Whole mount immunofluorescence analysis of placentas from normotensive versus preeclamptic pregnancies.

INTRODUCTION: Defects in placental angiogenesis and spiral artery remodeling have been proposed to play essential roles in the development of preeclampsia. However, the specific molecular mechanism(s) responsible for aberrant placental angiogenesis in preeclampsia are incompletely understood. The vascular endothelial growth factor receptors (VEGFR1, R2, R3) and STAT3 have critical functions in normal blood vessel development, but their potential roles in preeclampsia are currently unclear. In this study, we utilized a novel whole mount immunofluorescence (WMIF) method to compare expression of VEGFR1, R2, R3 and activated, phosphorylated STAT3 (pSTAT3) in placentas of preeclamptic (PE) versus normotensive (NT) pregnancies. METHODS: Placental biopsies collected from NT and PE pregnant women were fixed and stained with fluorochrome-conjugated antibodies to identify specific cell populations as follows: CD31 for blood vessel endothelial cells, cytokeratin-7 for trophoblast cells, and CD45 for immune cells. Expression of the VEGFRs and pSTAT3 were subsequently characterized by WMIF in conjunction with confocal microscopy. RESULTS: A total of 18 PE and 18 NT placentas were evaluated. No significant differences in the cell type-specific expression patterns or expression levels of VEGFR1, VEGFR2 or VEGFR3 were detected between NT and PE placentas. In contrast, statistically significant increases in pSTAT3 staining were detected in endothelial cells of PE placentas versus NT controls. DISCUSSION: Our study demonstrates that increased pSTAT3 expression in placental endothelial cells is associated with PE. We speculate that elevated pSTAT3 expression in the blood vessels of PE placentas may be due to aberrant angiogenesis, increased pro-inflammatory cytokine expression, and/or placental stress.

Uterine natural killer cells pace early development of mouse decidua basalis.

Pregnancy involves progressive relationship changes between conceptus-derived trophoblasts and maternal decidual vessels and leukocytes. Uterine natural killer (uNK) cells, the dominant leukocytes in early human and mouse decidua, have late gestational cardio-protective roles through mid-gestational initiation of decidual spiral arterial modification. The earlier gestational functions of uNK cells are unknown. Comparisons of gestation days (GD) 6.5-9.5 implant sites from allogeneically mated alymphoid or normal BALB/c mice (Rag2(-/-)Il2rg(-/-); NK-T-B- versus +/+) by whole mount immunohistochemistry revealed delays in Rag2(-/-)Il2rg(-/-) uterine lumen closure, trophoblast invasion and conceptus development. Also delayed were onset of mesometrial angiogenesis and pruning of neo-vascular networks in decidua basalis. This phenotype was fully reversed in BALB/c-Rag2(-/-)Il2rg(-/-) pregnancies that followed adoptive Rag2(-/-) (NK+B-T-) marrow transfer. These data suggest that uNK cells coordinate GD-appropriate phases of decidual angiogenesis, which in turn paces progressive changes in early implant sites that support normal fetal growth. Similar roles for human CD56(bright) decidual NK cells could explain the importance of CD56(bright) decidual NK cell activation to pregnancy success.

A novel role for RIP1 kinase in mediating TNFα production.

Receptor-interacting protein 1 (RIP1) is a Ser/Thr kinase with both kinase-dependent and kinase-independent roles in death receptor signaling. The kinase activity of RIP1 is required for necroptosis, a caspase-independent pathway of programmed cell death. In some cell types, the inhibition of caspases leads to autocrine production of TNFα, which then activates necroptosis. Here, we describe a novel role for RIP1 kinase in regulating TNFα production after caspase inhibition. Caspase inhibitors activate RIP1 kinase and another protein, EDD, to mediate JNK signaling, which stimulates Sp1-dependent transcription of TNFα. This pathway is independent of nuclear factor κB and also occurs after Smac mimetic/IAP antagonist treatment or the loss of TNF receptor-associated factor 2 (Traf2). These findings implicate cIAP1/2 and Traf2 as negative regulators of this RIP1 kinase-dependent TNFα production pathway and suggest a novel role for RIP1 kinase in mediating TNFα production under certain conditions.

Studies on the mechanism of RNAi-dependent heterochromatin assembly.

Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes double-stranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 (+) gene results in siRNA generation and RNAi- and heterochromatin-dependent silencing of the ura4 (+) gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).

Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts.

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP(+) vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-gamma). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.

Design and synthesis of substrate and internal standard conjugates for profiling enzyme activity in the Sanfilippo syndrome by affinity chromatography/electrospray ionization mass spectrometry.

We describe the design and synthesis of substrate and internal standard conjugates for application in profiling enzyme activity of the enzymes alpha-D-2-deoxy-2-N-sulfonamido-glucosamine sulfamidase, alpha-D-2-deoxy-2-N-acetyl-glucosamine hydrolase, acetyl-coenzymeA:alpha-D-2-deoxy-2-amino-glucosamine transferase, and alpha-D-2-deoxy-2-N-acetyl-glucosamine-6-sulfate sulfatase. Deficiency of any one of these enzymes results in a single clinical phenotype known as Sanfilippo syndrome. Such substrates have been proven effective in the confirmation of enzyme deficiency by a combination of affinity chromatography (AC) and electrospray ionization mass spectrometry (ESIMS), which forms the foundation for a new analytical technology (ACESIMS) of general interest and application to clinical and biomedical research.

Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: application to clinical enzymology.

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

AET-treated platelets: their usefulness for platelet antibody detection and an examination of their altered sensitivity to immune lysis.

2-Aminoethylisothiouronium bromide (AET) increases the sensitivity of blood cells to complement-mediated immune lysis. We compared the sensitivities of untreated or AET-treated platelets to immune lysis induced by different types of platelet antibody in the 51Cr platelet lysis test. AET platelets were 8-16 times more sensitive to autoantibody and alloantibody, but 8-16 times less sensitive to drug-dependent antibody. AET-platelets bound similar amounts of alloantibody but less drug-dependent antibody, and they lysed at higher complement dilutions than did untreated platelets. AET-platelets detected 10 of 25 autoantibodies, 9 of 9 alloantibodies, and 5 of 8 drug-dependent antibodies. Untreated platelets detected 1 of 25, 6 of 9, and 7 of 8 of these respective platelet antibodies. The use of AET-platelets in the 51Cr platelet lysis test increases its sensitivity for detecting non-drug-dependent platelet antibodies. AET-platelets resemble paroxysmal nocturnal hemoglobinuria (PNH) platelets in their enhanced sensitivity to complement-mediated lysis. They differ from PNH platelets in their insensitivity to immune lysis induced by drug-dependent antibodies and, in this respect, are similar to Bernard-Soulier syndrome platelets.