Phospholipase A(2) (PLA(2))-catalyzed hydrolysis of membrane phospholipids results in the stoichiometric production of a free fatty acid, most importantly arachidonic acid, and a lysophospholipid. Both of these phospholipid metabolites serve as precursors for inflammatory mediators such as eicosanoids or platelet-activating factor (PAF). Since it was initially discovered that non-steroidal anti-inflammatory drugs inhibit prostaglandin synthesis, a vast amount of drug development has been performed to selectively inhibit the production of the inflammatory metabolites of arachidonic acid while preserving their protective role. This research has culminated in the development of selective cyclooxygenase-2 (COX-2) inhibitors that act on the inducible, inflammatory COX enzyme, but do not affect the constitutive prostaglandin synthesis in cells that is mediated via COX-1. The development of PLA(2) inhibitors as potential anti-inflammatory agents has also been extensively pursued since the release of arachidonic acid from membrane phospholipids by PLA(3) is one of the rate-limiting factors for eicosanoid production. In addition to the production of eicosanoids, PLA(2)-catalyzed membrane phospholipid hydrolysis is also the initiating step in the generation of PAF, a potent inflammatory agent. Thus, inhibition of PLA(2) activity should, in theory, be a more effective anti-inflammatory approach. However, developing an inhibitor that would be selective for the production of inflammatory metabolites and not inhibit the beneficial properties of PLA(2) has so far proved to be elusive. This review will focus on agents used currently to inhibit PLA(2) activity and will explore their possible therapeutic use.

Recent research suggests that activation of protease-activated receptors (PARs) on the surface of endothelial and epithelial cells may play a role in general mechanisms of inflammation. We hypothesized that mast cell tryptase activation of endothelial cell PAR-2 is coupled to increased calcium-independent PLA2 (iPLA2) activity and increased platelet-activating factor (PAF) production that may play a role in inflammatory cell recruitment at sites of vascular injury. Stimulation of human coronary artery endothelial cells (HCAEC) with 20 ng/ml tryptase increased iPLA2 activity, arachidonic acid release, and PAF production. These tryptase-stimulated responses were inhibited by pretreatment with the iPLA2-selective inhibitor bromoenol lactone (BEL; 5 microM, 10 min). Similar patterns of increased iPLA2 activity and PAF production were also seen when HCAEC were treated with SLIGKV, which represents the tethered ligand sequence for the human PAR-2 once the receptor is cleaved by tryptase. Tryptase stimulation also increased cell surface expression of P-selectin, decreased electrical resistance, and increased neutrophil adherence to the endothelial cell monolayer. The tryptase-stimulated increases in both cell surface P-selectin expression and neutrophil adhesion were also inhibited with BEL pretreatment. We conclude that tryptase stimulation of HCAEC contributes importantly to early inflammatory events after vascular injury by activation of iPLA2, leading to arachidonic acid release, PAF production, cell surface P-selectin expression, and increased neutrophil adherence.

IntroductionWe developed a service to identify potential study participants through electronic medical records and deliver study invitations through patient portals.MethodsThe service was piloted in a cohort study that used multiple recruitment methods.ResultsPatient portal messages were sent to 1303 individuals and the enrollment rate was 10% (n=127). The patient portal enrollment rate was significantly higher than email and post mail (4%) strategies.ConclusionPatient portal messaging was an effective recruitment strategy.

. Calcium-independent phospholipase A 2 is regulated by a novel protein kinase C in human coronary artery endothelial cells. Am J Physiol Cell Physiol 288: C475-C482, 2005; doi:10.1152/ ajpcell.00306.2004.-We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca 2ϩ -independent phospholipase A2 (iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 Ϯ 0.1 vs. 5.3 Ϯ 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 Ϯ 1.0 vs. 12.0 Ϯ 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 Ϯ 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 Ϯ 1.0 nmol PO 4/mg of protein). Incubation of HCAEC with the selective iPLA 2 inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 Ϯ 0.3 vs. 1.1 Ϯ 0.1%) and increased production of lysoplasmenylcholine (1.4 Ϯ 0.2 vs. 0.6 Ϯ 0.1 nmol PO 4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 Ϯ 0.6 to 0.5 Ϯ 0.1%) and lysoplasmenylcholine production (2.0 Ϯ 0.1 to 0.2 Ϯ 0.1 nmol PO 4/mg of protein). These data suggest that PKC activates iPLA 2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production. lysophospholipids; cell signaling; phospholipid metabolism; arachidonic acid WE DEMONSTRATED PREVIOUSLY that thrombin stimulation of human coronary artery endothelial cells (HCAEC) activates a membrane-associated, Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ). This enzyme selectively hydrolyzes membrane plasmalogen phospholipids, leading to an increase in the production of lysoplasmalogens and arachidonic acid. Activation of endothelial cell (EC) membrane-associated iPLA 2 is also observed when EC are incubated for short intervals with phorbol 12-myristate 13-acetate (PMA), suggesting that the increase in iPLA 2 activity is mediated by protein kinase C (PKC). In addition, downregulation of PKC activity by prolonged incubation with PMA also results in complete inhibition of thrombin-stimulated iPLA 2 activity (6).

Meyer MC, McHowat J. Calcium-independent phospholipase A2-catalyzed plasmalogen hydrolysis in hypoxic human coronary artery endothelial cells. Am J Physiol Cell Physiol 292: C251-C258, 2007. First published August 30, 2006; doi:10.1152/ajpcell.00120.2006.-Thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in activation of a membrane-associated, calcium-independent phospholipase A2 (iPLA2) that selectively hydrolyzes membrane plasmalogen phospholipids. Rupture of an atherosclerotic plaque and occlusion of the coronary vasculature results in a coronary ischemic event in which HCAEC in the ischemic area would be exposed to dramatic decreases in oxygen tension in addition to thrombin exposure. We exposed HCAEC to hypoxia in the presence or absence of thrombin stimulation and measured iPLA 2 activation, membrane phospholipid hydrolysis, and the accumulation of biologically active phospholipid metabolites. HCAEC exposed to hypoxia, thrombin stimulation, or a combination of the two conditions demonstrated an increase in iPLA2 activity and an increase in arachidonic acid release from plasmenylcholine. Thrombin stimulation of normoxic HCAEC did not result in an accumulation of choline lysophospholipids, but hypoxia alone and in combination with thrombin stimulation led to a significant accumulation of lysoplasmenylcholine (LPlsCho). We propose that the presence of hypoxia inhibits LPlsCho catabolism, at least in part, as a result of the accumulation of long-chain acylcarnitines. The combination of increased production and decreased catabolism of LPlsCho is necessary for its accumulation. Pretreatment with bromoenol lactone to inhibit iPLA2 blocked membrane phospholipid hydrolysis and production of membrane phospholipid-derived metabolites. The increase in iPLA2 activity and the subsequent accumulation of membrane phospholipid-derived metabolites in HCAEC exposed to hypoxia or thrombin stimulation alone, and particularly in combination, have important implications in inflammation and arrhythmogenesis in atherosclerosis/thrombosis and subsequent myocardial ischemia. myocardial ischemia; arrhythmogenesis; thrombosis SUDDEN CARDIAC DEATH in humans invariably results from malignant ventricular arrhythmias secondary to acute myocardial ischemia precipitated by the evolution of an intracoronary thrombus (3,7,32). We have demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in hydrolysis of membrane plasmalogen phospholipids by a Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) that leads to the generation of several phospholipid metabolites that may play an important role in inflammation or arrhythmogenesis in the heart (21).Once blood flow is interrupted or severely reduced by increased or complete occlusion of a coronary artery precipitating an ischemic event, the endothelial cells in the ischemic area would be exposed to dramatic decreases in oxygen tension in addition to exposure to thrombin. Results from studies involving release of arachido...