eNOS/iNOS and endoplasmic reticulum stress-induced apoptosis in the placentas of patients with preeclampsia

Abstract

Disruption of nitric oxide pathway and endoplasmic reticulum (ER) stress had been observed in preeclampsia (PE). However, the correlation and overall detailed expression profiles of ER stress-related markers and endothelial nitric oxide synthase/inducible nitric oxide synthase (eNOS/iNOS) in patients with PE were poorly understood. In this study, placental protein expression of ER stress-related markers as well as eNOS/iNOS in normotensive control (n=32) and PE pregnancies (n=32) was examined by western blot. In addition, apoptosis was detected by terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) staining in placentas. Compared with control, we found elevated ER stress response was agreeable with iNOS upregulation in placenta tissue of PE patients. Placental protein expression of ER stress-related markers, including GRP78, GRP94, p-PERK, eIF2a, p-eIF2a, XBP1, CHOP, Ire1, p-Ire1 and iNOS, was higher, and eNOS expression was lower in PE (P<0.05 for all); however, the expression of ATF6 and PERK was similar in the PE and control groups. Upregulation of CHOP and iNOS was consistent of apoptosis increasing indicated by TUNEL staining and caspase 4 expression upregulation in PE placenta. Our datas suggest that the exaggerated ER stress response and upregulated iNOS are probably associated with increased apoptosis in placenta of PE patients and may contribute to the pathophysiology of PE.

Figure 1

Immunohistochemical staining and western blot analyses of eNOS and iNOS in placental tissues from normal women and those with PE (n=32 each). (a) Representative immunostaining of iNOS and eNOS (brown) in the normal and PE groups. (b) Western blot analyses of eNOS and iNOS in placental tissues from normal women and those with PE. *P<0.05 compared with a normal pregnancy.

Figure 2

Western blot analyses of ER stress markers in placental tissues from normal women and those with PE (n=32 each). Representative western blots of PERK, phospho-PERK, IRE1a, IRE1 (phospho-S724), eIF2α, phospho-eIF2a, XBP1s, GRP78, GRP94, CHOP, ATF6 and the loading control (GAPDH). Data are presented as mean±s.d. *P<0.05, PE versus control.

Figure 3

Apoptosis assay with TUNEL staining and western blot analyses of caspase 4. (a) PE placenta; DAPI staining (blue). (b) PE placenta; TUNEL staining (green) indicates many apoptotic nuclei. (c) PE placenta; the merged image of TUNEL (green) and DAPI (blue) staining. (d) Control placenta; DAPI staining (blue). (e) Control placenta; TUNEL staining (green) indicates few apoptotic nuclei. (f) Control placenta; the merged image of TUNEL (green) and DAPI (blue) staining. (g) The positive ratio of TUNEL staining in PE and control placentas. (h, i) Western blot analyses of caspase 4 in placentas from normal women and those with PE (n=32 each). Scale bars=100 μm. *P<0.05, PE versus control.