Abstract P523: Circulating Endothelial-Cell Derived Microvesicles Associated With Insufficient Sleep Impairs Cerebral Microvascular Fibrinolytic Capacity

Abstract

Chronic insufficient sleep (< 7 hour/night) is associated with increased risk of ischemic stroke. Endothelial cell dysfunction, specifically impairment endothelial release of tissue-type plasminogen activator (t-PA), represents an important mechanism underlying cerebrovascular events such as ischemic stroke. Endothelial cells are the primary site of synthesis and release of t-PA, the plasminogen activator in fibrinolysis and, thus, a key regulator of endogenous thrombolysis. We have previously demonstrated that insufficient sleep is associated with impaired endothelial capacity to release t-PA, however the mechanisms for the sleep-related fibrinolytic impairment are not well understood. Clinical interest in endothelial cell-derived microvesicles (EMVs) has intensified due to their involvement in the development of endothelial dysfunction and, in turn, cardiovascular and cerebrovascular events. The aim of this study was to determine, in vitro, the effects of circulating EMVs isolated from adults with chronic insufficient sleep on brain endothelial cell t-PA release. Considering EMVs are known to cross the blood brain barrier and are associated with ischemic stroke incidence, severity and outcome; circulating EMVs associated with insufficient sleep may be a mediator of stroke risk. Twenty-four healthy, non-obese, normotensive, sedentary adults (age: 23-69 years) were studied: 12 normal nightly sleep duration (7M/5F; age: 47±5 yr; nightly sleep: 7.4±0.1 hour/night) and 12 with short nightly sleep duration (9M/3F; 53±4 yr; 5.9±0.2 hour/night). EMV identification (CD144+) and isolation from peripheral blood was performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Intracellular expression of t-PA was significantly lower (~30%) in hCMECs treated with EMVs from adults with insufficient sleep (31±3 AU) compared with cells treated with EMVs from normal sleep adults (40±3 AU). Although the effect of EMVs on basal t-PA release was not different between the groups; thrombin-stimulated t-PA release was significantly lower in cells treated with EMVs from adults with insufficient sleep (from 57±3 to 59±4 ng/mL) compared with EMVs from adults with normal sleep (from 53±3 to 66±4 ng/mL). The capacity of hCMECs to release t-PA in response to thrombin was ~75% lower when treated with EMVs from adults with insufficient sleep. These results indicate that circulating EMVs associated with chronic insufficient sleep diminishes brain endothelial cell t-PA production and impairs t-PA release. Circulating EMVs represent a mechanistic factor in insufficient sleep-related fibrinolytic dysfunction and ischemic stroke risk.