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Abstract WMP3: Exosomes Derived From Stroke Activated Platelets Impair Cerebral Endothelial Permeability


 Introduction:
 Stroke activated platelets damage the blood brain barrier (BBB). Mechanisms underlying this process remain to be investigated. The present study tested a hypothesis that exosomes, nano size extracellular vesicles, derived from stroke-activated platelets promote BBB leakage.
 
 
 Methods:
 Platelets were harvested from blood samples collected from non-stroke adult and aged male rats and from respective rats subjected to 4h of middle cerebral artery occlusion (MCAO) (n=6/group). Exosomes were then isolated from the platelets by means of differential ultracentrifugation. Primary cerebral endothelial cells harvested from healthy adult rats were treated with platelet exosomes. Endothelial permeability and trans-endothelial electrical resistance (TEER) were assayed. Western blot analysis was performed to measure pro-coagulation and tight junction proteins.
 
 
 Results:
 Platelet exosomes (P-Exos) had an average size from 70 to 82 nm among adult and aged rats and these exosomes contained platelet exosomes markers: CD41, CD63, CD9 and Alix. P-Exos derived from non-ischemic adult and aged rats exhibited TEER at 154±18 and 159 ±16 Ω.cm
 2
 , respectively, and did not increase FITC-dextran leakage. However, P-Exos from ischemic adult rats significantly (p<0.05) decreased TEER (118 ±11 vs 154±18Ω.cm
 2
 in non-ischemia) and increased FITC-dextran leakage by 178%. P-Exos from aged ischemic rats further significantly decreased TEER (98 ±9 Ω.cm
 2
 ) and augmented dextran leakage to 216%. Western blot analysis of endothelial cells showed that P-Exos from ischemic adult rats significantly increased pro-inflammation proteins, ICAM-1, TNF-a and NFkB p65 and decreased tight junction proteins, ZO-1 and occludin, whereas P-Exos from ischemic aged rats further significantly increased and decreased pro-inflammation proteins and junction proteins.
 
 
 Conclusion:
 These data provide new insights into how stroke activated platelets mediate BBB disruption and promote endothelial proinflammatory protein expression via their released exosomes and how aging further negatively impacts cerebral endothelial cells.
