Diana M. Stafforini

Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.

We previously reported that high density lipoprotein (HDL) protects against the oxidative modification of low density lipoprotein (LDL) induced by artery wall cells causing these cells to produce pro-inflammatory molecules. We also reported that enzyme systems associated with HDL were responsible for this anti-inflammatory property of HDL. We now report studies comparing HDL before and during an acute phase response (APR) in both humans and a croton oil rabbit model. In rabbits, from the onset of APR the protective effect of HDL progressively decreased and was completely lost by day three. As serum amyloid A (SAA) levels in acute phase HDL (AP-HDL) increased, apo A-I levels decreased 73%. Concomitantly, paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-AH) levels in HDL declined 71 and 90%, respectively, from days one to three. After day three, there was some recovery of the protective effect of HDL. AP-HDL from human patients and rabbits but not normal or control HDL (C-HDL) exhibited increases in ceruloplasmin (CP). This increase in CP was not seen in acute phase VLDL or LDL. C-HDL incubated with purified CP and re-isolated (CP-HDL), lost its ability to inhibit LDL oxidation. Northern blot analyses demonstrated enhanced expression of MCP-1 in coculture cells treated with AP-HDL and CP-HDL compared to C-HDL. Enrichment of human AP-HDL with purified PON or PAF-AH rendered AP-HDL protective against LDL modification. We conclude that under basal conditions HDL serves an anti-inflammatory role but during APR displacement and/or exchange of proteins associated with HDL results in a pro-inflammatory molecule.

Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.

The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis.

ObjectivesTo review the platelet-activating factor (PAF) signaling system, its regulation, and its dysregulation in acute inflammation and thrombosis and in syndromes that involve these cascades, including sepsis. Data SourcesA summary of published literature from MEDLINE search files and published reviews. Data Extraction, Synthesis, and SummaryPAF, a phospholipid signaling molecule, transmits outside-in signals to intracellular transduction systems and effector mechanisms in a variety of cell types, including key cells of the innate immune and hemostatic systems: neutrophils, monocytes, and platelets. Thus, the PAF signaling system is a point of convergence at which injurious stimuli can trigger and amplify both acute inflammatory and thrombotic cascades. The biological activities of PAF are regulated by several precise mechanisms that, together, constrain and control its action in physiologic inflammation. Unregulated synthesis of PAF or defects in the mechanisms that limit its biological activities have the potential to cause pathologic inflammation and thrombosis. In addition, nonenzymatic generation of oxidized phospholipids that are recognized by the PAF receptor can trigger inflammatory and thrombotic events. There is evidence that the PAF signaling system is dysregulated in sepsis, shock, and traumatic injury and that interruption or termination of its effector responses leads to beneficial outcomes. Plasma PAF acetylhydrolase, an enzyme that hydrolyzes PAF and structurally related oxidized phospholipids, yielding products that are no longer recognized by the PAF receptor, may be a particularly important signal terminator. ConclusionThe PAF signaling system can trigger inflammatory and thrombotic cascades, amplify these cascades when acting with other mediators, and mediate molecular and cellular interactions (cross talk) between inflammation and thrombosis. Evidence from in vitro experiments, studies of experimental animals, and clinical observations in humans indicates that the PAF signaling system is important in sepsis and other syndromes of inflammatory injury and that therapeutic strategies to interrupt or terminate signaling via the PAF signaling system may be useful in these conditions.

Platelet-activating Factor Acetylhydrolases

Platelet-activating factor acetylhydrolases are structurally diverse isoenzymes that catalyze the hydrolysis of the acyl group at the second position of glycerol in unusual, bioactive phospholipids (Fig. 1). Thus, as categorized by enzymatic activity, they are phospholipases A2 (Groups VII and VIII, Ref. 1), which often initiate signal transduction and are regulated by the state of the cell activation. However, the platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) acetylhydrolases have the opposite role; they were discovered by investigators who focused on the inactivation of PAF (2). The phospholipid PAF has diverse physiological and pathological functions, and its inactivation was identified as an important step in regulating the overall biological function; indeed PAF acetylhydrolase has been termed a signal terminator (3). However, an immediate problem is apparent; phospholipases A2 are a common component of venoms, and their role in this context is to facilitate the spread of toxins by degrading membrane phospholipids; thus, how could there be such an active enzyme circulating in blood and present in the cytoplasm of cells? The answer came when it was shown that the PAF acetylhydrolases have marked selectivity for phospholipids with short acyl chains at the sn-2 position; with chains longer than nine carbons there was essentially no measurable activity (Fig. 2) (4, 5). Thus, normal membrane phospholipids are protected from hydrolysis by an intrinsic property of the enzyme. Subsequently, it was discovered that certain forms of PAF acetylhydrolases have a broader spectrum of action; they hydrolyze phospholipids containing relatively long sn-2 acyl chains (up to 9 methylene groups, Ref. 5). However, in this case, the suitability of a phospholipid as a substrate depends on another form of unusual sn-2 acyl group, one that contains a carbonyl group at the v-end of the acyl chain (5). The markedly restricted substrate specificity of PAF acetylhydrolases is unusual among phospholipases A2, and the unifying feature of the substrates utilized is that they have potent biological actions, which can lead to pathological events when they accumulate inappropriately. The nature of the substrates hydrolyzed by PAF acetylhydrolases points at key roles for these activities in physiology and pathology, and it has provided important clues into what is currently thought to be the main function of these enzymes, which is to act as scavengers of bioactive phospholipids. The initial studies on PAF acetylhydrolases were performed on the secreted form found in mammalian plasma; this isoform circulates in blood as a complex with lipoproteins (6, 7). In addition, PAF acetylhydrolase activities were identified in the cytosolic fraction of various mammalian tissues (8, 9) and human blood cells (9, 10). These activities have substrate specificities quite similar to that of the plasma PAF acetylhydrolase and thus belong to the same group of calcium-independent phospholipases A2. However, the plasma (11) and intracellular (12–14) forms of PAF acetylhydrolase are encoded by individual genes, and the identity among these varies widely, depending on the intracellular isoform. It is not yet precisely known how each domain affects the function of each PAF acetylhydrolase, but the diversity in gene structure most likely serves to determine specific roles played by each member of the group. PAF, the phospholipid that led to the discovery of PAF acetylhydrolase, is a mediator of a wide range of immune and allergic reactions (15, 16). It activates inflammatory cells at very low concentrations (10–10 M) through a G-protein-linked, serpentine receptor, and it is synthesized in a regulated pathway(s) in response to a variety of agonists. If the mechanism for PAF inactivation is impaired in some way, the return to basal conditions may be compromised, resulting in prolonged inflammation and inappropriately long recruitment of effector cells to sites of injury. The second group of compounds hydrolyzed by PAF acetylhydrolases, the oxidatively fragmented phospholipids (17, 18), also has short acyl groups at the sn-2 position of glycerol, but they are derived from oxidation of polyunsaturated fatty acids that occupy this position in the phospholipids of cellular membranes. These compounds apparently mimic the structure of PAF closely enough to bind to its receptor and thereby elicit the same responses. The similarities between PAF and the oxidatively fragmented phospholipids are contrasted by one essential difference: the synthesis of PAF is highly controlled (16, 19, 20) whereas oxidized phospholipids are produced in an unregulated manner. Therefore, the extent to which products of phospholipid oxidation will accumulate depends very heavily on the rate at which they are catabolized. The fact that PAF acetylhydrolases are maximally active in the basal state and do not require calcium for activity ensures that these activities provide an immediate defense mechanism against toxic effects mediated by fragmented phospholipids.

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Human Plasma Platelet-activating Factor Acetylhydrolase

Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A, activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 207’0, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an W-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had mlz ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Platelet-Activating Factor, a Pleiotrophic Mediator of Physiological and Pathological Processes

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects. This bioactive phospholipid mediates processes as diverse as wound healing, physiological inflammation, apoptosis, angiogenesis, reproduction and long-term potentiation. Recent progress has demonstrated the participation of MAP kinase signaling pathways as modulators of the two critical enzymes, phospholipase A2 and acetyltransferase, involved in the remodeling pathway of PAF biosynthesis. The unregulated production of structural analogs of PAF by non-specific oxidative reactions has expanded this superfamily of signaling molecules to include “PAF-like” lipids whose mode of action is identical to that of authentic PAF. The action of members of this family is mediated by the PAF receptor, a G protein-coupled membrane-spanning molecule that can engage multiple signaling pathways in various cell types. Inappropriate activation of this signaling pathway is associated with many diseases in which inflammation is thought to be one of the underlying features. Inactivation of all members of the PAF superfamily occurs by a unique class of enzymes, the PAF acetylhydrolases, that have been characterized at the molecular level and that terminate signals initiated by both regulated and unregulated PAF production.

Biology of Platelet-activating Factor Acetylhydrolase (PAF-AH, Lipoprotein Associated Phospholipase A2)

IntroductionThis article is focused on platelet-activating factor acetylhydrolase (PAF-AH), a lipoprotein bound, calcium-independent phospholipase A2 activity also referred to as lipoprotein-associated phospholipase A2 or PLA2G7. PAF-AH catalyzes the removal of the acyl group at the sn-2 position of PAF and truncated phospholipids generated in settings of inflammation and oxidant stress.DiscussionHere, I discuss current knowledge related to the structural features of this enzyme, including the molecular basis for association with lipoproteins and susceptibility to oxidative inactivation. The circulating form of PAF-AH is constitutively active and its expression is upregulated by mediators of inflammation at the transcriptional level. This mechanism is likely responsible for the observed up-regulation of PAF-AH during atherosclerosis and suggests that increased expression of this enzyme is a physiological response to inflammatory stimuli. Administration of recombinant forms of PAF-AH attenuate inflammation in a variety of experimental models. Conversely, genetic deficiency of PAF-AH in defined human populations increases the severity of atherosclerosis and other syndromes. Recent advances pointing to an interplay among oxidized phospholipid substrates, Lp(a), and PAF-AH could hold the key to a number of unanswered questions.

Platelet-activating factor acetylhydrolases in health and disease

The platelet-activating factor (PAF) acetylhydrolases catalyze hydrolysis of the sn-2 ester bond of PAF and related pro-inflammatory phospholipids and thus attenuate their bioactivity. One secreted (plasma) and four intracellular isozymes have been described. The intracellular isozymes are distinguished by differences in primary sequence, tissue localization, subunit composition, and substrate preferences. The most thoroughly characterized intracellular isoform, Ib, is a G-protein-like complex with two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause Miller-Dieker lissencephaly. Isoform II is a single polypeptide that is homologous to the plasma PAF acetylhydrolase and has antioxidant activity in several systems. Plasma PAF acetylhydrolase is also a single polypeptide with a catalytic triad of amino acids that is characteristic of the alpha/beta hydrolases. Deficiency of this enzyme has been associated with a number of pathologies. The most common inactivating mutation, V279F, is found in >30% of randomly surveyed Japanese subjects (4% homozygous, 27% heterozygous). The prevalence of the mutant allele is significantly greater in patients with asthma, stroke, myocardial infarction, brain hemorrhage, and nonfamilial cardiomyopathy. Preclinical studies have demonstrated that recombinant plasma PAF acetylhydrolase can prevent or attenuate pathologic inflammation in a number of animal models. In addition, preliminary clinical results suggest that the recombinant enzyme may have pharmacologic potential in human inflammatory disease as well. These observations underscore the physiological importance of the PAF acetylhydrolases and point toward new approaches for controlling pathologic inflammation.