title: PP003. Antagonism in A2A and A2B adenosine receptor on fetal endothelium proliferation involves a nitric oxide-depended intracellular pathway in early and late-onset pre-eclampsia.
abstract: BACKGROUND
  Adenosine A2A and A2B receptor intracellular pathway is associated with either increasing endothelial nitric oxide (NO) synthase (eNOS) expression or eNOS activation (i.e., tyrosine 1177 phosphorylation); a mechanism linked to pro or anti-proliferative effects depending of the cell type. However, there are no reports in pre-eclampsia.
  
  
  OBJECTIVE
  Investigate whether NO signaling pathway is involved in fetal endothelium proliferation induced by adenosine receptor activation in early and late-onset pre-eclampsia.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVEC) were isolated from normal (n=25), late-onset pre-eclampsia (n=11) and early-onset pre-eclampsia (n=22). Adenosine A2A and A2B expression was evaluated by immunocytochemistry and Western blot. Cell proliferation was analyzed using MTS-assay in absence or presence of non-selective adenosine receptor agonist (NECA 10μM), adenosine A2A receptor selective agonist (CGS-21680, 100nM), and/or the antagonists ZM-241385 (0-100μM) or MRS-1754 (0-1μM) for A2A and A2B receptors during 24h. In parallel experiments NOS inhibitor (L-NAME, 100μM) was used in co-incubation by either adenosine receptor agonist or antagonists. Nitrite concentration in the culture medium and protein nitration assessed by Western blot were measured in cells exposed to CGS-21680 (30min).
  
  
  RESULTS
  Early-onset pre-eclampsia was associated to low A2A (∼70%), but high (∼2-fold) A2B adenosine receptor protein abundance compared with normal or late-onset pre-eclampsia. Basally, HUVEC from early-onset showed a low (∼42%), whereas late-onset exhibited high proliferation (∼1.5-fold) compared with normal pregnancy. Cell proliferation was increased by CGS-21680 (∼2-fold) in late-onset or normal pregnancy and ∼5-fold in early-onset pre-eclampsia compared with respective control. NECA increased cell proliferation only in normal cells. Stimulatory effect of CGS-21680, was inhibited by ZM-241385 in normal pregnancies (Ki, 25nM) and late-onset (Ki 50nM) but not in early-onset (Ki ambiguous). Interestingly, MRS-1754 showed an increase in cell proliferation in a dose-response manner only in early-onset group. L-NAME partially blocked (∼25%) the stimulatory effect of CGS-21680 in late-onset and normal pregnancy. Interestingly, L-NAME revert the maximal stimulatory effect of MRS-1754 observed in early-onset. Total and phosphorylated eNOS protein was reduced (∼50%) in early-onset pre-eclampsia compared to late-onset or normal pregnancy. In turn, cells from late-onset pre-eclampsia exhibited high (∼2-fold) eNOS phosphorylation compared with normal pregnancy. In normal pregnancy, CGS-21680 (30min) increased (∼2-fold) the eNOS phosphorylation and nitrotyrosine formation, without changes in nitrite levels, but non-significant changes were observed in early or late-onset pre-eclamptic cells.
  
  
  CONCLUSIONS
  Fetal endothelium from early-onset exhibits a predominant anti-proliferative effect mediated by adenosine A2B receptors activation, whereas the stimulatory effect of adenosine A2A receptors prevails in cells from late-onset pre-eclampsia. Both pro and anti-proliferative effects seem mediated by a nitric oxide-depended intracellular pathway. Supported by FONDECYT 1100684, Conicyt Anillo ACT73.
authors.affiliation: Basic Science Department, Universidad del Bio Bio, Chillan, Chile.
authors.forename: J
authors.initials: J
authors.lastname: Acurio
authors.affiliation: Basic Science Department, Universidad del Bio Bio, Chile.
authors.forename: A
authors.initials: A
authors.lastname: Escudero
authors.affiliation: Basic Science Department, Universidad del Bio Bio, Chile.
authors.forename: C
authors.initials: C
authors.lastname: Celis
authors.affiliation: Obstetric and Gynecology, Hospital Clinico Herminda Martin, Chillan, Chile.
authors.forename: P
authors.initials: P
authors.lastname: Bertoglia
authors.affiliation: Basic Science Department, Universidad del Bio Bio, Chile.
authors.forename: C
authors.initials: C
authors.lastname: Escudero