Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-derived Factor Receptor (PEDF-R)

PurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.MethodsMice in which PNPLA2 was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POS were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.ResultsThe RPE of the cKO mice accumulated lipids as well as more abundant and larger rhodopsin particles compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knock down also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy

Early blood retinal barrier (BRB) dysfunction induced by hyperglycemia was related to increased pro-inflammatory activity of phospholipase A2 (PLA2) and the upregulation of vascular endothelial growth factor A (VEGF-A). Here, we tested the role of VEGF-A in high glucose (HG)-induced damage of human retinal endothelial cells (HRECs) mediated by Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) PLA2s. HRECs were treated with normal glucose (5 mM, NG) or high glucose (25 mM, HG) for 48 h with or without the VEGF-trap Aflibercept (Afl, 40 µg/mL), the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3; 15 µM), the iPLA2 inhibitor bromoenol lactone (BEL; 5 µM), or VEGF-A (80 ng/mL). Both Afl and AACOCF3 prevented HG-induced damage (MTT and LDH release), impairment of angiogenic potential (tube-formation), and expression of VEGF-A mRNA. Furthermore, Afl counteracted HG-induced increase of phospho-ERK and phospho-cPLA2 (immunoblot). VEGF-A in HG-medium increased glucose toxicity, through upregulation of phospho-ERK, phospho-cPLA2, and iPLA2 (about 55%, 45%, and 50%, respectively); immunocytochemistry confirmed the activation of these proteins. cPLA2 knockdown by siRNA entirely prevented cell damage induced by HG or by HG plus VEGF-A, while iPLA2 knockdown produced a milder protective effect. These data indicate that VEGF-A mediates the early glucose-induced damage in retinal endothelium through the involvement of ERK1/2/PLA2 axis activation.

Possible Involvement of Intracellular Calcium-Independent Phospholipase A2 in the Release of Secretory Phospholipases from Mast Cells—Increased Expression in Ileal Mast Cells of Crohn’s Disease

Increased activity of secretory phospholipases A2 (sPLA2) type-II was previously observed in ileum of Crohn’s disease (CD). Our aims were to explore the involvement of calcium-independent (i)PLA2β in the release of sPLA2s from the human mast cell (MC) line (HMC-1) and investigate expressions of cytosolic (c)PLA2α, iPLA2β, sPLA2-IIA and sPLA2-V in MCs of CD ileum. The release of sPLA2 was investigated in HMC-1 by immunocytochemistry and ELISA. The expression intensities of PLA2s in mucosal MCs, and the proportion of PLA2-positive MCs, were investigated in normal ileum and in ileum from patients with CD by immunohistochemistry. The calcium ionophore-stimulated release of sPLA2-IIA and sPLA2-V from HMC-1 was reduced by the iPLA2-inhibitor bromoenol lactone. All four PLA2s were detectable in mucosal MCs, both in normal ileum and in CD, but the proportion of iPLA2β-containing mucosal MCs and the expression intensity of sPLA2-IIA was increased in CD. Results indicate that iPLA2β is involved in the secretion of sPLA2s from HMC-1, and suggest that iPLA2β-mediated release of sPLA2 from intestinal MCs may contribute to CD pathophysiology. Ex vivo studies on isolated mucosal mast cells are however needed to clarify the precise role of MC PLA2s in the inflammatory processes of CD.

Bromoenol Lactone, an Inhibitor of Calcium-Independent Phospholipase A2, Suppresses Carrageenan-Induced Prostaglandin Production and Hyperalgesia in Rat Hind Paw

Prostaglandin (PG) E2and PGI2are essential to hyperalgesia in inflammatory tissues. These prostaglandins are produced from arachidonic acid, which is cleaved from membrane phospholipids by the action of phospholipase A2(PLA2). Which isozyme of PLA2is responsible for the cleavage of arachidonic acid and the production of prostaglandins essential to inflammation-induced hyperalgesia is not clear. In this study, we examined the effects of two PLA2isozyme-specific inhibitors on carrageenan-induced production of PGE2and PGI2in rat hind paw and behavioral nociceptive response to radiant heat. Local administration of bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2(iPLA2), significantly reduced carrageenan-induced elevation of prostaglandins in the inflamed foot pad 3 h after injection. It also ameliorated the hyperalgesic response between 1 h and 3 h after carrageenan injection. On the other hand, AACOCF3, an inhibitor of cytosolic PLA2, suppressed neither prostaglandin production nor the hyperalgesic response. BEL did not suppress the mRNA levels of iPLA2β, iPLA2γ, cyclooxygenase-2, microsomal prostaglandin E synthase, prostaglandin I synthase, or proinflammatory cytokines in the inflamed foot pad, indicating that BEL did not suppress inflammation itself. These results suggest that iPLA2is involved in the production of prostaglandins and hyperalgesia at the inflammatory loci.

Group VIA Phospholipase A 2 Mediates Enhanced Macrophage Migration in Diabetes Mellitus by Increasing Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4

Objective— We previously demonstrated that nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) mediates increased monocyte priming and chemotaxis under conditions of diabetic metabolic stress, and emerging data indicate that group VIA phospholipase A 2 (iPLA 2 β) also participates in regulating monocyte chemotaxis. Here, we examined relationships between iPLA 2 β expression and Nox4 action in mouse peritoneal macrophages subjected to diabetic metabolic stress. Approach and Results— Increased iPLA 2 β expression and activity were observed in macrophages from low-density lipoprotein receptor knockout mice that were fed a high-fat diet, and this was associated with time-dependent increases in atherosclerotic lesion size and macrophage content. Incubating macrophages with 30 mmol/L D-glucose, 100 μg/mL low-density lipoprotein, or both (D-glucose+low-density lipoprotein) induced a robust increase in iPLA 2 β expression and activity and in cell migration in response to monocyte chemoattractant protein-1. The increases in iPLA 2 β activity and cell migration were prevented by a bromoenol lactone iPLA 2 β suicide inhibitor or an iPLA 2 β antisense oligonucleotide. Incubating macrophages under conditions that mimic diabetic metabolic stress ex vivo resulted in increased Nox4 expression and activity and hydrogen peroxide generation compared with controls. Bromoenol lactone prevented those effects without affecting Nox2 expression. Nox4 inhibition eliminated diabetic metabolic stress–induced acceleration of macrophage migration. Lysophosphatidic acid restored Nox4 expression, hydrogen peroxide generation, and migration to bromoenol lactone–treated cells, and a lysophosphatidic acid receptor antagonist abrogated iPLA 2 β-mediated increases in Nox4 expression. Conclusions— Taken together, these observations identify iPLA 2 β and lysophosphatidic acid derived from its action as critical in regulating macrophage Nox4 activity and migration in the diabetic state in vivo and under similar conditions ex vivo.

Mitochondrial reactive oxygen species: which ROS signals cardioprotection?

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa AD, Garlid KD. Am J Physiol Heart Circ Physiol 295, H874–H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O2·−), hydrogen peroxide (H2O2), and hydroxyl radical (HO˙), each of which has been proposed as the signaling ROS. Feedback activation of mitoKATP provides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO˙ excludes H2O2 as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO˙ and that it is probably a product of phospholipid oxidation.