By cleaving sphingomyelin into ceramide, which is an essential component of plasma membrane microdomains, acid sphingomyelinase (Asm) pivotally controls cell signaling. To define how the activation of the Asm/ceramide pathway, which occurs within seconds to minutes upon stress stimuli, influences brain ischemia/reperfusion (I/R) injury, we exposed male and female wildtype mice carrying both alleles of Asm’s gene sphingomyelinase phosphodiesterase-1 (Smpd1+/+), heterozygously Asm-deficient mice (Smpd1+/−) and homozygously Asm-deficient mice (Smpd1−/−) of different age (8, 12 or 16 weeks) to 30, 60 or 90 min intraluminal middle cerebral artery occlusion (MCAO). For studying the contribution of brain-invading polymorphonuclear neutrophils (PMN) to I/R injury, PMNs were depleted by delivery of a PMN-specific Ly6G antibody. In male and female mice exposed to 30 min, but not 60 or 90 min MCAO, homozygous Smpd1−/− consistently increased I/R injury, blood–brain barrier permeability and brain leukocyte and PMN infiltration, whereas heterozygous Smpd1+/− reduced I/R injury. Increased abundance of the intercellular leukocyte adhesion molecule ICAM-1 was noted on cerebral microvessels of Smpd1−/− mice. PMN depletion by anti-Ly6G delivery prevented the exacerbation of I/R injury in Smpd1−/− compared with wildtype mice and reduced brain leukocyte infiltrates. Our results show that Asm tempers leukocyte entry into the reperfused ischemic brain, thereby attenuating I/R injury.

Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen–glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]−/−) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood–brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified.

Functional inhibitors of acid sphingomyelinase are clinically used as anti-depressants since ~60 years. Here, we show that acid sphingomyelinase inhibition by the antidepressants amitriptyline, fluoxetine and desipramine protects from ischemia/reperfusion and elicits a profound brain remodeling response with increased angiogenesis, improved blood-brain barrier integrity, reduced brain leukocyte infiltration and increased neuronal survival. Angiogenesis is promoted by small extracellular vesicles with bona fide characteristics of exosomes, which are released from endothelial cells and which constitute an elegant target for the amplification of stroke recovery.