Artículos de revistas: "HCMEC"

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Zakrzewicz, A., M. Gräfe, D. Terbeek, M. Bongrazio, W. Auch-Schwelk, B. Walzog, K. Graf, E. Fleck, K. Ley y P. Gaehtgens. "L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System". Blood 89, n.º 9 (1 de mayo de 1997): 3228–35. http://dx.doi.org/10.1182/blood.v89.9.3228.

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Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

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Marquez-Curtis, Leah A., Reid Bokenfohr, Locksley E. McGann y Janet A. W. Elliott. "Cryopreservation of human cerebral microvascular endothelial cells and astrocytes in suspension and monolayers". PLOS ONE 16, n.º 4 (14 de abril de 2021): e0249814. http://dx.doi.org/10.1371/journal.pone.0249814.

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The blood–brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.

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Meli, Athinoula, Ann McCormack, Ianina Conte, Qu Chen, James Streetley, Marlene L. Rose, Ruben Bierings et al. "Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts". International Journal of Molecular Sciences 24, n.º 5 (25 de febrero de 2023): 4553. http://dx.doi.org/10.3390/ijms24054553.

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The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel–Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

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Omatsu, Tatsushi, Gediminas Cepinskas, Cheril Clarson, Eric K. Patterson, Ibrahim M. Alharfi, Kelly Summers, Pierre-Olivier Couraud, Ignacio A. Romero, Babette Weksler y Douglas D. Fraser. "CXCL1/CXCL8 (GROα/IL-8) in human diabetic ketoacidosis plasma facilitates leukocyte recruitment to cerebrovascular endothelium in vitro". American Journal of Physiology-Endocrinology and Metabolism 306, n.º 9 (1 de mayo de 2014): E1077—E1084. http://dx.doi.org/10.1152/ajpendo.00659.2013.

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Diabetic ketoacidosis (DKA) in children is associated with intracranial vascular complications, possibly due to leukocyte-endothelial interactions. Our aim was to determine whether DKA-induced inflammation promoted leukocyte adhesion to activated human cerebrovascular endothelium. Plasma was obtained from children with type 1 diabetes either in acute DKA or in an insulin-controlled state (CON). Plasma concentrations of 21 inflammatory analytes were compared between groups. DKA was associated with altered circulating levels of ↑CXCL1 (GROα), ↑CXCL8 (IL-8), ↑IL-6, ↑IFNα2, and ↓CXCL10 (IP-10) compared with CON. These plasma analyte measurements were then used to create physiologically relevant cytokine mixtures (CM). Human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with either plasma (DKA-P or CON-P) or CM (DKA-CM or CON-CM) and assessed for polymorphonuclear leukocyte (PMN) adhesion. Stimulation of hCMEC/D3 with DKA-P or DKA-CM increased PMN adhesion to hCMEC/D3 under “flow” conditions. PMN adhesion to hCMEC/D3 was suppressed with neutralizing antibodies to CXCL1/CXCL8 or their hCMEC/D3 receptors CXCR1/CXCR2. DKA-P, but not DKA-CM, initiated oxidative stress in hCMEC/D3. Expression of ICAM-1, VCAM-1, and E-selectin were unaltered on hCMEC/D3 by either DKA-P or DKA-CM. In summary, DKA elicits inflammation in children associated with changes in circulating cytokines/chemokines. Increased CXCL1/CXCL8 instigated PMN adhesion to hCMEC/D3, possibly contributing to DKA-associated intracranial vascular complications.

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Khalyfa, Abdelnaby, David Gozal y Leila Kheirandish-Gozal. "Plasma Extracellular Vesicles in Children with OSA Disrupt Blood–Brain Barrier Integrity and Endothelial Cell Wound Healing In Vitro". International Journal of Molecular Sciences 20, n.º 24 (10 de diciembre de 2019): 6233. http://dx.doi.org/10.3390/ijms20246233.

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Pediatric obstructive sleep apnea (P-OSA) is associated with neurocognitive deficits and endothelial dysfunction, suggesting the possibility that disruption of the blood–brain barrier (BBB) may underlie these morbidities. Extracellular vesicles (EVs), which include exosomes, are small particles involved in cell–cell communications via different mechanisms and could play a role in OSA-associated end-organ injury. To examine the roles of EVs in BBB dysfunction, we recruited three groups of children: (a) absence of OSA or cognitive deficits (CL, n = 6), (b) OSA but no evidence of cognitive deficits (OSA-NC(−), n = 12), and (c) OSA with evidence of neurocognitive deficits (OSA-NC(+), n = 12). All children were age-, gender-, ethnicity-, and BMI-z-score-matched, and those with OSA were also apnea–hypopnea index (AHI)-matched. Plasma EVs were characterized, quantified, and applied on multiple endothelial cell types (HCAEC, HIAEC, human HMVEC-D, HMVEC-C, HMVEC-L, and hCMEC/D3) while measuring monolayer barrier integrity and wound-healing responses. EVs from OSA children induced significant declines in hCMEC/D3 transendothelial impedance compared to CL (p < 0.001), and such changes were greater in NC(+) compared to NC(−) (p < 0.01). The effects of EVs from each group on wound healing for HCAEC, HIAEC, HMVED-d, and hCMEC/D3 cells were similar, but exhibited significant differences across the three groups, with evidence of disrupted wound healing in P-OSA. However, wound healing in HMVEC-C was only affected by NC(+) (p < 0.01 vs. NC(−) or controls (CO). Furthermore, no significant differences emerged in HMVEC-L cell wound healing across all three groups. We conclude that circulating plasma EVs in P-OSA disrupt the integrity of the BBB and exert adverse effects on endothelial wound healing, particularly among OSA-NC(+) children, while also exhibiting endothelial cell type selectivity. Thus, circulating EVs cargo may play important roles in the emergence of end-organ morbidity in pediatric OSA.

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Samak, Mostafa, Diana Kaltenborn, Andreas Kues, Ferdinand Le Noble, Rabea Hinkel y Giulia Germena. "Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes". Biomedicines 10, n.º 1 (28 de diciembre de 2021): 58. http://dx.doi.org/10.3390/biomedicines10010058.

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Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

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Roig-Carles, David, Eduard Willms, Ruud D. Fontijn, Sarai Martinez-Pacheco, Imre Mäger, Helga E. de Vries, Mark Hirst et al. "Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation". Pharmaceutics 13, n.º 9 (21 de septiembre de 2021): 1525. http://dx.doi.org/10.3390/pharmaceutics13091525.

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Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

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Cucullo, Luca, Pierre-Olivier Couraud, Babette Weksler, Ignacio-Andres Romero, Mohammed Hossain, Edward Rapp y Damir Janigro. "Immortalized Human Brain Endothelial Cells and Flow-Based Vascular Modeling: A Marriage of Convenience for Rational Neurovascular Studies". Journal of Cerebral Blood Flow & Metabolism 28, n.º 2 (4 de julio de 2007): 312–28. http://dx.doi.org/10.1038/sj.jcbfm.9600525.

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In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood—Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope = 0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1β) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.

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Chen, Yanlan, Wen Huang, Wenlin Jiang, Xianghong Wu, Biao Ye y Xiaoting Zhou. "HIV-1 Tat Regulates Occludin and AβTransfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway". Oxidative Medicine and Cellular Longevity 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/4196572.

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HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβtransfer receptors.

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Chandrasekar, Bysani, Kirankumar Vemula, Rama Mohan Surabhi, Min Li-Weber, Laurie B. Owen-Schaub, Liselotte E. Jensen y Srinivas Mummidi. "Activation of Intrinsic and Extrinsic Proapoptotic Signaling Pathways in Interleukin-18-mediated Human Cardiac Endothelial Cell Death". Journal of Biological Chemistry 279, n.º 19 (11 de febrero de 2004): 20221–33. http://dx.doi.org/10.1074/jbc.m313980200.

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Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-κB DNA binding activity; 2) induces κB-driven luciferase activity; 3) induces IL-1β and TNF-α expression via NF-κB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-XL; 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-XSexpression; 6) inducesfasand Fas-L promoter activities via NF-κB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochromecrelease into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-XLchimeric phosphorothioated 2′-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-α, and NF-κB p65 knockdown or dominant negative IκB-α and dominant negative IκB-β or kinase dead IKK-β significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1β, tumor necrosis factor-α, or interferon-γ. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.

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Ito, Shingo, Mariko Oishi, Seiryo Ogata, Tatsuki Uemura, Pierre-Olivier Couraud, Takeshi Masuda y Sumio Ohtsuki. "Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro". Pharmaceutics 12, n.º 6 (23 de junio de 2020): 579. http://dx.doi.org/10.3390/pharmaceutics12060579.

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Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood–brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases.

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Zougbédé, Sergine, Florence Miller, Philippe Ravassard, Angelita Rebollo, Liliane Cicéron, Pierre-Olivier Couraud, Dominique Mazier y Alicia Moreno. "Metabolic Acidosis Induced by Plasmodium Falciparum Intraerythrocytic Stages Alters Blood—Brain Barrier Integrity". Journal of Cerebral Blood Flow & Metabolism 31, n.º 2 (4 de agosto de 2010): 514–26. http://dx.doi.org/10.1038/jcbfm.2010.121.

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The pathogenesis of cerebral malaria (CM) remains largely unknown. There is growing evidence that combination of both parasite and host factors could be involved in blood–brain barrier (BBB) breakdown. However, lack of adequate in vitro model of human BBB so far hampered molecular studies. In this article, we propose the use of hCMEC/D3 cells, a well-established human cerebral microvascular endothelial cell (EC) line, to study BBB breakdown induced by Plasmodium falciparum-parasitized red blood cells and environmental conditions. We show that coculture of parasitized erythrocytes with hCMEC/D3 cells induces cell adhesion and paracellular permeability increase, which correlates with disorganization of zonula occludens protein 1 expression pattern. Permeability increase and modification of tight junction proteins distribution are cytoadhesion independent. Finally, we show that permeability of hCMEC/D3 cell monolayers is mediated through parasite induced metabolic acidosis, which in turns correlates with apoptosis of parasitized erythrocytes. This new coculture model represents a very useful tool, which will improve the knowledge of BBB breakdown and the development of adjuvant therapies, together with antiparasitic drugs.

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Tai, Leon M., A. Jane Loughlin, David K. Male y Ignacio A. Romero. "P-Glycoprotein and Breast Cancer Resistance Protein Restrict Apical-to-Basolateral Permeability of Human Brain Endothelium to Amyloid-β". Journal of Cerebral Blood Flow & Metabolism 29, n.º 6 (15 de abril de 2009): 1079–83. http://dx.doi.org/10.1038/jcbfm.2009.42.

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The clearance of amyloid beta (Aβ) from the brain represents a novel therapeutic target for Alzheimer's disease. Conflicting data exist regarding the contribution of adenosine triphosphatebinding cassette transporters to the clearance of Aβ through the blood-brain barrier. Therefore, we investigated whether Aβ could be a substrate for P-glycoprotein (P-gp) and/or for breast cancer resistance protein (BCRP) using a human brain endothelial cell line, hCMEC/D3. Inhibition of P-gp and BCRP increased apical-to-basolateral, but not basolateral-to-apical, permeability of hCMEC/D3 cells to 125l Aβ 1–40. Our in vitro data suggest that P-gp and BCRP might act to prevent the blood-borne Aβ 1–40 from entering the brain.

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Dal Magro, Roberta, Agostina Vitali, Stefano Fagioli, Alberto Casu, Andrea Falqui, Beatrice Formicola, Lorenzo Taiarol et al. "Oxidative Stress Boosts the Uptake of Cerium Oxide Nanoparticles by Changing Brain Endothelium Microvilli Pattern". Antioxidants 10, n.º 2 (9 de febrero de 2021): 266. http://dx.doi.org/10.3390/antiox10020266.

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Vascular oxidative stress is considered a worsening factor in the progression of Alzheimer’s disease (AD). Increased reactive oxygen species (ROS) levels promote the accumulation of amyloid-β peptide (Aβ), one of the main hallmarks of AD. In turn, Aβ is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aβ on brain capillary endothelial cells was observed to compromise the blood–brain barrier functionality. In this context, antioxidant compounds might provide therapeutic benefits. To this aim, we investigated the antioxidant activity of cerium oxide nanoparticles (CNP) in human cerebral microvascular endothelial cells (hCMEC/D3) exposed to Aβ oligomers. Treatment with CNP (13.9 ± 0.7 nm in diameter) restored basal ROS levels in hCMEC/D3 cells, both after acute or prolonged exposure to Aβ. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aβ. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aβ or H2O2), might be involved in the enhancement of CNP uptake. This finding opens the possibility to exploit the modulation of endothelial microvilli pattern to improve the uptake of anti-oxidant particles designed to counteract ROS-mediated cerebrovascular dysfunctions.

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Becker, Guillaume, Sylvia Da Silva, Amelia-Naomi Sabo, Maria Cristina Antal, Véronique Kemmel y Laurent Monassier. "Blood–Brain Barrier Permeability: Is 5-Hydroxytryptamine Receptor Type 4 a Game Changer?" Pharmaceutics 13, n.º 11 (3 de noviembre de 2021): 1856. http://dx.doi.org/10.3390/pharmaceutics13111856.

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Serotonin affects many functions in the body, both in the central nervous system (CNS) and the periphery. However, its effect on the blood–brain barrier (BBB) in separating these two worlds has been scarcely investigated. The aim of this work was to characterize the serotonin receptor 5-HT4 in the hCMEC/D3 cell line, in the rat and the human BBB. We also examined the effect of prucalopride, a 5-HT4 receptor agonist, on the permeability of the hCMEC/D3 in an in vitro model of BBB. We then confirmed our observations by in vivo experiments. In this work, we show that the 5-HT4 receptor is expressed by hCMEC/D3 cells and in the capillaries of rat and human brains. Prucalopride increases the BBB permeability by downregulating the expression of the tight junction protein, occludin. This effect is prevented by GR113808, a 5-HT4 receptor antagonist, and is mediated by the Src/ERK1/2 signaling pathway. The canonical G-protein-dependent pathway does not appear to be involved in this phenomenon. Finally, the administration of prucalopride increases the diffusion of Evans blue in the rat brain parenchyma, which is synonymous with BBB permeabilization. All these data indicate that the 5-HT4 receptor contributes to the regulation of BBB permeability.

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Luissint, Anny-Claude, Christian Federici, François Guillonneau, Fabrice Chrétien, Luc Camoin, Fabienne Glacial, Kayathiri Ganeshamoorthy y Pierre-Olivier Couraud. "Guanine Nucleotide-Binding Protein Gαi2: A New Partner of Claudin-5 that Regulates Tight Junction Integrity in Human Brain Endothelial Cells". Journal of Cerebral Blood Flow & Metabolism 32, n.º 5 (15 de febrero de 2012): 860–73. http://dx.doi.org/10.1038/jcbfm.2011.202.

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The blood—brain barrier (BBB) selectively controls the exchanges between the blood and the brain: it is formed by tight junctions (TJs) between adjacent microvascular endothelial cells. The transmembrane protein claudin-5 is known as a key TJ protein at the BBB, although, the molecular mechanisms by which it regulates TJ tightness are poorly understood. To identify putative claudin-5 partners that contribute to TJ integrity, claudin-5-enriched membrane microdomains were prepared by cell fractionation, using the human brain endothelial cell line hCMEC/D3 and claudin-5 immunoprecipitates were submitted to tandem mass spectrometry. Because a high concentration of mannitol is known to transiently destabilize TJs, this analysis was performed in basal conditions, after mannitol treatment, and after recovery of TJ integrity. We here demonstrate that the G-protein subunit αi2 (Gαi2) interacts with claudin-5 and that association is correlated with TJ integrity in hCMEC/D3 cells; also, a selective expression of Gαi2 is observed in human brain vasculature in situ. Moreover, small interfering RNA-mediated depletion of Gαi2 or claudin-5 in hCMEC/D3 cells similarly increases their paracellular permeability and delays TJ recovery after mannitol treatment. Altogether, our results identify Gαi2 as a novel claudin-5 partner required for TJ integrity in brain endothelial cells.

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Pinto, Miguel, Carlos Fernandes, Eva Martins, Renata Silva, Sofia Benfeito, Fernando Cagide, Ricardo F. Mendes et al. "Boosting Drug Discovery for Parkinson’s: Enhancement of the Delivery of a Monoamine Oxidase-B Inhibitor by Brain-Targeted PEGylated Polycaprolactone-Based Nanoparticles". Pharmaceutics 11, n.º 7 (12 de julio de 2019): 331. http://dx.doi.org/10.3390/pharmaceutics11070331.

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The current pharmacological treatments for Parkinson’s disease only offer symptomatic relief to the patients and are based on the administration of levodopa and catechol-O-methyltransferase or monoamine oxidase-B inhibitors (IMAO-B). Since the majority of drug candidates fail in pre- and clinical trials, due largely to bioavailability pitfalls, the use of polymeric nanoparticles (NPs) as drug delivery systems has been reported as an interesting tool to increase the stealth capacity of drugs or help drug candidates to surpass biological barriers, among other benefits. Thus, a novel potent, selective, and reversible IMAO-B (chromone C27, IC50 = 670 ± 130 pM) was encapsulated in poly(caprolactone) (PCL) NPs by a nanoprecipitation process. The resulting C27-loaded PEGylated PCL NPs (~213 nm) showed high stability and no cytotoxic effects in neuronal (SH-SY5Y), epithelial (Caco-2), and endothelial (hCMEC/D3) cells. An accumulation of PEGylated PCL NPs in the cytoplasm of SH-SY5Y and hCMEC/D3 cells was also observed, and their permeation across Caco-2 and hCMEC/D3 cell monolayers, used as in vitro models of the human intestine and blood-brain barrier, respectively, was demonstrated. PEGylated PCL NPs delivered C27 at concentrations higher than the MAO-B IC50 value, which provides evidence of their relevance to solving the drug discovery pitfalls.

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Marqués, Javier, Joaquín Fernández-Irigoyen, Elena Ainzúa, María Martínez-Azcona, Adriana Cortés, Carmen Roncal, Josune Orbe, Enrique Santamaría y Guillermo Zalba. "NADPH Oxidase 5 (NOX5) Overexpression Promotes Endothelial Dysfunction via Cell Apoptosis, Migration, and Metabolic Alterations in Human Brain Microvascular Endothelial Cells (hCMEC/D3)". Antioxidants 11, n.º 11 (29 de octubre de 2022): 2147. http://dx.doi.org/10.3390/antiox11112147.

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NADPH oxidases (NOX) constitute the main reactive oxygen species (ROS) source in blood vessels. An oxidative stress situation due to ROS overproduction can lead into endothelial dysfunction, a molecular mechanism that precedes cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, and stroke. NOX5 is the last discovered member of the NOX family, studied in a lesser extent due to its absence in the rodent genome. Our objective was to describe the phenotypic alterations produced by an oxidative stress situation derived from NOX5 overexpression in an endothelial in vitro model. The in vitro model consists of the hCMEC/D3 cell line, derived from brain microvascular endothelium, infected with a recombinant NOX5-b adenovirus. After an initial proteomic analysis, three phenotypic alterations detected in silico were studied: cell proliferation and apoptosis, general and mitochondrial metabolism, and migration capacity. NOX5 infection of hCMEC/D3 generates a functional protein and an increase in ROS production. This model produced changes in the whole cell proteome. The in silico analysis together with in vitro validations demonstrated that NOX5 overexpression inhibits proliferation and promotes apoptosis, metabolic alterations and cell migration in hCMEC/D3 cells. NOX5 overexpression in endothelial cells leads to phenotypic changes that can lead to endothelial dysfunction, the onset of atherosclerosis, myocardial infarction, and stroke.

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Haarmann, Axel, Michael Schuhmann, Christine Silwedel, Camelia-Maria Monoranu, Guido Stoll y Mathias Buttmann. "Human Brain Endothelial CXCR2 is Inflammation-Inducible and Mediates CXCL5- and CXCL8-Triggered Paraendothelial Barrier Breakdown". International Journal of Molecular Sciences 20, n.º 3 (30 de enero de 2019): 602. http://dx.doi.org/10.3390/ijms20030602.

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Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood–brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood–brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood–brain barrier stabilization.

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20

Wu, Dongsheng, Camilla Cerutti, Miguel A. Lopez-Ramirez, Gareth Pryce, Josh King-Robson, Julie E. Simpson, Susanne MA van der Pol et al. "Brain Endothelial miR-146a Negatively Modulates T-Cell Adhesion through Repressing Multiple Targets to Inhibit NF-κB Activation". Journal of Cerebral Blood Flow & Metabolism 35, n.º 3 (marzo de 2015): 412–23. http://dx.doi.org/10.1038/jcbfm.2014.207.

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Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.

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21

Grossen, Philip, Gabriela Québatte, Dominik Witzigmann, Cristina Prescianotto-Baschong, Le-Ha Dieu y Jörg Huwyler. "Functionalized Solid-Sphere PEG-b-PCL Nanoparticles to Target Brain Capillary Endothelial CellsIn Vitro". Journal of Nanomaterials 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/7818501.

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Nanoparticles are increasingly used to implement drug targeting strategies. In the present study, solid-sphere nanoparticles (SNPs) made of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) were covalently linked to a monoclonal antibody (83-14 mAb) targeted against the human insulin receptor that is highly expressed on human brain microvascular endothelial cells. Resulting targeted SNPs were characterized using transmission electron microscopy (TEM), cryo-TEM, dynamic light scattering, and fluorescence correlation spectroscopy. The critical aggregation concentration was determined using a fluorescence approach. Interaction with a well-characterized humanin vitromodel of the blood-brain barrier (hCMEC/D3) was analysed using an array of methods (flow cytometry, confocal laser scanning microscopy, and TEM). The toxicity on hCMEC/D3 cells and in addition on the human liver cell line HepG2 was assessed using the MTT assay. SNPs with a diameter of 80 nm and a homogeneous size distribution were obtained. Successful conjugation of 83-14 mAb using a heterobifunctional linker resulted in 5-6 molecules of fluorescently labeled 83-14 mAb per SNP. Functionalized SNPs were taken up by hCMEC/D3 cells efficiently without showing a significant toxic effect on cells of the blood-brain barrier and HepG2 cells. These results indicate that functionalized PEG-b-PCL SNPs are a promising candidate to deliver drugs to the CNS.

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Sorosina, Melissa, Silvia Peroni, Elisabetta Mascia, Silvia Santoro, Ana Maria Osiceanu, Laura Ferrè, Ferdinando Clarelli et al. "Involvement of NINJ2 Protein in Inflammation and Blood–Brain Barrier Transmigration of Monocytes in Multiple Sclerosis". Genes 13, n.º 11 (25 de octubre de 2022): 1946. http://dx.doi.org/10.3390/genes13111946.

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Multiple sclerosis (MS) is an inflammatory neurodegenerative disorder of the central nervous system (CNS). The migration of immune cells into the CNS is essential for its development, and plasma membrane molecules play an important role in triggering and maintaining the inflammation. We previously identified ninjurin2, a plasma membrane protein encoded by NINJ2 gene, as involved in the occurrence of relapse under Interferon-β treatment in MS patients. The aim of the present study was to investigate the involvement of NINJ2 in inflammatory conditions and in the migration of monocytes through the blood–brain barrier (BBB). We observed that NINJ2 is downregulated in monocytes and in THP-1 cells after stimulation with the pro-inflammatory cytokine LPS, while in hCMEC/D3 cells, which represent a surrogate of the BBB, LPS stimulation increases its expression. We set up a transmigration assay using an hCMEC/D3 transwell-based model, finding a higher transmigration rate of monocytes from MS subjects compared to healthy controls (HCs) in the case of an activated hCMEC/D3 monolayer. Moreover, a positive correlation between NINJ2 expression in monocytes and monocyte migration rate was observed. Overall, our results suggest that ninjurin2 could be involved in the transmigration of immune cells into the CNS in pro-inflammatory conditions. Further experiments are needed to elucidate the exact molecular mechanisms.

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Hasseli, R., M. M. Fürst, P. Singh, U. Müller-Ladner, M. Kaps, F. Blaes, T. Gerriets y M. Tschernatsch. "AB0157 IGG FROM PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND SYSTEMIC SCLEROSIS HAVE AN INFLUENCE ON COAGULATION FACTORS IN HUMAN CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS IN-VITRO". Annals of the Rheumatic Diseases 79, Suppl 1 (junio de 2020): 1379. http://dx.doi.org/10.1136/annrheumdis-2020-eular.2631.

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Background:Endothelial cells from the microvasculature (hBMEC) of the brain show significant morphological and functional differences compared to EC from other anatomical areas. They are characterized by tight junctions, are not fenestrated and show less active transport mechanisms. On the other hand, the mitochondrial density is relatively high in hBMEC due to the high cerebral glucose metabolism.It could be already observed that interferon-α from SLE-sera induces the expression of MHC class I molecules on human dermal microendothelial cell line, but it is not known whether this also occurs on hBMEC. hBMECs can synthesize pro-inflammatory cytokines and chemokines such as IL-1β, but in lower concentrations than human umbilical vein endothelial cells.Patients suffering from systemic lupus erythematosus (SLE) or systemic sclerosis (SSc) show a wide spectrum of central nervous symptoms. Both, SLE and SSc, are characterised by different autoantibodies and endothelial vascular damage, especially in microvessels. 10-40% of patients with SLE suffer from lupus vasculopathy. Vascular dysfunction is one of the earliest pathological changes in SSc. Anti-endothelial autoantibodies (AECA) appear in SLE as well as in SSc and other connective tissue diseases. Research within the last years revealed that AECA play a critical role within the vascular pathogenesis of SLE and SSc. So far there is no evidence that AECA bind to hBMEC and it is not clear whether they have an effect on this special endothelial class.Objectives:In this project, we investigated if autoantibodies against hBMEC are detectable in SLE and SSc patients and if they have an influence on the activation of the endothelium by inducing adhesion molecules and on haemostasis by inducing factors of the clotting cascade.Methods:HiTrap Protein G HP antibody purification columns were used to purify IgG antibodies. Flow cytometry was used for analysis of autoantibodies against human cerebral microvascular endothelial cell line (hCMEC/D3). 26 sera of patients with SLE and 29 sera of patients with SSc were tested for presence of autoantibodies against hCMEC/D3. To analyse in vitro effects on hCMEC/D3, we measured changes in the expression of the following surface proteins: ICAM-1, VCAM-1, MHC class I and II, tissue factor, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA, each before and after three- and 24-hours incubation with IgG-fractions. IgG fractions of 12 SLE patients, 13 SSc patients and 13 healthy control persons (HC) were tested.Results:Autoantibodies against hCMEC/D3 were found in 21 of 26 patients with SLE (81%) and in 19 of 29 patients with SSc (66%) (p > 0.05) but not in healthy donors. After three hours incubation of hCMEC/D3 IgG-fractions, an upregulation of tissue factor by SSc-IgG (6.7% ± 5.2%) compared to HC-IgG (1.1% ± 2.8%, p < 0.01) and to SLE-IgG (1.6% ± 3.9%, p < 0.05), was detectable.There was no significant correlation between changes in surface protein expression and detection of ANA or of anti-hCMEC/D3 antibodies (p > 0.05).No change in expression of ICAM-1, VCAM-1, MHC class I and II, von-Willebrand-Factor, E-Selectin, P-Selectin, Thrombomodulin, CD73 and t-PA could be detected after incubation with IgG-fractions.Conclusion:Both, patients with SLE and patients with SSc showed autoantibodies against hBMEC. IgG fractions of patients with SSc, but not with SLE, induced an upregulation of tissue factor on the cell surface of hCMEC/D3. This could be an indicator for a direct pathogenic effect of AECA on hBMEC and might have an influence on haemostasis by activating the clotting cascade. Inhibition of these antibodies could reduce cerebral involvement of SSc.References:[1]Weksler BB, Subileau EA, Perriere N, et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. Faseb J 2005;19:1872-1874.Disclosure of Interests:Rebecca Hasseli: None declared, Magdalena Maria Fürst: None declared, Pratibha Singh: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Manfred Kaps: None declared, Franz Blaes: None declared, Tibo Gerriets: None declared, Marlene Tschernatsch: None declared

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Lu, Yan, Zhendong Xu, Fuyi Shen, Rong Lin, Haibing Li, Xiang Lv y Zhiqiang Liu. "Propofol Protects Against TNF-α-induced Blood-brain Barrier Disruption via the PIM-1/eNOS/NO Pathway". Current Neurovascular Research 17, n.º 4 (14 de diciembre de 2020): 471–79. http://dx.doi.org/10.2174/1567202617999200819142021.

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Background: The Inflammatory cytokine, tumor necrosis factor-α (TNF-α), disrupts blood-brain barrier (BBB). Propofol reportedly exerts an anti-inflammatory effect in the central nervous system. Objective: We hypothesized that propofol could provide a protective effect against TNF-α-induced disruption in human cerebral microvascular endothelial cells (hCMEC/D3 cells) and explored the underlying mechanisms. Methods: The hCMEC/D3 cell monolayers were pretreated with propofol, followed by TNF-α treatment. The integrity of BBB was reflected by assessing the trans-endothelial electrical resistance (TEER) and determining the expression of proteins within tight junctions (TJs). The effect of propofol on TNF-α-modulated nitric oxide production was measured by a nitrate reductase assay kit. The expression of ZO-1, claudin-5, occludin, TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), proviral-integration site for Moloney murine leukaemia virus (PIM)-1kinase, the phosphorylation of endothelial nitric oxide synthase at ser633 (peNOS-ser633) were detected by western blot. Results: In hCMEC/D3 cells, TNF-α treatment markedly disrupted the integrity of BBB. Further, we found TNF-α treatment could increase the expression of PIM-1, then activate the phosphorylation of eNOS and induce the release of nitric oxide (NO). More importantly, we found that TNF- α-impaired BBB integrity could be reversed by propofol. Conclusion: These results suggest that the PIM-1/eNOS/NO pathway plays a vital role, in which Propofol protects against TNF-α-induced blood-brain barrier disruption.

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Goeritzer, Madeleine, Eva Bernhart, Ioanna Plastira, Helga Reicher, Christina Leopold, Thomas O. Eichmann, Gerald Rechberger et al. "Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro". International Journal of Molecular Sciences 21, n.º 3 (9 de febrero de 2020): 1143. http://dx.doi.org/10.3390/ijms21031143.

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During inflammation, activated leukocytes release cytotoxic mediators that compromise blood–brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO−/− compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

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Ajikumar, Anjana, Merete B. Long, Paul R. Heath, Stephen B. Wharton, Paul G. Ince, Victoria C. Ridger y Julie E. Simpson. "Neutrophil-Derived Microvesicle Induced Dysfunction of Brain Microvascular Endothelial Cells In Vitro". International Journal of Molecular Sciences 20, n.º 20 (22 de octubre de 2019): 5227. http://dx.doi.org/10.3390/ijms20205227.

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The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.

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Park, Jee Hyun, Oriana E. Hawkins, Navin K. Chintala, Jon A. Weidanz y Ulrich Bickel. "Internalization of T-Cell Receptor Mimic Antibody RL6A by Brain Derived Endothelial Cells Analyzed by Flow Cytometry". Journal of Immunology 196, n.º 1_Supplement (1 de mayo de 2016): 214.12. http://dx.doi.org/10.4049/jimmunol.196.supp.214.12.

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Abstract T-cell receptor mimic monoclonal antibodies (TCRm) recognize peptide-MHC class I complexes. They combine the specificity of T-cell receptors with high binding affinities in the low nanomolar to picomolar range characteristic of monoclonal antibodies. The TCRm RL6A targets YLLPAIVHI peptide-HLA-A2 complexes, where the peptide is derived from the p68 RNA helicase protein. Previous work showed that this target is expressed by brain endothelial cells forming the blood-brain barrier (BBB). Upon binding, RL6A crosses the BBB via a receptor-mediated transcytosis mechanism. Confocal microscopy indicated that, after internalization, RL6A co-localized with the early endosomal marker (EEA1). Here we used conventional flow cytometry and imaging flow cytometry to quantitatively analyze the internalization process. Endothelial cell monolayers formed by hCMEC/D3 cells were incubated with unlabeled RL6A for time periods up to 120 min. Surface bound RL6A was detected by a secondary, PE-labeled antibody. RL6A disappeared in a biphasic pattern from the surface of hCMEC/D3 cells, with an initial half-life (t1/2) of internalization of about 3 min and a second, much slower phase (half life about 3 h) likely reflecting recycling to the membrane. In a complementary approach using imaging flow cytometry (Amnis ImagestreamX) and IDEAS analysis software, the internalization of AlexaFluor488-RL6A could be directly visualized and evaluated. The results confirmed that RL6A internalizes rapidly into hCMEC/D3 cells. In conclusion, flow cytometry is a suitable tool to analyze trafficking of specific peptide-HLA complexes in endothelial cells.

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Marcelo, Gonçalo, Jessica Ariana-Machado, Maria Enea, Helena Carmo, Benito Rodríguez-González, José Luis Capelo, Carlos Lodeiro y Elisabete Oliveira. "Toxicological Evaluation of Luminescent Silica Nanoparticles as New Drug Nanocarriers in Different Cancer Cell Lines". Materials 11, n.º 8 (28 de julio de 2018): 1310. http://dx.doi.org/10.3390/ma11081310.

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Luminescent mesoporous silica nanoparticles, CdTeQDs@MNs@PEG1, SiQDs@Isoc@MNs and SiQDs@Isoc@MNs@PEG2, were successfully synthetized and characterized by SEM, TEM, XRD, N2 nitrogen isotherms, 1H NMR, IR, absorption, and emission spectroscopy. Cytotoxic evaluation of these nanoparticles was performed in relevant in vitro cell models, such as human hepatoma HepG2, human brain endothelial (hCMEC/D3), and human epithelial colorectal adenocarcinoma (Caco-2) cell lines. None of the tested nanoparticles showed significant cytotoxicity in any of the three performed assays (MTT/NR/ LDH) compared with the respective solvent and/or coating controls, excepting for CdTeQDs@MNs@PEG1 nanoparticles, where significant toxicity was noticed in hCMEC/D3 cells. The results presented reveal that SiQDs-based mesoporous silica nanoparticles are promising nanoplatforms for cancer treatment, with a pH-responsive drug release profile and the ability to load 80% of doxorubicin.

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Dimova, Ivanka, Svetla Danova, Ekaterina Nikolova y Miglena Koprinarova. "Aloe-emodin triggers ROS and Ca 2+ production and decreases the levels of mitochondrial membrane potential of human brain capillary endothelial cells". International Journal of Phytomedicine 9, n.º 3 (2 de septiembre de 2017): 511. http://dx.doi.org/10.5138/09750185.2138.

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<p>The aim of this work was to investigate the mechanisms of cytotoxicity of phyto-hydroxyanthraquinone aloe-emodin (AE) on human brain microvascular endothelial cell line hCMEC/D3 and to assess the cellular response in the early stage of treatment in order to extend the knowledge of AE’s anti-angiogenic properties. The immortalized human brain capillary endothelial cells hCMEC/D3 were treated with a series of AE concentrations (5 - 200 μM) for a period of 24 hours. The cell viability was determined by MTS assay. The cellular adenosine triphosphate (ATP) levels were evaluated by CellTiter-Glo® luminescent assay. The intracellular reactive oxygen species (ROS) were determined by 2’,7’-dichlorofluorescein (CM- H2DCFDA) fluorescence assay. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine methyl ester (TMRM) staining, while Fluo-4 was used to measure 2 the intracellular free Ca 2+ concentrations inside living cells analysed by High Content Analysis using the Arrayscan VTI 740. Twenty-four- hour treatment of hCMEC/D3 cells with AE, in concentrations between 50 and 200 µM, decreased the cell viability as well as the intracellular ATP levels in a dose- dependent manner. Increased ROS production and disruption of the mitochondrial membrane potential have also been detected. Notably, AE at a concentration greater than 5 µM dramatically increased intracellular calcium levels. Our results collectively indicate that AE inhibits proliferation of human brain microvascular cells via a mechanism involving ROS generation, disruption of Ca 2+ homeostasis and mitochondrial damage.</p>

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Huang, Wen, Geun Bae Rha, Lei Chen, Melissa J. Seelbach, Bei Zhang, Ibolya E. András, Dennis Bruemmer, Bernhard Hennig y Michal Toborek. "Inhibition of telomerase activity alters tight junction protein expression and induces transendothelial migration of HIV-1-infected cells". American Journal of Physiology-Heart and Circulatory Physiology 298, n.º 4 (abril de 2010): H1136—H1145. http://dx.doi.org/10.1152/ajpheart.01126.2009.

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Telomerase, via its catalytic component telomerase reverse transcriptase (TERT), extends telomeres of eukaryotic chromosomes. The importance of this reaction is related to the fact that telomere shortening is a rate-limiting mechanism for human life span that induces cell senescence and contributes to the development of age-related pathologies. The aim of the present study was to evaluate whether the modulation of telomerase activity can influence human immunodeficiency virus type 1 (HIV-1)-mediated dysfunction of human brain endothelial cells (hCMEC/D3 cells) and transendothelial migration of HIV-1-infected cells. Telomerase activity was modulated in hCMEC/D3 cells via small interfering RNA-targeting human TERT (hTERT) or by using a specific pharmacological inhibitor of telomerase, TAG-6. The inhibition of hTERT resulted in the upregulation of HIV-1-induced overexpression of intercellular adhesion molecule-1 via the nuclear factor-κB-regulated mechanism and induced the transendothelial migration of HIV-1-infected monocytic U937 cells. In addition, the blocking of hTERT activity potentiated a HIV-induced downregulation of the expression of tight junction proteins. These results were confirmed in TERT-deficient mice injected with HIV-1-specific protein Tat into the cerebral vasculature. Further studies revealed that the upregulation of matrix metalloproteinase-9 is the underlying mechanisms of disruption of tight junction proteins in hCMEC/D3 cells with inhibited TERT and exposed to HIV-1. These results indicate that the senescence of brain endothelial cells may predispose to the HIV-induced upregulation of inflammatory mediators and the disruption of the barrier function at the level of the brain endothelium.

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Kurosawa, Toshiki, Yuma Tega, Yasuo Uchida, Kei Higuchi, Hidetsugu Tabata, Takaaki Sumiyoshi, Yoshiyuki Kubo, Tetsuya Terasaki y Yoshiharu Deguchi. "Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood–Brain Barrier". Pharmaceutics 14, n.º 8 (12 de agosto de 2022): 1683. http://dx.doi.org/10.3390/pharmaceutics14081683.

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A proton-coupled organic cation (H+/OC) antiporter working at the blood–brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.

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Vu, Kiem, Babette Weksler, Ignacio Romero, Pierre-Olivier Couraud y Angie Gelli. "Immortalized Human Brain Endothelial Cell Line HCMEC/D3 as a Model of the Blood-Brain Barrier Facilitates In Vitro Studies of Central Nervous System Infection by Cryptococcus neoformans". Eukaryotic Cell 8, n.º 11 (18 de septiembre de 2009): 1803–7. http://dx.doi.org/10.1128/ec.00240-09.

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ABSTRACT Cryptococcus neoformans cells must cross the blood-brain barrier prior to invading the central nervous system. Here we demonstrate that the immortalized human brain endothelial cell line HCMEC/D3 is a useful alternative to primary brain endothelial cells as a model of the blood-brain barrier for studies of central nervous system infection.

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Stephan, Delphine, Anais Roger, Jehanne Aghzadi, Sylvie Carmona, Christophe Picard, Jean-Philippe Dales y Sophie Desplat-Jégo. "TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells". Genes 13, n.º 10 (24 de septiembre de 2022): 1714. http://dx.doi.org/10.3390/genes13101714.

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Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood–brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

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Bay, Cindy, Gzona Bajraktari-Sylejmani, Walter E. Haefeli, Jürgen Burhenne, Johanna Weiss y Max Sauter. "Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine". International Journal of Molecular Sciences 23, n.º 7 (26 de marzo de 2022): 3637. http://dx.doi.org/10.3390/ijms23073637.

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The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

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Piazzini, Vieri, Elisa Landucci, Giulia Graverini, Domenico Pellegrini-Giampietro, Anna Bilia y Maria Bergonzi. "Stealth and Cationic Nanoliposomes as Drug Delivery Systems to Increase Andrographolide BBB Permeability". Pharmaceutics 10, n.º 3 (13 de agosto de 2018): 128. http://dx.doi.org/10.3390/pharmaceutics10030128.

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(1) Background: Andrographolide (AG) is a natural compound effective for the treatment of inflammation-mediated neurodegenerative disorders. The aim of this investigation was the preparation of liposomes to enhance the penetration into the brain of AG, by modifying the surface of the liposomes by adding Tween 80 (LPs-AG) alone or in combination with Didecyldimethylammonium bromide (DDAB) (CLPs-AG). (2) Methods: LPs-AG and CLPs-AG were physically and chemically characterized. The ability of liposomes to increase the permeability of AG was evaluated by artificial membranes (PAMPA) and hCMEC/D3 cells. (3) Results: Based on obtained results in terms of size, homogeneity, ζ-potential and EE%. both liposomes are suitable for parenteral administration. The systems showed excellent stability during a month of storage as suspensions or freeze-dried products. Glucose resulted the best cryoprotectant agent. PAMPA and hCMEC/D3 transport studies revealed that LPs-AG and CLPs-AG increased the permeability of AG, about an order of magnitude, compared to free AG without alterations in cell viability. The caveolae-mediated endocytosis resulted the main mechanism of up-take for both formulations. The presence of positive charge increased the cellular internalization of nanoparticles. (4) Conclusions: This study shows that developed liposomes might be ideal candidates for brain delivery of AG.

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Marinovic, Iva, Maria Bartosova, Rebecca Herzog, Juan Manuel Sacnun, Conghui Zhang, Robin Hoogenboom, Markus Unterwurzacher et al. "Understanding Cell Model Characteristics—RNA Expression Profiling in Primary and Immortalized Human Mesothelial Cells, and in Human Vein and Microvascular Endothelial Cells". Cells 11, n.º 19 (5 de octubre de 2022): 3133. http://dx.doi.org/10.3390/cells11193133.

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In vitro studies are essential in pre-clinical research. While choice of cell lines is often driven by handling and cost-effectiveness, in-depth knowledge on specific characteristics is scant. Mesothelial cells, which interact with endothelial cells, are widely used in research, including cancer and drug development, but have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared them to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC). Seventy-seven per cent of 12,760 genes were shared between the 4 cell lines, 1003 were mesothelial and 969 were endothelial cell specific. The transcripts reflected major differences between HPMC and MeT-5A in DNA-related processes, extracellular matrix, migration, proliferation, adhesion, transport, growth factor- and immune response, and between HUVEC and HCMEC in DNA replication, extracellular matrix and adhesion organization. Highly variable shared genes were related to six clusters, cell tissue origin and immortalization, but also cell migration capacity, cell adhesion, regulation of angiogenesis and response to hypoxia. Distinct, cell type specific biological processes were further described by cellular component-, molecular function- and Reactome pathway analyses. We provide crucial information on specific features of the most frequently used mesothelial and endothelial cell lines, essential for appropriate use.

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Kim, Yiyoung, Eun Ji Roh, Hari Prasad Joshi, Hae Eun Shin, Hyemin Choi, Su Yeon Kwon, Seil Sohn y Inbo Han. "Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model". International Journal of Molecular Sciences 22, n.º 20 (12 de octubre de 2021): 11012. http://dx.doi.org/10.3390/ijms222011012.

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In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

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Hoshi, Yutaro, Yasuo Uchida, Takashi Kuroda, Masanori Tachikawa, Pierre-Olivier Couraud, Takashi Suzuki y Tetsuya Terasaki. "Distinct roles of ezrin, radixin and moesin in maintaining the plasma membrane localizations and functions of human blood–brain barrier transporters". Journal of Cerebral Blood Flow & Metabolism 40, n.º 7 (14 de agosto de 2019): 1533–45. http://dx.doi.org/10.1177/0271678x19868880.

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The purpose of this study was to clarify the roles of ERM proteins (ezrin/radixin/moesin) in the regulation of membrane localization and transport activity of transporters at the human blood–brain barrier (BBB). Ezrin or moesin knockdown in a human in vitro BBB model cell line (hCMEC/D3) reduced both BCRP and GLUT1 protein expression levels on the plasma membrane. Radixin knockdown reduced not only BCRP and GLUT1, but also P-gp membrane expression. These results indicate that P-gp, BCRP and GLUT1 proteins are maintained on the plasma membrane via different ERM proteins. Furthermore, moesin knockdown caused the largest decrease of P-gp and BCRP efflux activity among the ERM proteins, whereas GLUT1 influx activity was similarly reduced by knockdown of each ERM protein. To investigate how moesin knockdown reduced P-gp efflux activity without loss of P-gp from the plasma membrane, we examined the role of PKCβI. PKCβI increased P-gp phosphorylation and reduced P-gp efflux activity. Radixin and moesin proteins were detected in isolated human brain capillaries, and their protein abundances were within a 3-fold range, compared with those in hCMEC/D3 cell line. These findings may mean that ezrin, radixin and moesin maintain the functions of different transporters in different ways at the human BBB.

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Artus, Cédric, Fabienne Glacial, Kayathiri Ganeshamoorthy, Nicole Ziegler, Maeva Godet, Thomas Guilbert, Stefan Liebner y Pierre-Olivier Couraud. "The Wnt/Planar Cell Polarity Signaling Pathway Contributes to the Integrity of Tight Junctions in Brain Endothelial Cells". Journal of Cerebral Blood Flow & Metabolism 34, n.º 3 (18 de diciembre de 2013): 433–40. http://dx.doi.org/10.1038/jcbfm.2013.213.

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Wnt morphogens released by neural precursor cells were recently reported to control blood–brain barrier (BBB) formation during development. Indeed, in mouse brain endothelial cells, activation of the Wnt/ β-catenin signaling pathway, also known as the canonical Wnt pathway, was shown to stabilize endothelial tight junctions (TJs) through transcriptional regulation of the expression of TJ proteins. Because Wnt proteins activate several distinct β-catenin-dependent and independent signaling pathways, this study was designed to assess whether the noncanonical Wnt/Par/aPKC planar cell polarity (PCP) pathway might also control TJ integrity in brain endothelial cells. First we established, in the hCMEC/D3 human brain endothelial cell line, that the Par/aPKC PCP complex colocalizes with TJs and controls apicobasal polarization. Second, using an siRNA approach, we showed that the Par/aPKC PCP complex regulates TJ stability and reassembling after osmotic shock. Finally, we provided evidence that Wnt5a signals in hCMEC/D3 cells through activation of the Par/aPKC PCP complex, independently of the Wnt canonical β-catenin-dependent pathway and significantly contributes to TJ integrity and endothelial apicobasal polarity. In conclusion, this study suggests that the Wnt/Par/aPKC PCP pathway, in addition to the Wnt/ β-catenin canonical pathway, is a key regulator of the BBB.

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Veszelka, Szilvia, Mária Mészáros, Gergő Porkoláb, Ágnes Rusznyák, Katalin Szászné Réti-Nagy, Mária A. Deli, Miklós Vecsernyés, Ildikó Bácskay, Judit Váradi y Ferenc Fenyvesi. "Effects of Hydroxypropyl-Beta-Cyclodextrin on Cultured Brain Endothelial Cells". Molecules 27, n.º 22 (10 de noviembre de 2022): 7738. http://dx.doi.org/10.3390/molecules27227738.

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The application of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the treatment of the rare cholesterol and lipid storage disorder Niemann–Pick disease type C opened new perspectives in the development of an efficient therapy. Even if the systemic administration of HPBCD was found to be effective, its low permeability across the blood–brain barrier (BBB) limited the positive neurological effects. Nevertheless, the cellular interactions of HPBCD with brain capillary endothelial cells have not been investigated in detail. In this study, the cytotoxicity, permeability, and cellular internalization of HPBCD on primary rat and immortalized human (hCMEC/D3) brain capillary endothelial cells were investigated. HPBCD shows no cytotoxicity on endothelial cells up to 100 µM, measured by impedance kinetics. Using a fluorescent derivative of HPBCD (FITC-HPBCD) the permeability measurements reveal that on an in vitro triple co-culture BBB model, FITC-HPBCD has low permeability, 0.50 × 10−6 cm/s, while on hCMEC/D3 cell layers, the permeability is higher, 1.86 × 10−5 cm/s. FITC-HPBCD enters brain capillary endothelial cells, is detected in cytoplasmic vesicles and rarely localized in lysosomes. The cellular internalization of HPBCD at the BBB can help to develop new strategies for improved HPBCD effects after systemic administration.

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Liu, Haochen, Yixuan Zhang, Hong Zhang, Sheng Xu, Huimin Zhao y Xiaoquan Liu. "Aβ-Induced Damage Memory in hCMEC/D3 Cells Mediated by Sirtuin-1". International Journal of Molecular Sciences 21, n.º 21 (3 de noviembre de 2020): 8226. http://dx.doi.org/10.3390/ijms21218226.

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It is well accepted by the scientific community that the accumulation of beta-amyloid (Aβ) may be involved in endothelial dysfunction during Alzheimer’s disease (AD) progression; however, anti-Aβ anti-bodies, which remove Aβ plaques, do not improve cerebrovascular function in AD animal models. The reasons for these paradoxical results require investigation. We hypothesized that Aβ exposure may cause persistent damage to cerebral endothelial cells even after Aβ is removed (referred to as cerebrovascular endothelial damage memory). In this study, we aimed to investigate whether cerebrovascular endothelial damage memory exists in endothelial cells. hCMEC/D3 cells were treated with Aβ1–42 for 12 h and then Aβ1–42 was withdrawn for another 12 h incubation to investigate whether cerebrovascular endothelial damage memory exists in endothelial cells. A mechanism-based kinetics progression model was developed to investigate the dynamic characters of the cerebrovascular endothelial damage. After Aβ1–42 was removed, the sirt-1 levels returned to normal but the cell vitality did not improve, which suggests that cerebrovascular endothelial damage memory may exist in endothelial cells. Sirt-1 activator SRT2104 and NAD+ (Nicotinamide Adenine Dinucleotide) supplement may dose-dependently relieve the cerebrovascular endothelial damage memory. sirt-1 inhibitor EX527 may exacerbate the cerebrovascular endothelial damage memory. Kinetics analysis suggested that sirt-1 is involved in initiating the cerebrovascular endothelial damage memory; otherwise, NAD+ exhaustion plays a vital role in maintaining the cerebrovascular endothelial damage memory. This study provides a novel feature of cerebrovascular endothelial damage induced by Aβ.

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Fasler-Kan, Elizaveta, Claudia Suenderhauf, Natasha Barteneva, Birk Poller, Daniel Gygax y Jörg Huwyler. "Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3". Brain Research 1354 (octubre de 2010): 15–22. http://dx.doi.org/10.1016/j.brainres.2010.07.077.

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Yin, Yujie, Qian Zhang, Qifei Zhao, Guoyuan Ding, Cong Wei, Liping Chang, Hongrong Li et al. "Tongxinluo Attenuates Myocardiac Fibrosis after Acute Myocardial Infarction in Rats via Inhibition of Endothelial-to-Mesenchymal Transition". BioMed Research International 2019 (16 de junio de 2019): 1–13. http://dx.doi.org/10.1155/2019/6595437.

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Endothelial-to-mesenchymal transition (EndMT) is an essential mechanism in myocardial fibrosis (MF). Tongxinluo (TXL) has been confirmed to protect the endothelium against reperfusion injury after acute myocardial infarction (AMI). However, whether TXL can inhibit MF after AMI via inhibiting EndMT remained unknown. This study aims to identify the role of EndMT in MF after AMI as well as the protective effects and underlying mechanisms of TXL on MF. The AMI model was established in rats by ligating left anterior descending coronary artery. Then, rats were administered with high- (0.8 g·kg−1·d−1), mid- (0.4 g·kg−1·d−1), and low- (0.2 g·kg−1·d−1) dose Tongxinluo and benazepril for 4 weeks, respectively. Cardiac function, infarct size, MF, and related indicators of EndMT were measured. In vitro, human cardiac microvascular endothelial cells (HCMECs) were pretreated with TXL for 4 h and then incubated in hypoxia conditions for 3 days to induce EndMT. Under this hypoxic condition, neuregulin-1 (NRG-1) siRNA were further applied to silence NRG-1 expression. Immunofluorescence microscopy was used to assess expression of endothelial marker of vWF and fibrotic marker of Vimentin. Related factors of EndMT were determined by Western blot analysis. TXL treatment significantly improved cardiac function, ameliorated MF, reduced collagen of fibrosis area (types I and III collagen) and limited excessive extracellular matrix deposition (mmp2 and mmp9). In addition, TXL inhibited EndMT in cardiac tissue and hypoxia-induced HCMECs. In hypoxia-induced HCMECs, TXL increased the expression of endothelial markers, whereas decreasing the expression of fibrotic markers, partially through enhanced expressions of NRG-1, phosphorylation of ErbB2, ErbB4, AKT, and downregulated expressions of hypoxia inducible factor-1a and transcription factor snail. After NRG-1 knockdown, the protective effect of TXL on HCMEC was partially abolished. In conclusion, TXL attenuates MF after AMI by inhibiting EndMT and through activating the NRG-1/ErbB- PI3K/AKT signalling cascade.

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Qin, Lan-hui, Wen Huang, Xue-an Mo, Yan-lan Chen y Xiang-hong Wu. "LPS Induces Occludin Dysregulation in Cerebral Microvascular Endothelial Cells via MAPK Signaling and Augmenting MMP-2 Levels". Oxidative Medicine and Cellular Longevity 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/120641.

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Disrupted blood-brain barrier (BBB) integrity contributes to cerebral edema during central nervous system infection. The current study explored the mechanism of lipopolysaccharide- (LPS-) induced dysregulation of tight junction (TJ) proteins. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to LPS, SB203580 (p38MAPK inhibitor), or SP600125 (JNK inhibitor), and cell vitality was determined by MTT assay. The proteins expressions of p38MAPK, JNK, and TJs (occludin and zonula occludens- (ZO-) 1) were determined by western blot. The mRNA levels of TJ components and MMP-2 were measured with quantitative real-time polymerase chain reaction (qRT-PCR), and MMP-2 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). LPS, SB203580, and SP600125 under respective concentrations of 10, 7.69, or 0.22 µg/mL had no effects on cell vitality. Treatment with LPS decreased mRNA and protein levels of occludin and ZO-1 and enhanced p38MAPK and JNK phosphorylation and MMP-2 expression. These effects were attenuated by pretreatment with SB203580 or SP600125, but not in ZO-1 expression. Both doxycycline hyclate (a total MMP inhibitor) and SB-3CT (a specific MMP-2 inhibitor) partially attenuated the LPS-induced downregulation of occludin. These data suggest that MMP-2 overexpression and p38MAPK/JNK pathways are involved in the LPS-mediated alterations of occludin in hCMEC/D3; however, ZO-1 levels are not influenced by p38MAPK/JNK.

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Mkrtchyan, H., S. Scheler, I. Klein, A. Fahr, P. O. Couraud, I. A. Romero, B. Weksler y T. Liehr. "Molecular Cytogenetic Characterization of the Human Cerebral Microvessel Endothelial Cell Line hCMEC/D3". Cytogenetic and Genome Research 126, n.º 4 (2009): 313–17. http://dx.doi.org/10.1159/000253080.

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Burghardt, Isabel, Elisa Ventura, Tobias Weiss, Judith Johanna Schroeder, Katharina Seystahl, Christian Zielasek, Dorothee Gramatzki y Michael Weller. "Endoglin and TGF-β signaling in glioblastoma". Cell and Tissue Research 384, n.º 3 (20 de enero de 2021): 613–24. http://dx.doi.org/10.1007/s00441-020-03323-5.

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AbstractMicrovascular proliferation is a key feature of glioblastoma and neovascularization has been implicated in tumor progression. Glioblastomas use pro-angiogenic factors such as vascular endothelial growth factor (VEGF) for new blood vessel formation. Yet, anti-VEGF therapy does not prolong overall survival so that alternative angiogenic pathways may need to be explored as drug targets. Both glioma cells and glioma-associated endothelial cells produce TGF-β superfamily ligands which bind TGF-β receptors (TGF-βR). The TGF-βR type III endoglin (CD105), is a marker of proliferating endothelium that has already been studied as a potential therapeutic target. We studied endoglin expression in glioblastoma tissue and in glioma-associated endothelial cells in a cohort of 52 newly diagnosed and 10 recurrent glioblastoma patients by immunohistochemistry and by ex vivo single-cell gene expression profiling of 6 tumors. Endoglin protein levels were similar in tumor stroma and endothelium and correlated within tumors. Similarly, endoglin mRNA determined by ex vivo single-cell gene expression profiling was expressed in both compartments. There was positive correlation between endoglin and proteins of TGF-β superfamily signaling. No prognostic role of endoglin expression in either compartment was identified. Endoglin gene silencing in T98G glioma cells and in human cerebral microvascular endothelial cells (hCMEC) did not affect constitutive or exogenous TGF-β superfamily ligand-dependent signaling, except for a minor facilitation of pSmad1/5 signaling in hCMEC. These observations challenge the notion that endoglin might become a promising therapeutic target in glioblastoma.

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Forcaia, Greta, Beatrice Formicola, Giulia Terribile, Sharon Negri, Dmitry Lim, Gerardo Biella, Francesca Re, Francesco Moccia y Giulio Sancini. "Multifunctional Liposomes Modulate Purinergic Receptor-Induced Calcium Wave in Cerebral Microvascular Endothelial Cells and Astrocytes: New Insights for Alzheimer’s disease". Molecular Neurobiology 58, n.º 6 (29 de enero de 2021): 2824–35. http://dx.doi.org/10.1007/s12035-021-02299-9.

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AbstractIn light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).

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Ma, Xiaoqing, Ho Jun Yun, Kenneth Elkin, Yunliang Guo, Yuchuan Ding y Guangwen Li. "MicroRNA-29b Suppresses Inflammation and Protects Blood-Brain Barrier Integrity in Ischemic Stroke". Mediators of Inflammation 2022 (23 de agosto de 2022): 1–11. http://dx.doi.org/10.1155/2022/1755416.

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Objectives. Following cerebral ischemia, microRNA- (miR-) 29b in circulating blood is downregulated. This study investigates the underlying mechanism and implications of miR-29b in leukocyte induction. Methods. miR-29b from stroke patients and rats with middle cerebral artery occlusion (MCAO) were assessed using real-time polymerase chain reaction (PCR). miR-29b agomir was used to increase miR-29b expression in leukocytes via intravenous injection. C1q and tumor necrosis factor (C1QTNF) 6, interleukin- (IL-) 1β, zonula occludens- (ZO-) 1, occludin, and ischemic outcomes were assessed in MCAO rats. Additionally, hCMEC/D3 cells were subjected to oxygen–glucose deprivation (OGD) and cocultured with HL-60 cells. Results. miR-29b levels in neutrophils were found to be significantly lower in stroke patients compared with healthy controls, which may indicate its high diagnostic sensitivity and specificity for stroke. Moreover, miR-29b levels in leukocytes showed a negative correlation with National Institute of Health Stroke Scale (NIHSS) scores and C1QTNF6 levels. In MCAO rats, miR-29b overexpression reduced brain infarct volume and brain edema, decreasing IL-1β levels in leukocytes and in the brain 24 hours poststroke. miR-29b attenuated IL-1β expression via C1QTNF6 inhibition, leading to decreased blood-brain barrier (BBB) disruption and leukocyte infiltration. Moreover, miR-29b overexpression in HL-60 cells downregulated OGD-induced hCMEC/D3 cell apoptosis and increased ZO-1 and occludin levels in vitro. Conclusion. Leukocytic miR-29b attenuates inflammatory response by augmenting BBB integrity through C1QTNF6, suggesting a novel miR-29b-based therapeutic therapy for ischemic stroke.

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Jin, Liang, Roger L. Nation, Jian Li y Joseph A. Nicolazzo. "Species-Dependent Blood-Brain Barrier Disruption of Lipopolysaccharide: Amelioration by ColistinIn VitroandIn Vivo". Antimicrobial Agents and Chemotherapy 57, n.º 9 (24 de junio de 2013): 4336–42. http://dx.doi.org/10.1128/aac.00765-13.

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ABSTRACTThe aim of this study was to usein vitroandin vivomodels to assess the impact of lipopolysaccharide (LPS) from two different bacterial species on blood-brain barrier (BBB) integrity and brain uptake of colistin. Following repeated administration of LPS fromPseudomonas aeruginosa, the brain-to-plasma ratio of [14C]sucrose in Swiss outbred mice was not significantly increased. Furthermore, while the brain uptake of colistin in mice increased 3-fold following administration of LPS fromSalmonella enterica, LPS fromP. aeruginosahad no significant effect on colistin brain uptake. This apparent species-dependent effect did not appear to correlate with differences in plasma cytokine levels, as the concentrations of tumor necrosis factor alpha and interleukin-6 following administration of each LPS were not different (P> 0.05). To clarify whether this species-specific effect of LPS was due to direct effects on the BBB, human brain capillary endothelial (hCMEC/D3) cells were treated with LPS fromP. aeruginosaorS. entericaand claudin-5 expression was measured by Western blotting.S. entericaLPS significantly (P< 0.05) reduced claudin-5 expression at a concentration of 7.5 μg/ml. In contrast,P. aeruginosaLPS decreased (P< 0.05) claudin-5 expression only at the highest concentration tested (i.e., 30 μg/ml). Coadministration of therapeutic concentrations of colistin ameliorated theS. entericaLPS-induced reduction in claudin-5 expression in hCMEC/D3 cells and the perturbation in BBB function in mice. This study demonstrates that BBB disruption induced by LPS is species dependent, at least betweenP. aeruginosaandS. enterica, and can be ameliorated by colistin.

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Sreekanthreddy, Peddagangannagari, Radka Gromnicova, Heather Davies, James Phillips, Ignacio A. Romero y David Male. "A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions". F1000Research 4 (17 de noviembre de 2015): 1279. http://dx.doi.org/10.12688/f1000research.7142.1.

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The aim of this study was to develop a three-dimensional (3D) model of the human blood-brain barrier in vitro, which mimics the cellular architecture of the CNS and could be used to analyse the delivery of nanoparticles to cells of the CNS. The model includes human astrocytes set in a collagen gel, which is overlaid by a monolayer of human brain endothelium (hCMEC/D3 cell line). The model was characterised by transmission electron microscopy (TEM), immunofluorescence microscopy and flow cytometry. A collagenase digestion method could recover the two cell types separately at 92-96% purity. Astrocytes grown in the gel matrix do not divide and they have reduced expression of aquaporin-4 and the endothelin receptor, type B compared to two-dimensional cultures, but maintain their expression of glial fibrillary acidic protein. The effects of conditioned media from these astrocytes on the barrier phenotype of the endothelium was compared with media from astrocytes grown conventionally on a two-dimensional (2D) substratum. Both induce the expression of tight junction proteins zonula occludens-1 and claudin-5 in hCMEC/D3 cells, but there was no difference between the induced expression levels by the two media. The model has been used to assess the transport of glucose-coated 4nm gold nanoparticles and for leukocyte migration. TEM was used to trace and quantitate the movement of the nanoparticles across the endothelium and into the astrocytes. This blood-brain barrier model is very suitable for assessing delivery of nanoparticles and larger biomolecules to cells of the CNS, following transport across the endothelium.

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