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A1835 - Activation of Secretory A2 Phospholipase Isotypes and Surfactant Injury in Murine ARDS
Author Block: R. Hite1, M. V. Novotny2, J. Lee3, C. C. Smallcombe2, C. Furdui3, M. C. Seeds3; 1Respiratory Institute, Department of Critical Care, Cleveland Clinic, Cleveland, OH, United States, 2Lerner Research Institute, Department of Pathobiology, Cleveland Clinic, Cleveland, OH, United States, 3Department of Internal Medicine, Molecular Medicine, Wake Forest Baptist School of Medicine, Winston-Salem, NC, United States.
RATIONALE: Activation of secretory phospholipase A2 (sPLA2) in ARDS leads to hydrolysis of surfactant phospholipids (PL), phosphatidylglycerol (PG) depletion, and surfactant dysfunction. Multiple sPLA2 may contribute, but the time course for activation of specific isotypes is unknown. This study examines the changes in sPLA2 activity and isotypes (mRNA and protein) in a murine ARDS model caused by intra-tracheal lipopolysaccharide (IT-LPS). The goal is to identify potential therapeutic targets for mitigating surfactant degradation during ARDS recovery. METHODS: Murine ARDS lavage: IT-LPS (9µg/kg) was delivered to Balb/c mice, and bronchoalveolar lavage (BAL) was performed at times of peak injury (48 and 96 hours post) including sPLA2 activation and PG depletion. Results are compared to control mice prior to injury (baseline) and at 48 hours post sham injury (LPS vehicle). Sequential centrifugation of BAL produces a cell pellet, surfactant pellet and supernatant, which contains sPLA2 protein and activity. sPLA2 activity was measured by ex vivo hydrolysis of a radiolabeled (3H-PG) commercial surfactant. mRNA expression of sPLA2 was measured using RT-PCR of BAL cell pellets and lung tissue. sPLA2 proteins were identified by targeted proteomics with LC-MS/MS. Surfactant Function was measured using a pulsating bubble surfactometer. Surfactant PL composition was quantified by HPLC/ELSD. RESULTS: Between 48 - 96 hours after IT-LPS, sPLA2 activity is elevated (4-8 fold) in the BAL of mice relative to controls (baseline and sham). Peak sPLA2 activity corresponds to the peak of PG depletion in surfactant and peak of surfactant dysfunction. At 48 hours, RT-PCR of lung tissue identified increased mRNA for Group 2A sPLA2 (PLA2G2A, 6 fold), PLA2G5 (3-4 fold) and PLA2G10 (3-4 fold), and all 3 decreased by 96 hours with PLA2G5 and PLA2G10 returning to baseline. PLA2G2D mRNA decreased upon IT-LPS challenge, and PLA2G1B was unchanged. In the BAL cell pellet, PLA2G2A mRNA peaks (6-8 fold) at 24 hours, while PLA2G10 peaks (6 fold) at 48 hours and remains elevated (4-6 fold) at 96 hours. At 48 hours, targeted proteomic analysis of supernatants revealed peptide fragments consistent with PLA2G2A, PLA2G2D, PLA2G5 and PLA2G10 proteins, each with multiple daughter ions. CONCLUSION: In a murine ARDS model, cellular mRNA expression and protein levels of multiple sPLA2 are increased over 24-96 hours after IT-LPS. Their presence corresponds with peak hydrolysis of surfactant PL and surfactant dysfunction. These studies have potential to identify effective approaches to sPLA2 inhibition with therapeutic benefit in ARDS.