PP030. Transmission electron microscopy reveals leakage of laeverin into the villous capillaries and ectopic expression in the cytoplasm instead of cell membrane of the trophoblasts in preeclamptic placentas.

INTRODUCTION

Laeverin, a membrane-bound aminopeptidase is specifically expressed in human placenta. We previously reported that mRNA levels of laeverin-gene are up-regulated in the placentas of severely preeclamptic women compared to healthy controls. Furthermore, immunofluorescence studies indicated that laeverin is expressed in the cytoplasm rather than the cell membrane of preeclamptic placentas.

OBJECTIVE

To study differences in size and integrity of laeverin protein expressed in preeclamptic and normal placentas, and to investigate sub-cellular localization of laeverin, in the trophoblasts.

METHODS

Proteins from placental tissue of three severely preeclamptic women and three healthy controls were extracted using Magnabeads in MagNaLyser in T-PER solution. Western blot analysis was done by SDS-polyacrylamide gel electrophoresis and electro-blotting on PVDP membranes. Membranes were developed by Tropix CDP-Star and immuno-reactive bands were visualised. Immuno-electronmicroscopy was performed on high pressure freezed (Tokyasu method) tissue samples of three placentas (from1 healthy and 2 severely preeclamptic women). Ultrathin sections were fixed and labeled with primary antibody raised against laeverin, followed by antibody conjugated with 5nm gold particles (PAG5). Experiments were performed in triplicates and images were taken using a JEM-1010 transmission electron microscope at 50,000 and 70,000 magnifications.

RESULTS

Western blot analysis detected laeverin-protein of normal size (approximately 100kDa) both in normal and preeclamptic placentas. However, laeverin was overexpressed in preeclamptic placentas compared to healthy controls. Immuno-electronmicroscopy revealed presence of laeverin within the capillaries in preeclamptic placentas which was not seen in healthy controls. At a sub-cellular level laeverin was localized on the cell membrane of trophoblasts in healthy placentas. However, in preeclamptic placentas, laeverin was localized in the cytoplasm and in particular in the Golgi apparatus.

CONCLUSIONS

In preeclamptic placentas, laeverin is overexpressed and it appears to leak into the villous capillaries and localize in the cytoplasm instead of cell membrane of the trophoblasts.