The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver.

Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

Activation of complement and contact system in Alzheimer's disease.

beta-Amyloid protein (betaA) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and ability to trigger a local inflammatory response. Although assembly of betaA in particular aggregates seems to be crucial event in AD pathogenesis, soluble, non-fibrillar betaA may also be involved. Non-fibrillar betaA1-42, and truncated peptide 1-28, induced dose-dependent activation of C4 sparing C3. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar betaA can still activate C4 in plasma genetically deficient in C1q. A C1q independent mechanism of complement classical pathway activation could be via the activation of contact/kinin system. The possible involvement of contact system in AD is suggested by the finding that this system is massively activated in CSF of AD patients. The mechanism of activation of contact system could be the result of an anionic interaction of residues within the region 1-11 of betaA1-42 with factor XII, and of kallikrein generation. Concomitant incubation of a small cationic peptide (lysine4) with betaA abrogated its ability to trigger the cleavage of high molecular weight kininogen. In vivo, prevention of contact system activation beside the reduction of kallikrein generation, can also decrease the activation of complement system and the release of interleukin-6, both factors being considered to play an important role in the inflammatory reactions in AD brain.

The region 1-11 of Alzheimer amyloid-beta is critical for activation of contact-kinin system.

Amyloid-beta protein (Abeta) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and its ability to trigger a local inflammatory response. In the present study using truncated Abeta peptides, we identified the region between residues 1 and 11 as critical for the activation of the contact system in vitro through an ionic interaction of Abeta with factor XII and/or kallikrein. Concomitant incubation of a small cationic peptide (lysine(4)) with Abeta abrogated its ability to trigger the cleavage of high molecular weight kininogen, indicating that Abeta's activity can be blocked by an inhibitory peptide. These findings could be clinically important, since there is evidence that the contact system is activated in AD brain. Thus, prevention of contact system activation, beside diminishing the recruitment of glial cells and microvascular permeability, can also decrease the activation of complement system and the release of IL6, both factors being considered to play an important role in the inflammatory reactions in AD brain.