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

ETCHEVERRY, TOMÁS; LOUREIRO, FLORENCIA; MALBRÁN, MERCEDES NEGRI; LEGUIZAMÓN GF; MARTINEZ NORA; FARINA MARIANA

Congreso; ISRH 2021- International Symposium on Reproductive Health: Overcoming Barriers for Research in Reproduction; 2021

IntroductionIt 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 [1]. 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 [2].ObjectivesEvaluate if the changes in the ECS seen in preeclampsia are also produced by hypoxia-reoxygenation and/or HIF-1α stabilization.MethodsPlacental 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: 6h in 5% CO2, 95% air (Control group, Ctrl); 2h in 5% CO2 2% O2 93% N2 followed by 4h of re-oxygenation (Hypoxia-reoxygenation group, HR); or 6h in same gas mixture as Ctrl plus 250µM CoCl2 (a HIF-1α stabilizing agent, Co group).Cell culture: BeWo cell line was cultured for 48h in DMEM/F-12 + 10% FBS at 37°C in a 5% CO2, 95% air atmosphere in the absence or presence of 25µM Forskolin. For the hypoxia-reoxygenation group, cells were submitted to two hypoxic events: at 0h and 24h.FAAH and NAPE-PLD mRNA levels were quantified by RT-qPCR. FAAH, NAPE-PLD and HIF-1α protein levels were assessed by Western Blot. Data from 3-4 independent experiments were analyzed using paired t-test or ANOVA followed by Dunnett’s test.ResultsExplants 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.ConclusionsHR 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.