Rat lung pericytes demonstrate prolonged upregulation of lipopolysaccharide-induced secretory phospholipase A2 activity

Abstract

Introduction: Sepsis remains the leading cause of death in intensive care units in the United States. Secretory phospholipase A2 (SPLA2) is a key enzyme in the production of eicosanoids which has been shown to be upregulated in patients with sepsis. Recently, a clinical trial of a selective SPLA2 inhibitor resulted in decreased 28 day all-cause mortality rates by fifty percent in those patients who received the high dose of the inhibitor within 18 hours of sepsis-induced organ failure. Extensive studies indicate that the microvascular lung pericyte plays a key role in the regulation of capillary permeability in sepsis. We performed this study to elucidate the chronology of SPLA2 upregulation in rat lung pericytes with and without continued lipopolysaccharide (LPS) stimulation. Materials and Methods: Rat lung pericytes were harvested as previously described. Pericytes were incubated in 100 μg/ml of LPS for 18 hours. The supernatant was then measured for SPLA2 activity and new media with and without LPS was added. This supernatant was harvested one hour later and examined for SPLA2 activity. The Student’s t-test was utilized to compare groups with statistical significance assumed at an alpha level of 0.05. Results: Rat lung microvascular pericytes exposed to LPS for 18 hours showed a more than four-fold increase in SPLA2 activity over control. When placed in new media (without LPS) for one hour, previously exposed pericytes showed a ten-fold higher SPLA2 activity than control pericytes (p < 0.02). Those pericytes exposed again to 100 μg/ml LPS in the new media showed a further ten-fold increase in activity over the aforementioned pericytes, or 100 times greater than control (p < 0.05). Conclusion: Rat lung microvascular pericytes in vitro continue to secrete secretory phospholipase A2 more than eighteen hours after initial LPS exposure. This phospholipase activity is increased almost geometrically by continued LPS exposure. This greatly increased secretion of secretory phospholipase A2 with prolonged exposure to LPS has important clinical implications in patients with ongoing sepsis.