Role of Eicosanoids in Shock

Medical University Of South Carolina, Charleston SC

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Abstract

DESCRIPTION (provided by applicant): This competitive revision application is to support a significant expansion of the research protocol of the approved funded project GM2763-27 entitled The Role of Eicosanoids in Shock in response to Notice Number (NOT-OD-09-058) Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. Septic shock occurs in more than 750,000 patients each year in the US, nearly 215,000 of who die. Our competitively renewed grant is a continuing evolution of studies focused upon identifying molecular mechanisms that negatively regulate Toll-like receptor (TLR) function and inflammation in sepsis. During the past funding cycle several major findings have provided a new direction and focus. The new direction is, in part, derived from our findings that mice deficient in the heterotrimeric guanine nucleotide inhibitory protein (G)?i2 exhibit an enhanced pro- inflammatory phenotype to endotoxemia and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Activation of G?i protein by specific ligands is beneficial in endotoxemia and sepsis and suppresses TLR activation in vitro. However thus far we have not identified endogenous Gi protein ligands as potential candidates for Gi protein activation. Recently we have found that the endogenous Gi ligand stromal cell derived factor (SDF-1??{also known as CXCL12}), which is a Gai ligand, is beneficial in sepsis potentially through recruitment of endothelial progenitor cells (EPCs) and anti-inflammation. Recent studies demonstrate that circulating EPCs are increased in septic patients and that non-survivors have significantly lower numbers of EPCs than survivors. SDF-1??recruits EPCs to sites of injury and facilitates repair. SDF-1??also activates anti-inflammatory signaling through Gi protein-mediated pathways. Our preliminary studies employed the clinically relevant murine CLP model at moderate and severe levels. Our data demonstrate that plasma SDF-1??and circulating EPCs are increased in mice subjected to moderate CLP but not in mice subjected to severe CLP. Furthermore we demonstrated that the SDF-1??drug surrogate CTCE-0214 suppresses lipopolysaccharide (LPS) shock and zymosan peritonitis-induced plasma TNF??production and CLP-induced mortality in mice. This data demonstrate for the first time that SDF-1??is beneficial in sepsis. We also found that SDF-1??and CTCE-0214 suppress LPS-induced IL-6 production in bone marrow derived macrophages (BMDM). These findings lead us to propose the new hypothesis representing an expanded scope of the parent R01 that recruitment of endothelial progenitor cells and activation of anti-inflammatory signaling pathways are the mechanisms by which SDF-1??is beneficial in sepsis. Two expanded specific aims address this hypothesis: 1. Determine the effect of CTCE-0214 on the CLP-induced recruitment of EPCs and inflammatory response. CD-1 mice will be subjected to CLP followed by injection of CTCE-0214. EPCs levels and CLP-induced inflammation will be studied. 2. Investigate the mechanism by which SDF-1??and CTCE-0214 recruit EPCs and suppress the inflammatory response. EPCs, BMDM, mouse aortic endothelial cell (MAEC) and splenic CD4+ T cells will be isolated from wild type and the novel G?i2 knockout and G?i2 gain of function mice. Induction of chemotaxis of EPCs and inhibition of apoptosis of EPCs by SDF-1??and CTCE-0214 will be examined. SDF-1??and CTCE-0214-induced activation of anti-inflammatory pathway and suppression of inflammation will be examined in BMDM and MAECs and by examining CD4+ T cell apoptosis. Such studies may lead to innovative approaches to treat sepsis. Public Health relevance: Recently we have found that the endogenous heterotrimeric Gi ligand stromal cell derived factor (SDF-1??{also known as CXCL12}), which is a G?i2 ligand, is potentially beneficial in sepsis through recruitment of endothelial progenitor cells (EPCs) and antiinflammation. A new hypothesis representing an expanded scope of the parent R01 is recruitment of endothelial progenitor cells and activation of anti-inflammatory signaling pathways are the mechanisms by which SDF-1??is beneficial in sepsis. Two expanded specific aims address this hypothesis;1. Determine the effect of an SDF- 1??analog CTCE-0214 on CLP-induced recruitment of EPCs and inflammatory response and 2. Investigate the mechanisms by which SDF-1??and CTCE-0214 recruit EPCs and suppress the inflammatory response.