Bertoglia-Fuentes, Patricio

Placentas from gestational diabetes mellitus (GDM) are often hypervascularized; however, participation of vascular endothelial growth factor (VEGF) and its receptors in this placental adaptation is unclear. We aimed to test whether changes in phosphorylation of tyrosine 951 or tyrosine 1175 (pY951 or pY1175) of the vascular endothelial growth factor receptor 2 (KDR) are associated with the proangiogenic state observed in placentas from GDM. We obtained placental samples from women with normal pregnancies (n = 24) or GDM (n = 18). We measured the relative expression of markers for endothelial cell number (CD31, CD34), VEGF, vascular endothelial growth factor receptor 1 (Flt-1), KDR, pY951 and pY1175 of KDR in placental homogenate. Immunohistochemistry of placental blood vessels were performed using CD34. Proliferation and migration of human umbilical vein endothelial cells (HUVEC) obtained from normal pregnancy and GDM were determined in absence or presence of conditioned medium (CM) harvested from GDM or normoglycemic HUVEC cultures. GDM was associated with more CD31 and CD34 protein compared to normal pregnancy. High number, but reduced area of placental blood vessels was found in GDM. Reduced Flt1 levels (mRNA and protein) are associated with reduced KDR mRNA, but higher KDR protein levels in placentas from GDM. No significant changes in Y951-or Y1175-phosphorylation of KDR in placentas from GDM were found. GDM did not alter proliferation of HUVECs, but enhanced migration. Conditioned medium harvested from GDM HUVEC cultures enhanced KDR protein amount, tube formation capacity and cell migration in HUVEC isolated from normoglycemic pregnancies. The data indicate that GDM is associated with reduced expression of Flt-1 but high pro-migratory activation of KDR reflecting a proangiogenic state in GDM.

We aim to investigate whether A2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A2A agonist, 24 h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC50 10−9.2 M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (∼1.5-fold) and pre-eclamptic pregnancies (∼1.2-fold), an effect blocked by the A2A antagonist, ZM-241385 (10−5 M) and the inhibitor of NO synthase, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A2A. CGS-21680 also increased (∼1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A2A decrease (∼30 %) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.