Molecular Mechanism And Experimental Study Of Gadd45α-Mediated Signalling Transduction Pathway In The Pathogenesis Of Preeclampsia

Objective To evaluate the expression and location of both Gadd45αand p-p38 MAPK in human placenta in the third trimester of pregnancy; to determine the levels of soluble fms-like tyrosine kinase-1(sFlt-1) and soluble Endoglin (sEng) in maternal sera; to assay the microvessel density of human placenta. To disscuss the role of Gadd45α—p38 MAPK signaling pathway in the endothelial cells dysfunction in preeclampsia. HUVEC from the vein of normal human umbilical cord induced with hypoxia/reoxygenation exposure have been employed to mimic oxidative stress insult of placental endothelium and identify the molecular mechanisms in the current study. We have also attempted to give an insight into the therapeutic potentials including Gadd45αknockdown and p38 inhibitors, for treatment of PE.Methods (1) The mRNA expression of Gadd45αwas compared in the women with or without PE during the third trimester. Immunohistochemical ABC method and Western blotting were employed to detect the expression and localization of Gadd45αand phospho-P38 MAPK protein; placentas endothelial cells were labeled by CD31 to assay the MVD of each group; the levels of serum sFlt-1 and sEng were measured by ELISA. (2) Gadd45αknockdown with lentiviral vector-based short-hairpin RNA (shRNA) and p38 inhibitor SB203580 were undertaken to further decipher the possible molecular mechanism. Via detecting the protein levels of Gadd45αand phospho-P38 MAPK; together with evaluating the concentrations of sFlt-1and sEng in the supernatant of HUVEC which were suffered different treatments, we confirmed the signaling pathway H/R insult—Gadd45α↑—phospho-p38 MAPK↑—sFlt-1, sEng↑in vascular endothelial cells. (3) HUVEC induced with H/R exposure were employed to mimic ischemia/reperfusion insult of placental endothelium in the current study. Moreover, Gadd45αknockdown with lentiviral vector-based short-hairpin RNA (shRNA) and p38 inhibitor SB203580 were undertaken to further decipher the possible molecular and cellular mechanisms of the Gadd45α—p38 MAPK signalling pathway in apoptosis, oxidative stress injury and capability of in vitro angiogenesis in H/R-induced endothelial cells which were involved in the endothelial cells dysfunction in preeclampsia.Results (1) Gadd45αmRNA levels and placental MVD were significantly elevated in preeclamptic placentas compared to controls. In addition, Western blotting analysis showed overexpression of Gadd45αprotein was associated with robust activation of p38 MAPK in preeclamptic placentas. Moreover, consistent with previous findings, our data also identified the levels of sFlt-1 and sEng were dramatically higher in PE group than in normotensive controls. It was detected that positive stained cells were mostly syneytiotrophoblasts in normotensive placentas whereas in preeclamptic placentas the two proteins were detected in both syneytiotrophoblasts and vascular endothelial cells. Furthermore, there were positive correlations between Gadd45αprotein levels and the concentrations of serum sFlt-1, sEng in preeclampsism groups. (2) H/R exposure induced significant upregulation of Gadd45αand phosphorylation of p38, as well as elevated sFlt-1and sEng secretion in HUVEC. Moreover, inhibition of Gadd45αexpression scavenged both p38 activation and sFlt-1, sEng secretion, whereas the p38 inhibitor SB203580 had no effect on Gadd45αproduction, but inhibited sFlt-1 and sEng secretion were strongly demonstrated. Therefore, a regulatory signalling pathway that H/R-induced oxidative stress caused overproduction of Gadd45α, leading to its downstream effector p38 activation and ultimately inducing sFlt-1 and sEng secretion in HUVEC was established. (3) H/R exposure induced significant apoptosis, OS insult, as well as decreased migration and in vitro tube formation capabilities of H/R-induced HUVEC. Moreover, inhibition of Gadd45αexpression or p38 inhibition attenuates apoptosis, inhibits intracellular ROS accumulation and MDA production as well as up-regulates SOD and GSH-Px activities, enhanced migration and in vitro tube formation capabilities of H/R-induced HUVEC were also demonstrated.Conclusions (1) Gadd45α—p38 MAPK was involved in the development of PE, and correlated with the increased maternal sera levels of sFlt-1, sEng. (2) A regulatory signalling pathway that H/R intervention caused induction of Gadd45α, leading to p38 activation and ultimately increasing sFlt-1 and sEng secretion in HUVEC was concurrently established. The study opened up a promising new avenue of investigation for increasing understanding of the origination of sFlt-1 and sEng and provides novel therapeutic targets in the pathogenesis in PE. (3)The study also opened up promising new avenues of investigation for novel therapeutic targets including targeting Human Gadd45αgene therapy for enhancing angiogenesis in the ischemic placenta, and of development for new drugs based on the specific inhibitor of p38 MAPK signal transduction pathway.