Cadmium induces vascular permeability via activation of the p38 MAPK pathway

The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl₂) on human umbilical vein endothelial cells (HUVECs). At 4 μM, CdCl₂ induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24 h. This effect of CdCl₂ was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl₂-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and β-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.     

Effect of platelet-activating factor receptor expression on CHO cell motility

Tumor cell migration may favor local mass expansion and metastasis dissemination. Several tumors were found to express the receptor for platelet-activating factor (PAF), a potent mediator of leukocyte chemotaxis and endothelial cell migration. However, its functional role on tumor cells is largely unexplored. In the present study, we evaluated the motogenic effect of PAF on Chinese hamster ovarian (CHO) cancer cells transfected with the human PAF-receptor cDNA (CHO PAF-R). By using time-lapse recording, we detected a rapid motogenic response to PAF stimulation on CHO PAF-R, whereas no effect was evident on vector-only transfected cells. Such an effect was observed on scattered cell motility, on cells seeded on a fibronectin- or collagen-coated surface, and on migration of confluent monolayer cells. Cell speed increased at 1 h and was maximal 6-8 h after PAF stimulation on CHO PAF-R. Concomitantly, PAF induced marked changes in cytoskeleton actin distribution with cell contraction, assembling of stress fibers, and polar foci of adhesion. In conclusion, the present study demonstrates that PAF is a potent inducer of tumor cell motility, thus suggesting a role for this mediator in tumor growth and dissemination.     

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures

This study examines the hypothesis that PAF stimulates release of PGI₂ from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF_1α from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF_1α was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF_1α release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI₂ release.     

A yeast PAF acetylhydrolase ortholog suppresses oxidative death

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.     

Vascular permeability induced by VEGF family members in vivo: role of endogenous PAF and NO synthesis

We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet-activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF-induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF-A(165), VEGF-A(121), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased vascular permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME) abrogated protein extravasation mediated by VEGF-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR-2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF-A(165)-mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF-A(165) proinflammatory activities.     

Dynamics of PAF-induced conjunctivitis reveals differential expression of PAF receptor by macrophages and eosinophils in the rat

The action of platelet-activating factor (PAF) toward the PAF receptor (PAF-R) plays an important role in inflammation. We employed immunohistochemistry and quantitative confocal immunofluorescence microscopy to examine the dynamic changes of PAF-R expression in the conjunctiva in response to PAF-induced conjunctivitis in Brown Norway rats within the first 24 h after topical administration of PAF. Instillation of PAF caused an alteration in pattern of recruitment of macrophages and eosinophils into the conjunctiva, as was visualized by immunohistochemical staining for the antigens ED 1 and ED 2 (markers of macrophages) and MBP (a marker of eosinophils). An increase in the number of PAF-R-positive cells was also detected. Quantitative colocalization analysis revealed the strongest rise in the degree of PAF-R expression by macrophages within the first 6 h, whereas their infiltration increased throughout the period of observation. However, eosinophils showed a high degree of PAF-R expression during all 24 h of the experiment, although they infiltrated strongly only within the first 2 h. Thus, for the first time, the use of quantitative colocalization analysis software developed by us has revealed intrinsic details of the interaction of PAF and PAF-R in conjunctivitis, findings not otherwise obtainable by using qualitative approaches alone. Our results provide a theoretical basis for a definition of the proper time-frame in which to prevent and control the infiltration of macrophages and eosinophils into the conjunctiva.     

A Novel Platelet-Activating Factor Receptor Antagonist Inhibits Choroidal Neovascularization and Subretinal Fibrosis

Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration (AMD), the most common cause of blindness in developed countries. To date, the precise molecular and cellular mechanisms underlying CNV have not been elucidated. Platelet-activating factor (PAF) has been previously implicated in angiogenesis; however, the roles of PAF and its receptor (PAF-R) in CNV have not been addressed. The present study reveals several important findings concerning the relationship of the PAF-R signaling with CNV. PAF-R was detected in a mouse model of laser-induced CNV and was upregulated during CNV development. Experimental CNV was suppressed by administering WEB2086, a novel PAF-R antagonist. WEB2086-dependent suppression of CNV occurred via the inhibition of macrophage infiltration and the expression of proangiogenic (vascular endothelial growth factor) and proinflammatory molecules (monocyte chemotactic protein-1 and IL-6) in the retinal pigment epithelium–choroid complex. Additionally, WEB2086-induced PAF-R blockage suppresses experimentally induced subretinal fibrosis, which resembles the fibrotic subretinal scarring observed in neovascular AMD. As optimal treatment modalities for neovascular AMD would target the multiple mechanisms of AMD-associated vision loss, including neovascularization, inflammation and fibrosis, our results suggest PAF-R as an attractive molecular target in the treatment of AMD.     

Platelet-activating factor receptor (PAF-R) and acetylhydrolase (PAF-AH) are co-expressed in immature bovine trophoblast giant cells throughout gestation, but not at parturition

Platelet-activating factor (PAF) was associated with successful implantation in the cow, trophoblast invasiveness and angiogenesis. Bovine placentation is characterized by the limited invasion of trophoblast giant cells (TGC) into the maternal caruncular epithelium. TGC exhibit both endocrine activity and properties of tumor cells and may thus be targets of and mediators for the action of PAF. We examined PAF-receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) gene expression and localized mRNA and corresponding proteins in bovine placentomes throughout gestation and at parturition. PAF-R and PAF-AH protein and mRNA were highly expressed and colocalized in immature TGC from early gestation until near term, while mature TGC were negative. After the onset of parturition both PAF-R and PAF-AH were expressed in the maternal stroma, predominantly endothelial cells. The expression of PAF-R and PAF-AH in immature but not mature TGC during gestation implicates a role for PAF in the differentiation, maturation and function of bovine placentomal TGC. Placentomal angiogenesis could be mediated by binding of PAF to PAF-R present in endothelial cells. The parturition-related "switch" of PAF-R and PAF-AH from TGC to the maternal stroma suggests that PAF may participate in the regulation of parturition and in prepartum tissue programming.     

Platelet-activating factor receptor knockout mice are protected from MPTP-induced dopaminergic degeneration

Platelet-activating factor (PAF), a potent mediator of inflammatory and immune responses, plays various roles in neuronal functions. However, little is known about the role of PAF/platelet-activating factor receptor (PAF-R) in Parkinson’s disease. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in significant increases in PAF species in the striatum of wild-type mice. These increases paralleled PAF-R gene expression in wild-type mice. Although nuclear factor kappa B (NF-κB) DNA-binding activity was increased significantly in MPTP-treated wild-type mice, this increase was not significant in PAF-R antagonist ginkgolide B (GB)-treated mice or PAF-R knockout (PAF-R^−/−) mice. Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, significantly ameliorated the dopaminergic deficits induced by MPTP in wild-type mice. MPTP treatment significantly increased oxidative damage, the immunoreactivity of ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglial cells, and microglial differentiation of the M1 type in the striatum of wild-type mice. Consistently, PDTC significantly attenuated MPTP-induced behavioral impairments in wild-type mice. However, dopaminergic deficits, oxidative damage, reactive microglial cells, and behavioral impairments induced by MPTP were not significantly observed in GB-treated mice or PAF-R^−/− mice. PDTC did not significantly alter the attenuations evident in MPTP-treated PAF-R^−/− mice, indicating that NF-κB is a critical target for neurotoxic modulation of PAF-R. We propose for the first time that PAF/PAF-R can mediate dopaminergic degeneration via an NF-κB-dependent signaling process.     

Role of MSK1 in the signaling pathway leading to VEGF-mediated PAF synthesis in endothelial cells

Vascular endothelial growth factor (VEGF) inflammatory effects require acute platelet-activating factor (PAF) synthesis by endothelial cells (EC). We previously reported that VEGF-mediated PAF synthesis involves the activation of VEGF receptor-2/Neuropilin-1 complex, which is leading to the activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs) and group V secretory phospholipase A(2) (sPLA(2)-V). As the mechanisms regulating sPLA(2)-V remain unknown, we addressed the role of the mitogen- and stress-activated protein kinase-1 (MSK1), which can be rapidly and transiently activated by p38 or p42/44 MAPKs. In native bovine aortic endothelial cells (BAEC), we observed a constitutive protein interaction of MSK1 with p38, p42/44 MAPKs, and sPLA(2)-V. These protein interactions were maintained in BAEC transfected either with the empty vector pCDNA3.1, wild-type MSK1 (MSK1-WT) or N-terminal dead kinase MSK1 mutant (MSK1-D195A). However, in BAEC expressing C-terminal dead kinase MSK1 mutant (MSK1-D565A), the interaction between MSK1 and sPLA(2)-V was reduced by 82% and 90% under basal and VEGF-treated conditions as compared to native BAEC. Treatment with VEGF for 15 min increased basal PAF synthesis in native BAEC, pCDNA3.1, MSK1-WT, and MSK1-D195A by 166%, 139%, 125%, and 82%, respectively. In contrast, PAF synthesis was prevented in cells expressing MSK1-D565A mutant. These results demonstrate the essential role of the C-terminal domain of MSK1 for its constitutive interaction with sPLA(2)-V, which appears essential to support VEGF-mediated PAF synthesis.     

Hypoxia increases stimulus-induced PAF production and release from human umbilical vein endothelial cells.

Hypoxia alters endothelial cell function and metabolism. Since PAF is synthesized by endothelial cells and capable of modulating endothelial cell responses, we investigated the effect of hypoxia on synthesis and release of PAF from endothelial cells. We found: (1) Approx. 90% of the radylPAF derivative in stimulated endothelial cells is acylPAF. (2) Acute hypoxic (15 min-1 h) priming increased ionophore- and thrombin-induced radylPAF accumulation. (3) Long-term hypoxic exposure increased radylPAF accumulation at 24 and 48 h in the presence of ionophore. (4) Bioactive PAF was released into media and hypoxia and ionophore synergistically increased PAF release. (5) Hypoxia and ionophore stimulation increased phospholipase A2 activity and decreased acetylhydrolase activity in endothelial cells. We conclude that hypoxia and ionophore increase PAF synthesis and release from endothelial cells.     

PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo

The role of platelet-activating factor (PAF) inCa²⁺ signaling and Ca²⁺-related enhancement ofreactive oxygen intermediate (ROI) generation in neutrophils ofburn-injured rats was ascertained by evaluating the effect of treatmentof the rats with a PAF receptor antagonist. The treatment of rats withthe antagonist also allowed us to evaluate the role of PAF in thepriming of neutrophil ROI response with burn in vivo. A full skinthickness burn injury was produced in anesthetized rats by exposing30% of total body surface area to 98°C water for 10 s. Sham andburn rats were killed 1 day later, and their blood was collected toobtain neutrophils. Fluorescence-activated cell sorter analysis wasused to quantify ROI production by the neutrophils. Cytosolic-freeCa²⁺ concentration ([Ca²⁺]_i)imaging technique was employed to measure neutrophil[Ca²⁺]_i in individual cells andmicrofluorometry for the assessment of[Ca²⁺]_i responses in suspensions ofneutrophils. There was an overt enhancement of ROI generation by burnrat neutrophils. ROI release was accompanied by a marked elevation of[Ca²⁺]_i signaling. The treatment of rats withPAF receptor antagonist before burn prevented the upregulation of both[Ca²⁺]_i and ROI generation in neutrophils.These studies indicate that enhanced ROI production in neutrophils inthe early stages after burn injury results from a PAF-mediated primingof the [Ca²⁺]_i signaling pathways in vivo.

     

PMS777, A Bis-interacting Ligand for PAF Receptor Antagonism and AChE Inhibition,Attenuates PAF-induced Neurocytotoxicity in SH-SY5Y Cells

(1) HIV-1 and viral proteins-evoked chronic brain inflammation, which is characterized by microglial activation, is the pivotal neuropathogenesis of HIV-1-associated dementia (HAD). Platelet-activating factor (PAF), mainly released from activated microglia and acts as a high potent inflammatory mediator and a neurotoxin, is indicated to be a principle initiator of neuroinflammation, neuronal dysfunction, and apoptosis related to HAD. Thus, bis-interacting ligands of acetylcholinesterase (AChE) inhibition and PAF receptor antagonism would be of great interest in the therapeutic potential of HAD not only for improvement of cognitive performance, but also for disease-modifying. (2). We have previously reported that a novel tetrahydrofuran-derived bis-interacting ligand PMS777 had satisfying potencies for PAF receptor blockade and AChE inhibition, and markedly improved cholinergic dysfunction-induced cognitive impairment in mice. Continuing with our research, we further investigated the neuroprotective activities of PMS777 on PAF-triggered neuronal injury in human neuroblastoma SH-SY5Y cells. (3) The bis-interacting ligand PMS777 (10 μM) obviously alleviated PAF-induced cell apoptosis in SH-SY5Y cells. Pretreatment with PMS777 also markedly inhibited intracellular Ca²⁺ overload, down-regulation of anti-apoptotic bcl-2 mRNA, stimulation of pro-apoptotic bax mRNA expression and activation of caspase-3 pathway. Also, PMS777 could fine-tune pro-inflammatory cyclooxygenase-2 (cox-2) mRNA expression in PAF-treated cells. (4) These results suggest that PMS777 possesses a neuroprotective profile via anti-apoptotic/inflammatory signaling and warrant further investigations in connection with the potential value of this compound in HAD treatment.     

Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.

Platelet-activating factor (PAF), an alkylether phospholipid, is produced in the brain when it is subjected to various stimuli. Using a Xenopus oocyte expression system, we obtained evidence for functional PAF receptor mRNA expression in rat brain. The presence of the PAF receptor was confirmed and shown to be quite ubiquitous in the CNS by RNA blot and radioligand binding studies. To investigate the neuronal functions of PAF, intracellular Ca2+ increase elicited by nanomolar PAF application was analyzed in cultured rat hippocampal cells. Fractions of NMDA-responsive cells and non-NMDA-responsive cells were shown to respond to PAF, suggesting a potential role for PAF in the Ca2+ signaling pathway in the hippocampus.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

Alpha-bulnesene, a novel PAF receptor antagonist isolated from Pogostemon cablin

Alpha-bulnesene is a sesquiterpenoid isolated from the water extract of Pogostemon cablin. It showed a potent and concentration-dependent inhibitory effect on platelet-activating factor (PAF) and arachidonic acid (AA) induced rabbit platelet aggregation. In a radioligand binding assay for the PAF receptor, alpha-bulnesene competitively inhibited [(3)H]PAF binding to the PAF receptor with an IC(50) value of 17.62+/-5.68microM. alpha-Bulnesene also dose-dependently inhibited PAF-induced intracellular Ca(2+) increase in fluo-3/AM-loaded platelets (IC(50) values of 19.62+/-1.32microM). Furthermore, alpha-bulnesene inhibited AA-induced thromboxane B(2) (TXB(2)) formation and prostaglandin E(2) (PGE(2)) formation. These results indicate that the inhibitory effect of alpha-bulnesene on platelet aggregation was due to a dual activity; specifically the chemical blocked PAF-induced intracellular signal transduction and interfered with cyclooxygenase activity, which resulted in a decrease in thromboxane formation. This study is the first to demonstrate that alpha-bulnesene is a PAF receptor antagonist as well as an anti-platelet aggregation agent.     

Platelet-activating factor (PAF) increases NO production in human endothelial cells-real-time monitoring by DAR-4M AM

BACKGROUND: Platelet-activating factor (PAF) is a potent inflammatory lipid mediator that increases vascular permeability and vasodilation. Several studies have addressed the effect of PAF on nitric oxide (NO) production from microvessels in vivo. OBJECTIVE: The aim of present study was to evaluate the effect of PAF on NO production in primary cultured human vascular endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were loaded with diaminorhodamine-4M acetoxymethyl ester (DAR-4MAM), and the cells were stimulated with PAF. Intracellular NO production was monitored as increase in fluorescence intensity. Also, NO production was visualized at cellular levels using DAR-4M AM and fluorescence imaging. RESULTS: Significant increases in NO production in HUVECs were soon after the PAF stimulation, reaching a plateau after 10 min of the stimulation. The increase of NO production at 10 min after the stimulation was statistically significant (p<0.05) for 0.01-10 microM PAF. PAF-induced NO production was abolished by pretreatment of HUVECs with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or PAF receptor antagonist BN 52021. LysoPAF, the inactive metabolite of PAF, did not exert a significant effect on intracellular NO levels. CONCLUSIONS: These results provide direct evidence that PAF cause intracellular NO production via activation of PAF receptors in human vascular endothelial cells.     

Endotoxin induces PAF production in the rat ileum: Quantitation of tissue PAF by an improved method

PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consitent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70–80%. We found that LPS (2–10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.