Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury

Abstract

It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma. These results indicate that following TBI, the cerebral endothelium undergoes vascular remodeling through shedding of eMVs containing TJPs and endothelial markers. The detection of this shedding potentially allows for a novel methodology for real-time monitoring of cerebral vascular health (remodeling), BBB status and neuroinflammation following a TBI event.

Appearance of claudin-5+ leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles

“…Also, lending support are reports by Takahashi et al [40] and Haqqani et al [39] that cultures of human aortic endothelial cells and human BMEC, respectively, release EVs containing junctional proteins. Most recently, Andrews et al [41] described the release from cultured human BMECs of EVs containing the TJ protein occludin, following application of mechanical trauma to these cells, and additionally noted EVs bearing this protein were elevated in blood plasma of mice subject to traumatic brain injury (TBI). Further paralleling our results for CLN-5 + vesicles, these authors observed that occludin + EVs stemming from trauma were of both exosome and microvesicle size.…”

“…As mechanical trauma to the CNS microvasculature is also associated with endothelial activation, immune reaction, and neuroinflammation [77–79], BMEC release of EVs carrying TJ proteins might be a general phenomenon linked to CNS leukocyte infiltration. Notably, Andrews et al [41] also found the percent of occludin + EVs in the blood of TBI mice to be comparatively minor, e.g., <6% of total EVs, compared to ~1% of CLN-5 + EVs detected in mice with early-stage EAE. The slight discrepancies in the observed percentages could be due to differences in the experimental conditions employed, with TBI possibly being more disruptive to the vasculature.…”

“…The prospect of EVs acting as vehicles to transfer junctional proteins to leukocytes is particularly alluring for several reasons. EVs from varied sources have been reported to contain several junctional proteins [36–40] and mechanical injury to cultured brain microvascular endothelial cells (BMEC) found to stimulate release of EVs harboring the TJ protein occludin [41]. Moreover, several reports reinforce the idea that varied leukocyte subtypes are physiological targets of endothelial EVs.…”

Wheel running reduces ethanol seeking by increasing neuronal activation and reducing oligodendroglial/neuroinflammatory factors in the medial prefrontal cortex

“…In this context, platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is known to produce neuroinflammation and subsequent blood brain barrier disruption, and accumulating evidence suggests that it could be used as a potential marker for neurological disorders (Losy et al, 1999, Zaremba and Losy, 2002, Hwang et al, 2005, Kalinowska and Losy, 2006, Woodfin et al, 2007, Privratsky et al, 2010, Cheung et al, 2015, Andrews et al, 2016). However, it is not known whether withdrawal from chronic ethanol experience regulates PECAM-1 and if the alterations in PECAM-1 correlate with withdrawal induced increases in oligodendroglia and myelinating glia.…”

Traumatic Penumbra: Opportunities for Neuroprotective and Neurorestorative Processes

“…That is, MSCs-derived exosomes that carry and transfer their cargo such as miRNAs to parenchymal cells may mediate brain plasticity and improve functional recovery after TBI [204]. Furthermore, another potential application of brain endothelial-derived eMVs could be as biosignatures for monitoring the health of the BBB in CNS conditions associated with trauma and neuroinflammation [239].…”