Novel therapeutic strategies to prevent reperfusion injury progression following ischemic stroke – PIKSIP
Ischemic stroke continues to be one of the most frequent causes of death or disability. The only approved treatment is based on reperfusion therapy with recombinant tissue plasminogen activator (rtPA) either alone or combined with thrombectomy within a therapeutic window of respectively 4h30 and 6h, as beyond this time limit irreversible cerebral lesions develop that are associated with risk of bleeding. Even when complete cerebral arterial recanalization is possible, about 25% of patients develop a progressive cerebral infarction as a result of reperfusion injury. The underlying mechanisms are complex and involve endothelial dysfunction, the appearance of a prothrombotic phenotype and a pronounced inflammatory response that leads to rupture of the blood-brain barrier (BBB), leukocyte infiltration and neuronal death. Considering the increasing global incidence of stroke, new therapeutic options are urgently needed to reduce reperfusion injury progression.
Partner 1’s laboratory has been studying the roles of phosphoinositides (PI) and their metabolizing enzymes for many years. PI3-kinases (PI3Ks) are divided into three classes based upon their structure and regulation. Class II PI3Ks have been the least investigated. Among them, PI3K-C2b is mainly thought to convert PI to PI3P on endosomal membranes and to regulate vesicular trafficking that results in protein degradation or recycling. Although this enzyme has not yet been well-characterized, its described roles in the intracellular signalling of endothelial cells places it as a potentially key target for reducing the effects of reperfusion injury on BBB, and indeed our preliminary results strongly support this hypothesis. If this is true then a better control of this mechanism would have important therapeutic value in the prevention of endothelial injury by preserving junction integrity. Therefore the main objective of this project is to investigate the class II PI3K-C2b isoform as a promising target for reducing the effects of reperfusion injury following ischemic stroke.
We will analyze in detail the degree of reperfusion injury protection conferred by inhibition of PI3K-C2b using two complementary experimental stroke models (with different dynamics of post-ischemic recirculation) on PI3K-C2b kinase-dead knock-in mice. Lesion volume, BBB integrity and cerebrovascular inflammation will be then evaluated by high resolution imaging and MRI. Partner 2 has a strong expertise in the understanding of the mechanisms occurring at the acute phase of ischemic stroke and rtPA. rtPA is the only currently approved treatment, but despite its positive effects when infused early after stroke onset, it has also been shown to have a deleterious effect on the endothelium when delayed. Thus, we will compare the potential role of PI3K-C2b in the processes underlying early versus late administration of rtPA. Because of the complexities of studying the barrier function in vivo, human cerebral microvascular endothelial cell line hCMEC/D3 with PI3K-C2b knocked-down (by shRNA) will be used. We will seek to determine the mechanism by which the inhibition of PI3K-C2b protects the BBB. Does it play a role in the regulation of tight and adherens junctions or in the expression of adhesive proteins (VCAM-1 or P-selectin)? We will study the role of PI3K-C2b in the production of PI3P and its implication in endothelial cell signaling, including endosomal trafficking. Protecting endothelial cells may represent a promising therapeutic approach when combined with the established recanalizing strategies.
Moreover, due to the neuroprotective effect recently described for the PI3Kd-selective drug idelalisib on inflammatory disease and experimental stroke, we will evaluate whether co-targeting these two PI3K isoforms (PI3K-C2b and PI3Kd) could even more prevent reperfusion injury progression. We expect that the simultaneous targeting of immune (PI3Kd) and endothelial (PI3K-C2b) cells will improve protection.
Madame Marie-Pierre GRATACAP (Institut National de la Santé et de la Recherche Médicale)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Inserm UMR 1048 - I2MC Institut National de la Santé et de la Recherche Médicale
SP2U Inserm U919
Help of the ANR 319,364 euros
Beginning and duration of the scientific project: September 2016 - 36 Months