Human Brain Microvascular Endothelial Cells Research Articles

Rbpj Deficiency Disrupts Vascular Remodeling via Abnormal Apelin and Cdc42 (Cell Division Cycle 42) Activity in Brain Arteriovenous Malformation.

Brain arteriovenous malformations (bAVM) are characterized by enlarged blood vessels, which direct blood through arteriovenous shunts, bypassing the artery-capillary-vein network and disrupting blood flow. Clinically, bAVM treatments are invasive and not routinely applicable. There is critical need to understand mechanisms of bAVM pathologies and develop pharmacological therapies. We used an in vivo mouse model of Rbpj-mediated bAVM, which develops pathologies in the early postnatal period and an siRNA in vitro system to knockdown RBPJ in human brain microvascular endothelial cells (ECs). To understand molecular events regulated by endothelial Rbpj, we conducted RNA-Seq and chromatin immunoprecipitation-Seq analyses from isolated brain ECs. Rbpj-deficient (mutant) brain ECs acquired abnormally rounded shape (with no change to cell area), altered basement membrane dynamics, and increased endothelial cell density along arteriovenous shunts, compared to controls, suggesting impaired remodeling of neonatal brain vasculature. Consistent with impaired endothelial cell dynamics, we found increased Cdc42 (cell division cycle 42) activity in isolated mutant ECs, suggesting that Rbpj regulates small GTPase (guanosine triphosphate hydrolase)-mediated cellular functions in brain ECs. siRNA-treated, RBPJ-deficient human brain ECs displayed increased Cdc42 activity, disrupted cell polarity and focal adhesion properties, and impaired migration in vitro. RNA-Seq analysis from isolated brain ECs identified differentially expressed genes in mutants, including Apelin, which encodes a ligand for G protein-coupled receptor signaling known to influence small GTPase activity. Chromatin immunoprecipitation-Seq analysis revealed chromatin loci occupied by Rbpj in brain ECs that corresponded to G-protein and Apelin signaling molecules. In vivo administration of a competitive peptide antagonist against the Apelin receptor (Aplnr/Apj) attenuated Cdc42 activity and restored endothelial cell morphology and arteriovenous connection diameter in Rbpj-mutant brain vessels. Our data suggest that endothelial Rbpj promotes rearrangement of brain ECs during cerebrovascular remodeling, through Apelin/Apj-mediated small GTPase activity, and prevents bAVM. By inhibiting Apelin/Apj signaling in vivo, we demonstrated pharmacological prevention of Rbpj-mediated bAVM.

G1 Cell Cycle Arrest Is Induced by the Fourth Extracellular Loop of Meningococcal PorA in Epithelial and Endothelial Cells

Neisseria meningitidis is the most frequent cause of bacterial meningitis and is one of the few bacterial pathogens that can breach the blood-brain barrier (BBB). The 37/67 kDa laminin receptor (LamR) was previously identified as a receptor mediating meningococcal binding to rodent and human brain microvascular endothelial cells, which form part of the BBB. The meningococcal surface proteins PorA and PilQ were identified as ligands for this receptor. Subsequently, the fourth extracellular loop of PorA (PorA-Loop4) was identified as the LamR-binding moiety. Here, we show that PorA-Loop4 targets the 37 kDa laminin receptor precursor (37LRP) on the cell surface by demonstrating that deletion of this loop abrogates the recruitment of 37LRP under meningococcal colonies. Using a circularized peptide corresponding to PorA-Loop4, as well as defined meningococcal mutants, we demonstrate that host cell interaction with PorA-Loop4 results in perturbation of p-CDK4 and Cyclin D1. These changes in cell cycle control proteins are coincident with cellular responses including inhibition of cell migration and a G1 cell cycle arrest. Modulation of the cell cycle of host cells is likely to contribute to the pathogenesis of meningococcal disease.

Cerebral Malaria Model Applying Human Brain Organoids.

Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood-brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.

Zika virus NS1 suppresses VE-cadherin via hsa-miR-29b-3p/DNMT3b/MMP-9 pathway in human brain microvascular endothelial cells

Zika virus infection has been reported to cause microcephaly in newborns. ZIKV exploits various strategies to cross the blood-brain barrier. ZIKV NS1 may compromise the barrier integrity of endothelial cells by regulating expression of junctional proteins. MicroRNAs play an important role in post-transcriptional gene regulations. We demonstrated that ZIKV-NS1 affected the adherence junction protein in human brain microvascular endothelial cells via hsa-miR-29b-3p/DNMT3b/MMP-9 pathway. The hCMEC/D3 cells were exposed to ZIKV-NS1 with different doses (500 ng/mL and 1000 ng/mL) for 24 h. The expression pattern of DNTM3b, MMP-9, and VE-cadherin were studied using immunoblotting and the distribution of DNMT3b and MMP-9 were studied using immunofluorescence. The quantification of hsa-miR-29b-3p was done through qRT-PCR. Direct regulation of DNMT3b by hsa-miR-29b-3p was demonstrated by overexpression of hsa-miR-29b-3p using hsa-miR-29b-3p mimic, and knockdown of hsa-miR-29b-3p by using hsa-miR-29b-3p inhibitors. The ZIKV-NS1 affected the barrier function of endothelial cells through the increased expression of hsa-miR29b-3p, which suppressed the DNMT3b, thus enhanced expression of MMP-9, which finally suppressed the expression of VE-cadherin. These findings suggested that ZIKV-NS1 alters the expression of Adherens Junction protein in human brain microvascular endothelial cells through hsa-miR-29b-3p/DNMT3b/MMP-9 pathway, which compromised the barrier function of human brain microvascular endothelial cells.

Circulating Plasma Exosomal Proteins of Either SHIV-Infected Rhesus Macaque or HIV-Infected Patient Indicates a Link to Neuropathogenesis.

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50-60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood-brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers-possibly associated with viral reactivation and neuropathogenesis-that may elucidate the etiology of HAND.

Organic electrochemical transistor-based immuno-sensor using platinum loaded CeO2 nanosphere-carbon nanotube and zeolitic imidazolate framework-enzyme-metal polyphenol network

This work demonstrates an organic electrochemical transistor (OECT) immuno-sensor with a detection limit down to fg mL−1. The OECT device transforms the antibody-antigen interaction signal by using the zeolitic imidazolate framework-enzyme-metal polyphenol network nanoprobe, which can produce electro-active substance (H2O2) through the enzyme-catalytic reaction. The produced H2O2 is subsequently electrochemically oxidized at the platinum loaded CeO2 nanosphere-carbon nanotube modified gate electrode, resulting in an amplified current response of the transistor device. This immuno-sensor realizes the selective determination of vascular endothelial growth factor 165 (VEGF165) down to the concentration of 13.6 fg mL−1. It also shows good applicable capacity for determining the VEGF165 that human brain microvascular endothelial cells and U251 human glioblastomas cells secreted in the cell culture medium. The ultrahigh sensitivity of the immuno-sensor is derived from excellent performances of the nanoprobe for enzyme loading and the OECT device for H2O2 detection. This work may provide a general way to fabricate the OECT immuno-sensing device with high performances.

MiR-4432 Targets FGFBP1 in Human Endothelial Cells.

MicroRNAs (miRs) are small non-coding RNAs that modulate the expression of several target genes. Fibroblast growth factor binding protein 1 (FGFBP1) has been associated with endothelial dysfunction at the level of the blood-brain barrier (BBB). However, the underlying mechanisms are mostly unknown and there are no studies investigating the relationship between miRs and FGFBP1. Thus, the overarching aim of the present study was to identify and validate which miR can specifically target FGFBP1 in human brain microvascular endothelial cells, which represent the best in vitro model of the BBB. We were able to identify and validate miR-4432 as a fundamental modulator of FGFBP1 and we demonstrated that miR-4432 significantly reduces mitochondrial oxidative stress, a well-established pathophysiological hallmark of hypertension.

Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Circular RNA CircPDS5B impairs angiogenesis following ischemic stroke through its interaction with hnRNPL to inactivate VEGF-A

BackgroundIschemic stroke (IS) is the primary cause of mortality and disability worldwide. Circular RNAs (circRNAs) have been proposed as crucial regulators in IS. This study focused on the role of circPDS5B in IS and its underlying mechanism. MethodTransient middle cerebral artery occlusion (tMCAO) mice and glucose deprivation/reoxygenation (OGD/R)-exposed human brain microvascular endothelial cells (BMECs) were used as IS models. Expression levels of circPDS5B, heterogenous nuclear ribonucleoprotein L (hnRNPL), runt-related transcription factor-1 (Runx1), and Zinc finger protein 24 (ZNF24) were quantified by qRT-PCR. MTT, wound healing, transwell and tube formation assays were employed to evaluate the cell proliferation, migration, and angiogenesis, respectively. Moreover, RNA pull-down, and RIP assay were performed to investigate the interaction among circPDS5B, hnRNPL and vascular endothelial growth factor-A (VEGF-A). ResultscircPDS5B was significantly up-regulated in IS patients and tMCAO mice. Deficiency of circPDS5B relieved brain infarction and neuronal injury of tMCAO mice. OGD/R-induced apoptosis, inhibition in viability, migration, and angiogenesis in BMECs were dramatically abrogated by circPDS5B knockdown. Mechanistically, circPDS5B stabilized Runx1 and ZNF24 via recruiting hnRNPL, thereby suppressing the transcription and expression of VEGFA. hnRNPL silencing strengthened circPDS5B knockdown-mediated beneficial effect on IS. ConclusionAltogether, our study showed that high expression of circPDS5B exacerbated IS through recruitment of hnRNPL to stabilize Runx1/ZNF24 and subsequently inactivate VEGFA. Our findings suggest circPDS5B may be a novel therapeutic target for IS.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Protective effects of brain and muscle ARNT-like gene 1 on oxidized low-density lipoprotein-induced human brain microvascular endothelial cell injury by alleviating ferroptosis.

Ferroptosis plays an important role in atherosclerotic cerebrovascular diseases. The brain and muscle ARNT-like gene 1 (BMAL1) is an important mediator in the progression of cerebrovascular diseases. However, whether BMAL1 regulates ferroptosis in atherosclerotic cerebrovascular diseases remains obscure. Here, human brain microvascular endothelial cells (HBMECs) were exposed to oxidized low-density lipoprotein (ox-LDL) to imitate cerebrovascular atherosclerosis. It was found that ox-LDL treatment induced ferroptosis events and reduced BMAL1 expression in HBMECs, which could be reversed by ferroptosis inhibitor ferrostatin-1. Furthermore, BMAL1 overexpression markedly mitigated ox-LDL-induced ferroptosis events and cell damage. Moreover, BMAL1 overexpression significantly promoted nuclear factor erythroid 2-related factor 2 (Nrf2) expression in HBMECs under ox-LDL conditions. And, Nrf2 silencing attenuated the protective effects of BMAL1 on ox-LDL-stimulated HBMEC damage and ferroptosis. Altogether, our findings delineate the cerebrovascular protective role of BMAL1/Nrf2 by antagonizing ferroptosis in response to ox-LDL stimulation and provide novel perspectives for therapeutic strategies for atherosclerotic cerebrovascular diseases.

Abstract GS5-08: Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer with rapid onset and a strong propensity to spread to distant organs. Five-year overall survival (OS) rates remain poor, in part because of the high risk of brain metastasis: 19% of patients with IBC have brain metastases within the first 2 years after diagnosis. Our hypothesis for this study was that soluble E-cadherin (sEcad), an 80-kDa extracellular proteolytic fragment of full-length E-cadherin - a tumor promoter in IBC, is crucial for driving brain metastasis in IBC. Methods: We analyzed serum sEcad levels in from 348 IBC patients by ELISA. Four IBC cell lines [ER–/HER2+ (MDA-IBC3; SUM190) and ER–/HER2– (SUM149; BCX010)], human brain microvascular endothelial cells, and immortalized human astrocytes were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Mass spectrometry and Bio-ID-based proteomics assays, and RNA sequencing were used to identify sEcad-interacting proteins and potential mechanisms. In vivo, we studied tumor growth and brain metastasis in mice by injecting IBC cells into the mammary fatpad or tail vein, respectively, of SCID/Beige mice. Results: In IBC patients, higher serum sEcad levels correlated with poorer OS (p=0.02), earlier development of metastasis (p=0.006), and development of brain metastasis (p=0.04). On multivariable analysis, sEcad independently predicted OS (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). In vitro, sEcad overexpression in IBC cell lines promoted anchorage-independent growth, migration, invasion, and resistance to anoikis. In vivo, sEcad-overexpressing SUM149 cells promoted primary tumor growth (p=0.007). Mice injected with sEcad-overexpressing MDA-IBC3 cells also had higher incidence of brain metastasis (100% vs 50%, p=0.03), metastatic burden (p=0.02) and number of metastases per mouse (p=0.0009), and had worse OS (p=0.0016), and brain metastasis-free survival (p=0.04), relative to controls. We further found that sEcad increased cancer cell adhesion to brain endothelial cells (p=0.01) and promoted induction of reactive astrocytes (as identified by high glial fibrillary acidic protein levels) in vitro and in vivo. Mechanistically, mass spectrometry and Bio-ID assays identified X-linked inhibitor of apoptosis protein (XIAP), a potent inhibitor of apoptotic cell death, as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Further analysis showed that sEcad bound to the BIR2 domain of XIAP. XIAP is the most potent and best-defined anti-apoptotic IAP family member, and it could induce NF-κB activation to inhibit tumor cell apoptosis Gene set enrichment analysis of RNA-seq profiling data showed activation of NF-kB signaling and downregulation of apoptotic pathways in the sEcad-overexpressing SUM149 cells compared with controls. Immunoblotting revealed that sEcad enhanced XIAP expression, activated NF-κB signaling, and inhibited cleavage of caspase-3 in IBC cells. Conclusions: We found that higher serum sEcad correlates with development of brain metastases and independently predicts poor OS in patients with IBC. We further found that sEcad promotes tumor growth and brain metastasis, perhaps via activation of XIAP/NF-κB signaling in breast cancer cells and promotion of endothelial cell adhesion and reactive astrocytosis in the brain microenvironment. These findings uncover a novel and crucial role for sEcad in brain metastasis and provide new insights and potential therapeutic targets for patients with metastatic IBC. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S. Villodre, Juhee Song, Ganiraju C Manyam, Moises J Tacam, Jing Wang, Chandra Bartholomeusz, Debu Tripathy, Naoto T. Ueno, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-08.

Abstract P2-21-05: Vascular effects of Platelet-derived growth factor-B (PDGFB) in breast cancer metastasis to the brain

Abstract Brain metastases are associated with shortened survival and poorer quality of life in breast cancer patients. Recently, the importance of platelet-derived growth factor-B (PDGFB) and its stromally localized cognate receptor, platelet-derived growth factor receptor-beta (PDGFRβ) in promoting breast cancer metastasis to the brain was identified by our group. Historically, PDGFB-to-PDGFRβ signaling is known to promote angiogenesis and vessel maturation both in normal physiology and in cancer, but the mechanism(s) behind this tumor stromal interplay in brain metastasis is poorly understood. Based on our previous work and the work of others, we hypothesized that breast tumor-derived PDGFB directly modulates both brain endothelial and blood brain barrier (BBB) function by altering angiogenesis and endothelial permeability. To test this directly, we over-expressed PDGFB ligand in MDA-MB-231 and HCC1187 triple negative breast cancer cell lines and compared the angiogenic potential of the conditioned medium (CM) from these cells. We utilized both a 3D in vitro (human brain microvascular endothelial cell, HBMEC) spheroid angiogenesis model and a tri-culture BBB system (HBMECs in direct interaction with human astrocytes and human pericytes) to test the BBB vasculature. CM harvested from PDGFB overexpressing cells significantly increased the sprouting angiogenesis potential of 3D HBMECs and the migration of 3D tri-culture BBB spheroids compared to CM obtained from parental cells. In addition, CM from PDGFB overexpressing cells caused the surface of BBB spheroids to exhibit significantly increased permeability to high molecular weight dextran compared to CM from parental cells. In agreement with these findings, the tight junction protein ZO1 was diminished. Importantly, in vivo BBB permeability was also tested upon intracardiac injection of MDA-MB-231 cells with and without PDGFB overexpression. These studies revealed that Evans blue dye fluorescence intensity was significantly higher in the brains of mice injected with PDGFB overexpressing cells compared to parental cells. Overall, these studies reveal a previously unrecognized role for PDGFB-to-PDGFRβ signaling in brain vascular remodeling and BBB permeability, which has clinical implications for women with PDGFB-positive breast cancer. Citation Format: Sajita Shah, Rebecca Packard, Sarah Steck, Alexis Mossing, Yalini Ramamoorthy, Katie Thies, Alexander Didier, Jonathan Spehar, Dillon Richardson, Tasneem Arsiwala, Jonathan Godbout, Steven Sizemore, Paul Lockman, Gina Sizemore. Vascular effects of Platelet-derived growth factor-B (PDGFB) in breast cancer metastasis to the brain [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-05.

Circ_0000566 contributes oxygen-glucose deprivation and reoxygenation (OGD/R)-induced human brain microvascular endothelial cell injury via regulating miR-18a-5p/ACVR2B axis.

Circular RNAs (circRNAs) exert regulatory roles in cerebrovascular disease. Human brain microvascular endothelial cells (HBMECs) participated in brain vascular dysfunction in ischemic stroke. Herein, the functions of circ_0000566 in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced HBMECs were investigated. The expression of circ_0000566, miR-18a-5p, and Activin receptor type 2B (ACVR2B) was measured via quantitative real-time PCR (qRT-PCR). Cell Counting Kit-8 (CCK-8) and flow cytometry assays were utilized to detect cell viability and cell apoptosis. Western blot assay was employed to measure the levels of apoptotic-related proteins and ACVR2B. The secretion of IL-1β, IL-6, and TNF-α was detected via corresponding kits. The relationship between miR-18a-5p and circ_0000566 or ACVR2B was examined via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0000566 and ACVR2B were highly expressed, while miR-18a-5p was down-regulated in OGD/R-treated HBMECs. OGD/R treatment promoted HBMECs apoptosis and inflammation and suppressed cell viability, which could be attenuated by silencing of circ_0000566. Circ_0000566 acted as a miR-18a-5p sponge to contribute to OGD/R-induced HBMECs injury. ACVR2B served as a direct target of miR-18a-5p, and ACVR2B overexpression might abolish the inhibitory role of miR-18a-5p on OGD/R-treated HBMEC injury. Circ_0000566 sponged miR-18a-5p to regulate OGD/R-induced HBMECs injury via regulating ACVR2B expression.

Enhancing the stability, BBB permeability and neuroprotective activity of verbascoside in vitro using lipid nanocapsules in combination with menthol

Verbascoside (VER) shows promising neuroprotective activity. However, the instability and low permeability in crossing the blood–brain barrier (BBB) greatly hinder its application. In the present study, verbascoside was encapsulated into lipid nanocapsules (LNC) by reverse micelle (RM) to increase its stability. Besides, we used VER-RM-LNC combined with an envoy drug, menthol, to improve its BBB permeability and neuroprotective activity. VER-RM-LNC was prepared by the phase inversion temperature method, resulting in an encapsulation efficiency of nearly 85 %. The formulated VER-RM-LNC was stable for 6 months at 4 °C. VER encapsulated into LNC possessed enhanced stability and a reduced release profile. Menthol increased the cellular uptake and the permeability of VER-RM-LNC in the BBB model in vitro. In addition, the improved neuroprotective activity of VER through incubation with menthol and VER-RM-LNC was verified in the neurotoxic human brain microvascular endothelial cells model induced by Aβ25-35.

Circvrk1 downregulation attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation through modulating the miR-150-5p/MLLT1 axis.

The pivotal regulatory role of circular RNAs (circRNAs) in ischemic stroke (IS) has been expounded. The study aimed to probe the exact role and underlying mechanism of circVRK1 in oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs) injury. HBMECs challenged by OGD were used as in vitro models of IS. Quantitative real-time PCR was used to examine the levels of circVRK1, vaccinia-related kinase 1 (VRK1), miR-150-5p and MLLT1 mRNA. Cell viability, migration angiogenesis ability and death were evaluated by Cell counting kit-8 assay, transwell assay, wound-healing assay, tube formation assay and flow cytometry analysis. All the protein levels were monitored by western blot assay. Enzyme-linked immunosorbent assay was conducted for examining cell oxidative stress. Dual-luciferase reporter assay, RIP assay and RNA pull-down assay were performed to verify the combination between miR-150-5p and circVRK1 or MLLT1. CircVRK1 was upregulated in OGD-treated HBMECs. CircVRK1 knockdown alleviated OGD-caused effects on HBMECs migration, angiogenesis, death, inflammatory response and oxidative stress. Furthermore, circVRK1 could sponge miR-150-5p, and miR-150-5p silencing also mitigated the impact of circVRK1 deficiency on OGD-evoked injury. Besides, MLLT1 acted as a molecular target of miR-150-5p, and the protective influence of miR-150-5p on OGD-induced cell damage was overturned by MLLT1 introduction. CircVRK1 knockdown weakened OGD-evoked injury in HBMECs through modulating miR-150-5p/MLLT1 pathway, and this might supply new insights and probable targets for IS treatment.

Human umbilical cord mesenchymal stem cells-derived exosomal circDLGAP4 promotes angiogenesis after cerebral ischemia-reperfusion injury by regulating miR-320/KLF5 axis.

Accumulating evidence suggests that human umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSCs-Exos) are a promising therapeutic strategy for cerebral ischemia-reperfusion injury (CIRI). However, the underlying mechanism remains unclear. hUC-MSCs-Exos were identified by electron microscopy, NTA, and Western blotting. In the hypoxia/reoxygenation (H/R) cell model, human brain microvascular endothelial cells (HBMECs) were cocultured with hUC-MSCs-Exos. Then, cell viability, migration, apoptosis, and tube formation were measured by MTT, flow cytometry, transwell, and tube formation assays. RT-qPCR and Western blotting were used to detect the changes in RNA and protein. RNA pull-down and dual luciferase reporter assays confirmed the relationship between circDLGAP4, miR-320, and KLF5. Ischemia-reperfusion (I/R) rat model was established for in vivo experiments. hUC-MSCs-Exos increased the expression levels of circDLGAP4 and KLF5 but decreased miR-320 in H/R-treated HBMECs by transferring exosomal circDLGAP4. Knockdown of circDLGAP4 in hUC-MSCs-Exos reversed the promoting effects of hUC-MSCs-Exos on cell viability, migration, and tube formation in H/R-treated HBMECs in vitro and also abolished the protective effects of hUC-MSCs-Exos on cerebrovascular injury in I/R rats. Mechanistically, exosomal circDLGAP4 negatively regulated miR-320 in HBMECs, which directly bound to KLF5. In addition, the downregulation of miR-320 could reverse the regulatory effect of exosomal shcircDLGAL5 in H/R-treated HBMECs by upregulating KLF5. hUC-MSCs-Exos-derived circDLGAP4 reduced cerebrovascular injury by regulating miR-320/KLF5 signaling. These results provide a stem cell-based approach to treat CIRI.

Long-term administration of CU06-1004 ameliorates cerebrovascular aging and BBB injury in aging mouse model

BackgroundAge-related changes in the cerebrovasculature, including blood–brain barrier (BBB) disruption and vascular dementia, are emerging as potential risks for many neurodegenerative diseases. Therefore, the endothelial cells that constitute the cerebrovasculature may play key roles in preventing brain injury. Our previous study showed that CU06-1004, an endothelial cell dysfunction blocker, prevented vascular leakage, enhanced vascular integrity in ischemic reperfusion injury, and promoted the normalization of tumor vasculature. Here, we evaluated the effects of CU06-1004 on age-related cerebrovascular functional decline in the aged mouse brain.ResultsIn this study, we investigated the protective effects of CU06-1004 against oxidative stress–induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with hydrogen peroxide (H2O2) to establish an oxidative stress–induced model of cellular injury. Compared with H2O2 treatment alone, pretreatment of HBMECs with CU06-1004 considerably reduced oxidative stress–induced cytotoxicity, reactive oxygen species generation, senescence-associated β-galactosidase activity, senescence marker expression, and the expression levels of inflammatory proteins. Based on the observed cytoprotective effects of CU06-1004 in HBMECs, we examined whether CU06-1004 displayed protective effects against cerebrovascular aging in mice. Long-term administration of CU06-1004 alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged mouse brain. CU06-1004 supplementation also reduced the extravasation of plasma IgG by improving BBB integrity in the aged mouse brain, associated with reductions in neuronal injury. A series of behavioral tests also revealed improved motor and cognitive functions in aged mice that received long-term CU06-1004 administration.ConclusionsThese findings suggest that CU06-1004 may represent a promising therapeutic approach for delaying age-related cerebrovascular impairment and improving cognitive function in old age.

Abstract TP222: Aging-associated Medin Induces Human Brain Microvascular Endothelial Cell Pro-inflammatory And Pro-thrombotic Activation Partly Via NFκB

Background: Vascular aging is characterized by endothelial dysfunction and proinflammatory &amp; prothrombotic changes. It is responsible for aging-associated pathologies including stroke, vascular dementia (VaD) and Alzheimer’s disease (AD). The underlying mechanisms of vascular aging are poorly understood. Medin is an amyloidogenic protein that accumulates in aging vasculature, is associated with VaD and AD, and is a candidate mediator of vascular aging through unknown mechanisms. Our aim is to test the hypothesis that medin induces proinflammatory and prothrombotic changes in human brain microvascular endothelial cells (HBMVECs) through nuclear factor kappa B (NFκB). Methods: HBMVECs were exposed to vehicle, recombinant medin (5 μM, a physiologically relevant dose) without or with RO106-9920 (a small molecule selective NFκB inhibitor, 10 μM) or RO106-9920 alone for 20 hours (N=3-17/treatment). Cell lysates were measured for gene expression of inflammatory cytokines/chemokines (IL-8, IL-6, ICAM-1 and VCAM-1) and thrombotic factors (thrombomodulin (TM), a transmembrane glycoprotein and potent inhibitor of coagulation, plasminogen activator inhibitor-1 (PAI-1) which promotes thrombosis and tissue factor (TF) which initiates the coagulation cascade), using real time PCR. Results: See Figure. Medin increased gene expression of IL-8, IL-6, ICAM-1, VCAM-1 and PAI-1, reduced TM with no change in TF. Co-treatment of medin with RO106-9920 attenuated the increases in IL-8, IL-6, ICAM-1, VCAM-1 and the reduction in TM, with no change in PAI-1. Conclusions: Medin caused increased pro-inflammatory gene expression in HBMVECs. It also caused reduced TM and increased PAI-1 gene expression, signifying prothrombotic activation. Co-treatment with NFκB inhibitor prevented medin’s proinflammatory effect, and effect on TM, but not PAI-1. The effect of inducing endothelial cell activation supports medin as a potential novel target to treat vascular aging.

Detecting physiological concentrations of Alzheimer's associated amyloid-β oligomers with a cell-based response.

Alzheimer's disease (AD) is characterized by blood-brain barrier (BBB) breakdown and neuronal cell death, associated with aggregated amyloid-β (Aβ) plaques deposited around brain vasculature and within the brain. Changes in the brain occur up to 20 years before symptoms arise; however, there is currently no pre-symptomatic detection method available for AD. An intact BBB requires tight junctions (TJ) that render relatively high transendothelial electrical resistance (TEER). AD-associated breakdown of the BBB correlates with vascular Aβ deposition, suggesting that aggregated, pathological Aβ can modulate TEER. We explore leveraging this cellular response for early disease diagnosis via detection of early Aβ aggregates. Primary human brain microvascular endothelial cells (HBMVECs) were cultured on a suspended membrane and supplemented to mimic the BBB. Once TEER values reflected TJ formation, HBMVEC monolayers were treated with oligomeric Ab at picomolar to nanomolar concentrations, and TEER was monitored. Oligomeric Aβ at picomolar and nanomolar concentrations, but not monomer, induced a reduction of TEER and disruption of TJs. These results demonstrate that pathogenic Aβ aggregates are uniquely responsible for inducing endothelial monolayer permeability and reducing monolayer electrical resistance, and this response can be detected at physiological concentrations. This work also validates that introduction of physiologically active Aβ oligomers can increase permeability of an endothelial monolayer, while non-pathogenetic monomer is inert. These results provide evidence that oligomers, the pathogenic form of Aβ, selectively break down the BBB. Future work will include establishing a threshold of Aβ oligomer concentrations that reduce TEER to establish cellular sensitivity, and sensitizing cells to obtain a more pronounced response. Translation of this experimental system to a cell-based biosensor platform will lay the groundwork for the development of cost-effective, early AD detection.