Characterisation of the phospholipase A2 isoforms supporting prostacyclin production synthesis by endothelial cells
Endothelial cells line the luminal surface of blood vessels where they release hormones including prostacyclin (PGI2) that dilates blood vessels, inhibits platelet aggregation and protects against atherogenesis. PGI2 is produced by the sequential actions of three enzymes: phospholipase A2 (PLA2) cleaves membrane phospholipids to liberate arachidonic acid (AA), which is converted by cyclooxygenase (COX) into PGH2, which is then converted to PGI2 by PGI2 synthase. Although numerous PLA2 isoforms exist, it is thought to be the Ca2+-dependent cytosolic PLA2 (cPLA2) isoform that supports prostaglandin synthesis (1,2). However, it has been proposed that PGI2 release from endothelial cells induced by thrombin or tryptase is dependent upon Ca2+-independent iPLA2 (3). To test this proposal, we examined the contribution of cPLA2 and iPLA2 to PGI2 synthesis using blood-outgrowth endothelial cells (BOECs) from healthy volunteers and from a cPLA2-deficient patient (2), as well as intact mouse arteries.
BOECs were grown from blood of healthy donors (n=4) and a patient lacking cPLA2 (2). These cells are an established model of endothelium from vessels (4). BOECs were incubated with the cPLA2 inhibitor pyrrophenone (0.1-10µM) or vehicle (0.1% DMSO; 30min). They were then incubated with thrombin (1U/ml; PBS) or Ca2+ ionophore (A23187; 30µM; DMSO 0.1% final) to activate endogenous iPLA2 and cPLA2 enzymes, respectively, or with AA (50µM; ethanol 0.05% final), which stimulates PGI2 formation without the requirement of PLA2. In parallel, aortic rings prepared from mice (male, BALB/c, 10 weeks; n=6) were incubated with pyrrophenone, the iPLA2 inhibitor bromoenol lactone (BEL; 0.1-10µM) or vehicle (0.1% DMSO), as above. Mouse aortic rings do not respond robustly to thrombin, so for these experiments, PGI2 release was stimulated with acetylcholine (ACh; 1µM; saline). In this tissue Ach, as for thrombin, acts via a Gq-protein coupled receptor signalling pathway. In both assays, PGI2 release was determined after 30min incubation by measurement of its breakdown product, 6-ketoPGF1 α using ELISA.
BOECs released PGI2 in response to A23187, thrombin and AA (vehicle, 160±19pg/ml; thrombin, 1853±1089; A23187, 608±180pg/ml; AA, 2035±836pg/ml; all p<0.001 by Wilcoxon signed rank test). PGI2 release induced by thrombin was inhibited in a concentration-dependent manner by the cPLA2 inhibitor pyrrophenone (-logIC50; 6.8±0.3, p=0.02 by Kruskal-Wallis test), as was release of PGI2 induced by A23187 (-logIC50; 7.1±0.7, p=0.05 by Kruskil-Wallis test). Release of PGI2 from BOECs incubated with the COX substrate, AA, which bypasses the need for PLA2 activity, was not affected by pyrrophenone. In agreement, BOECs from a cPLA2-deficient individual did not produce PGI2 in response to thrombin or A23187 but responded normally to exogenous AA. In mouse aortic rings, ACh stimulated robust production of PGI2 which was inhibited in a concentration-dependent manner by pyrrophenone (-logIC50; 6.2±0.5; p=0.04 by Kruskil-Wallis test) but unaffected by the iPLA2 inhibitor, BEL (ACh; 1374±252pg/ml, ACh+10μM BEL; 1377±201pg/ml, p=0.94 by Mann-Whitney test).
These data demonstrate that cPLA2 and not iPLA2 is the dominant isoform driving PGI2 production by endothelial cells and intact arteries.
(1) Adler DH et al, JCI 118:2121, 2008. (2) Brooke MA et al, Gut In press, 2013 (3) Sharma J et al, Biochem 49:5273, 2010. (4) Starke RD et al, Blood, 121:2773, 2013.