Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia

Abstract

To investigate whether fetal endothelial cell proliferation and migration are modulated by the A 2A adenosine receptor (A 2A AR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n023), preterm delivery (n04), and late-onset (LOPE, n010) and early-onset preeclampsia (EOPE, n08). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A 2A AR. Also, the nitric oxide synthase (NOS) inhibitor, L-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A 2A AR and VEGF and NO synthesis (i.e., total and phosphorylated serine 1177 endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LO-PE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC 50 for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age.L-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC 50 036.2± 2.5 and 8.6±2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A 2A AR/NO/ VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.

Adenosine A2A receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies

“…Pregnant women who attended to the Obstetric and Gynecology Department of the Herminda Martin Clinical Hospital (HCHM), Chillan, Chile, for their delivery were included as described previously by our group [14]. Exclusion criteria included chronic hypertension, altered renal function, diabetes, chronic disease, twin pregnancies, recurrent miscarriages, and abruption placenta.…”

“…Contrary to this last view, stimulation of A 2A triggers nitric oxide (NO) synthesis, enhancing VEGF protein expression in human umbilical vein endothelial cells (HUVECs) from normal pregnancy [14]. Also, the inhibitor of NO synthase, N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME), blocked A 2A -mediated upregulation of VEGF expression (protein and messenger RNA (mRNA)) suggesting that NO is down stream of A 2A -mediated VEGF in HUVEC from normal pregnancies.…”

“…Briefly, human placental microvascular endothelial cells (hPMECs) [28] and HUVEC [14] were isolated from normal and pre-eclamptic placentas as previously described by our group. Experiments were performed at standard culture condition (37°C, 5 % CO 2 ) without control of oxygen tension.…”

“…RNA was quantified by spectrophotometry (A 260 nm), and its integrity was assessed by the A 260/280 nm ratio and directly observed in an agarose gel (2 %). Aliquots of 1 μg of total RNA were used in the reverse transcription reaction by using oligo dT, random primers, and the enzyme Moloney Murine Leukemia Virus-reverse transcriptase (MMLV-RT, Promega, USA) in a cycle of reverse transcription at 37°C as described elsewhere [14]. …”

“…In particular in LOPE, stereological studies evidenced no changes [22], while estimation of endothelial markers such as CD31 [18] or CD34 [17] denoted elevated placental angiogenesis compared to normotensive controls. Despite this discrepancy, previous in vitro studies also supported a pro-angiogenic behavior of HUVEC isolated from LOPE [14]. Then, it is feasible that increased vessel formation in placentas from LOPE would facilitate oxygen/nutrient transfer between mother and fetus [23] as a compensatory mechanism looking for adequate fetus growth.…”