Differences in cord blood extracellular vesicle cargo in preterm and term births.

OBJECTIVE: This study determined the cord plasma-derived extracellular vesicle (exosomes; 30-160 nm particles) proteomic profile in patients who had spontaneous preterm birth (PTB) or preterm premature rupture of membranes (pPROM), compared to those who delivered at term regardless of labor status. METHODS: This is a cross-sectional analysis of a retrospective cohort that quantified and determined the proteomic cargo content of exosomes present in cord blood plasma samples in PTB or pPROM, and normal term in labor (TL) or term not in labor (TNIL) pregnancies. Exosomes were isolated by differential centrifugation followed by size exclusion chromatography. Exosomes were characterized by nanoparticle tracking analysis (quantity and size) and markers (dot blots for exosome markers). The exosomal proteomic profile was identified by liquid chromatography-mass spectrometry (LC-MS/MS). Ingenuity pathway analysis determined canonical pathways and biofunctions associated with dysregulated proteins. RESULTS: Cord plasma exosomes have similar quantity and exhibit both tetraspanin and ESCRT protein markers specific of exosomes regardless of the conditions. Proteomics analysis exhibited several similar markers as well as very unique markers in exosomes from each condition; however, bioinformatics analysis revealed a generalized and non-specific inflammatory condition represented in exosomes from different condition that is not indicative of any specific underlying biological functions indicative of an underlying pathology. CONCLUSIONS: Compared to maternal plasma and amniotic fluid exosomes, the value of cord plasma derived exosomes is limited. Quantity, character, and proteomic cargo contents in exosomes or the pathways and functions represented by differentially expressed proteins do not distinguish specific conditions regarding normal and abnormal parturition. The value of cord plasma exosome proteomic cargo has limited value as an indicator of an underlying physiology or as a biomarker of fetal well-being.

Quantitative Proteomics by SWATH-MS of Maternal Plasma Exosomes Determine Pathways Associated With Term and Preterm Birth.

Exosomes are membrane-bound nanovesicles that transport molecular signals between cells. This study determined changes in maternal plasma exosome proteomics contents in term and preterm births. Maternal plasma (MP) samples were collected from group 1: term not in labor (TNIL, n = 13); group 2: term in labor (TL, n = 11); group 3: preterm premature rupture of membranes (pPROM, n = 8); and group 4: preterm birth (PTB, n = 13). Exosomes isolated from plasma by differential density centrifugation followed by size exclusion chromatography were characterized by morphology (electron microscopy), quantity and size (nanoparticle tracking analysis), and markers (western blot). A quantitative, information-independent acquisition [sequential windowed acquisition of all theoretical mass spectra (SWATH-MS)] approach was used to determine the protein profile in exosomes. Ingenuity Pathway Analysis determined pathways associated with the protein profile identified in exosomes. MP exosomes were spherical, had a mean diameter of 120 nm, and were positive for exosomal proteins CD63 and TSG101 irrespective of pregnancy status. No distinct changes in exosome quantities were seen in maternal circulation across the groups. SWATH-MS identified 72 statistically significant proteins across the groups studied. Bioinformatics analysis showed the proteins within the exosomes in TNIL, TL, pPROM, and PTB target pathways mainly associated with inflammatory and metabolic signals. Exosomal data suggest that homeostatic imbalances, specifically inflammatory and endocrine signaling, might disrupt pregnancy maintenance resulting in labor-related changes both at term and preterm. Reflection of physiologic changes in exosomes is suggestive of its usefulness as biomarkers and cellular function indicators.

Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells.

Term and preterm parturition are associated with oxidative stress (OS)-induced p38 mitogen-activated protein kinase (p38MAPK)-mediated fetal tissue (amniochorion) senescence. p38MAPK activation is a complex cell- and stimulant-dependent process. Two independent pathways of OS-induced p38MAPK activation were investigated in amnion epithelial cells (AECs) in response to cigarette smoke extract (CSE: a validated OS inducer in fetal cells): (1) the OS-mediated oxidation of apoptosis signal-regulating kinase (ASK)-1 bound Thioredoxin (Trx[SH]2) dissociates this complex, creating free and activated ASK1-signalosome and (2) transforming growth factor-mediated activation of (TGF)-beta-activated kinase (TAK)1 and TGF-beta-activated kinase 1-binding protein (TAB)1. AECs isolated from normal term, not-in-labor fetal membranes increased p38MAPK in response to CSE and downregulated it in response to antioxidant N-acetylcysteine. In AECs, both Trx and ASK1 were localized; however, they remained dissociated and not complexed, regardless of conditions. Silencing either ASK1 or its downstream effectors (MKK3/6) did not affect OS-induced p38MAPK activation. Conversely, OS increased TGF-beta's release from AECs and increased phosphorylation of both p38MAPK and TAB1. Silencing of TAB1, but not TAK1, prevented p38MAPK activation, which is indicative of TAB1-mediated autophosphorylation of p38MAPK, an activation mechanism seldom seen. OS-induced p38MAPK activation in AECs is ASK1-Trx signalosome-independent and is mediated by the TGF-beta pathway. This knowledge will help to design strategies to reduce p38MAPK activation-associated pregnancy risks.

A distinct mechanism of senescence activation in amnion epithelial cells by infection, inflammation, and oxidative stress.

PROBLEM: We investigated p38MAPK activation-induced fetal membrane cell senescence in response to inflammation (tumour necrosis factor-alpha [TNF-α]) and infection (lipopolysaccharide [LPS]), factors associated with spontaneous preterm birth. METHOD OF STUDY: Primary amnion epithelial cells (AECs) were exposed to TNF-α, 50 ng/mL and LPS, 100 ng/mL. Cigarette smoke extract (CSE), a known OS inducer, was used as positive control. AECs were cotreated with the antioxidant N-acetyl cysteine (NAC) and p38MAPK inhibitor SB203580 to determine the effect of OS and p38MAPK. Western blot analysis was performed for active (Phospho-p38MAPK) and total p38MAPK. Senescence was determined by flow cytometry, and culture supernatants were tested for IL-6 using ELISA. RESULTS: TNF-α, but not LPS, increased p38MAPK activation compared to untreated cells (P = .01). The number of senescent cells and senescence-associated IL-6 was higher in both TNF-α and LPS-treated cells compared to control (P = .001, P = .01, respectively). Antioxidant NAC inhibited p38MAPK activation by TNF-α. p38MAPK inhibitor SB203580 reduced the development of senescence and IL-6 by TNF-α and LPS. CSE treatment validated our current data. CONCLUSION: TNF-α caused OS-mediated p38MAPK induction, senescence, and IL-6 increase from AECs. LPS also induced senescence and IL-6 increase. Inflammatory and infectious factors may cause premature fetal cell senescence contributing to preterm birth pathophysiology.