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Group V phospholipase A2 (gVPLA2) mediates responses to methicillin-resistant Staph aureus (MRSA) in lung endothelium
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A2895 - Group V phospholipase A2 (gVPLA2) mediates responses to methicillin-resistant Staph aureus (MRSA) in lung endothelium
Author Block: Y. M. Htwe, L. Meliton, T. Kawasaki, S. M. Dudek; Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States.
RATIONALE: Infections are the most common cause of the acute respiratory distress syndrome (ARDS), which is characterized by inflammatory disruption of the alveolar-vascular barrier secondary to activation of the lung endothelium cells (EC). Previous work has demonstrated that the 14-kDa mammalian enzyme group V phospholipase A2 (gVPLA2) is activated after LPS and increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. In the current study we characterized the functional role of gVPLA2 expression in mediating lung endothelial responses to methicillin-resistant Staph aureus (MRSA), an important and clinically relevant stimulus.
METHODS: Human pulmonary artery or microvascular lung EC (HPAEC or HLMVEC) were cultured and exposed to heat-killed MRSA (1-3 x 10*8 CFU). In some experiments, EC also were treated with LY 311727 (50mM), a specific group II sPLA2 small molecule inhibitor, either pre or post MRSA exposure. Barrier function was determined by transendothelial resistance (TER) using the ECIS assay, as well as labeled dextran transwell permeability. Cytokine release was determined by ELISA. Expression of gVPLA2 was determined by RT-PCR. Mouse pulmonary vascular EC (mPVEC) were isolated from gVPLA2 KO and C57/BL6 WT mice, sorted by flow cytometry, and then passaged several times until sufficient numbers were obtained for ECIS studies.
RESULTS: MRSA significantly increases gVPLA2 expression in HPAEC within one hour as measured by RT-PCR. MRSA induces IL-8 secretion from HLMVEC, which is attenuated by LY 311727. Heat-killed MRSA significantly increases permeability in human lung EC in a dose-dependent fashion. LY311727 attenuates these effects after either pre or post treatment. In general, isolated mPVEC required substantially higher concentrations of thrombin, LPS, or HK-MRSA to increase permeability than needed for cultured human lung EC. mPVEC isolated from gVPLA2 KO mice exhibited less permeability after these barrier-disrupting agents than mPVEC from WT mice.
CONCLUSION: These results demonstrate that gVPLA2 is upregulated in lung EC by MRSA and participates in mediating inflammatory and permeability effects caused by this clinically relevant stimulus. Strategies targeting this enzyme may have therapeutic potential in ARDS caused by MRSA infection.
Author Block: Y. M. Htwe, L. Meliton, T. Kawasaki, S. M. Dudek; Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States.
RATIONALE: Infections are the most common cause of the acute respiratory distress syndrome (ARDS), which is characterized by inflammatory disruption of the alveolar-vascular barrier secondary to activation of the lung endothelium cells (EC). Previous work has demonstrated that the 14-kDa mammalian enzyme group V phospholipase A2 (gVPLA2) is activated after LPS and increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. In the current study we characterized the functional role of gVPLA2 expression in mediating lung endothelial responses to methicillin-resistant Staph aureus (MRSA), an important and clinically relevant stimulus.
METHODS: Human pulmonary artery or microvascular lung EC (HPAEC or HLMVEC) were cultured and exposed to heat-killed MRSA (1-3 x 10*8 CFU). In some experiments, EC also were treated with LY 311727 (50mM), a specific group II sPLA2 small molecule inhibitor, either pre or post MRSA exposure. Barrier function was determined by transendothelial resistance (TER) using the ECIS assay, as well as labeled dextran transwell permeability. Cytokine release was determined by ELISA. Expression of gVPLA2 was determined by RT-PCR. Mouse pulmonary vascular EC (mPVEC) were isolated from gVPLA2 KO and C57/BL6 WT mice, sorted by flow cytometry, and then passaged several times until sufficient numbers were obtained for ECIS studies.
RESULTS: MRSA significantly increases gVPLA2 expression in HPAEC within one hour as measured by RT-PCR. MRSA induces IL-8 secretion from HLMVEC, which is attenuated by LY 311727. Heat-killed MRSA significantly increases permeability in human lung EC in a dose-dependent fashion. LY311727 attenuates these effects after either pre or post treatment. In general, isolated mPVEC required substantially higher concentrations of thrombin, LPS, or HK-MRSA to increase permeability than needed for cultured human lung EC. mPVEC isolated from gVPLA2 KO mice exhibited less permeability after these barrier-disrupting agents than mPVEC from WT mice.
CONCLUSION: These results demonstrate that gVPLA2 is upregulated in lung EC by MRSA and participates in mediating inflammatory and permeability effects caused by this clinically relevant stimulus. Strategies targeting this enzyme may have therapeutic potential in ARDS caused by MRSA infection.
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