Tariq Syed

Histological Evidence of Oxidative Stress and Premature Senescence in Preterm Premature Rupture of the Human Fetal Membranes Recapitulated in Vitro

Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes (<34 weeks) and the inducibility fetal membrane senescence phenotype by oxidative stress in vitro. IHC was performed for p53, p21, and phospho (p)-p38 mitogen-activated protein kinase (MAPK) as markers of senescence phenotype in pPROM, PTBs, and term births. Term fetal membranes were exposed to cigarette smoke extract to induce oxidative stress. Western blots documented p-p53 and p-p38 MAPK. Transmission electron microscopy assessed cellular morphologic features in clinical and cigarette smoke extract-treated membranes. A total of 80% of pPROM cells and >60% of term cells were positive for all three senescence phenotype markers, and concentrations were higher than in PTBs (P < 0.05). p53 staining was comparable in membranes from PTB and term birth pregnancies, whereas only <30% and <45% of cells were positive for p21 and p38 MAPK, respectively. In vitro cigarette smoke extract exposure increased p-p38 MAPK without any detectable change in p-p53 MAPK. Enlargement of organelles consistent with senescence phenotype was evident in pPROM and term membranes in vivo and after cigarette smoke extract treatment in vitro but was less apparent in PTBs. Histologic and biochemical resemblance of pPROM and term membranes suggests premature senescence of the membranes is a mechanistic feature in pPROM, and this can be phenocopied in an in vitro model.

Fetal DNA Methylation Associates with Early Spontaneous Preterm Birth and Gestational Age

Spontaneous preterm birth (PTB, <37 weeks gestation) is a major public health concern, and children born preterm have a higher risk of morbidity and mortality throughout their lives. Recent studies suggest that fetal DNA methylation of several genes varies across a range of gestational ages (GA), but it is not yet clear if fetal epigenetic changes associate with PTB. The objective of this study is to interrogate methylation patterns across the genome in fetal leukocyte DNA from African Americans with early PTB (241/7–340/7 weeks; N = 22) or term births (390/7–406/7weeks; N = 28) and to evaluate the association of each CpG site with PTB and GA. DNA methylation was assessed across the genome with the HumanMethylation450 BeadChip. For each individual sample and CpG site, the proportion of DNA methylation was estimated. The associations between methylation and PTB or GA were evaluated by fitting a separate linear model for each CpG site, adjusting for relevant covariates. Overall, 29 CpG sites associated with PTB (FDR<.05; 5.7×10−10<p<2.9×10−6) independent of GA. Also, 9637 sites associated with GA (FDR<.05; 9.5×10−16<p<1.0×10−3), with 61.8% decreasing in methylation with shorter GA. GA-associated CpG sites were depleted in the CpG islands of their respective genes (p<2.2×10−16). Gene set enrichment analysis (GSEA) supported enrichment of GA-associated CpG sites in genes that play a role in embryonic development as well as the extracellular matrix. Additionally, this study replicated the association of several CpG sites associated with gestational age in other studies (CRHBP, PIK3CD and AVP). Dramatic differences in fetal DNA methylation are evident in fetuses born preterm versus at term, and the patterns established at birth may provide insight into the long-term consequences associated with PTB.

Senescence of Primary Amniotic Cells via Oxidative DNA Damage

Objective Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells. Methods Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2′7′-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19arf. Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ. Results ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19Arf were not significantly affected by NAC. Conclusions The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.

HMGB1 promotes a p38MAPK associated non-infectious inflammatory response pathway in human fetal membranes.

Objective Spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (pPROM) are major pregnancy complications often associated with a fetal inflammatory response. Biomolecular markers of this fetal inflammatory response to both infectious and non-infectious risk factors and their contribution to PTB and pPROM mechanism are still unclear. This study examined fetal membrane production, activation and mechanistic properties of high mobility group box 1 (HMGB1) as a contributor of the non-infectious fetal inflammatory response. Materials and Methods HMGB1 transcripts and active HMGB1 were profiled in fetal membranes and amniotic fluids collected from PTB and normal term birth. In vitro, normal term not in labor fetal membranes were exposed to lipopolysaccharide (LPS) and water soluble cigarette smoke extract (CSE). HMGB1-transcripts and its protein concentrations were documented by RT-PCR and ELISA. Recombinant HMGB1 treated membranes and media were subjected to RT-PCR for HMGB1 receptors, mitogen activated protein kinase pathway analysis, cytokine levels, and Western blot for p38MAPK. Results HMGB1 expression and its active forms were higher in PTB and pPROM than normal term membranes and amniotic fluid samples. Both LPS and CSE enhanced HMGB1 expression and release in vitro. Fetal membrane exposure to HMGB1 resulted in increased expression of TLR2 and 4 and dose-dependent activation of p38MAPK-mediated inflammation. Conclusions HMGB1 increase by fetal membrane cells in response to either oxidative stress or infection can provide a positive feedback loop generating non-infectious inflammatory activation. Activation of p38MAPK by HMGB1 promotes development of the senescence phenotype and senescence associated sterile inflammation. HMGB1 activity is an important regulator of the fetal inflammatory response regardless of infection.

Expression of 8-oxoguanine Glycosylase in Human Fetal Membranes

The most common DNA lesion generated by oxidative stress (OS) is 7, 8‐dihydro‐8‐oxoguanine (8‐oxoG) whose excision repair is performed by 8‐oxoguanine glycosylase (OGG1). We investigated OGG1 expression changes in fetal membranes from spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) and its changes in vitro in normal fetal membranes exposed to OS inducer water‐soluble cigarette smoke extract (CSE).

Fetal membrane biomarker network diversity and disease functions induced by intra-amniotic pathogens.

Intra‐amniotic pathogens and by‐products activate innate immune responses encompassing multitudes of signaling molecules and pathways that can result in spontaneous preterm birth (PTB). This study investigates fetal membrane response to bacterial stimulation using a bioinformatics approach.

Expression profiles of fetal membrane nicotinamide adenine dinucleotide phosphate oxidases (NOX) 2 and 3 differentiates spontaneous preterm birth and pPROM pathophysiologies

INTRODUCTION Nicotinamide adenine dinucleotide phosphate oxidases (NOX 1-5) are enzymes that generate cellular reactive oxygen species (ROS) besides mitochondria and might be important ROS sources associated with pregnancy complications, particularly preterm premature rupture of membranes (pPROM), that has been related to ROS. OBJECTIVE To characterize NOX enzymes expression in human fetal membranes. METHODS Differential expression and localization of NOX isoforms in human fetal membranes collected from women with uncomplicated pregnancies at term, preterm birth (PTB) or pPROM and in vitro in normal term membranes maintained in an organ explant system stimulated with water-soluble cigarette smoke extract (wsCSE) were documented by real time PCR and immunohistochemistry. RESULTS Fetal membranes from term deliveries, PTB and pPROM expressed NOX 2, 3 and 4 mRNAs whereas NOX 1 and 5 were not detected. NOX 2 expression was 2.3-fold higher in PTB than pPROM (p = 0.005) whereas NOX 3 was 2.2-fold higher in pPROM compared to PTB (p = 0.04). NOX 2 and 3 expressions at term mimicked pPROM and PTB, respectively. No difference in NOX 4 expression was observed among the studied groups. NOX 2, 3 and 4 were localized to both amniotic and chorionic cells. Expression of NOX 2, 3 and 4 were not significant in wsCSE-stimulated membranes compared to untreated controls. DISCUSSION/CONCLUSIONS NOX enzymes are present in the fetal membranes and are differentially expressed in PTB and pPROM. Absence of any changes in NOXs expression after wsCSE stimulation suggests ROS generation in the membranes does not always correlate with NOX expression.

Screening of lysyl oxidase (LOX) and lysyl oxidase like (LOXL) enzyme expression and activity in preterm prelabor rupture of fetal membranes.

Abstract Objective: Lysyl oxidase (LOX) and LOX like enzymes (LOXL1–4) physiologically remodel extracellular matrix and pathologically contribute to cellular senescence under oxidative stress (OS). We characterized LOX and LOXL expressions and activity in human fetal membranes. Methods: Human fetal membranes from women with uncomplicated pregnancies at term, preterm birth with intact membranes (PTB) or preterm prelabor rupture of membranes (pPROM), and in vitro fetal membranes stimulated with water-soluble cigarette smoke extract (CSE), an OS inducer, were analyzed by real-time PCR and immunohistochemistry for LOX and LOXL (1–4) expression and localization. LOX activity was measured by fluorometric assay. Results: LOX gene expression was ∼2.5-fold higher in fetal membranes from pPROM compared to PTB and term (P=0.02). LOX and LOXL1, 2 and 4 were localized to both amniotic and chorionic cells, whereas LOXL3 was limited to chorion. LOX and LOXL isoform expressions were not different between CSE treated and untreated groups, while LOX activity was increased in the presence of an antioxidant (P=0.02). Conclusions: Increase of LOX expression in pPROM, an OS-related disease, and the apparent inhibition of LOX activity by CSE restored by antioxidant treatment suggest that reactive oxygen species might influence LOX-mediated tissue remodeling in fetal membranes. Balanced antioxidant supplementation during pregnancy may reduce the risk of pPROM by increasing LOX activity.