Arachidonate 5 lipoxygenase-derived leukotrienes are primary signals of leukocyte activation and inflammation in response to ischemic cardiac injury (MI; myocardial infarction). Using risk-free male C57BL/6J and arachidonate 5-lipoxygenase (ALOX5) null mice (8-12 weeks), we quantitated leukocytes and ALOX5-derived bioactive lipids of the infarcted left ventricle (LV) and spleen to measure the physiological inflammation and cardiac repair. Our results showed that ALOX5 endogenously generates specialized pro-resolving mediators (SPMs) that facilitate cardiac repair post-MI. Deficiency of ALOX5 lead to increase cyclooxygenase gene expression, 6-keto prostaglandin F1α, and neutrophil abundance with signs of unresolved inflammation post-MI. Consequently, ALOX5 deficiency impaired the resolution of inflammation and cardiac repair, including increased myocardium rupture post-MI in acute heart failure. On-time ALOX5 activation is critical for leukocyte abundance from the infarcted heart, indicating an essential role of ALOX5 in the resolution of inflammation. In addition, to balance the inflammatory responses, ALOX5 is also necessary for fibroblast signaling, as the ALOX5-deficient fibroblast are prone to fibroblast-to-myofibroblast differentiation leading to defective scar formation in post-MI cardiac repair. Consistent with these findings, ALOX5-null mice showed an overly inflammatory response, defective fibrotic signaling, and unresolved inflammation. These findings are indicative of a critical role of ALOX5 in myocardium healing, inflammation-resolution signaling, cardiac repair, and fibroblast pathophysiology.

Phospholipase A2 (PLA2) is a membrane lytic enzyme that is present in many organisms. Human PLA2 has emerged as a potential biomarker as well as a therapeutic target for several diseases including cancer, cardiovascular diseases, and some inflammatory diseases. The current study focuses on the development of lipo-beads that are very reactive and highly sensitive to PLA2. To develop the best supported lipid bilayer formulation, several lipid combinations were investigated using 10 μm porous silica beads. The reactivity of PLA2 was monitored via the decrease in particle fluorescence because of the release of entrapped fluorescent dye from the particle pores or the disintegration of a fluorescent lipid constituted on the bilayer upon lipid hydrolysis using flow cytometry. The enzyme binding studies indicate that lipo-beads with bulky fluorescent tags in the lipid head group and anionic lipids produce a more pronounced response. The kinetic studies suggest that these lipo-beads are very reactive with PLA2 and can generate a detectable signal in less than 5 min. The enzyme inhibition studies were also conducted with two known PLA2 inhibitors, varespladib and quercetin. We find that quercetin can hydrolyze the supported membrane, and thus inhibition of PLA2 is not observed; however, varespladib has shown significant PLA2 inhibition on lipo-beads. We have demonstrated that our lipo-bead-based approach can detect annexin-3, a known disease biomarker, as low as 10 nM within 5 min after incubation.