Plasma platelet activating factor-acetylhydrolase (PAF-AH)

The platelet-activating factor-acetylhydrolase (PAF-AH) is an enzyme which catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. The family of PAF-AHs consists of two intracellular isoforms (Ib and II), and one secreted isoform (plasma). These PAF-AHs show different biochemical characteristics and molecular structures. Plasma PAF-AH and intracellular isoform, II degrade not only PAF but also oxidatively fragmented phospholipids with potent biological activities. Among these PAF-AHs, plasma PAF-AH has been the target of many clinical studies in inflammatory diseases, such as asthma, sepsis, and vascular diseases, because the plasma PAF-AH activity in the patients with these diseases is altered when compared with normal individuals. Finding a genetic deficiency in the plasma PAF-AH opened the gate in elucidating the protecting role of this enzyme in inflammatory diseases. The most common loss-of-function mutation, V279F, is found in more than 30% of Japanese subjects (4% homozygous, 27% heterozygous). This single nucleotide polymorphism in plasma PAF-AH and the resulting enzymatic deficiency is thought to be a genetic risk factor in various inflammatory diseases in Japanese subjects. Administration of recombinant plasma PAF-AH or transfer of the plasma PAF-AH gene improves pathology in animal models. Therefore, substitution of plasma PAF-AH would be an effective in the treatment of the patients with the inflammatory diseases and a novel clinical approach. In addition, the detection of polymorphisms in the plasma PAF-AH gene and abnormalities in enzyme activity would be beneficial in the diagnosis of the inflammatory diseases.     

Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus

Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 associated with lipoproteins that hydrolyzes platelet-activating factor (PAF) and oxidized phospholipids. We have developed an ELISA for PAF-AH that is more sensitive than previous methods, and have quantified HDL-associated and non-HDL-associated PAF-AH in healthy, hyperlipidemic, and diabetic subjects. In healthy subjects, plasma total PAF-AH concentration was positively correlated with PAF-AH activity and with plasma total cholesterol, triacylglycerol, LDL cholesterol and apolipoprotein B (apoB) concentrations (all P < 0.01). HDL-associated PAF-AH concentration was correlated positively with plasma apoA-I and HDL cholesterol. Subjects with hyperlipidemia (n = 73) and diabetes mellitus (n = 87) had higher HDL-associated PAF-AH concentrations than did controls (P < 0.01). Non-HDL-associated PAF-AH concentration was lower in diabetic subjects than in controls (P < 0.01). Both hyperlipidemic and diabetic subjects had lower ratios of PAF-AH to apoB (P < 0.01) and higher ratios of PAF-AH to apoA-I (P < 0.01) than did controls. Our results show that the distribution of PAF-AH mass between HDLs and LDLs is determined partly by the concentrations of the lipoproteins and partly by the mass of enzyme per lipoprotein particle, which is disturbed in hyperlipidemia and diabetes mellitus.     

Lung production of platelet-activating factor acetylhydrolase in oleic acid-induced acute lung injury

Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAF- acetylhydrolases (PAF-AHs) terminate PAF's signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0=0.001+/-0.001 nmol/ml/min/g vs Time 6=0.031+/-0.018 nmol/ml/min/g, p=0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation.     

N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.     

Human macrophages secret platelet-activating factor acetylhydrolase

When monocytes mature to macrophages, their ability to accumulate the pro-inflammatory lipid autacoid, platelet-activating factor (PAF), is markedly decreased (Elstad, M. R. Stafforini, D. M., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1989) J. Biol. Chem. 264, 8467-8470) in conjunction with a 260-fold increase in the activity of intracellular PAF acetylhydrolase (PAF-AH). We now demonstrate that macrophages also secrete PAF-AH and that the secreted enzyme is biochemically and immunologically identical to the human plasma PAF-AH. It is sensitive to the same active-site-directed inhibitors, has the same electrophoretic mobility, is associated with lipoprotein particles, and transfers between low density lipoprotein and high density lipoprotein in a pH-dependent manner like the plasma PAF-AH. In addition, both activities hydrolyze oxidatively fragmented phospholipids and PAF. These data indicate that macrophages are a cellular source of the plasma PAF-AH. Thus, macrophages secrete an enzyme that inactivates lipid mediators at sites of inflammation and in plasma. These changes during the maturation of monocytes to macrophages may serve to limit the acute inflammatory response.     

Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.     

Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF-releasing factor in serum.

The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators.     

The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2

Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.     

Altered distribution of platelet-activating factor- acetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia

Platelet-activating factor-acetylhydrolase (PAF-AH) is a lipoprotein-associated phospholipase A2 capable of hydrolyzing platelet-activating factor (PAF) and oxidatively modified phospholipids. We studied the plasma- and lipoprotein-associated PAF-AH activity in patients with primary hypercholesterolemia. Thirty-eight unrelated patients with heterozygous familial hypercholesterolemia (HeteroFH), five patients with homozygous FH (HomoFH), and 33 patients with primary non-FH hypercholesterolemia (NonFH) participated in the study. In all patient groups the plasma PAF-AH activity was significantly elevated compared with 33 normolipidemic controls, the HomoFH having the highest and the NonFH patients showing the lowest enzyme activity. Gradient ultracentrifugation studies showed that this increase is not only due to the elevation in the plasma LDL but also to the increase in the PAF-AH activity associated with each LDL subfraction, being more profound in the small-dense LDL-5. Unlike LDL, no difference in the HDL-associated PAF-AH activity was observed among all groups. Consequently, an altered distribution of enzyme activity among apolipoprotein B (apoB)- and apolipoprotein A-I (apoA-I)-containing lipoproteins is observed in hypercholesterolemic patients, resulting in a significant decrease in the ratio of the HDL-associated PAF-AH to the total plasma enzyme activity compared with controls. This reduction is proportional to the increase of the plasma LDL-cholesterol (LDL-C) levels and consequently to the severity of the hypercholesterolemia. Thus, the ratio of HDL-associated PAF-AH-total plasma enzyme activity may be useful as a potential marker of atherogenicity in subjects with primary hypercholesterolemia.     

Characterization of a platelet activating factor acetylhydrolase from rat adipocyte

Platelet activating factor-acetylhydrolases (PAF-AHs) are a family of enzymes with the common property of hydrolyzing and inactivating PAF and thus regulating its levels. In the course of studying the role of PAF in rat adipocytes and its possible implication in body weight regulation and immune response, conditions in which adipocytes are involved, we investigated the existence of PAF-AH in these cells. We detected PAF-AH activity in rat adipocytes which is mainly distributed in the cytosol. The behaviour of the enzyme during hydrophobic chromatography, together with the fact that part of the enzyme activity was found in the fat cake of adipocyte homogenate suggests the hydrophobic nature of rat adipocyte PAF-AH. The enzyme activity was distinct from the Ca2+-dependent and independent phospholipase A2, the lysophospholipase, the lecithin:cholesterol acyltransferase (LCAT) and from non-specific acetylhydrolases. We partially purified PAF-AH from rat adipocyte cytosolic fraction and the purified enzyme revealed a major protein band of 66 kDa and a minor one of 37 kDa on SDS/PAGE. The purified PAF-AH has an apparent Km value of 4.9 microM and the enzyme activity was inactivated by PMSF and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and moderately stimulated by dithiothreitol (DTT). Furthermore, in this study we identified PAF in rat adipocytes and determined its concentration.     

Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure

An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.     

Platelet-activating factor acetylhydrolase (PAF-AH)

Platelet-activating factor (PAF) is one of the most potent lipid messengers involved in a variety of physiological events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity, and its deacetylation induces loss of activity. The deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH). A series of biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Type I PAF-AH is a G-protein-like complex consisting of 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 type I lissencephaly. Recent studies indicate that LIS1/beta is important in cellular functions such as induction of nuclear movement and control of microtubule organization. Although substantial evidence is accumulating supporting the idea that the catalytic subunits are also involved in microtubule function, it is still unknown what role PAF plays in the process and whether PAF is an endogenous substrate of this enzyme. Type II PAF-AH is a single polypeptide and shows significant sequence homology with plasma PAF-AH. Type II PAF-AH is myristoylated at the N-terminus and like other N-myristoylated proteins is distributed in both the cytosol and membranes. Plasma PAF-AH is also a single polypeptide and exists in association with plasma lipoproteins. Type II PAF-AH as well as plasma PAF-AH may play a role as a scavenger of oxidized phospholipids which are thought to be involved in diverse pathological processes, including disorganization of membrane structure and PAF-like proinflammatory action. In this review, we will focus on the structures and possible biological functions of intracellular PAF-AHs.     

To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase

Mounting ambiguity persists around the functional role of the plasma form of platelet-activating factor acetylhydrolase (PAF-AH). Because PAF-AH hydrolyzes PAF and related oxidized phospholipids, it is widely accepted as an anti-inflammatory enzyme. On the other hand, its actions can also generate lysophosphatidylcholine (lysoPC), a component of bioactive atherogenic oxidized LDL, thus allowing the enzyme to have proinflammatory capabilities. Presence of a canonical lysoPC receptor has been seriously questioned for a multitude of reasons. Animal models of inflammation show that elevating PAF-AH levels is beneficial and not deleterious and overexpression of PAF receptor (PAF-R) also augments inflammatory responses. Further, many Asian populations have a catalytically inert PAF-AH that appears to be a severity factor in a range of inflammatory disorders. Correlation found with elevated levels of PAF-AH and CVDs has led to the design of a specific PAF-AH inhibitor, darapladib. However, in a recently concluded phase III STABILITY clinical trial, use of darapladib did not yield promising results. Presence of structurally related multiple ligands for PAF-R with varied potency, existence of multi-molecular forms of PAF-AH, broad substrate specificity of the enzyme and continuous PAF production by the so called bi-cycle of PAF makes PAF more enigmatic. This review seeks to address the above concerns.     

Platelet-activating factor acetylhydrolases: An overview and update

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique members of the phospholipase A₂ family that can hydrolyze the acetyl group of PAF, a signaling phospholipid that has roles in diverse (patho)physiological processes. Three types of PAF-AH have been identified in mammals, one plasma type and two intracellular types [PAF-AH (I) and PAF-AH (II)]. Plasma PAF-AH and PAF-AH (II) are monomeric enzymes that are structurally similar, while PAF-AH (I) is a multimeric enzyme with no homology to other PAF-AHs. PAF-AH (I) shows a strong preference for an acetyl group, whereas plasma PAF-AH and PAF-AH (II) also hydrolyze phospholipids with oxidatively modified fatty acids. Plasma PAF-AH has been implicated in several diseases including cardiovascular disease. PAF-AH (I) is required for spermatogenesis and is increasingly recognized as an oncogenic factor. PAF-AH (II) was recently shown to act as a bioactive lipid-producing enzyme in mast cells and thus could be a drug target for allergic diseases. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.     

Bovine platelet-activating factor acetylhydrolase (PAF-AH) activity related to fertility

Plasma platelet-activating factor acetylhydrolase (PAF-AH), the enzyme characterized by the association with plasma lipoproteins, degrades platelet-activating factor (PAF) as well as PAF-like oxidatively fragmented phospholipids produced during oxidative stress. Apart from pro-inflammatory properties, PAF is also related to reproductive processes and successful fertility. In order to get a better insight into the involvement of PAF-AH in the fertility of cows, the aim of the study was to determine the PAF-AH activity as well as the C-reactive protein, cholesterol and high density lipoprotein-cholesterol (HDL-C) in the serum of dairy cows throughout the pregnancy and lactation, as well as in infertile cows. The results showed that serum PAF-AH activity changes throughout pregnancy and lactation with a lower level during periparturient period. It is also found higher PAF-AH activity in lactating cows with reproductive disorders compared to high lactating cows without reproductive disorders. Strong correlation between PAF-AH activity and HDL-C concentration indicates that HDL could have considerable influence on PAF-AH activity in bovine plasma. CRP concentration was also lower during transition period suggesting that lactation might stimulate CRP synthesis in bovine. A higher CRP concentration in cows with reproductive disorders compared to fertile cows at the peak of lactation, demonstrates that milk production is not the only factor influencing CRP in cows. A significant correlation between PAF-AH activity and CRP level shows that both parameters could be influenced by reproductive status of dairy cows.     

Anti-apoptotic actions of the platelet-activating factor acetylhydrolase I alpha2 catalytic subunit

Platelet-activating factor (PAF) is an important mediator of cell loss following diverse pathophysiological challenges, but the manner in which PAF transduces death is not clear. Both PAF receptor-dependent and -independent pathways are implicated. In this study, we show that extracellular PAF can be internalized through PAF receptor-independent mechanisms and can initiate caspase-3-dependent apoptosis when cytosolic concentrations are elevated by approximately 15 pM/cell for 60 min. Reducing cytosolic PAF to less than 10 pM/cell terminates apoptotic signaling. By pharmacological inhibition of PAF acetylhydrolase I and II (PAF-AH) activity and down-regulation of PAF-AH I catalytic subunits by RNA interference, we show that the PAF receptor-independent death pathway is regulated by PAF-AH I and, to a lesser extent, by PAF-AH II. Moreover, the anti-apoptotic actions of PAF-AH I are subunit-specific. PAF-AH I alpha1 regulates intracellular PAF concentrations under normal physiological conditions, but expression is not sufficient to reduce an acute rise in intracellular PAF levels. PAF-AH I alpha2 expression is induced when cells are deprived of serum or exposed to apoptogenic PAF concentrations limiting the duration of pathological cytosolic PAF accumulation. To block PAF receptor-independent death pathway, we screened a panel of PAF antagonists (CV-3988, CV-6209, BN 52021, and FR 49175). BN 52021 and FR 49175 accelerated PAF hydrolysis and inhibited PAF-mediated caspase 3 activation. Both antagonists act indirectly to promote PAF-AH I alpha2 homodimer activity by reducing PAF-AH I alpha1 expression. These findings identify PAF-AH I alpha2 as a potent anti-apoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.     

Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage

Platelet activating factor (PAF) is a key molecule for inflammation. To examine a role of peroxisome proliferator-activated receptor gamma (PPARgamma) in inflammatory reactions of atherosclerosis, we investigated the effects of 15-deoxy-(Delta12,14)-Prostaglandin J2 (15d-PGJ2) and pioglitazone, PPARgamma ligands, on plasma PAF-acetylhydrolase (PAF-AH) expression in THP-1 macrophages. PAF-AH mRNA and protein were up-regulated by the PPARgamma ligands. Prostaglandin F2alpha (PGF2alpha), a PARgamma inhibitor, abrogated the up-regulation of PAF-AH mRNA by pioglitazone, suggesting that PPARgamma activation is involved in the induction of PAF-AH by pioglitazone. As PAF promotes the cell motility with cytoskeletal reorganization, we investigated the effect of pioglitazone on PAF-mediated morphological changes in THP-1 macrophages. In the absence of pioglitazone, PAF promoted the elongation of actin cytoskeleton, which was inhibited by pretreatment with pioglitazone. In contrast, pioglitazone was not able to inhibit the morphological changes induced by C-PAF, a non-hydrolyzable PAF agonist. Thus, it is suggested that PAF-induced morphological changes could be inhibited by pioglitazone through PAF-AH, which rapidly hydrolyzed PAF. These data propose that PPARgamma/PAF-AH pathway is a clinical target for the prevention against atherosclerosis.     

PAF and its metabolic enzymes in healthy volunteers: interrelations and correlations with basic characteristics

PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent inflammatory mediator, is synthesized via the remodeling and the de novo route, key enzymes of which are acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), respectively. PAF-acetylhydrolase (PAF-AH) and its extracellular isoform lipoprotein-associated phospholipase-A(2) (Lp-PLA(2)) catabolize PAF. This study evaluated PAF levels together with leukocyte PAF-CPT, lyso-PAF-AT, PAF-AH and Lp-PLA(2) activities in 106 healthy volunteers. Men had lower PAF levels and higher activity of both catabolic enzymes and lyso-PAF-AT than women (P-values <0.05). Age was inversely correlated with PAF levels in men (r=-0.279, P=0.06) and lyso-PAF-AT in women (r=-0.280, P=0.05). In contrast, Lp-PLA(2) was positively correlated with age (r=0.201, P=0.04). Moreover, PAF-CPT was positively correlated with glucose (r=0.430, P=0.002) in women. In addition, Principal Component Analysis revealed three PAF metabolic patterns: (i) increased activities of PAF-CPT and PAF-AH, (ii) increased activities of PAF-CPT and lyso-PAF-AT and (iii) increased activity of Lp-PLA(2). The present study underlines the complexity of PAF's metabolism determinants.     

Clinical aspects of plasma platelet-activating factor-acetylhydrolase

Plasma platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), which is characterized by tight association with plasma lipoproteins, degrades not only PAF but also phospholipids with oxidatively modified short fatty acyl chain esterified at the sn-2 position. Production and accumulation of these phospholipids are associated with the onset of inflammatory diseases and preventive role of this enzyme has been evidenced by many recent studies including prevalence of the genetic deficiency of the enzyme in the patients and therapeutic effects of treatment with recombinant protein or gene transfer. With respect to the atherosclerosis, however, it is not fully cleared whether this enzyme plays an anti-atherogenic role or pro-atherogenic role because plasma PAF-AH also might produce lysophosphatidylcholine (LysoPC) and oxidatively modified nonesterified fatty acids with potent pro-inflammatory and pro-atherogenic bioactivities. These dual roles of plasma PAF-AH might be regulated by the altered distribution of the enzyme between low density lipoprotein (LDL) and high density lipoprotein (HDL) particles because HDL-associated enzymes are considered to contribute to the protection of LDL from oxidative modification. This review focuses on the recent findings which address the role of this enzyme in the human diseases especially including asthma, septic shock and atherosclerosis.     

Paraoxonase gene Gln192Arg (Q192R) polymorphism is associated with coronary artery spasm

We recently reported that oxidative stress is involved in the pathogenesis of coronary spasm. We hypothesized that oxidative-stress-related genetic factors and certain polymorphisms in the paraoxonase gene (PON1) and platelet-activating factor acetylhydrolase (PAF-AH) might influence the pathogenesis of coronary spasm. We therefore examined the possible association between the PON1 Q192R or PAF-AH V279F polymorphisms and coronary spasm in 214 patients with coronary spasm and 212 control subjects. Genotypes were determined by polymerase chain reaction/restriction fragment length polymorphism analysis. The incidence of the PON1-192R allele was significantly higher in the coronary spasm group than in the control group (65% vs 53%; P=0.0005). The PAF-AH-279F allele was not associated with coronary spasm (15% vs. 16%; P=0.8781). Multiple logistic regression analysis with forward stepwise selection involving the PON1-192R allele and the environmental risk factors revealed that the most predictive independent risk factor for coronary spasm was the PON1-192R allele (significance=0.0016, OR=2.52), followed by cigarette smoking (significance=0.0007, OR=2.01). We also measured plasma levels of TBARS (thiobarbituric acid-reactive substances) as a marker of oxidative stress. TBARS levels were higher in R/R types than in Q/Q types (2.115+/-0.086 nmol/ml [ n=25] vs 1.676+/-0.102 nmol/ml [ n=11], P<0.01). Thus, there is a significant association between the PON1-192R allele and coronary spasm; the PON1-192R allele may play an important role in the genesis of coronary spasm, probably by attenuating the suppression of oxidative stress.