Hypoxia-reoxygenation, one of the factors responsible for the alteration of the endocannabinoid system in the placenta

ETCHEVERRY, TOMAS; LOUREIRO, FLORENCIA; MALBRAN, NEGRI; LEGUIZAMÓN, GUSTAVO; MARTÍNEZ, NORA; FARINA, MARIANA

Simposio; International Symposium on Reproductive Health: overcoming barriers for research in reproduction; 2021

Introduction: It has been described that the endocannabinoid system (ECS) has several implications in human placentation and that alterations in this system are associated with placental pathologies. We previously demonstrated that the ECS is dysregulated in preeclampsia, with elevated levels of NAPE-PLD and decreased levels of FAAH, the main enzymes responsible for the synthesis and degradation pathways, respectively.Objectives: Evaluate if the changes in the ECS seen in preeclampsia are also produced by hypoxia-reoxygenation and/or HIF-1a stabilization.Methods: Placental explants: Term placentas obtained from elective cesareans were cut into 50mg explants and cultured for 6h in DMEM/F-12 + 10% FBS at 37 °C under the following conditions: 6 h in 5% CO2, 95% air (Control group, Ctrl); 2 h in 5% CO2 2% O2 93% N2 followed by 4 h of re-oxygenation (Hypoxia-reoxygenation group, HR); or 6 h in same gas mixture as Ctrl plus 250 µ M CoCl2 (a HIF-1a stabilizing agent, Co group).Cell culture: BeWo cell line was cultured for 48 h in DMEM/F-12 + 10% FBS at 37 °C in a 5% CO2, 95% airatmosphere in the absence or presence of 25 µ M Forskolin. For the hypoxia-reoxygenation group, cells were submitted to two hypoxic events: at 0 h and 24 h.FAAH and NAPE-PLD mRNA levels were quantified by RT-qPCR. FAAH, NAPE-PLD and HIF-1a proteinlevels were assessed by Western Blot. Data from 3?4 independent experiments were analyzed using paired t-test or ANOVA followed by Dunnett?s test.Results: Explants from HR group showed higher NAPE-PLD mRNA levels, while the Co group did not present any changes. In the case of FAAH, both conditions significantly diminished FAAH mRNA. BeWo cells incubated with forskolin and exposed to HR showed an increase in NAPE-PLD protein levels.Conclusions: HR explants but not Co presented an ECS profile similar to preeclampsia. Changes detected in the synthesis and degradation pathways are observed mainly in the syncytiotrophoblast.