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58 Effect of Varespladib-Protected Surfactant in Cultured Rat Alveolar Macrophages Stimulated with LPS
  1. D De Luca1,
  2. F Vendittelli1,
  3. H Fraser2,
  4. L Gentile1,
  5. J Trias2,
  6. A Minucci1,
  7. ED Capoluongo1
  1. 1PICU and Lab of Clinical Molecular Biology, Catholic University of the Sacred Heart, Rome, Italy
  2. 2ANthera Pharmaceuticals, R&D, Hayward, CA, USA

Abstract

Background Secretory phospholipase A2 (sPLA2) is a crucial enzyme for inflammatory response and surfactant catabolism. Acute lung injury (ALI) is a life-threating syndrome characterized by surfactant dysfunction and raised levels of sPLA2. Varespladib is a potent selective sPLA2 inhibitor that is effective in animal models of ALI. Nothing is known about the joint administration surfactant+varespladib and we aimed at studying the effect on the sPLA2 pathway.

Methods 1x106 normal alveolar macrophages (from Rattus Norvegicus) were cultured in Ham’sF12 medium + 2% fetal bovine serum. Cultures were incubated with 15 ng/mL LPS for 24h, then treated with 200 µg poractant-α, 90 µM varespladib, both or nothing. These concentrations were those achieving 50% sPLA2 activity reduction in previous experiments. After 24h, culture supernatants were assayed for sPLA2 activity, free fatty acids (FFA) and total proteins concentrations.

Results sPLA2 activity corrected for the protein level is 0.26±0.02, 0.24±0.02, 0.24±0.02 and 0.28±0.02 IU/µg in cultures treated with surfactant, varespladib, both or nothing (overall p=0.016; Dunnett post-hoc between cultures treated with varespladib and varespladib+surfactant against untreated cultures p=0.01). FFA are higher in untreated cultures (394±82 µM), than in surfactant- (219±70 µM) and in varespladib-treated ones (148±51 µM). Combined treatment reduced FFA to 206±47 µM (overall p=0.017; Sidak post-hoc p=0.036 and p=0.023 for the varespladib and combined treatment against control cultures).

Conclusions The joined administration of varespladib and poractant-α significantly reduce sPLA2 activity and FFA production. Surfactant+varespladib affect sPLA2 pathway significantly more than the surfactant alone.

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