Effect of Platelet Activating Factor-Acetyihydrolase on the Formation and Action of Minimally Oxidized Low Density Lipoprotein

Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10-2 U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL. (J. Clin. Invest. 1995. 95:774-782) DFP, diisopropyl fluorophosphate; HAEC, human aortic endothelial cells; HASMC, human aortic smooth muscle cells; LPC, lysophosphatidylcholine; MM-LDL, mildly oxidized LDL; MTM, monocyte transmigration; Ox-LDL, highly oxidized LDL; PAF-AH, platelet activating factor-acetylhydrolase; PAPC, l-palmitoyl-2-arachidonoyl-sn-glycero- 3-phosphorylcholine; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acids. 3-phosphorylcholine (PAPC) and l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphorylcholine, were obtained from Avanti Polar Lipids, Alabaster, AL. TLC silica plates and HPLC analytical and guard columns were obtained from Alltech Associates, Inc., Deerfield, IL. Aminopropyl and octadecyl solid phase extraction columns were obtained from J. T. Baker Inc., Phillipsburg, NJ. Transwells and chamber slides were obtained from Costar Corp., Cambridge, MA. The fluorescent probe ,1,'-diocta- decyl-3,3,3',3' tetramethyl-indocarbocyanine perchlorate (DiI) was purchased from Molecular Probes Inc., Eugene, OR. Centricons were obtained from Amicon Corp., Beverly, MA. PAF-AH was isolated from erythrocytes as described (36), using a solvent mixture of chloroformlisopropanol/ triethylamine/methanol/0.25% KC1 (90:75:54:27:18). Lipids were vis-ualized by spraying the developed plate with 10% sulfuric acid in ethanol and charring at 120'C for 15 min. For HPLC analysis, phospholipids were resuspended in chloroform and injected onto a silica column (Spherisorb, 150 mm x 4.6 mm, 5 pm; Alltech Associates, Inc.) and eluted isocratically with a mobile solvent of acetonitrile/methanol/water (79:8:13) at a flow rate of 1.0 ml/min. In some cases, fractions were collected from the column under sterile conditions, dried to a residue under nitrogen at 370C, resuspended in tissue culture medium, and incubated with endothelial cells to test for the induction of binding activity. native LDL ,ug/ml) in the or of various test compounds for 24 h. The culture supernatants were subsequently transferred to untreated cocultures and were incubated for an additional 24 h. Monocytes were labeled with the fluorescent probe DiI at 40C for 10 min, were centrifuged to remove the excess DiI, and the cell pellet was resuspended in medium 199 at the desired monocyte density. At the end of the second 24-h treatment, Dil-labeled monocytes were and In pilot experiments culture were screened for chemotactic activity using cell line THP-1 which using human peripheral blood monocytes (data not shown). Data were analyzed using model 1 ANOVA. from labeled substrate by solid phase chromatography and quantitated by liquid scintillation as previously (40). Enzymic activity is expressed in micromoles per milliliter per hour after appropriate corrections. There was complete agreement between the values obtained using the two assay methods. Other procedures. Measurement of cell protein content and those of lipoproteins was performed by microtiter plate assay based on the method of et al.