Brain Vascular Endothelial Cells Research Articles

Oxytocin transported from the blood across the blood-brain barrier by receptor for advanced glycation end-products (RAGE) affects brain function related to social behavior

Oxytocin (OT) is a neuropeptide that primarily functions as a hormone controlling female reproductive processes. Since numerous recent studies have shown that single and repetitive administrations of OT increase trust, social interaction, and maternal behaviors in humans and animals, OT is considered a key molecule that regulates social memory and behavior. Furthermore, OT binds to receptors for advanced glycation end-products (RAGE), and it has been demonstrated that loss of RAGE in the brain vascular endothelial cells of mice fails to increase brain OT concentrations following peripheral OT administration. This leads to the hypothesis that RAGE is involved in the direct transport of OT, allowing it access to the brain by transporting it across the blood–brain barrier; however, this hypothesis is only based on limited evidence. Herein, we review the recent results related to this hypothesis, such as the mode of transport of OT in the blood circulation to the brain via different forms of RAGE, including membrane-bound full-length RAGE and soluble RAGE. We further review the modulation of brain function and social behavior, which seem to be mediated by RAGE-dependent OT. Overall, this review mostly confirms that RAGE enables the recruitment of circulating OT to the brain, thereby influencing social behavior. The requirement for further studies considering the physiological aspects of RAGE is also discussed.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Abstract C116: Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Oligomeric tau enters brain vascular endothelial cells and induces cellular senescence and brain microvascular dysfunction in tauopathy

AbstractBackgroundVascular mechanisms of Alzheimer’s disease (AD) may constitute the most therapeutically addressable biological pathway underlying dementia. Soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, particularly in microvascular endothelial cells.MethodsPrimary human brain microvascular endothelial cells (HBEC) were exposed to tau oligomers. Microtubule stability, eNOS activation, and markers of senescence were measured. Mice expressing P301S‐mutant human tau (P301S(PS19) mice), a model of tauopathy, were given immunotherapy to selectively remove tau oligomers, or non‐specific IgGs. After measurements of vascular reactivity, eNOS activity and markers of senescence were measured in microvasculature purified from brain.ResultOligomeric tau significantly decreased microtubule stability and eNOS activity, and induced cellular senescence in primary HBECs. Microtubule destabilization, reduced eNOS activity, and increased markers of cellular senescence in brain microvasculature of P301S(PS19) mice were associated with impaired vascular reactivity, and the latter was negated by immunotherapy that specifically removed oligomeric tau from brain.ConclusionBrain microvascular deficits in P301S(PS19) mice modeling tauopathy, mediated by tau oligomers, may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of tau oligomers. Pathogenic tau oligomers and senescent brain microvascular endothelial cells may thus be targets for intervention in AD and other tauopathies.

Modulation of Heme-Induced Inflammation Using MicroRNA-Loaded Liposomes: Implications for Hemolytic Disorders Such as Malaria and Sickle Cell Disease.

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.

Thyrotropin receptor autoantibody (TRAb) enhance the expression of thyrotropin receptor on mouse brain vascular endothelial cells: in vivo and in vitro.

The possibility that thyrotropin receptor (TSHR) expression in non-thyroid tissue is well-documented. However, there is insufficient data on the expression of TSHR in medulla oblongata regions, particularly when focusing on the background of encephalopathy associated with hyperthyroidism. In this study, we explored the expression of the functional TSHR in Graves' disease (GD) mouse cerebral vascular endothelial cells and the effects of thyrotropin receptor autoantibody (TRAb) on its expression. A mouse model of GD was constructed with an adenovirus overexpressing TSHR289. The location and expression of the TSHR gene and protein in vivo were determined via RT-qPCR, Western blot, and immunofluorescence techniques. The effect of TRAb on the expression of functional TSHR in vitro was investigated using bEnd.3 cells. Our results show that medulla oblongata vascular endothelial cells from GD mice expressed higher levels of TSHR compared to control mice. In an in vitro experiment, novel results demonstrated that after treatment with a monoclonal TSHR-specific agonistic antibody (M22), the expression of TSHR on the bEnd.3 cells increased at both the protein and mRNA levels. Furthermore, compared with bEnd.3 cells were treated with IBMX only, those treated with M22 showed increased cAMP production. This study suggested that TSHR is expressed and functionally active in the mouse medulla oblongata and in vitro-cultured bEnd.3 cells and TRAb (M22) increased the expression of TSHR on bEnd.3 cells.

Natalizumab Treatment Induces Proinflammatory CD4 T Cells Preferentially in the Integrin β7+ Compartment.

Natalizumab, a monoclonal humanized antibody targeting integrin α4, inhibits the transmigration of lymphocytes into the CNS by preventing the interaction of integrin α4β1 with V-CAM expressed on brain vascular endothelial cells. Although natalizumab treatment reduces the clinical relapse rate in patients with relapsing-remitting MS, its discontinuation after reactivation of the JC virus is associated with a rebound of the disease in 20% of patients. The mechanisms of this rebound are not elucidated, but natalizumab increases the frequencies of circulating CD4 T cells expressing proinflammatory cytokines as well as the proportion of circulating Th17/Th1 cells (Th1-like Th17 cells). Gut-derived memory CD4 T cells are a population of growing interest in the pathogenesis of MS, but whether and how their properties are affected by natalizumab is not known. Here, we studied the phenotype and cytokine expression profile of circulating gut-derived memory CD4 T cells in patients with relapsing-remitting MS under natalizumab. We identified gut-derived memory CD4 T cells by their expression of integrin β7 and compared their properties and those of integrin β7- memory CD4 T cells across healthy donors and patients with relapsing-remitting MS treated or not with natalizumab. We also compared the capacity of integrin β7- and integrin β7+ CD4 T-cell subsets to transmigrate in vitro across a model of blood-brain barrier. The proportions of proinflammatory Th17/Th1 cells as well as of IL-17A+IFNγ+ and IL-17A+GM-CSF+ cells were higher in memory CD4 T cells expressing integrin β7 in patients receiving natalizumab compared with healthy donors and patients with relapsing-remitting MS not receiving natalizumab. By contrast, integrin β7 negative memory CD4 T cells only presented a modest increased in their proportion of Th17/Th1 cells under natalizumab. We further observed that integrin β7+ Th17/Th1 cells migrated as efficiently as integrin β7- Th17/Th1 across a monolayer of brain microvascular endothelial cells. Our study shows that circulating integrin β7+ memory CD4 T cells of patients with relapsing-remitting MS under natalizumab are enriched in proinflammatory cells supporting the hypothesis that integrin β7+ memory CD4 T cells could play a pathogenic role in the disease rebound observed at natalizumab discontinuation.

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

Vascular mechanisms of Alzheimer’s disease (AD) may constitute a therapeutically addressable biological pathway underlying dementia. We previously demonstrated that soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, including prominently in microvascular endothelial cells. Here we show that soluble pathogenic tau accumulates in brain microvascular endothelial cells of P301S(PS19) mice modeling tauopathy and drives AD-like brain microvascular deficits. Microvascular impairments in P301S(PS19) mice were partially negated by selective removal of pathogenic soluble tau aggregates from brain. We found that similar to trans-neuronal transmission of pathogenic forms of tau, soluble tau aggregates are internalized by brain microvascular endothelial cells in a heparin-sensitive manner and induce microtubule destabilization, block endothelial nitric oxide synthase (eNOS) activation, and potently induce endothelial cell senescence that was recapitulated in vivo in microvasculature of P301S(PS19) mice. Our studies suggest that soluble pathogenic tau aggregates mediate AD-like brain microvascular deficits in a mouse model of tauopathy, which may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of soluble tau aggregates.

Oleracone F Alleviates Cognitive Impairment and Neuropathology in APPswe/PSEN1dE9 Mice by Reducing the Expression of Vascular Cell Adhesion Molecule and Leukocyte Adhesion to Brain Vascular Endothelial Cells.

Alzheimer's disease (AD) is the most common neurodegenerative disease and the blood-brain barrier dysfunction has been suggested as a key pathological feature of the disease. Our research group successfully established a synthetic protocol for oleracones, a novel series of flavonoids isolated from the plant extract of Portulaca oleracea L. (PO). PO extract was reported to have anti-inflammatory and antioxidant effects, enhancing cognitive function. Thus, we investigated the effects and mechanism of oleracones on cognition using AD model transgenic mice (Tg; APPswe/PSEN1dE9). Oleracone F treatment significantly improved memory dysfunction in Tg mice. Oleracone F decreased the number, burden, and immunoreactivity of amyloid plaques and amyloid precursor protein (APP) protein levels in the brains of Tg mice compared to wild-type mice. Oleracone F also alleviated inflammation observed in Tg mice brains. In vitro studies in human microvascular endothelial cells (HBMVECs) demonstrated that oleracones D, E, and F blocked the elevations in VCAM-1 protein induced by tumor necrosis factor-α (TNF-α), hindering leukocyte adhesion to HBMVECs. Taken together, our results suggest that oleracones ameliorated cognitive impairment by blocking TNF-α-induced increases in VCAM-1, thereby reducing leukocyte infiltration to the brain and modulating brain inflammation.

Fruquintinib/HMPL-013 ameliorates cognitive impairments and pathology in a mouse model of cerebral amyloid angiopathy (CAA)

Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular amyloid-β (Aβ) accumulation, and always accompanied by Alzheimer's disease (AD). The mechanisms revealing CAA pathogenesis are still unclear, and it is challenging to develop an efficient therapeutic strategy for its treatment. Vascular endothelial growth factor (VEGF) and its receptors including VEGFR-1,-2,-3 activation are involved in Aβ processing, and modulate numerous cellular events associated with central nervous system (CNS) diseases. In the present study, we attempted to explore the regulatory function of fruquintinib (also named as HMPL-013), a highly selective inhibitor of VEGFR-1,-2,-3 tyrosine kinases, on CAA progression in Tg-SwDI mice. Here, we found that HMPL-013-rich diet consumption for 12 months significantly improved the behavioral performances and cerebral blood flow (CBF) of Tg-SwDI mice compared with the vehicle group. Importantly, HMPL-013 administration considerably reduced Aβ1-40 and Aβ1-42 burden in cortex and hippocampus of Tg-SwDI mice through regulating Aβ metabolism process. Congo red staining confirmed Aβ deposition in vessel walls, reflecting CAA formation, which was, however, strongly ameliorated after HMPL-013 treatment. Neuron death, aberrant glial activation and pro-inflammatory response in brain tissues of Tg-SwDI mice were dramatically alleviated after HMPL-013 consumption. More studies showed that the protective effects of HMPL-013 against CAA might be partially attributed to its regulation on the expression of genes associated with blood vasculature. Intriguingly, VEGF and phosphorylated VEGFR-1,-2 protein expression levels were remarkably decreased by HMPL-013 in cortex and hippocampus of Tg-SwDI mice, which were validated in HMPL-013-treated brain vascular endothelial cells (BVECs) under hypoxia. Finally, we found that VEGF-induced human umbilical vein endothelial cells (HUVEC) proliferation and tube formation were strongly abolished upon HMPL-013 exposure. Collectively, all these findings demonstrated that oral administration of HMPL-013 had therapeutic potential against CAA by reducing Aβ deposition, inflammation and neuron death via suppressing VEGF/VEGFR-1,-2 signaling.

EICOSAPENTAENOIC ACID (EPA) DECREASES CYTOKINE RELEASE AND EXPRESSION OF INFLAMMATORY AND PRO-THROMBOTIC PROTEINS IN BRAIN VASCULAR ENDOTHELIUM

Brain vascular endothelial cell (EC) dysfunction contributes to ischemic stroke due to inflammation and release of pro-thrombotic factors. Treatment with icosapent ethyl (IPE), the ethyl ester of the omega-3 fatty acid, eicosapentaenoic acid (EPA), reduced first and total ischemic strokes each by 36%, in statin-treated patients with elevated cardiovascular risk (REDUCE-IT). We tested the effects of EPA on cytokine release and expression of inflammatory proteins from brain microvascular ECs during inflammation.

Rab27a-dependent exosomes protect against cerebral ischemic injury by reducing endothelial oxidative stress and apoptosis.

IntroductionMulticellular crosstalk within the brain tissue has been suggested to play a critical role in maintaining cerebral vascular homeostasis. Exosomes (EXs) mediated cell–cell communication, but its role in cerebral ischemic injury is largely unknown. Rab27a is one of the major genes controlling EX release. Here, we explored the role of Rab27a in regulating brain EXs secretion, and the effects of Rab27a‐mediated EXs on ischemia evoked cerebral vascular disruption and brain injury.MethodsCerebral ischemia was induced in Rab27a knockout (Rab27a−/−) and wide type (WT) mice by transient middle cerebral artery occlusion (tMCAO). Differential gene expression analysis was performed in ischemic brain tissue by using mRNA sequencing. EXs isolated from brain tissue of Rab27a−/− and WT mice (EXWT or EXRab27a−/−) were pre‐administrated into tMCAO operated Rab27a−/− mice or oxygen and glucose deprivation (OGD) treated primary brain vascular endothelial cells (ECs).ResultsWe demonstrated that Rab27a expression in the peri‐infarct area of brain was significantly elevated, which was associated with local elevation in EXs secretion. Rab27a deficiency dramatically decreased the level of EXs in brain tissue of normal and tMCAO‐treated mice, and Rab27a−/− mice displayed an increase in infarct volume and NDS, and a decrease in cMVD and CBF following tMCAO. Pre‐infusion of EXWT increased the brain EXs levels in the tMCAO operated Rab27a−/− mice, accompanied with an increase in cMVD and CBF, and a decrease in infarct volume, NDS, ROS production, and apoptosis. The effects of EXRab27a−/− infusion were much diminished although in a dose‐dependent manner. In OGD‐treated ECs, EXRab27a−/− showed less effectivity than EXWT in decreasing ROS overproduction and apoptosis, paralleling with down‐regulated expression of NOX2 and cleaved caspase‐3.ConclusionOur study demonstrates that Rab27a controls brain EXs secretion and functions, contributing to cerebral vascular protection from ischemic insult by preventing oxidative stress and apoptosis via down‐regulating NOX2 and cleaved caspase‐3 expression.

Structurally related (−)-epicatechin metabolites and gut microbiota derived metabolites exert genomic modifications via VEGF signaling pathways in brain microvascular endothelial cells under lipotoxic conditions: Integrated multi-omic study

Dysfunction of blood-brain barrier formed by endothelial cells of cerebral blood vessels, plays a key role in development of neurodegenerative disorders. Epicatechin exerts vasculo-protective effects through genomic modifications, however molecular mechanisms of action, particularly on brain endothelial cells, are largely unknow. This study aimed to use a multi-omic approach (transcriptomics of mRNA, miRNAs and lncRNAs, and proteomics), to provide novel in-depth insights into molecular mechanisms of how metabolites affect brain endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological concentrations. We showed that metabolites can simultaneously modulate expression of protein-coding, non-coding genes and proteins. Integrative analysis revealed interactions between different types of RNAs and form functional groups of genes involved in regulation of processing like VEGF-related functions, cell signaling, cell adhesion and permeability. Molecular modeling of genomics data predicted that metabolites decrease endothelial cell permeability, increased by lipotoxic stress. Correlation analysis between genomic modifications observed and genomic signature of patients with vascular dementia and Alzheimer's diseases showed opposite gene expression changes. Taken together, this study describes for the first time a multi-omic mechanism of action by which (−)-epicatechin metabolites could preserve brain vascular endothelial cell integrity and reduce the risk of neurodegenerative diseases. SignificanceDysfunction of the blood-brain barrier (BBB), characterized by dysfunction of endothelial cells of cerebral blood vessels, result in an increase in permeability and neuroinflammation which constitute a key factor in the development neurodegenerative disorders. Even though it is suggested that polyphenols can prevent or delay the development of these disorders, their impact on brain endothelial cells and underlying mechanisms of actions are unknow. This study aimed to use a multi-omic approach including analysis of expression of mRNA, microRNA, long non-coding RNAs, and proteins to provide novel global in-depth insights into molecular mechanisms of how (−)-epicatechin metabolites affect brain microvascular endothelial cells under lipid-stressed (as a model of BBB dysfunction) at physiological relevant conditions. The results provide basis of knowledge on the capacity of polyphenols to prevent brain endothelial dysfunction and consequently neurodegenerative disorders.

Differential Expression Profile of Sphingosine‐1‐Phosphate Receptors in Human Brain Vascular Smooth Muscle and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

Sphingosine‐1‐phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand activation, S1PRs 1‐5 couple with diverse heterotrimeric G‐protein subunits (Gαi, Gαq/11, Gα12/13) initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti‐inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, S1PR expression in the brain vasculature following ischemic injury has not yet been investigated. Therefore, the present study characterized the expression profile of S1PRs 1‐5 in human brain microvascular endothelial cells (ECs) and brain vascular smooth muscle cells (VSMCs) following hypoxia plus glucose deprivation (HGD). Male ECs and VSMCs were exposed to either HGD (1% O2) or normoxia (21% O2) for 3h. Semi‐quantitative RT‐PCR was used to examine gene expression and concomitantly, protein levels were assessed with standard western blot and/or FIJI‐mediated ICC analysis. We hypothesized that ECs and VSMCs differentially express S1PRs 1‐5 both basally and during ischemia‐like conditions. At the mRNA level, S1PRs 1‐5 gene expression was observed in VSMCs with S1PR4 and 5 being just above the level of detection. S1PRs 1‐4 expression was detected in ECs with S1PR4 having the lowest expression. During normoxic conditions, we observed that S1PRs 1 and 3 mRNA levels were most prominently expressed in both the ECs and VSMCs. In ECs, HGD exposure did not alter the gene expression of S1PRs 1‐3. At the protein level, we observed that HGD increased S1PR1 levels, however S1PR2 and 3 protein levels were unaltered. In VSMCs, HGD increased both mRNA and protein levels of S1PR2 but did not alter S1PRs 1 and 3 mRNA expression and protein levels. In conclusion, acute exposure to HGD appears to differentially regulate S1PRs in VSMCs and ECs at the transcriptional and translational level, respectively. In VSMCs, HGD increased S1PR2 gene expression and protein levels, whereas in ECs, HGD increased S1PR1 protein levels. The differential expression in S1PRs both basally and following HGD exposure may suggest differential signaling mechanisms within the two brain vascular cell types. Furthermore, differential expression of S1PR1 and 2 highlight the potential beneficial and/or detrimental involvement of S1PRs in the pathophysiology of ischemic injury within the cerebrovasculature. Further investigation will be necessary to determine the signaling role of S1PRs and their impact on cerebrovascular cell function following acute ischemic injury in the brain.

High BMP4 expression in low/intermediate risk BCP-ALL identifies children with poor outcomes

AbstractPediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) outcome has improved in the last decades, but leukemic relapses are still one of the main problems of this disease. Bone morphogenetic protein 4 (BMP4) was investigated as a new candidate biomarker with potential prognostic relevance, and its pathogenic role was assessed in the development of disease. A retrospective study was performed with 115 pediatric patients with BCP-ALL, and BMP4 expression was analyzed by quantitative reverse transcription polymerase chain reaction in leukemic blasts at the time of diagnosis. BMP4 mRNA expression levels in the third (upper) quartile were associated with a higher cumulative incidence of relapse as well as a worse 5-year event-free survival and central nervous system (CNS) involvement. Importantly, this association was also evident among children classified as having a nonhigh risk of relapse. A validation cohort of 236 patients with BCP-ALL supported these data. Furthermore, high BMP4 expression promoted engraftment and rapid disease progression in an NSG mouse xenograft model with CNS involvement. Pharmacological blockade of the canonical BMP signaling pathway significantly decreased CNS infiltration and consistently resulted in amelioration of clinical parameters, including neurological score. Mechanistically, BMP4 favored chemoresistance, enhanced adhesion and migration through brain vascular endothelial cells, and promoted a proinflammatory microenvironment and CNS angiogenesis. These data provide evidence that BMP4 expression levels in leukemic cells could be a useful biomarker to identify children with poor outcomes in the low-/intermediate-risk groups of BCP-ALL and that BMP4 could be a new therapeutic target to blockade leukemic CNS disease.

Transcription Factor HEY1 Improves Brain Vascular Endothelial Cell Function and Alleviates Ischemic Stroke by Upregulating NOTCH3.

To investigate the function of hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) and Notch receptor 3 (NOTCH3) in ischemic stroke. Stroke models were established by middle cerebral artery occlusion (MCAO) and oxygen glucose deprivation (OGD) in rats and rat brain microvascular endothelial cells (BMVECs), respectively. Neurological deficit evaluation and 2,3,5-triphenyltetrazolium chloride staining were used to assess cerebral injury. The expression of HEY1 and NOTCH3 was manipulated using gain and loss of function approaches. Terminal deoxynucleotidyl transferase dUTP nick end labeling and Western blotting analysis of cleaved caspase-3 and B-cell lymphoma-2 (Bcl2) were used to evaluate apoptosis. Enzyme-linked immunosorbent assay was performed to measure the expression levels of interleukin (IL)-1β, IL-6 and IL-18. The proliferation and migration of BMVECs were analyzed by Ki-67 immunofluorescence and scratch assay, respectively. Tube formation assay was conducted to measure the length of capillary-like tubes formed by BMVECs. Co-immunoprecipitation was used to testify the relationship between HEY1 and NOTCH3. HEY1 and NOTCH3 were upregulated in MCAO and OGD models. HEY1 ameliorated ischemic injuries in MCAO rats. Knockdown of HEY1 or NOTCH3 promoted OGD-induced apoptosis and inflammation and inhibited proliferation and migration in BMVECs. NOTCH3 was a binding protein of HEY1. Overexpression of HEY1 offset the disease-promoting effect of NOTCH3 silencing. HEY1 suppresses apoptosis and inflammation and promotes proliferation and migration in BMVECs by upregulating NOTCH3, thereby ameliorating ischemic stroke.

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction

ABSTRACT A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan’s blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.

Abstract WP210: Eicosapentaenoic Acid (EPA) Decreases Cytokine Release And Inflammatory Proteins In Brain Vascular Endothelium During Inflammation

Background: Brain vascular endothelial cell (EC) dysfunction is characterized by release of pro-inflammatory mediators that contribute to atherosclerosis and ischemic stroke. Treatment with icosapent ethyl (IPE), the ethyl ester of the omega-3 fatty acid, eicosapentaenoic acid (EPA), reduced first and total ischemic strokes each by 36%, without increasing hemorrhagic stroke, in statin-treated patients with elevated cardiovascular risk (REDUCE-IT). We tested the effects of EPA on cytokine release and expression of inflammatory proteins from brain microvascular ECs during inflammation. Methods: Human brain microvascular endothelial cells (HBECs) were pretreated with the cytokine IL-6 at 12 ng/ml for 2 h before treatment with EPA (40 μM) for 24 h. Proteomic analysis was performed using LC/MS to capture relative expression levels. Only significant changes in protein expression between treatment groups >1-fold were analyzed. Gene set enrichment analysis was also performed on all significantly regulated proteins to identify groups of proteins (pathways) that were affected. Levels of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-α (TNF-α) were measured by immunochemistry (ELISA). Results: IL-6 exposure produced increased levels of sICAM-1 and TNF-α by 102% and 147% (p<0.001), respectively, in brain ECs compared with vehicle. EPA treatment reduced release of sICAM-1 by 43% (p<0.001) and TNF-α by 52% (p<0.001) compared to IL-6 alone. EPA also downregulated 43 proteins involved in the “neutrophil degranulation” pathway in brain ECs (p-adjusted = 2.63 х 10 -12 ), including heat shock protein 90-alpha and beta by 1.1-fold each relative to IL-6 alone. Conclusions: In brain vascular ECs, EPA significantly reduced expression of pro-inflammatory mediators and proteins associated with neutrophil degranulation under inflammatory conditions. The ability of EPA to reverse brain EC dysfunction during inflammatory may contribute to reductions in stroke risk.

Zebularine protects against blood-brain-barrier (BBB) disruption through increasing the expression of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin

ABSTRACT Blood-brain-barrier (BBB) disruption is an important pathological characteristic of ischemic stroke (IS) and mainly results from dysfunction of brain vascular endothelial cells and tight junctions. Zebularine is a novel inhibitor of DNA methyltransferase (DNMT). Here, we assessed its effects on BBB disruption in IS. Firstly, we reported that Zebularine maintained BBB integrity in middle cerebral artery occlusion (MCAO) mice by increasing the expressions of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin. Importantly, we found that Zebularine reduced the production of pro-inflammatory cytokines, attenuated brain edema, and improved neurological deficits. In in vitro experiments, the bEnd.3 brain endothelial cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R), and the protective effects of Zebularine were assessed. Our findings demonstrated that Zebularine prevented OGD/R-induced cytotoxicity by reducing the release of lactate dehydrogenase (LDH). Additionally, Zebularine protected bEnd.3 cells against OGD/R-induced hyper-permeability and reduction of trans-endothelial electrical resistance (TEER). Notably, we found that treatment with Zebularine activated the Adenosine 5ʹ-monophosphate (AMP)-activated protein kinase (AMPK) pathway by increasing the phosphorylation of adenosine monophosphate-activated protein kinase α (AMPKα). Blockage of AMPKα using its specific inhibitor compound C abolished the beneficial effects of Zebularine in mitigating endothelial hyper-permeability by reducing the expressions of ZO-1 and VE-cadherin. These findings suggest that the protective effects of Zebularine against OGD/R-induced endothelial hyper-permeability are mediated by the activation of AMPKα. In conclusion, our study sheds light on the potential application of Zebularine in the treatment of IS.