Roles of Ceramide Glucosyltransferase in Controlling Cerebral Microvascular Endothelial Integrity and Angiogenesis Post-Ischemia/Reperfusion

Sphingolipids critically regulate microvascular integrity and function, but the role of glycosphingolipids in endothelial survival and angiogenesis remains poorly defined. Herein, we experimentally deactivated or activated UDP-glucose ceramide glucosyltransferase (UGCG), which converts ceramide to glucosylceramide, by the pharmacological inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), siRNA-mediated knockdown or the pharmacological activator L-PDMP. Sphingolipid and glycosphingolipid profiles were examined by liquid chromatography-tandem-mass spectrometry. Effects on endothelial survival, proliferation, transwell migration, extracellular vesicle (EV) release and tube formation were assessed in human cerebral microvascular endothelial cells (hCMEC/D3). In vitro, pharmacological UGCG deactivation near-completely suppressed hexosylceramide levels and at high dose increased ceramide and sphingosine-1-phosphate (S1P), a known pro-angiogenic sphingolipid, while UGCG knockdown moderately decreased mostly short (C16, C18) hexosylceramides. UGCG activation increased hexosylceramide without significantly altering ceramide and S1P. Pharmacological UGCG deactivation increased endothelial tube formation, a marker of angiogenesis, but at high dose decreased endothelial survival, whereas UGCG knockdown and UGCG activation reduced endothelial tube formation and migration or proliferation, respectively. Pharmacological UGCG deactivation and activation, but not UGCG knockdown increased endothelial release of EVs with anti-angiogenic activity. In mice exposed to transient middle cerebral artery occlusion, pharmacological UGCG deactivation and activation reduced the length and branch density of small-sized (< 4 µm) and intermediate (4-5.4 µm) cerebral microvessels in the reperfused striatum as revealed by 3D light-sheet microscopy, indicative of microvascular endothelial degeneration. Our results suggest that pharmacological UGCG deactivation promotes angiogenesis in vitro probably via S1P elevation. In vivo, UGCG deactivation failed to stabilize microvascular network integrity post-ischemia/reperfusion, presumably due to ceramide-associated cell stress.