Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

Abstract

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.

Activation of Transcription Factor EB Alleviates Tubular Epithelial Cell Injury via Restoring Lysosomal Homeostasis in Diabetic Nephropathy

“…Ultrathin sections were examined using a Philips CM100 electron microscope (Eindhoven, The Netherlands). Primary lysosomes (electron-dense organelles filled with very tiny granules and usually spheres in three-dimensional shape) and secondary lysosomes (less electron-dense and large heterogeneous more or less spherical organelles) [ 32 ] were identified by two individuals, blinded to the study.…”

Dichotomy in hypoxia-induced mitochondrial fission in placental mesenchymal cells during development and preeclampsia: consequences for trophoblast mitochondrial homeostasis

“…6B ). TEM was employed to visualize and quantify primary and secondary lysosomes as previously described [ 20 ] as well as lysosome-mitochondria interactions in isolated pMSCs. While primary lysosome-to-mitochondria interactions were found in pMSCs from early 1 st trimester placentae, numerous secondary lysosome-to-mitochondria interactions (Fig.…”

“…Specifically, pMSCs isolated from 6 to 8-week placenta and PE placenta were cultured at 3% O 2 , while pMSCs isolated from 10 to 12-week and term placenta were cultured at 8% O 2 . Exosomes were isolated from 24 h conditioned media of term and PE pMSCs as previously reported [ 20 ]. For details on isolation, characterization and uptake of exosomes see Supplementary Methods .…”

“…Characterization of isolated pMSCs from 1st trimester, term and PE placentae was performed using FACS analysis as reported [ 20 ] (details in Supplementary Methods ).…”

“…Cells were grown under standard conditions (ambient air, 5% CO 2 ) in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% (v/v) FBS and 1% (v/v) P/S. JEG3 cells were treated with 25 μM Imipramine (Sigma-Aldrich, St. Louis, USA #I7379) or 10 μM Fluoxetine (Sigma-Aldrich, St. Louis, USA-# F132) for 6–12 h prior to fixation for IF or PLA measurements as previously described [ 20 ]. JEG3 cells seeded onto 6-well plates were exposed to 5–20 μg/mL sphingomyelin 18:0 (SM-18:0 (d18:1/18:0) Sigma-Aldrich, USA, #860586 P) or 90% EtOH vehicle for 6 h prior to immunofluorescence (IF) and western blot (WB) analyses.…”