Endothelial therapy for ischemic stroke. – ETHERISCH

We will use ex vivo modeling of thromboinflammation and microvascular occlusion and in vivo modeling of ischemic stroke to address our objectives. This will be combined with genetic and pharmacological tools to address mechanisms of vascular occlusion and to identify potential drug targets. Various imaging techniques will be employed to visualize temporal aspects of vascular occlusion both ex vivo and in vivo.

Our observations show that after large vessel occlusion, there is rapid onset of microvascular thrombosis in the downstream vascular territory. This is partly offset by near immediate retrograde perfusion through collateral anastomoses with neighboring arterial territories. However, expansion of microvascular thrombosis reduces the area perfused by the collateral vasculature within hours. Ex vivo modeling of human blood flowed over fibrin shows a stepwise process of platelet adhesion, spreading, aggregation, fibrin formation and neutrophil recruitment and activation that culminates in capillary obstruction and the cessation of flow. This process, in particular neutrophil recruitment and activation, is exacerbated by glucose. Diabetes and hyperglycemia also exacerbate brain injury in murine models of ischemic stroke, while vascular reactivity appears to be dispensable for retrograde perfusion and brain injury. Use of specific inhibitors in the ex vivo perfusion chamber model demonstrates essential roles for the platelet GPVI receptor, polyphosphates, the intrinsic cascade of blood coagulation, and glucose transporters in the thromboinflammatory cascade, presenting potential therapeutic targets. Experimental stroke modeling confirms this sequence of events and shows that endothelial cell signaling counteracts the process to preserve microvascular patency and blood flow. Collectively, our results support the notion that thromboinflammation and endothelial cell signaling are promising targets to reduce brain injury from ischemic stroke. However, it will be important to test and validate these mechanisms further in experimental settings before evaluating the potential for clinical translation.

This project has identified key mechanistic stages that drive the propagation of microvascular thrombosis after fibrin is deposited in the microvasculature. Useful inhibitors of these stages have been identified in a flow chamber model. These will be further tested in rodent models of ischemic stroke to assess their potential to reduce infarct volumes without increasing the risk of bleeding. Refinements of the flow chamber model will be conducted to better mimic how microvascular occlusion is initiated in vivo. Combination therapies will be tested with therapeutics that act in parallel and show promise in mono therapy.

Ischemic stroke is a major cause of death and morbidity worldwide and therefore an important public health issue. Current treatment, limited to removal of the large vessel occlusion at origin of ischemia, is offered to a minority of patients and not always effective. Novel adjunct therapies are therefore needed. There is potential promise in therapeutic approaches aimed to optimize microvascular function in the affected brain regions so as to limit the expansion of the core of the ischemic infarct before recanalization and to reduce the risk of malignant edema and hemorrhagic transformation. Pharmacological approaches to this end include the stimulation of endogenous endothelial protective pathways and the prevention of inflammation-induced thrombosis, also known as thromboinflammation. This proposal aims to explore the benefit of endothelial cell stimulation with sphingosine-1 phosphate receptor-1 (S1P1) agonists either alone or in conjunction with inhibition of thromboinflammation in mouse models of ischemic stroke. We hypothesize that these approaches can be safely combined to optimize tissue perfusion and possibly preserve blood-brain barrier function in the ischemic cortex, thus mitigating the impact of large artery occlusion and improving the efficacy of standard therapy. In order to better appreciate the potential benefit of targeting these pathways we will first use genetic, pharmacological, molecular and imaging tools to explore their spatial and temporal engagement, mechanism of action, and inter-play in the ischemic cerebral cortex. We will then attempt combinatorial therapy in mouse models of ischemic stroke and address if S1P1 stimulation either alone or in combination with inhibitors of thromboinflammation can offer benefit in the context of endothelial dysfunction triggered by hyperglycemia in diabetes mellitus, a major risk factor for and disease modifier of ischemic stroke. We expect that this project will inform on the potential benefits of pharmacological stimulation of microvascular perfusion for ischemic stroke and assist the design of future clinical trials in this domain.