191: Punicalagin attenuates hypoxia-induced apoptosis by down regulating p53 activity in cultured human placental syncytiotrophoblasts

factor expression in pregnancy Amy Wong, Hau Kwaan, William Grobman, Ivy Weiss, Cynthia Wong Northwestern University, Feinberg School of Medicine, Department of Obstetrics and Gynecology, Chicago, IL, Northwestern University, Feinberg School of Medicine, Department of Medicine, Chicago, IL, Northwestern University, Feinberg School of Medicine, Department of Anesthesiology, Chicago, IL OBJECTIVE: Microparticles (MPs) are potent activators of the coagulation system. To investigate whether MPs originating from platelets or trophoblast cells that express tissue factor (TF) contribute to coagulation changes in pregnancy, we aimed to characterize whether pregnancy, labor, and delivery are associated with changes in the source and composition of MPs. STUDY DESIGN: Blood samples were collected in 20 non-pregnant women, 20 term pregnant women not in labor (presenting for induction or scheduled cesarean delivery), and 20 term pregnant women in labor. Two samples were collected in the pregnant groups, one prior to delivery and the second one hour after delivery. Using flow cytometry, we used CD41a and NDOG2 antibodies to identify MPs from platelets and trophoblasts, respectively, and TF antigen to identify MPs expressing tissue factor. Comparisons were made between the non-pregnant and pregnant groups, non-laboring and laboring groups, and pre-delivery and post-delivery groups within the pregnant groups. RESULTS: There was no difference among the non-pregnant, pregnant pre-delivery non-laboring, and pregnant pre-delivery laboring groups with regard to the proportion of MPs originating from platelets or expressing TF. Also, the presence of labor did not affect the proportion of MPs originating from trophoblasts. Conversely, the proportion of platelet-derived MPs present among women in labor increased after delivery (8.5 vs. 20.5%, p 0.02). CONCLUSION: In the current study, pregnancy was not associated with changes in cell origin of MPs or in the number of TF-expressing MPs. However, delivery appears to be associated with an increase in the number of platelet-derived MPs. Further investigation may determine whether this increase contributes to the clinically meaningful coagulation changes in pregnancy and the puerperium, as well as to identify other possible MP sources or antigens that may affect coagulation. 191 Punicalagin attenuates hypoxia-induced apoptosis by down regulating p53 activity in cultured human placental syncytiotrophoblasts Baosheng Chen, Mark S Longtine, D Michael Nelson Washington University School of Medicine, Department of Obstetrics and Gynecology, St. Louis, MO OBJECTIVE: Punicalagin is a promiment polyphenol in pomegranate juice (PJ), and both punicalagin and PJ reduce oxidative stress and apoptosis in human placental trophoblasts (Chen et al. Am J Physiol 302:E1142, 2012). The mechanism for this effect is unknown. We tested the hypothesis that punicalagin attenuates apoptosis in cultures of syncytiotrophoblasts exposed to hypoxia by regulating the expression of p53, MDM2, Hif-1 , and Bcl-2 family member proteins. STUDY DESIGN: Primary human trophoblasts were cultured in 5% CO2/air for 28 h in DMEM with 10% FBS and then for 24 h in phenolred free DMEM with 10% charcoal-stripped FBS. Syncytiotrophoblasts formed during this time, and at 52 h the cultures were exposed to 24 h of 1% oxygen with 5% CO2, 10% H2, and 84% N2, in medium containing 33.8 mM punicalagin or 7.5 mM glucose as control. Protein extracts of cultures were harvested at 76 h and western blotting quantified expression levels of p53, MDM2, Hif-1 , Bcl-2, Bcl-XL, Bak and Bax. RESULTS: Levels of p53 were significantly decreased after exposure to punicalagin compared to control in the syncytiotrophoblasts under hypoxia (p 0.05). Moreover, in hypoxic syncytiotrophoblast punicalagin exposure increased expression (p 0.05) of MDM2, the major negative regulator of p53 levels, and reduced expression of Hif-1 , which interacts with p53. There were no differences in expression between punicalagin-exposed and control exposed syncytiotrophoblasts for any of the four proteins of the Bcl-2 family examined. CONCLUSION: Punicalagin, a prominent polyphenol in pomegranate juice, reduces p53 activity and modulates the p53 pathway to limit, in part, hypoxia-induced apoptosis in syncytiotrophoblasts. NIH RO1 HD 29190.