Stroke is a leading cause of death worldwide. Possible treatments that can be administered in the sub-acute phase and stimulate restorative mechanisms by enhancing vascular remodeling are clinically not available. Amitriptyline has been used for treatment of anxiety and depressive disorders for decades. In recent years, amitriptyline and other antidepressants have been demonstrated to functionally inhibit the activity of acid sphingomyelinase (human protein: ASM; murine protein: Asm) which catalyzes the production of the bioactive sphingolipid ceramide. Promising pre-clinical and clinical studies demonstrated that ASM inhibitors induce neuroprotection and ameliorate behavioral outcome after stroke. The underlying mechanism has not been identified.

Herein, we studied the effect of amitriptyline on brain remodeling in a model of transient intraluminal middle cerebral artery occlusion (tMCAO). By means of high-resolution light sheet fluorescence microscopy, we provide evidence that amitriptyline promotes angiogenesis in an Asm/ ceramide-dependent way in previously ischemic, reperfused brain tissue. In cerebral microvascular endothelial cells exposed to ischemia/ reperfusion injury, mimicked by oxygen-glucose deprivation (OGD) and subsequent reoxygenation/ glucose recultivation, ceramide accumulated in intracellular vesicles that were characterized as multivesicular bodies/ late endosomes. These vesicles were released from the cells in response to amitriptyline. Extracellular vesicles (EVs) derived from amitriptyline-treated cells induced angiogenesis and mimicked the effect of direct amitriptyline administration in vitro. In vivo, the promotion of angiogenesis by amitriptyline was ensued by a robust surrogate of successful brain tissue remodeling, that is, increased blood-brain barrier integrity, reduced inflammation and enhanced neuronal survival. These data provide a rationale for the use of ASM inhibitors in the treatment of ischemic stroke.