Amnion-Epithelial-Cell-Derived Exosomes Demonstrate Physiologic State of Cell under Oxidative Stress

Abstract

At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i) exosomes act as carriers of signals in utero-placental compartments and ii) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05). Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined AEC exosome characteristics and their cargo reflected the physiologic status of the cell of origin and suggests that AEC-derived exosomal p38 MAPK plays a major role in determining the fate of pregnancy. Understanding the propagation of fetal signals and their mechanisms in normal term pregnancies can provide insights into pathologic activation of such signals associated with spontaneous preterm parturitions.

Feto-Maternal Trafficking of Exosomes in Murine Pregnancy Models

“…No subjects were recruited or consented for this study since we used discarded placenta from normal term, not-in-labor cesarean sections that were de-identified before they were received by lab staff, as described previously (Sheller et al, 2016). Placental samples obtained for this study were from the John Sealy Hospital at The University of Texas Medical Branch (UTMB) at Galveston, TX, USA.…”

“…The amniotic membrane was processed within 15 min after delivery as described previously (Lim et al, 2013; Menon et al, 2013; Sheller et al, 2016). Primary AECs ( n = 4) were cultured in T75 flasks containing complete media consisting of Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 media (DMEM/F12; Mediatech Inc., Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 10% Penicillin/Streptomycin (Mediatech Inc.) and 100 μg/mL epidermal growth factor (EGF; Sigma-Aldrich) at 37°C, 5% CO2, and 95% air humidity to 60–65% confluence.…”

“…Culture media were collected and stored at -80°C until exosome isolation. Media was thawed overnight then isolated using differential ultracentrifugation as described previously, (Sheller et al, 2016) with the following modifications. After the 2 h 100,000 × g centrifugation, the supernatant was removed and the exosome pellet was resuspended in PBS.…”

“…Distant senescent signaling is likely facilitated through intercellular signaling vesicles called exosomes (Sheller et al, 2016). Exosomes are 30–100 nm endosome-derived vesicles with specific characteristics that separate them from other larger particles such as microvesicles and apoptotic bodies (György et al, 2011; Akers et al, 2013).…”

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

“…Exposure to CSE, PM, and other air pollutants was found to alter transcriptomic (microRNA) and, in one study, proteomic EV composition (Héliot et al 2017;Lim et al 2016;Pavanello et al 2016;Pergoli et al 2017;Rodosthenous et al 2016;Sheller et al 2016). Based on biological pathway analysis, these compositional changes were predicted to promote inflammation, although this was not experimentally confirmed.…”