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Platelet-activating factor acetylhydrolase IB subunit beta (PAFAH1B2) plays important roles in inflammation and anaphylaxis. However, its primary function in pancreatic cancer remains unclear. In the current study, we report that PAFAH1B2 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and correlated inversely with patient survival. PAFAH1B2 overexpression induced epithelial–mesenchymal transition (EMT), migration and invasion in vitro and metastasis in vivo. Conversely, silencing PAFAH1B2 inhibited these aggressive phenotypes. Moreover, PAFAH1B2 overexpression in PDAC cells was directly mediated by HIF1a. PAFAH1B2 expression in PDAC clinical specimens correlated positively with HIF1a expression. Overall, our results defined PAFAH1B2 as a target gene of HIF1a and a critical driver of PDAC metastatic behaviors.

To explore the regulation of PLA2G7 silencing on myocardial fibrosis in mice with coronary atherosclerosis, we established model of atherosclerosis using ApoE knockout mice, and set up a normal group. The successfully modeled mice were assigned into the following three groups: PLA2G7 silencing (shRNA) group, negative control (NC) group and blank group. Peripheral blood and myocardial tissue of each group of mice were harvested. The expressions of serum total cholesterol, triglyceride, low density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in mice were determine using Elisa, and the atherosclerosis index was calculated. The pathological changes of myocardial tissue were observed after HE staining. The collagen volume fraction in myocardium was determined with the use of Masson staining. The expression of PLA2G7 in myocardial tissue as well as myocardial fibrosis markers C-reactive protein, interleukin-6 and intercellular adhesion molecule-1 in each group were detected by qRT-PCR and Western blot. The area of thoracic aorta injury was detected after oil red O staining. Compared with the normal group, the levels of total cholesterol, triglyceride and LDL-C were increased, HDL-C levels were decreased, and atherosclerosis index was increased in the PLA2G7 shRNA group, NC group and blank group (all P 0.05). The pathological state of myocardial tissue in the other three groups (except for the normal group) was obvious, but the PLA2G7 shRNA group showed certain improvement as compared with the blank group and the NC group (all P 0.05). Compared with the NC group, the PLA2G7 shRNA group had significantly decreased collagen volume fraction, myocardial fibrosis and area of thoracic aorta injury (all P 0.05). In conclusion, PLA2G7 silencing can improve the myocardial fibrosis in mice with coronary atherosclerosis, and PLA2G7 is expected to be a potential target for coronary atherosclerosis.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

Atherosclerosis is a disease of the large- and medium-size arteries that is characterized by the formation of atherosclerotic plaques, in which foam cells are the characteristic pathological cells. However, the key underlying pathomechanisms are still not fully elucidated. In this study, we investigated the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in ox-LDL-induced oxidative stress and cell apoptosis, and further, elucidated the potential machanisms in human THP1 macrophages. Flow cytometry and western blot analyses showed that both cell apoptosis and Lp-PLA2 expression were dose-dependently elevated after ox-LDL treatment for 24 h and also time-dependently increased after 50 mg/L ox-LDL incubation in THP1 macrophages. In addition, Lp-PLA2 silencing decreased ox-LDL-induced Lp-PLA2 and CD36 expression in THP1 macrophages. We also found that the levels of oil red O-staining, triglyceride (TG) and total cholesterol (TC) were significantly upregulated in ox-LDL-treated THP1 cells, but inhibited by Lp-PLA2 silencing. Furthermore, ox-LDL treatment resulted in significant increases of ROS and MDA but a marked decrease of SOD, effects that were reversed by Lp-PLA2 silencing in THP1 cells. Lp-PLA2 silencing reduced ox-LDL-induced cell apoptosis and caspase-3 expression in THP1 cells. Moreover, Lp-PLA2 siRNA transfection dramatically lowered the elevated levels of p-Akt and p-mTOR proteins in ox-LDL-treated THP1 cells. Both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin decreased the augmented caspase-3 expression and TC content induced by ox-LDL, respectively. Taken together, these results revealed that Lp-PLA2 silencing protected against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages.

Approximately 4% of the Japanese population genetically lack plasma platelet activating factor acetylhydrolase (PAF-AH) and show a higher prevalence of thromboembolic disease, but whether they are susceptible to another PAF-related disease, asthma, remains controversial. To determine the role of plasma PAF-AH in airway physiology, we performed PAF bronchoprovocation tests in 8 plasma PAF-AH-deficient subjects and 16 control subjects. Serial inhalation of PAF (1-1000 microg/ml) concentration-dependently induced acute bronchoconstriction, but there was no significant difference between PAF-AH-deficient and control subjects (11.7 +/- 4.6% vs. 9.6 +/- 2.8% decrease in forced expiratory volume in 1 s). Transient neutropenia after single inhalation of PAF (1000 microg/ml) showed no significant difference between the groups either in its magnitude (72 +/- 11% vs. 65 +/- 9% decrease) or duration (4.1 +/- 1.0 vs. 3.3 +/- 0.8 min). In conclusion, a lack of plasma PAF-AH activity alone does not augment physiological responses to PAF in the airway.

Platelet activating factor (PAF) is immediately degraded and inactivated in the bloodstream by plasma PAF acetylhydrolase (PAF-AH). Although plasma PAF-AH-like activity was secreted in vitro from various cell types such as macrophages and hepatocytes, the exact cellular source(s) of this enzyme activity in vivo remains unclear. There is a naturally-occurring missense mutation (V279F) in the plasma PAF-AH gene in the Japanese population which results in complete loss of the enzyme activity. We analyzed 52 Japanese who had received an allogeneic bone marrow transplant and maintained donor-derived hematopoiesis. Ten recipients had chimeric plasma PAF-AH genotypes between the donor-derived peripheral blood leukocytes and the recipient-derived epithelial cells of buccal mucosa. Multiple regression analysis demonstrated that PAF-AH activity in plasma depended on the donor's genotype (standardized regression coefficient = 0.68, P < 0.0001), but not on the recipient's genotype (p = 0.48). One recipient who was a V279F homozygote in leukocytes and wild type homozygote in buccal mucosa had undetectable PAF-AH activity in plasma. We conclude that most of the PAF-AH activity in human plasma originates from hematopoietic lineage cells.

Human and murine high density lipoprotein (HDL) has previously been shown to decrease the accumulation of lipid peroxides on low density lipoprotein (LDL) under oxidising conditions. Several lines of evidence, including the ineffectiveness of HDL from paraoxonase knockout mice, suggest that paraoxonase (PON1) located on HDL is responsible for its protective effect against lipid peroxidation. In this report we compare the effect of HDL from chicken, turkey and ostrich with human HDL on lipid peroxidation of LDL. Avian serum lacked PON1 activity and PON1 immunoactivity was also absent by ELISA and Western blotting whereas all three techniques detected PON1 in a variety of non-avian species (cow, guinea-pig, rat, sheep, mouse, hamster, monkey and rabbit). Platelet activating factor acetyl hydrolase (PAFAH) activity was also absent from avian serum. Avian HDL isolated from plasma when incubated with human LDL was ineffective in preventing the Cu2+-induced accumulation of lipid peroxides on this lipoprotein whereas human HDL under the same conditions was highly effective in this respect. Avian LDL was much more resistant to oxidation than human LDL, perhaps explaining the lack of HDL-PON1 and PAFAH. We conclude that these findings provide further evidence than PON1 has an important role in the antiatherogenic/anti-inflammatory effects of HDL and that avian HDL can provide a valuable model which complements the use of HDL from paraoxonase knockout mice in the investigation of PON1 and PAFAH.

The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994--T mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001--G missense mutation, resulting in a Gln281--Arg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281--Arg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity.

The cDNA for human platelet-activating factor acetylhydrolase (PAF-AH) beta-subunit was cloned. The complete amino acid sequence deduced from cDNA contains 229 amino acids and is completely identical with that of the bovine subunit. Moreover, the sequence of the human beta-subunit protein shows 62.4% identity with the human gamma-subunit at the amino acid level. Southern blot analysis suggested the presence of multiple genes and we indeed found another closely related pseudo gene. Northern blot analysis showed that the 4.0 kb transcript was expressed in all tissues tested, suggesting the ubiquitous distribution of the subunit protein. Differential distribution of beta- and gamma-subunits might suggest that the oligomeric structure of PAF-AH is different from tissue to tissue.

The cDNA for human cytosolic platelet-activating factor acetylhydrolase (PAF-AH) gamma-subunit was isolated and the gene structure was elucidated. The complete amino acid sequence deduced from cDNA contains 231 amino acid residues. Southern blot analysis indicated the presence of a single gene. Northern blot analysis showed that 1.0 kb transcript was expressed in a tissue specific manner. Comparison of the expression pattern between adult and fetal tissues suggests that the enzyme plays an important role during the development of brain.

Overexpression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is implicated in atherosclerosis. We tested the hypothesis that lentivirus-mediated Lp-PLA(2) silencing could inhibit atherosclerosis in apolipoprotein E-deficient mice. Sixty eight apolipoprotein E-deficient mice were fed a high-fat diet and a constrictive collar was placed around the left carotid artery to induce plaque formation. The mice were randomly divided into control, negative control (NC) and RNA interference (RNAi) groups. Lp-PLA(2) RNAi or scrambled NC lentivirus viral suspensions were constructed and transfected into the carotid plaques 8 weeks after surgery; the control group was administered saline. The carotid plaques were assessed 7 weeks later using hematoxylin and eosin, Masson's trichrome and oil red O staining; plasma and lesion inflammatory gene expression were examined using ELISAs and real-time PCR. Seven weeks after transfection, the serum concentration and plaque mRNA expression of Lp-PLA(2) was significantly lower in the RNAi group, and lead to reduced local and systemic inflammatory gene expression. Lp-PLA(2) RNAi also ameliorated plaque progression, reduced the plaque lipid content and increased the plaque collagen content. The effects of Lp-PLA(2) RNAi were independent of serum lipoprotein levels, as the triglyceride and total cholesterol levels of the control, NC and RNAi groups were not significantly different. These findings support the hypothesis that lentivirus-mediated Lp-PLA(2) gene silencing has therapeutic potential to inhibit atherosclerosis and increase plaque stability, without altering the plasma lipoprotein profile.

The human lissencephaly-1 gene (LIS1) is a disease gene responsible for Miller-Dieker lissencephaly syndrome (MDL). LIS1 gene is located in the region of chromosome 17p13.3 that is frequency deleted in MDL patients and in human liver cancer cells. However, the expression and significance of LIS1 in liver cancer remain unknown. Here, we investigated the expression of LIS1 in hepatocellular carcinoma (HCC) tissues by real-time PCR, Western blot, and immunohistochemistry. The results indicated that the mRNA and protein levels of LIS1 were downregulated in about 70% of HCC tissues, and this downregulation was significantly associated with tumor progression. Functional studies showed that the reduction of LIS1 expression in the normal human liver cell line QSG7701 or the mouse fibroblast cell line NIH3T3 by shRNA resulted in colony formation in soft agar and xenograft tumor formation in nude mice, demonstrating that a decrease in the LIS1 level can promote the oncogenic transformation of cells. We also observed that the phenotypes of LIS1-knockdown cells displayed various defective mitotic structures, suggesting that the mechanism by which reduced LIS1 levels results in tumorigenesis is associated with its role in mitosis. Furthermore, we demonstrated that ectopic expression of LIS1 could significantly inhibit HCC cell proliferation and colony formation. Our results suggest that LIS1 plays a potential tumor suppressor role in the development and progression of HCC.

Paf-acether (paf) synthesis was previously shown to be impaired in 24 hr-adherent and Bacillus Calmette-Guerin-activated murine peritoneal macrophages as compared to resident macrophages. We report here that the induction of acetylhydrolase was responsible for the decreased paf output in 24 hr-adherent macrophages. The kinetic analysis of the enzymes derived from 2 hr-, 24 hr- and BOG-activated adherent macrophages and from plasma revealed that the Km for paf was similar whatever the source of the acetylhydrolase whereas the Vmax was five-fold increased in 24 hr- cultured macrophages. The acetylhydrolase activity was Ca2+-independent and was not inhibited by addition of alkyl-acyl(long chain)-glycerophosphocholine suggesting that the enzyme was not a phospholipase A2.

We prepared unlabeled and 3H-labeled analogs of platelet-activating factor (PAF) containing a N-methylcarbamyl residue at the sn-2 position. PAF and its methylcarbamyl analog competed for binding to high affinity receptors on human polymorphonuclear neutrophils; their respective dissociation constants for these receptors were 0.2 and 1.1 nM. The binding affinities of the two analogs correlated precisely with their capacities to stimulate neutrophil degranulation responses. Unlike PAF, however, the methylcarbamyl analog completely resisted metabolic inactivation by neutrophils and by human sera. Thus, these compounds' biological potencies are determined predominantly by receptor binding: cellular metabolism of the ligands neither contributes to nor appreciably limits their stimulating actions.

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent Km of 2.5 microM and a Vmax of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca2+. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals (P less than 0.01).