The receptor for activated complement factor 5 (C5aR) conveys myocardial ischemic damage by mediating neutrophil transmigration

Abstract

Tissue loss after myocardial ischemia with reperfusion (MI/R) is in part conveyed by neutrophil recruitment to post-ischemic myocardium. Strategies to prevent reperfusion injury would help to limit myocardial damage. The receptor for activated complement factor 5 (C5aR) plays a prominent role in inflammation. We examine the effects of C5aR-deficiency on reperfusion injury after MI/R. C5aR(-/-)-mice and their C57BL/6- (WT) littermates underwent transient myocardial ischemia followed by different time points of reperfusion. Infarct size and leukocyte infiltration were determined. Expression of C5aR, inflammatory cytokines and adhesion molecules were analyzed by real-time RT-PCR. Leukocyte-endothelial interactions were assessed by low-shear adhesion- and transmigration-assays in vitro. Myocardial C5aR mRNA expression was 2.8-fold increased by ischemia. Infarct size per area-at-risk and leukocyte recruitment into infarctions were reduced in C5aR(-/-)-compared to WT-mice as well as in WT mice treated with the C5aR-antagonist JPE1375. IL-6, IL-1β, ICAM-1 and VCAM-1 expression were not different, while TNFα expression was reduced in C5aR(-/-)-mice after MI/R. In vitro, C5aR on leukocytes is required for effective transendothelial migration but not adhesion. Expression of MMP9 and JAM-A, molecules that are involved in leukocyte transmigration, were reduced in C5aR(-/-) mice in vivo. Genetic C5aR deficiency blunts the inflammatory response in murine MI/R resulting in reduced inflammatory cell recruitment, which is due to a C5aR-dependent effect on leukocyte transmigration across inflamed endothelium into the ischemic myocardium. This effect could be related to MMP9- and JAM-A expression in response to ischemia and reperfusion.

Keywords: 2,3,5-triphenyl-tetrazolium chloride; AAR; C5L2; C5a-like receptor 2; C5aR; C5aR (CD 88); CTG; Cell Tracker Green(®); DMEM; DNA; Dulbecco's modified eagle medium; ELISA; GAPDH; HBSS; HE; HPRT; Hank's balanced salt solution; I/R; ICAM-1; IL-1β; IL-6; IV; Immortalized murine endothelial cells; JAM-A; KO; LAD; LFA-1; MI/R; MMP9; Myocardial ischemia; Neutrophil recruitment; OCT; PBS; PMN; RT-PCR; Reperfusion; TNFα; TTC; Transmigration; VCAM-1; WT; area-at-risk; deoxyribonucleic acid; enzyme linked immunosorbent assay; fEnd.5; glycerinaldehyd-3-phosphat-dehydrogenase; hematoxylin-eosin; high power field; hpf; hypoxanthin-phosphoribosyl-transferase; intercellular adhesion molecule-1; interleukin-1beta; interleukin-6; intravenous; ischemia with following reperfusion; junctional adhesion molecule-A; knock out; left anterior descending coronary artery; leukocyte-function-associated antigen-1; mRNA; matrix metalloproteinase 9; messenger ribonucleic acid; myocardial ischemia with following reperfusion; phosphate buffered saline; polymorphonuclear neutrophils; receptor for activated complement factor 5; reverse transcriptase - polymerase chain reaction; tissue freezing medium (Tissue-Tek(®) OCT™); tumor necrosis factor alpha; vascular cell adhesion molecule-1; wild type.