Increased placental angiogenesis in late and early onset pre-eclampsia is associated with differential activation of vascular endothelial growth factor receptor 2.

INTRODUCTION: Placentas from both early-onset (EOPE) and late-onset pre-eclampsia (LOPE) exhibit signs of underperfusion, which in turn, may be associated with altered angiogenesis. Tyrosine 951 (Y951) and Y1175 phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2) induced by VEGF triggers the angiogenesis process. Endothelial markers such as CD31 and CD34 have been used for estimating angiogenic processes in several tissues, including placenta. We asked whether vascular density in placental villi was related to Y951/Y1175 phosphorylation of VEGFR2 in LOPE or EOPE. METHODS: We obtained placental samples from women with normal pregnancies (n = 22), LOPE (n = 13), EOPE (n = 15) and preterm deliveries (n = 10). Slices from placental tissue were used for CD31 immunostaining. We estimated the expression of CD31, CD34, VEGF, and VEGFR2 by western blot and quantitative PCR. Y951 phosphorylation of VEGFR2 was estimated by western blot, whereas Y1175 phosphorylation was analyzed by ELISA. RESULTS: Vessel density in terminal villi and CD31 and CD34 protein abundance were increased in LOPE and EOPE compared to normal pregnancy. However, mRNA levels for CD31 and CD34 were lower in LOPE than in normal pregnancy and VEGF mRNA was higher in EOPE. VEGFR2 protein concentration was not different among the studied groups. Y951 and Y1175 phosphorylation of VEGFR2 was higher in LOPE than in the normotensive group, but only Y951 exhibited greater phosphorylation in EOPE compared to normal pregnancy. DISCUSSION: Changes in vessel formation in the pre-eclamptic placenta are controversial. Our study suggests a pro-angiogenic state in both LOPE and EOPE. These changes are however, associated with differential expression of endothelial markers and VEGFR2 activation. CONCLUSION: There is evidence of increased placental angiogenesis in LOPE and EOPE that is associated with differential activation of VEGFR2.

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.

INTRODUCTION: 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. OBJECTIVES: 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. CONCLUSION: 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.

PP026. Hyperuricemia in the prognosis of adverse perinatal outcomes.

INTRODUCTION: Elevated uric acid levels during the first or third trimester of gestation have been associated with poor perinatal outcomes in women with hypertensive pregnancies. OBJECTIVES: Investigate whether uric acid levels are related to adverse perinatal outcomes in Chilean hypertensive women. METHODS: It is a post-hoc analysis from a retrospective study including clinical records (n=416) of women with diagnosis of hypertension in pregnancy treated in the Hospital Clínico Herminda Martin, Chillán, Chile. From these records, 86 showed quantification of uric acid plasma levels at the moment of hypertension diagnosis ((3) 140/90mmHg; at 34±5weeks). Women were divided into three groups, considering uric acid levels below 25th percentile (Low group, n=27, <3.7mg/dl), from 25th to 75th percentile (Middle group, n=38, 3.8 to 5.7mg/dl) and above 75th percentile (High group, n=21, >5.8mg/dl) for the studied population. RESULTS: In the entire group of hypertensive women, the uric acid/creatinine ratio was positively related to hospitalization days (p=0.04), and negatively associated with newborn weight (p=0.02) and size (p=0.01). ANOVA analysis did not show statistical differences in age, parity, systolic, diastolic or media blood pressure, body mass index, proteinuria, hepatic enzymes or hypoxia perinatal in women with low, middle or high uric acid levels. However, women with high uric acid levels showed a longer-hospitalization period (∼1.2days more), less platelet count (∼40×10(3)/ml) and high creatinine plasma levels (∼0.2mg/dl) and their babies showed less birth weight (∼800g) and were smaller (∼3cm) compared with women with low uric acid levels. Relative risk of intrauterine growth restriction in women with high levels of uric acid was 1.3 (CI, 0.96 to 1.73) compared with women with low levels. CONCLUSION: These data reinforce the general agreement about the utility of hyperuricemia in the prognosis of adverse perinatal outcomes in hypertensive pregnancies.

Resultados clínicos y perinatales de los embarazos con hipertensión arterial en un hospital de referencia de la VIII Región de Chile / Clinical and perinatal outcomes associated with hypertension syndrome in pregnancy

Objetivo: Conocer los resultados clínicos, bioquímicos y perinatales asociados al síndrome de hipertensión del embarazo (SHE) en el Hospital Herminda Martín de Chillan. Métodos: Se realizó un estudio retrospectivo de registros clínicos (n=416) con diagnóstico de SHE en el periodo 2006 a 2008. Los registros disponibles fueron divididos en tres grupos de acuerdo al nivel de presión arterial: Grupo I (n=124) <140/90 mmHg; Grupo II (n=98) ³ 140-159/³ 90-109 mmHg y Grupo III (n=41)³ 160/110 mmHg. Adicionalmente, un subgrupo (n=85) fue dividido considerando el percentil de distribución del nivel de ácido úrico materno en: SHE con niveles bajos (p75). Se analizaron y compararon los grupos estudiados y se correlacionó las variables estudiadas con los resultados perinatales. Resultados: La prevalence de SHE fue de 3,8 por ciento. Las mujeres del grupo II y III muestran peores resultados clínicos y neonatales que las mujeres del grupo I. El índice de masa corporal (IMC), la presión arterial materna y el nivel de ácido úrico están relacionados negativamente con la antropometría neonatal. Además, la antropometría neonatal fue menor en las mujeres con niveles más altos de ácido úrico, situación que no obedece a la severidad de la hipertensión o el IMC materno. Conclusión: La presencia de SHE esta asociada a mayor morbilidad materna y neonatal. Este estudio permitió detectar deficiencias (e.L, falta de cumplimiento en criterio diagnóstico) y hacer recomendaciones sobre probables marcadores de riesgo perinatal (e.L, nivel de ácido úrico).

Aims: To know the clinical and biochemical spectra and perinatal outcomes associated with syndrome of hypertension in pregnancy (HP) in the Herminda Martin Hospital from Chilian. Methods: It is a retrospective study using clinical records with HP diagnosis (N=416) during 2006 to 2008. The available records were divided in three different groups according with arterial blood pressure: Group I (n=124) <140/90 mmHg; Group II (n=98) ³ 140-159/³ 90-109 mmHg and Group III (n=41)³ 160/110 mmHg. Additionally, a subset (n=85) was divided considering the percentile of the uric acid level in the mother as: HP with low (p75th) uric acid levels. The studied groups were analyzed and compared each other and quantitative variables were correlated with perinatal outcomes. Results: The prevalence of HP was 3.8 percent. Women in the groups II and III showed worse clinical and neonatal outcomes compared with women in the group I. Body mass index (BMI), maternal blood pressure and uric acid levels were negatively correlated with neonatal anthropometry. Moreover, neonatal anthropometry was lower in women with high uric acid levels, a situation that was independent of maternal hypertension or BMI. Conclusion: Hypertension in pregnancy was associated with high maternal and neonatal morbidity. This study allowed identifies some weaknesses (e.i., lack of compliance in diagnosis criteria) as well as suggest the potential role of perinatal risk markers (e.i., uric acid levels).