Endothelial type I interferon signaling modulates the vascular response to ischemic brain injury.

Vascular normalization and restoration of blood-brain barrier (BBB) function are critical to reestablish a microenvironment that supports tissue repair and functional recovery after ischemic stroke. During development, central nervous system (CNS) angiogenesis and barriergenesis are coupled via canonical Wnt/β-catenin signaling. Following ischemic insult, brain endothelial cells (BECs) upregulate Wnt signaling on a background of acute inflammation. How distinct immune signaling pathways cooperate with developmental pathways to differentially regulate and temporally coordinate post-stroke vascular remodeling remains poorly understood. Here, we identify a unique BEC type I interferon (IFNI) signature in human and mouse ischemic stroke tissue using immunohistochemistry and RNA in situ hybridization. By leveraging two clinically relevant murine ischemic stroke models, single-cell transcriptomic technology, and Biocytin-TMR tracer leakage assays, we identify that genetic deletion of endothelial IFNI receptor (IFNAR1) exacerbates post-stroke barrier disruption and results in an expansion of BECs characterized by marker genes associated with angiogenesis and immature BBB. Conversely, acute administration of exogenous IFNI ameliorates post-stroke BBB disruption. Functional in vitro assays (trans-endothelial electrical resistance / wound healing) and western blotting reveal that BEC IFNI signaling modulates VE-Cadherin stabilization and vascular endothelial growth factor (VEGF) signaling to enhance BEC barrier properties and suppress angiogenic features, respectively. Together, our work supports that endogenous BEC IFNI signaling regulates acute BEC function after ischemic brain injury through the 1) restriction of angiogenesis onset and/or 2) augmentation of vascular maturation to promote barrier function. Moreover, these findings may have broader implications for understanding CNS vascular pathology and normalization in the setting of brain states characterized by acute (i.e. stroke, traumatic brain injury) or chronic (i.e. multiple sclerosis, neurodegeneration, aging) sterile inflammation.

Testing of a hypothesis for osmotic opening of the blood-brain barrier.

Background and purpose: Our previous study found adoptively transferred regulatory T cell (Treg) protected against ischemic stroke via attenuating blood brain barrier (BBB) disruption. Here, we examined the role of CCR5, a chemokine receptor in the Tregs’ recruitment and protection to the ischemic BBB after stroke. Methods: Transient focal cerebral ischemia was induced in widetype C57/BL6 or CCR5(-/-) mice by unilateral middle cerebral artery occlusion (MCAO) for 60 minutes. Tregs (2x10 6 /mouse) in 200 μl PBS or PBS control were injected intravenously at 2 hours after MCAO. Chemo-attractant migration of Tregs to the ischemic BBB was examined by two photon in vivo imaging and cell migration test in vitro. RT-PCR, Immunofluorescence staining and confocal microscopy were used to assess the chemokine expression, endothelial ICAM-1 expression and peripheral immune cell infiltration following cerebral ischemic injury. In vivo endogenous IgG leakage and in vitro BBB integrity were examined to determine the important role of CCR5 in Tregs’ protection on the ischemic BBB. Results: We found that cerebral ischemia reduced Treg number in the peripheral but the expression of CCR5 on Tregs in the blood was increased. In the ischemic brain, the expression of CCR5 ligands were significantly upregulated, and the expression of CCL5 co-localized with ischemic brain endothelial cells. Genetic depletion of CCR5 on the donor Tregs significantly impaired the transferred Tregs’ ability to migrate to the ischemic BBB and attenuated Tregs’ protection against BBB disruption and peripheral immune cell infiltration as determined by two photon in vivo imaging, immunofluorescence staining and MMP-9 ELISA. In vitro migration test further revealed that depletion of CCR5 hindered Tregs to migrate across endothelial cell layer. Conclusions: In conclusion, CCR5 plays an indispensible role in Tregs’ recruitment and protection against ischemic BBB disruption. Therapeutic strategies targeting CCR5 warrants further investigation to improve Treg based cell therapy in stroke treatment.

During ischemic stroke, the integrity of blood-brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged amplifying ischemic neuronal damage. There have been attempts to identify the exact mechanism ischemic BBB disruption to minimize brain damage under ischemic stroke. Autophagy is catabolic process which involves degradation and recycling of damaged or unnecessary organelles. However, excessive autophagy can induce cell damage and death under pathological conditions such as ischemia. In this study, we evaluated if autophagy is a key mechanism of BBB dysfunction under ischemic stroke. In vitro BBB model of bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), an ischemic mimic condition. After exposure to OGD for 18 hours, cell viability was significantly decreased and cellular permeability was impaired. The conversion of LC3-I to LC3-II and puncta of LC3 in bEnd.3 were increased, demonstrating that autophagy is induced under ischemic condition. Modulation of autophagy by 3-methyladenine, an autophagy inhibitor, reversed the conversion of LC3 as well as decreased cell viability, suggesting that autophagy involves in ischemic BBB damage. The level of occludin, a tight junction protein in BBB, was decreased after OGD, and this was reversed by inhibition of autophagy. Our findings showed that induction of autophagy might contribute to increased permeability through occludin degradation in brain endothelial cells under ischemia, providing a new mechanism of BBB disruption in ischemic stroke.

AimsIschemic stroke is a life‐threatening disease with limited therapeutic strategies. Blood‐brain barrier (BBB) disruption is a critical pathological process that contributes to poor outcomes in ischemic stroke. We previously showed that the microglial inhibition of the inflammasome sensor absent in melanoma 2 (AIM2) suppressed the inflammatory response and protected against ischemic stroke. However, whether AIM2 is involved in BBB disruption during cerebral ischemia is unknown.MethodsMiddle cerebral artery occlusion (MCAO) and oxygen‐glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice and brain microvascular endothelial cells (HBMECs), respectively. The infarct volume, neurological deficits, and BBB permeability were measured in mice after MCAO. Transendothelial electrical resistance (TEER) and neutrophil adhesion to the HBMEC monolayer were assessed after OGD/R treatment. Western blot and immunofluorescence analyses were conducted to evaluate the expression of related proteins.ResultsAIM2 was shown to be expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. AIM2 deletion reduced the infarct volume, improved neurological and motor functions, and decreased BBB disruption. In vitro, OGD/R significantly increased the protein levels of AIM2 and ICAM‐1 and decreased those of the tight junction (TJ) proteins ZO‐1 and occludin. AIM2 knockdown effectively protected BBB integrity by promoting the expression of TJ proteins and decreasing ICAM‐1 expression and neutrophil adhesion. Mechanistically, AIM2 knockdown reversed the OGD/R‐induced increases in ICAM‐1 expression and STAT3 phosphorylation in brain endothelial cells. Furthermore, treatment with the p‐STAT3 inhibitor AG490 mitigated the effect of AIM2 on BBB breakdown.ConclusionOur findings indicated that inhibiting AIM2 preserved the BBB integrity after ischemic stroke, at least partially by modulating STAT3 activation and that AIM2 may be a promising therapeutic target for cerebral ischemic stroke.

Preeclampsia and Cerebrovascular Disease.

Interferon-β alleviates delayed tPA-induced adverse effects via modulation of MMP3/9 production in ischemic stroke

Diabetes mellitus (DM) is one of the most common diseases in the world. Among its effects are an increase in the risk of cognitive impairment, including Alzheimer's disease, and blood-brain barrier (BBB) dysfunction. DM is characterized by high blood glucose levels that are caused by either lack of insulin (Type I) or resistance to the actions of insulin (Type II). The phenotypes of these two types are dramatically different, with Type I animals being thin, with low levels of leptin as well as insulin, whereas Type II animals are often obese with high levels of both leptin and insulin. The best characterized change in BBB dysfunction is that of disruption. The brain regions that are disrupted, however, vary between Type I vs Type II DM, suggesting that factors other than hyperglycemia, perhaps hormonal factors such as leptin and insulin, play a regionally diverse role in BBB vulnerability or protection. Some BBB transporters are also altered in DM, including P-glycoprotein, lowdensity lipoprotein receptor-related protein 1, and the insulin transporter as other functions of the BBB, such as brain endothelial cell (BEC) expression of matrix metalloproteinases (MMPs) and immune cell trafficking. Pericyte loss secondary to the increased oxidative stress of processing excess glucose through the Krebs cycle is one mechanism that has shown to result in BBB disruption. Vascular endothelial growth factor (VEGF) induced by advanced glycation endproducts can increase the production of matrix metalloproteinases, which in turn affects tight junction proteins, providing another mechanism for BBB disruption as well as effects on P-glycoprotein. Through the enhanced expression of the redox-related mitochondrial transporter ABCB10, redox-sensitive transcription factor NF-E2 related factor-2 (Nrf2) inhibits BEC-monocyte adhesion. Several potential therapies, in addition to those of restoring euglycemia, can prevent some aspects of BBB dysfunction. Carbonic anhydrase inhibition decreases glucose metabolism and so reduces oxidative stress, preserving pericytes and blocking or reversing BBB disruption. Statins or N-acetylcysteine can reverse the BBB opening in some models of DM, fibroblast growth factor-21 improves BBB permeability through an Nrf2-dependent pathway, and nifedipine or VEGF improves memory in DM models. In summary, DM alters various aspects of BBB function through a number of mechanisms. A variety of treatments based on those mechanisms, as well as restoration of euglycemia, may be able to restore BBB functions., including reversal of BBB disruption.

Ischaemic stroke is a leading cause of death and disability. Circulating extracellular vesicles (EVs) post‐stroke may help brain endothelial cells (BECs) counter ischaemic injury. However data on how EVs from ischaemic stroke patients, considering injury severity, affect these cells are limited. The aims were to characterize the inflammatory and angiogenic components of circulating EVs in acute ischaemic stroke patients, considering stroke severity, and to investigate whether these circulating EVs differentially influence the proangiogenic properties and blood–brain barrier (BBB) integrity of human BECs. Eighteen ischaemic stroke patients (acute phase: 24–48 h) and nine controls matched by age, sex, and blood pressure were studied. Stroke severity was classified as severe (n = 9) or mild (n = 9). Plasma EVs were analysed for size, concentration, and protein markers (CD63, Alix, CD81, TSG101, HSP70), as well as proinflammatory and angiogenic proteins. EV uptake, cell viability, proangiogenic capacity, electrical resistance [TEER (transendothelial electrical resistance)], and dextran‐70 kD permeability were assessed using human brain microvascular endothelial cells (hCMEC/D3). Stroke patients had lower EV concentrations than controls (p = 0.075), with mild‐stroke patients having the smallest EVs. Stroke‐derived EVs had higher levels of interleukin 6 (IL‐6), tumour necrosis factor α (TNF‐α), nitrotyrosine, and vascular endothelial growth factor (VEGF) but lower placental growth factor (PLGF) compared to controls. IL‐6 was higher in mild strokes (p = 0.0025), and VEGF was higher in severe strokes (p = 0.048). EVs from severe‐stroke cases enhanced proangiogenic capacity and minimally disrupted the BBB. Stroke severity influences EV number, size, and composition. EVs from severe strokes may promote BBB restoration and cerebral angiogenesis, suggesting their role in intercellular communication and homeostasis in ischaemic tissue. imageKey points Ischaemic stroke is one of the leading causes of death worldwide. After an ischaemic stroke several physiological processes are triggered to recover the injured tissue. Increasing evidence has suggested that extracellular vesicles (EVs) present in the bloodstream could play a role in brain recovery, but their specific impact, especially concerning stroke severity, was unclear. This study demonstrates that plasma‐derived EVs from first‐ever ischaemic stroke patients have distinctive characteristics and effects over brain angiogenesis and blood–brain barrier (BBB) integrity. Our study proposes that circulating EVs from patients with severe stroke may carry protective factors to initiate brain endothelial cell recovery after acute episodes. These findings underscore the role of EVs as potential effectors of BBB recovery and biomarkers in severe ischaemic stroke.

Background: Blood Brain Barrier (BBB) disruption is central to vasogenic edema development after ischemia in preclinical studies. We investigated the relationship between BBB disruption and cerebral edema in patients receiving reperfusion therapies for anterior circulation large vessel occlusion. Methods: In a post-hoc pooled analysis of the Tenecteplase versus Alteplase before Endovascular Therapy for Ischemic Stroke (EXTEND-IA TNK) part 1 and 2 multicenter RCTs, the DWI lesion on 24h post-treatment MRI and peri-infarct salvaged penumbra derived from pre-treatment CT-Perfusion (CTP) were segmented and registered to 24h MR Perfusion. A validated permeability measure (percentage of measured cerebral blood volume lost due to T1 effect from gadolinium leakage across the BBB) was calculated for each ROI. The association between post-treatment BBB disruption in the infarct and cerebral edema assessed on a trichotomized ordinal scale of negligible (<1mm midline shift), mild (≥1 to <5mm) or severe (≥5mm) was analyzed using ordinal logistic regression. Results: Of 220 patients analyzed, median (IQR) BBB disruption was 2.36(1.4-4.1)% in the infarct, 1.61(1.0-2.6)% in salvaged penumbra and 0.98(0.7-1.5)% in normal brain (p<0.001). There were 119 (54.1%) patients with negligible, 90 (40.8%) mild and 11 (5.0%) severe cerebral edema at 24h. In multivariable analysis, infarct BBB disruption was associated with increased cerebral edema (cOR=1.11 per%, 95%CI 1.02-1.21;p=0.012, adjusted for age, admission NIHSS and reperfusion (mTICI2b/3 or >50% reperfusion on early CTP 2h post-lysis). Infarct BBB disruption correlated with follow-up infarct volume (rho=0.37;p<0.001), and was associated with parenchymal hematoma (aOR=1.18, 95%CI 1.00-1.40;p=0.048) and worse outcome assessed on 90-day modified Rankin Scale (cOR=1.16 per%, 95%CI 1.08-1.26;p<0.001, adjusted for age, admission NIHSS and reperfusion). Conclusions: BBB disruption after reperfusion treatment extends beyond the infarct lesion, and is associated with cerebral edema development, hemorrhagic transformation and poor outcome. Further studies to evaluate BBB integrity as an imaging biomarker and potential therapeutic target in malignant cerebral edema after ischemic stroke are needed.

The physiopathology of life-threatening cerebrovascular complications in preeclampsia is unknown. We investigated whether disruption of the blood-brain barrier, generated using circulating small extracellular vesicles (sEVs) from women with preeclampsia or placentae cultured under hypoxic conditions, impairs the expression of tight junction proteins, such as CLDN5 (claudin-5), mediated by VEGF (vascular endothelial growth factor), and activation of KDR (VEGFR2 [VEGF receptor 2]). We perform a preclinical mechanistic study using sEVs isolated from plasma of pregnant women with normal pregnancy (sEVs-NP; n=9), sEVs isolated from plasma of women with preeclampsia (sEVs-PE; n=9), or sEVs isolated from placentas cultured in normoxia (sEVs-Nor; n=10) or sEVs isolated from placentas cultured in hypoxia (sEVs-Hyp; n=10). The integrity of the blood-brain barrier was evaluated using in vitro (human [hCMEC/D3] and mouse [BEND/3 (brain endothelial cell 3)] brain endothelial cell lines) and in vivo (nonpregnant C57BL/6J mice [4-5 months old; n=13] injected with sEVs-Hyp) models. sEVs-PE and sEVs-Hyp reduced total and membrane-associated protein CLDN5 levels (P<0.05). These results were negated with sEVs-PE sonication. sEVs-Hyp injected into nonpregnant mice generated neurological deficits and blood-brain barrier disruption, specifically in the posterior area of the brain, associated with brain endothelial cell uptake of sEVs, sEVs-Hyp high extravasation, and reduction in CLDN5 levels in the brain cortex. Furthermore, sEVs-PE and sEVs-sHyp had higher VEGF levels than sEVs-NP and sEVs-Nor. Human brain endothelial cells exposed to sEVs-PE exhibited a reduction in the activation of KDR. Reduction in CLDN5 observed in cells treated with sEVs-Hyp was further enhanced in cells treated with KDR selective inhibitor. sEVs-PE disrupts the blood-brain barrier, an effect replicated with sEVs-Hyp, and involves reduced CLDN5 and elevated VEGF contained within these vesicles. However, our results do not support the participation of KDR activation in the downregulation of CLDN5 observed with sEVs-Hyp. These findings will improve our understanding of the pathophysiology of cerebrovascular alterations in women with preeclampsia.

The Protective Mechanisms of Intravenous Immunoglobulin (IVIg) in Ischaemic Stroke

Restoration of blood-brain barrier (BBB) dysfunction, which drives worse outcomes of ischemic stroke, is a potential target for therapeutic opportunities, whereas a sealed BBB blocks the therapeutics entrance into the brain, making the BBB protection strategy paradoxical. Post ischemic stroke, hypoxia/hypoglycemia provokes the up-regulation of transmembrane glucose transporters and iron transporters due to multiple metabolic disorders, especially in brain endothelial cells. Herein, we develop a myricetin oligomer-derived nanostructure doped with Ce to bypass the BBB which is cointermediated by glucose transporters and iron transporters such as glucose transporters 1 (GLUT1), sodium/glucose cotransporters 1 (SGLT1), and transferrin(Tf) reporter (TfR). Moreover, it exhibits BBB restoration capacity by regulating the expression of tight junctions (TJs) through the activation of protective autophagy. The myricetin oligomers scaffold not only acts as targeting moiety but is the prominent active entity that inherits all diverse pharmacological activities of myricetin. The suppression of oxidative damage, M1 microglia activation, and inflammatory factors makes it a multitasking nanoagent with a single component as the scaffold, targeting domain and curative components.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.

Blood brain barrier (BBB) disruption is an integral feature of numerous neurological diseases with infectious, inflammatory, neoplastic and vascular components. It is of particular interest in the immune mediated neurological diseases multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and acute hemorrhagic leukoencephalitis (AHLE). In particular, AHLE is associated with very high mortality. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. This lack of understanding currently undermines therapeutic approaches to ameliorate uncontrolled BBB disruption. In this review, we highlight the current experimental model systems available to address the contribution of inflammatory cells in BBB disruption. The contribution of T cells and other immune cell subsets in altering blood brain barrier tight junctions in the experimental autoimmune encephalomyelitis (EAE), lipopolysaccharide (LPS), and virus induced CNS vascular permeability model systems is also addressed. Results obtained from the use of these model systems have put forward a defined role for neutrophils, CD4 and CD8 T cells and vascular endothelial growth factor (VEGF) in BBB disruption. Based on these findings, we describe models in which immune cells engage and alter CNS cell types of the neurovascular unit and define future avenues of research. Acute disseminating encephalomyelitis (ADEM) and acute hemorrhagic leukoencephalitis (AHLE) are demyelinating diseases that usually present shortly after infections or vaccination (Callen et al., 2009; Gibbs et al., 2005). ADEM typically has an overall favorable prognosis, with 60-80% of patients showing a complete recovery (Dale et al., 2000; Gibbs et al., 2005; Rust, 2000; Tenembaum et al., 2002). AHLE, which is also known as Hurst’s disease, has been considered a severe form of ADEM and has a very poor prognosis, usually resulting in death in 2-14 days (Geerts et al., 1991; Hart & Earle 1975; Pinto et al., 2011; Posey et al., 1994; Suchowersky et al., 1983). This disease is characterized by extensive BBB disruption and microhemorrhage formation. Typical acute symptoms of AHLE include fever, malaise, and headache, followed by a rapid progression of multifocal neurologic signs