Abstract
In ischemic stroke (IS) impairment of the blood–brain barrier (BBB) has an important role in the secondary deterioration of neurological function. BBB disruption is associated with ischemia-induced inflammation, brain edema formation, and hemorrhagic infarct transformation, but the underlying mechanisms are incompletely understood. Dysfunction of endothelial cells (EC) may play a central role in this process. Although neuronal NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome upregulation is an established trigger of inflammation in IS, the contribution of its expression in EC is unclear. We here used brain EC, exposed them to oxygen and glucose deprivation (OGD) in vitro, and analyzed their survival depending on inflammasome inhibition with the NLRP3-specific drug MCC950. During OGD, EC death could significantly be reduced when targeting NLRP3, concomitant with diminished endothelial NLRP3 expression. Furthermore, MCC950 led to reduced levels of Caspase 1 (p20) and activated Gasdermin D as markers for pyroptosis. Moreover, inflammasome inhibition reduced the secretion of pro-inflammatory chemokines, cytokines, and matrix metalloproteinase-9 (MMP9) in EC. In a translational approach, IS was induced in C57Bl/6 mice by 60 mins transient middle cerebral artery occlusion and 23 hours of reperfusion. Stroke volume, functional outcome, the BBB integrity, and—in good agreement with the in vitro results—MMP9 secretion as well as EC survival improved significantly in MCC950-treated mice. In conclusion, our results establish the NLRP3 inflammasome as a critical pathogenic effector of stroke-induced BBB disruption by activating inflammatory signaling cascades and pyroptosis in brain EC.
Highlights
Endothelial cells (EC) play a central role in maintaining blood–brain barrier (BBB) integrity
NLR-family pyrin domaincontaining protein 3 (NLRP3) inhibition leads to a higher endothelial survival after oxygen and glucose deprivation (OGD) Our aim was to characterize the influence of the NLRP3-specific inflammasome-inhibitor MCC950 on EC
It could be clearly shown that a MCC950 concentration of 100 μmol/l leads to the highest bEnd5 survival rate within the OGD setting while higher concentrations seemed to have a toxic and lower concentration an insufficient effect (Fig. 1, Figure S1)
Summary
Endothelial cells (EC) play a central role in maintaining blood–brain barrier (BBB) integrity. There is increasing evidence that inflammatory processes contribute to stroke development and bleeding complications [3–7], but the role of EC within these has been largely neglected. Inflammasomes in general, and the NLR-family pyrin domaincontaining protein 3 (NLRP3) inflammasome in particular, are molecular protein complexes that sense cellular deviation from homeostasis as a danger signal and subsequently initiate inflammatory responses [8]. They are responsible for the activation of Caspase 1 and its downstream pro-inflammatory cytokines interleukin (IL) 1b, IL18, and the pyroptosis-inducing Gasdermin D (GSDMD). NLRP3-induced inflammation plays a detrimental role in systemic endothelial dysfunction (e.g., large vessel disease, arteriosclerosis) [10], but the role of endothelial
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