The co‐culture of HMVEC‐Ls with Y. pestis (CO92pgm − ) infected PBMCs had no effect on permeability. About 10 genes that lead to increased permeability when knocked down were identified in the initial screen and need to be confirmed. In another approach to identify siRNA's that block vascular leakage, a pathogenic strain of bacteria that leads to vascular collapse was used. Experiments have been performed to optimize the Y. pestis infected PBMC/HMVEC‐L ratio to determine the best conditions that lead to an increase in vascular permeability. This information and the siRNA knock down results will help identify genes with a potential for therapeutic roles. Research support is through DTRA, TMTI.DRUG.02.10.WR.023.