Hypoxia and oxidative stress induce sterile placental inflammation in vitro.

Fetal growth restriction (FGR) and stillbirth are associated with placental dysfunction and inflammation and hypoxia, oxidative and nitrative stress are implicated in placental damage. Damage-associated molecular patterns (DAMPs) are elevated in pregnancies at increased risk of FGR and stillbirth and are associated with increase in pro-inflammatory placental cytokines. We hypothesised that placental insults lead to release of DAMPs, promoting placental inflammation. Placental tissue from uncomplicated pregnancies was exposed in vitro to hypoxia, oxidative or nitrative stress. Tissue production and release of DAMPs and cytokines was determined. Oxidative stress and hypoxia caused differential release of DAMPs including uric acid, HMGB1, S100A8, cell-free fetal DNA, S100A12 and HSP70. After oxidative stress pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-8, TNFα, CCL2) were increased both within explants and in conditioned culture medium. Hypoxia increased tissue IL-1α/ß, IL-6, IL-8 and TNFα levels, and release of IL-1α, IL-6 and IL-8, whereas CCL2 and IL-10 were reduced. IL1 receptor antagonist (IL1Ra) treatment prevented hypoxia- and oxidative stress-induced IL-6 and IL-8 release. These findings provide evidence that relevant stressors induce a sterile inflammatory profile in placental tissue which can be partially blocked by IL1Ra suggesting this agent has translational potential to prevent placental inflammation evident in FGR and stillbirth.

Increased placental macrophages and a pro-inflammatory profile in placentas and maternal serum in infants with a decreased growth rate in the third trimester of pregnancy.

PROBLEM: There is growing evidence for the role of placental inflammation in the pathophysiology of pregnancy complications including fetal growth restriction (FGR). This study aimed to characterize the inflammatory profile in the maternal circulation and the placenta of infants who were growth restricted and those that were small for gestational age (SGA). METHOD OF STUDY: Placental villous tissue and maternal serum were obtained from pregnancies where infants were SGA at birth or who had a decreasing growth rate (≥25 centiles) across the third trimester. Immunohistochemical and histological analyses of placental samples were conducted for macrophage number, alongside vascular and cell turnover analysis. Inflammatory profile was analyzed in maternal and placental compartments via ELISAs and multiplex assays. RESULTS: There were significantly more CD163+ macrophages in placentas of infants with a decreased growth rate compared to controls, but not in SGA infants (median 8.6/ nuclei vs 3.8 and 2.9, P = .008 and P = .003, respectively). Uric acid (P = .0007) and IL-8 (P = .0008) were increased in placentas, and S100A8 (P < .0002) was increased in maternal serum of infants with decreased growth rate. No changes in the maternal serum or placental lysates of SGA infants were observed. CONCLUSION: The evidence of an altered inflammatory profile in infants with a decreasing growth rate, but not in those that were born SGA, provides further evidence that inflammation plays a role in true FGR. It remains unclear whether the increased placental macrophages occur as a direct result, or as a consequence of the pro-inflammatory environment observed in fetal growth restriction.

Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Cell free hemoglobin impairs vascular function and blood flow in adult cardiovascular disease. In this study, we investigated the hypothesis that free fetal hemoglobin (fHbF) compromises vascular integrity and function in the fetoplacental circulation, contributing to the increased vascular resistance associated with fetal growth restriction (FGR). Women with normal and FGR pregnancies were recruited and their placentas collected freshly postpartum. FGR fetal capillaries showed evidence of erythrocyte vascular packing and extravasation. Fetal cord blood fHbF levels were higher in FGR than in normal pregnancies ( P < 0.05) and the elevation of fHbF in relation to heme oxygenase-1 suggests a failure of expected catabolic compensation, which occurs in adults. During ex vivo placental perfusion, pathophysiological fHbF concentrations significantly increased fetal-side microcirculatory resistance ( P < 0.05). fHbF sequestered NO in acute and chronic exposure models ( P < 0.001), and fHbF-primed placental endothelial cells developed a proinflammatory phenotype, demonstrated by activation of NF-κB pathway, generation of IL-1α and TNF-α (both P < 0.05), uncontrolled angiogenesis, and disruption of endothelial cell flow alignment. Elevated fHbF contributes to increased fetoplacental vascular resistance and impaired endothelial protection. This unrecognized mechanism for fetal compromise offers a novel insight into FGR as well as a potential explanation for associated poor fetal outcomes such as fetal demise and stillbirth.-Brook, A., Hoaksey, A., Gurung, R., Yoong, E. E. C., Sneyd, R., Baynes, G. C., Bischof, H., Jones, S., Higgins, L. E., Jones, C., Greenwood, S. L., Jones, R. L., Gram, M., Lang, I., Desoye, G., Myers, J., Schneider, H., Hansson, S. R., Crocker, I. P., Brownbill, P. Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction.

Increased vascular resistance and reduced fetoplacental blood flow are putative aetiologies in the pathogenesis of fetal growth restriction (FGR); however, the regulating sites and mechanisms remain unclear. We hypothesised that placental vessels dictate fetoplacental resistance and in FGR exhibit endothelial dysfunction and reduced flow-mediated vasodilatation (FMVD). Resistance was measured in normal pregnancies (n = 10) and FGR (n = 10) both in vivo by umbilical artery Doppler velocimetry and ex vivo by dual placental perfusion. Ex vivo FMVD is the reduction in fetal-side inflow hydrostatic pressure (FIHP) following increased flow rate. Results demonstrated a significant correlation between vascular resistance measured in vivo and ex vivo in normal pregnancy, but not in FGR. In perfused FGR placentas, vascular resistance was significantly elevated compared to normal placentas (58 ± 7.7 mmHg and 36.8 ± 4.5 mmHg, respectively; 8 ml min(-1) ; means ± SEM; P < 0.0001) and FMVD was severely reduced (3.9 ± 1.3% and 9.1 ± 1.2%, respectively). In normal pregnancies only, the highest level of ex vivo FMVD was associated with the lowest in vivo resistance. Inhibition of NO synthesis during perfusion (100 µm l-NNA) moderately elevated FIHP in the normal group, but substantially in the FGR group. Human placenta artery endothelial cells from FGR groups exhibited increased shear stress-induced NO generation, iNOS expression and eNOS expression compared with normal groups. In conclusion, fetoplacental resistance is determined by placental vessels, and is increased in FGR. The latter also exhibit reduced FMVD, but with a partial compensatory increased NO generation capacity. The data support our hypothesis, which highlights the importance of FMVD regulation in normal and dysfunctional placentation.

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA.

Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.