Silencing of C5a receptor gene with siRNA for protection from Gram-negative bacterial lipopolysaccharide-induced vascular permeability.
Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions. Whereas these changes have been shown to be associated with activation of the complement system, leading to the release of C5a and interaction of C5a-C5a receptor (C5aR), the role of C5aR in endothelial cells remain(s) ill-defined. Here, we report an essential role of C5aR in endothelial cell injury and vascular permeability through silencing of the C5aR gene using siRNA. In the cultured mouse dermal microvascular endothelial cells (MEMECs) monolayer transfected with C5aR-siRNA, endotoxin-induced cell injury by evaluated as transendothelial flux, cell detachment, and cytoskeletal disorganization was inhibited. Upregulation of vascular cell adhesion molecule-1 (VCAM-1) was also suppressed. Studies exploring the underlying mechanism of siRNA-mediated suppression in VCAM-1 expression were related to reduction of NF-kappaB activation and nuclear localization of both p50 and p65. The effect was associated with inhibition in activation of protein kinase Cdelta(PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. In the model of mice administrated with C5aR-siRNA, endotoxin-induced plasma leakage was inhibited in local abdominal skin. Systemic administration of endotoxin to mice resulted in increased microvascular permeability in multiple organs was reduced. These studies demonstrate that the C5aR responsible for vascular endothelial cell injury and plasma permeability is an important factor, and that blockade of C5aR may be useful therapeutic targets for the prevention of vascular permeability in pathogenic condition.
Liu
Zi-ming
ZM
West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.
Zhu
Shi-ming
SM
Qin
Xiang-jing
XJ
Cheng
Zhi-de
ZD
Liu
Meng-yuan
MY
Zhang
Hai-mou
HM
Liu
Dong-xu
DX
D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects*; D006090:Gram-Negative Bacteria / Q000737:chemistry*; D051550:I-kappa B Kinase / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics* / Q000378:metabolism; D012867:Skin / Q000098:blood supply / Q000187:drug effects; D051996:Transcription Factor RelA / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; D034741:RNA, Small Interfering; D044087:Receptor, Anaphylatoxin C5a; D051996:Transcription Factor RelA; D019010:Vascular Cell Adhesion Molecule-1; D051550:I-kappa B Kinase