Antisense Oligonucleotide Inhibition Of Type II Phospholipase A2Expression, Release And Activity In Vitro

Lappas, Martha; Munns, Melinda J; King, Roger G; Rice, Gregory E.

doi:10.1053/plac.2001.0673

Cited by 9 publications

(6 citation statements)

References 30 publications

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“…Another possible approach one could undertake in order to study the role of specific sPLA 2 s in spinal pain processing would be to knock down the protein expression of specific sPLA 2 forms in the spinal cord. This has successfully been performed in macrophage-like cell line (Balboa et al, 1996;Shinohara et al, 1999), fibroblasts (Kuwata et al, 1998) and human placenta explants (Lappas et al, 2001) and the results from these studies indeed indicate a role for group II and V sPLA 2 in eicosanoid biosynthesis.…”

Section: Discussionmentioning

confidence: 86%

Spinal phospholipase A2 in inflammatory hyperalgesia: Role of the small, secretory phospholipase A2

et al. 2005

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Current work emphasizes that peripheral tissue injury and inflammation results in a heightened sensitivity to subsequent noxious input (hyperalgesia) that is mediated in large part by the spinal synthesis and release of eicosanoids, in particular prostaglandins. Secreted phospholipase A(2)s (sPLA(2)s) form a class of structurally related enzymes that release arachidonic acid from cell membranes that is further processed to produce eicosanoids. We hypothesized that spinal sPLA(2)s may contribute to inflammation-induced hyperalgesia. Spinal cord tissue and cerebrospinal fluid were collected from rats for assessment of sPLA(2) protein expression and sPLA(2) activity. A basal sPLA(2) protein expression and activity was detected in spinal cord homogenate (87+/-17 pmol/min/mg), though no activity could be detected in cisternal cerebrospinal fluid, of naive rats. The sPLA(2) activity did not change in spinal cord tissue or cerebrospinal fluid assessed over 8 h after injection of carrageenan into the hind paw. However, the sPLA(2) activity observed in spinal cord homogenates was suppressed by addition of LY311727, a selective sPLA(2) inhibitor. To determine the role of this spinal sPLA(2) in hyperalgesia, we assessed the effects of lumbar intrathecal (IT) administration of LY311727 in rats with chronic IT catheters in three experimental models of hyperalgesia. IT LY311727 (3-30 microg) dose-dependently prevented intraplantar carrageenan-induced thermal hyperalgesia and formalin-induced flinching, at doses that had no effect on motor function. IT LY311727 also suppressed thermal hyperalgesia induced by IT injection of substance P (30 nmol). Using in vivo spinal microdialysis, we found that IT injection of LY311727 attenuated prostaglandin E(2) release into spinal dialysate otherwise evoked by the IT injection of substance P. Taken together, this work points to a role for constitutive sPLA(2)s in spinal nociceptive processing.

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Section: Discussionmentioning

confidence: 86%

Spinal phospholipase A2 in inflammatory hyperalgesia: Role of the small, secretory phospholipase A2

et al. 2005

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“…In this model, villous cytotrophoblast continues to proliferate and structural organization of the villous trophoblast layer stays intact for several days, 32,33 even in the presence of antisense oligonucleotides. 34 The explant model allows the observation of cytotrophoblast fusion in the living complex placental tissue. It is superior to the use of primary trophoblast cells, which upon isolation irreversibly leave the cell cycle and undergo apoptosis within a few days.…”

Section: Methodological Approachmentioning

confidence: 99%

Syncytial fusion of human trophoblast depends on caspase 8

et al. 2003

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Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.

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“…Separate tissue samples (n ϭ 9) were incubated for 6 h, and both the tissue and incubation medium was collected and assayed for type II PLA 2 as previously described (20,21). The concentration of type II PLA 2 was quantified by a noncompetitive sandwich ELISA using two monoclonal antibodies raised against recombinant human type II PLA 2 as described previously (21), and data were corrected for protein and expressed as nanograms per milligram protein.…”

Section: Experimental Assaysmentioning

confidence: 99%

Section: Experimental Assaysmentioning

confidence: 99%

“…The concentration of type II PLA 2 was quantified by a noncompetitive sandwich ELISA using two monoclonal antibodies raised against recombinant human type II PLA 2 as described previously (21), and data were corrected for protein and expressed as nanograms per milligram protein. The concentration of PGF 2␣ in the incubation medium was determined by a RIA as previously described (21), and data were corrected for total protein and expressed as picomole per milligram protein. The release of 13,14-dihydro-15-keto PGF 2␣ into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Cayman Chemical Co., Ann Arbor, MI), data were corrected for total protein and expressed as picogram per milligram protein.…”

Section: Experimental Assaysmentioning

confidence: 99%

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Regulation of Phospholipase Isozymes by Nuclear Factor-κB in Human Gestational Tissuesin Vitro

Lappas¹,

Permezel²,

Georgiou³

et al. 2004

The Journal of Clinical Endocrinology & Metabolism

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Phospholipid-derived mediators are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. We previously demonstrated that, in human intrauterine tissues, lipopolysaccharide (LPS)-stimulated nuclear factor-kappaB (NF-kappaB) DNA binding activity, and subsequent cytokine release can be suppressed by sulfasalazine (SASP) concentrations greater than 5 mM. The aim of this study was to elucidate the effect the SASP on secretory type II phospholipase A(2) (PLA(2)), cytosolic PLA(2) (cPLA(2)), cyclooxygenase (COX) isozymes, and subsequent prostaglandin F(2alpha) (PGF(2alpha)) production in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 4-9 separate placentas) were incubated in the presence of SASP (0.1, 1, 5, and/or 10 mM) under either basal or LPS (10 microg/ml) conditions. After 6 h incubation, the tissues were collected and assayed for type II PLA(2) by ELISA and cPLA(2), COX-1, and COX-2 content by Western blotting. The incubation medium was collected and assayed for type II PLA(2) and 13,14-dihydro-15-keto PGF(2alpha) release by ELISA and PGF(2alpha) by RIA. Treatment of placenta, amnion, and choriodecidua with SASP concentrations greater than 5 mM significantly inhibited basal and/or LPS-stimulated type II PLA(2) content and release, 13,14-dihydro-15-keto PGF(2alpha) release, and cPLA(2) protein content (ANOVA, P < 0.05); however, no effect of SASP was observed on basal or LPS-stimulated COX-1 or COX-2 protein. Although no effect of SASP was observed on basal and LPS-stimulated PGF(2alpha) release from placenta and amnion, it significantly increased both basal and LPS-stimulated PGF(2alpha) release from choriodecidua. In addition, SASP concentrations of 5 mM or greater significantly suppressed NF-kappaB DNA binding activity. These data are consistent with the hypothesis that NF-kappaB regulates the expression and release of phospholipase isozymes.

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Antisense Oligonucleotide Inhibition Of Type II Phospholipase A2Expression, Release And Activity In Vitro

Cited by 9 publications

References 30 publications

Spinal phospholipase A2 in inflammatory hyperalgesia: Role of the small, secretory phospholipase A2

Spinal phospholipase A2 in inflammatory hyperalgesia: Role of the small, secretory phospholipase A2

Syncytial fusion of human trophoblast depends on caspase 8

Regulation of Phospholipase Isozymes by Nuclear Factor-κB in Human Gestational Tissuesin Vitro

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