Pharmacological UGCG deactivation promotes angiogenesis in vitro probably via S1P elevation, but in vivo UGCG deactivation failed to stabilize microvascular network integrity post-ischemia/reperfusion, presumably due to ceramide-associated cell stress.
Key Findings
Results
Pharmacological UGCG deactivation using D-PDMP near-completely suppressed hexosylceramide levels in human cerebral microvascular endothelial cells.
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) was used as the pharmacological inhibitor of UDP-glucose ceramide glucosyltransferase (UGCG).
Sphingolipid and glycosphingolipid profiles were examined by liquid chromatography-tandem-mass spectrometry.
At high dose, D-PDMP increased ceramide and sphingosine-1-phosphate (S1P), a known pro-angiogenic sphingolipid.
UGCG knockdown via siRNA only moderately decreased mostly short (C16, C18) hexosylceramides, in contrast to the near-complete suppression achieved pharmacologically.
Results
UGCG activation using L-PDMP increased hexosylceramide levels without significantly altering ceramide and S1P.
L-PDMP was used as the pharmacological activator of UGCG.
Hexosylceramide levels increased following L-PDMP treatment.
Ceramide and sphingosine-1-phosphate levels were not significantly altered by UGCG activation.
This lipid profile differed substantially from the profile produced by pharmacological UGCG deactivation.
Results
Pharmacological UGCG deactivation increased endothelial tube formation but at high dose decreased endothelial survival.
Endothelial tube formation is described as a marker of angiogenesis.
Effects were assessed in human cerebral microvascular endothelial cells (hCMEC/D3).
High-dose D-PDMP decreased endothelial survival, consistent with ceramide-associated cell stress.
The pro-angiogenic effect was attributed to elevated S1P levels resulting from UGCG inhibition.
Results
UGCG knockdown reduced endothelial tube formation and migration, while UGCG activation reduced proliferation.
siRNA-mediated UGCG knockdown reduced both endothelial tube formation and transwell migration.
Effects on tube formation, transwell migration, and proliferation were assessed in hCMEC/D3 cells.
These findings indicate that both loss and gain of UGCG function can impair distinct aspects of endothelial angiogenic behavior.
Results
Pharmacological UGCG deactivation and activation, but not UGCG knockdown, increased endothelial release of extracellular vesicles with anti-angiogenic activity.
Extracellular vesicle (EV) release was assessed in hCMEC/D3 cells.
Both D-PDMP and L-PDMP treatments increased EV release.
siRNA-mediated UGCG knockdown did not increase EV release.
The EVs released had anti-angiogenic activity, representing a potential mechanism by which UGCG modulation affects angiogenesis.
Results
In mice subjected to transient middle cerebral artery occlusion, both pharmacological UGCG deactivation and activation reduced the length and branch density of small-sized and intermediate cerebral microvessels in the reperfused striatum.
3D light-sheet microscopy was used to assess cerebral microvessel architecture in vivo.
Reductions were observed in small-sized (<4 µm) and intermediate (4–5.4 µm) cerebral microvessels.
Both vessel length and branch density were reduced in the reperfused striatum.
These changes were interpreted as indicative of microvascular endothelial degeneration.
The in vivo model used was transient middle cerebral artery occlusion (ischemia/reperfusion) in mice.
Discussion
Pharmacological UGCG deactivation failed to stabilize microvascular network integrity post-ischemia/reperfusion in vivo, presumably due to ceramide-associated cell stress.
Despite promoting angiogenesis in vitro, D-PDMP did not protect or restore the microvascular network after ischemia/reperfusion in vivo.
High-dose UGCG inhibition increased ceramide levels, which are associated with cell stress and reduced endothelial survival.
Authors attribute the in vivo failure to ceramide-associated cell stress rather than insufficient angiogenic signaling.
This discrepancy between in vitro and in vivo findings highlights the complexity of sphingolipid signaling in the ischemic brain.
Background
Glycosphingolipids, specifically hexosylceramides including glucosylceramide, play a role in regulating endothelial survival, migration, proliferation, and angiogenic behavior in cerebral microvascular endothelial cells.
UGCG converts ceramide to glucosylceramide, the first step in glycosphingolipid synthesis.
Both deactivation and activation of UGCG produced measurable effects on multiple endothelial functions.
The study used hCMEC/D3 cells as the primary in vitro model for human cerebral microvascular endothelial cells.
Glycosphingolipid profiles were quantified by liquid chromatography-tandem-mass spectrometry.
What This Means
This research suggests that an enzyme called UGCG, which controls the production of certain fat molecules (glycosphingolipids) in brain blood vessel cells, plays an important role in how these vessels survive and grow after a stroke. The researchers tested what happens when they block or boost this enzyme in both laboratory-grown human brain blood vessel cells and in mice that had a stroke. When they blocked the enzyme using a drug called D-PDMP, the blood vessel cells showed signs of increased new vessel formation (angiogenesis) in the lab, likely because blocking the enzyme caused a buildup of another fat molecule called sphingosine-1-phosphate (S1P), which promotes vessel growth. However, high doses of the drug also caused cell stress and death by increasing ceramide levels, a different fat molecule known to be harmful to cells.
In the stroke mouse model, both blocking and boosting the UGCG enzyme led to reduced length and branching of small blood vessels in the damaged brain area, suggesting that the blood vessel network actually deteriorated rather than recovered. The researchers propose that while blocking UGCG can encourage new vessel growth in a controlled lab setting, the harmful ceramide buildup from high-dose drug treatment likely overwhelms any beneficial effects in a living organism dealing with the additional stress of stroke and reperfusion injury.
This research suggests that manipulating glycosphingolipid metabolism to treat stroke-related blood vessel damage is more complicated than it might seem in laboratory conditions. Simply blocking or activating UGCG does not straightforwardly protect or restore the brain's tiny blood vessels after a stroke, and any future therapeutic strategies targeting this pathway would need to carefully balance these competing effects on ceramide, S1P, and glycosphingolipid levels.
Mohamud Yusuf A, Zafar M, Hagemann N, Brockmeier U, Schumacher F, Kleuser B, et al.. (2026). Roles of Ceramide Glucosyltransferase in Controlling Cerebral Microvascular Endothelial Integrity and Angiogenesis Post-Ischemia/Reperfusion.. Translational stroke research. https://doi.org/10.1007/s12975-026-01488-9