Primary Cerebral Endothelial Cells Research Articles

Molecular mechanisms of naringenin modulation of mitochondrial permeability transition acting on F1FO-ATPase and counteracting saline load-induced injury in SHRSP cerebral endothelial cells

Naringenin (NRG) was characterized for its ability to counteract mitochondrial dysfunction which is linked to cardiovascular diseases. The F1FO-ATPase can act as a molecular target of NRG. The interaction of NRG with this enzyme can avoid the energy transmission mechanism of ATP hydrolysis, especially in the presence of Ca2+ cation used as cofactor. Indeed, NRG was a selective inhibitor of the hydrophilic F1 domain displaying a binding site overlapped with quercetin in the inside surface of an annulus made by the three α and the three β subunits arranged alternatively in a hexamer. The kinetic constant of inhibition suggested that NRG preferred the enzyme activated by Ca2+ rather than the F1FO-ATPase activated by the natural cofactor Mg2+. From the inhibition type mechanism of NRG stemmed the possibility to speculate that NRG can prevent the activation of F1FO-ATPase by Ca2+. The event correlated to the protective role in the mitochondrial permeability transition pore opening by NRG as well as to the reduction of ROS production probably linked to the NRG chemical structure with antioxidant action. Moreover, in primary cerebral endothelial cells (ECs) obtained from stroke prone spontaneously hypertensive rats NRG had a protective effect on salt-induced injury by restoring cell viability and endothelial cell tube formation while also rescuing complex I activity.

Abstract WP296: Human Vascular Smooth Muscle Cells Derived Small Extracellular Vesicles Reduce the Neurovascular Damage After Stroke in Diabetic Mice

Introduction: Stroke patients with type-2 diabetes exhibit exacerbated cerebrovascular dysfunction and increased neurological deficits compared to non-diabetic stroke patients. Vascular smooth muscle cells (VSMCs) play an important role in mediating neurovascular function, however, how VSMCs communicate with cerebral endothelial cells (CECs) has not been studied. In this study, we tested the hypothesis that small extracellular vesicles (sEVs) derived from healthy human VSMCs (VSMC-sEVs) reduce diabetes- and ischemia-neurovascular damage. Methods and Results: VSMC-sEVs were characterized. In vitro CEC permeability assay showed that treatment of hypoxia-challenged human brain microvascular endothelial cells (BMECs) or primary CECs isolated from type-2 diabetic mice (n=6/group) with VSMC-sEVs significantly reduced and augmented cell permeability and trans-endothelial electrical resistance, respectively, compared to non VSMC-sEVs control. Inhibition of sEV endocytosis in BMECs with clathrin (CPZ) or caveolin (Nystatin) inhibitors abrogated the effect of VSMC-sEVs on CEC proliferation and migration. We then examined the effect of VSMC-sEVs on diabetic ischemia in which diabetic mice, with diabetes induced by a high fat diet and streptozotocin, were subjected to permanent MCAo. Middle-age diabetic male mice (10-12m old) were treated with intranasal administration of VSMC-sEVs (1x10 9 particles per injection) or saline (n=12/group) at 24h after cerebral ischemia. We found that VSMC-sEVs treatment significantly increased regional cerebral blood flow and FITC-dextran perfused functional microvessels (17.55% vs 13.32% control) as well as decreased brain hemorrhage and BBB leakage (1.51% vs 1.76% of albumin, p<0.05) in the ischemic border zone 28 days after MCAo compared with the saline treatment mice. Moreover, compared to the saline treatment, VSMC-sEVs significantly improved neurological function after stroke as measured by modified neurological severity score, and foot-fault and adhesive removal tests, respectively. Conclusion: This study suggests that VSMC-sEVs have a therapeutic effect to improve neurological outcome after diabetic stroke, likely by reducing diabetes and ischemia impaired neurovascular function.

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner.

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.

Beneficial Effects of Citrus Bergamia Polyphenolic Fraction on Saline Load-Induced Injury in Primary Cerebral Endothelial Cells from the Stroke-Prone Spontaneously Hypertensive Rat Model.

High salt load is a known noxious stimulus for vascular cells and a risk factor for cardiovascular diseases in both animal models and humans. The stroke-prone spontaneously hypertensive rat (SHRSP) accelerates stroke predisposition upon high-salt dietary feeding. We previously demonstrated that high salt load causes severe injury in primary cerebral endothelial cells isolated from SHRSP. This cellular model offers a unique opportunity to test the impact of substances toward the mechanisms underlying high-salt-induced vascular damage. We tested the effects of a bergamot polyphenolic fraction (BPF) on high-salt-induced injury in SHRSP cerebral endothelial cells. Cells were exposed to 20 mM NaCl for 72 h either in the absence or the presence of BPF. As a result, we confirmed that high salt load increased cellular ROS level, reduced viability, impaired angiogenesis, and caused mitochondrial dysfunction with a significant increase in mitochondrial oxidative stress. The addition of BPF reduced oxidative stress, rescued cell viability and angiogenesis, and recovered mitochondrial function with a significant decrease in mitochondrial oxidative stress. In conclusion, BPF counteracts the key molecular mechanisms underlying high-salt-induced endothelial cell damage. This natural antioxidant substance may represent a valuable adjuvant to treat vascular disorders.

Abstract TP11: G-quadruplexes Regulate The Senescence Factor Cysteine-rich Angiogenic Inducer-61 In Primary Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. Whether CCN1 contributes to senescence of CEC and how it is regulated require further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. G4 stabilization regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and exhibited cognitive deficits. The expression of human CCN1 can be upregulated via the stabilization of G4s. A sequence in the 3’- untranslated region (UTR) of the CCN1 mRNA has the ability to fold in to G4 in vitro . Thus, we hypothesize that CCN1 is upregulated with aging and induces senescence, and that G4 stabilization upregulates CCN1 in cultured primary CEC. We used whole brain lysates, cerebral vessel fractions, and cultured primary CEC from young (4-months old, mo) and aged (18-mo) female mice to determine the CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA G4s for fluorescence spectra. We found that CCN1 levels were increased in the brains of aging mice (p=0.0314, n=5), in cerebral vessel fractions (p=0.001, n=5), and in cultured primary CEC derived from aged mice (p=0.012, n=3), compared with those derived from young mice. In cultured CEC, a recombinant CCN1 protein induced SA phenotypes, such as SA beta-galactosidase activity (p=0.003, n=3), and double-strand DNA damage (p=0.016, n=3). Furthermore, CCN1 levels were upregulated by a G4 ligand (p=0.0103, n=3), and a G-rich motif in the 3’-UTR of the Ccn1 mRNA folded into a G4 (p=0.008, n=4). In conclusion , we demonstrate that CCN1 induces senescence in cultured primary CEC, and we propose G4 stabilization as a novel mechanism that regulates the SASP component CCN1.

G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells.

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3′-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3′-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

Disrupted blood-brain barrier in 5×FAD mouse model of Alzheimer’s disease can be mimicked and repaired in vitro with neural stem cell-derived exosomes

Alzheimer’s disease (AD) is a devastating neurodegenerative disease and is associated with blood-brain barrier (BBB) disruption. AD mice and cell culture models play an essential role in understanding AD pathogenesis and validation of therapeutic reagents. One of the commonly used AD mice is the 5 × FAD mouse and previous studies have shown that BBB leakage occurs at 9 months of age in the mice. However, it remains unknown whether disrupted BBB also occurs in young animals and whether AD-caused BBB impairment can be replicated and further corrected in a cell culture model. Here, we examine BBB breakdown in the 5 × FAD mouse model at different ages including both pre-symptomatic and post-symptomatic ages and test an in vitro BBB model established with the 5 × FAD primary cerebral endothelial cells. Moreover, with the BBB in vitro model, we also examined the therapeutic effect of human neural stem cells (NSCs)-derived exosomes on AD-caused BBB leakage. Our result indicated that BBB breakdown in the 5 × FAD mice occurred at 4 months of age, which could be mimicked with an in vitro BBB model. Importantly, we further demonstrated that treatment of the in vitro BBB model with NSCs-derived exosomes reversed AD-caused BBB deficiency. The information should be useful for researchers to determine which ages of the AD mice should be employed in specific in vivo and in vitro studies and the data also suggest that AD-caused BBB disruption can be corrected at least by NSC-derived exosomes.

Abstract WMP3: Exosomes Derived From Stroke Activated Platelets Impair Cerebral Endothelial Permeability

Introduction: Stroke activated platelets damage the blood brain barrier (BBB). Mechanisms underlying this process remain to be investigated. The present study tested a hypothesis that exosomes, nano size extracellular vesicles, derived from stroke-activated platelets promote BBB leakage. Methods: Platelets were harvested from blood samples collected from non-stroke adult and aged male rats and from respective rats subjected to 4h of middle cerebral artery occlusion (MCAO) (n=6/group). Exosomes were then isolated from the platelets by means of differential ultracentrifugation. Primary cerebral endothelial cells harvested from healthy adult rats were treated with platelet exosomes. Endothelial permeability and trans-endothelial electrical resistance (TEER) were assayed. Western blot analysis was performed to measure pro-coagulation and tight junction proteins. Results: Platelet exosomes (P-Exos) had an average size from 70 to 82 nm among adult and aged rats and these exosomes contained platelet exosomes markers: CD41, CD63, CD9 and Alix. P-Exos derived from non-ischemic adult and aged rats exhibited TEER at 154±18 and 159 ±16 Ω.cm 2 , respectively, and did not increase FITC-dextran leakage. However, P-Exos from ischemic adult rats significantly (p<0.05) decreased TEER (118 ±11 vs 154±18Ω.cm 2 in non-ischemia) and increased FITC-dextran leakage by 178%. P-Exos from aged ischemic rats further significantly decreased TEER (98 ±9 Ω.cm 2 ) and augmented dextran leakage to 216%. Western blot analysis of endothelial cells showed that P-Exos from ischemic adult rats significantly increased pro-inflammation proteins, ICAM-1, TNF-a and NFkB p65 and decreased tight junction proteins, ZO-1 and occludin, whereas P-Exos from ischemic aged rats further significantly increased and decreased pro-inflammation proteins and junction proteins. Conclusion: These data provide new insights into how stroke activated platelets mediate BBB disruption and promote endothelial proinflammatory protein expression via their released exosomes and how aging further negatively impacts cerebral endothelial cells.

Abstract TP96: MiR-34a Targets Cytochrome c and Shapes Stroke Outcomes

Background: The blood-brain barrier (BBB) is a crucial interface between the central nervous system (CNS) and the circulatory system that maintains cerebral homeostasis by selectively allowing entry of blood solutes into the CNS. BBB dysfunction has been observed in cerebrovascular diseases and neurodegenerative disorders such as stroke. The BBB is opened during stroke, having negative impacts on stroke outcomes. Mitochondria are key players in this abnormal opening of the BBB, and decreased cytochrome c (CYC) levels have been shown in the mitochondrial fraction in stroke. We have recently demonstrated miR-34a regulates the BBB by targeting CYC in vitro . Objective: To investigate the role of miR-34a in stroke and verify its target CYC in vivo . Methods and Results: Using a murine transient middle cerebral artery occlusion (tMCAO) model, we demonstrate elevated miR-34a expression in serum and primary cerebral endothelial cells (CECs) from stroke mice. We report that deficiency of miR-34a significantly reduces BBB permeability and improves stroke outcomes. CYC is decreased in the ischemic hemisphere from wild-type (WT) but not miR-34a -/- mice following stroke reperfusion. Uncoupling of electron flow by a pharmacological inhibitor Carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compromises mitochondrial oxidative phosphorylation (MOP) in cultured CECs and worsens infarction in stroke mice. Conclusions: Our study provides the first description of miR-34a and electron flow affecting stroke outcomes, which could lead to a revision of current miR-34a targets and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke and Alzheimer’s disease. Key words: Blood-brain barrier; Stroke; MiR-34a; Mitochondria; Cytochrome c. Non-standard Abbreviations and Acronyms: CEC: cerebral endothelial cell CYC: cytochrome c FCCP: Carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone MOP: mitochondrial oxidative phosphorylation tMCAO: transient middle cerebral artery occlusion

Modulation of Amyloid-β1-40 Transport by ApoA1 and ApoJ Across an in vitro Model of the Blood-Brain Barrier.

Amyloid-β (Aβ) accumulation in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) is likely caused by the impairment of its brain clearance that partly occurs through the blood-brain barrier (BBB). In this context, an in vitro BBB model is a valuable tool for studying the molecular mechanisms that regulate this process. This study assessed brain Aβ elimination across the BBB and its modulation by the natural chaperones Apolipoprotein A1 (ApoA1) and Apolipoprotein J/Clusterin (ApoJ). The model was based on primary cerebral endothelial cells that were cultured on Matrigel-coated Transwells and treated with fluorescently labeled-Aβ1-40 to track its efflux across the BBB, which corresponds to trafficking from the basolateral (brain) to apical (blood) compartments. We observed that the transport of basolateral Aβ1-40 was enhanced when it was complexed to rApoJ, whereas the complex formed with rApoA1 did not influence Aβ1-40 efflux. However, the presence of rApoA1 in the apical compartment was able to mobilize Aβ1-40 from the basolateral side. We also observed that both rApoA1 and rApoJ moderately crossed the monolayer (from blood to brain) through a mechanism involving the LDL receptor-related protein family. In contrast to the increased rApoJ efflux when complexed to Aβ1-40, rApoA1 trafficking was restricted when it was bound to the Aβ peptide. In summary, the present study highlights the role of ApoJ and ApoA1 in the in vitro modulation of Aβ elimination across the BBB.

Complement component C3a plays a critical role in endothelial activation and leukocyte recruitment into the brain.

BackgroundThe complement system is becoming increasingly recognized as a key participant in many neurodegenerative diseases of the brain. Complement-deficient animals exhibit reduced neuroinflammation.MethodsIn the present study, we administered intracerebroventricularly lipopolysaccharide (LPS) to mimic local infection of the brain and investigated the role of key complement component C3 in brain vasculature endothelial activation and leukocyte recruitment. The degree of neutrophil infiltration was determined by esterase staining. Leukocyte-endothelial interactions were measured using intravital microscopy. Cerebral endothelial activation was evaluated using real-time PCR and Western blotting.ResultsNeutrophil infiltration into the brain cortex and hippocampus was significantly reduced in C3−/− mice and C3aR−/− mice but not in C6−/− mice. We detected markedly attenuated leukocyte-endothelial interactions in the brain microvasculature of C3−/− mice. Accordingly, in response to LPS administration, the brain microvasculature in these mice had decreased expression of P-selectin, E-selectin, intercellular cell adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Depletion of C3 from the circulation also caused reduction in VCAM-1 and E-selectin expression and leukocyte recruitment, suggesting that C3 in the circulation contributed to brain endothelial activation. Furthermore, C3−/− mice exhibited decreased leukocyte recruitment into the brain upon tumor necrosis factor-α (TNF-α) stimulation. C3a activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and induced the upregulation of VCAM-1 and ICAM-1 expression in murine primary cerebral endothelial cells in vitro.ConclusionsOur study provides the first evidence that C3a plays a critical role in cerebral endothelial activation and leukocyte recruitment during inflammation in the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0485-y) contains supplementary material, which is available to authorized users.

Andrographolide stimulates p38 mitogen-activated protein kinase–nuclear factor erythroid-2-related factor 2–heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats

Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could be a therapeutic agent for treating ischemic stroke or neurodegenerative diseases.

Abstract W MP52: Treatment With N-acetyl-seryl-aspartyl-lysyl-proline Improves Neurological Functional Recovery in Diabetic Middle Aged Rats After Embolic Stroke

Background and Purpose: Diabetes mellitus (DM) is a common metabolic disease among middle-aged and older populations, which leads to an increase of stroke incidence and poor stroke recovery. The present study investigated the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on stroke recovery in middle aged DM rats after embolic stroke. Methods: Male Wistar rats at age of 13 months were subjected to nicotinamide (NTM) and streptozotocin (STZ) injection . Rats with confirmed hyperglycemia status were subjected to embolic middle cerebral artery occlusion (MCAO) 30d after STZ-NTM injection. AcSDKP (2.4mg/kg) or saline were subcutaneously infused for 3d starting at 1d after stroke (n=12/group). Results: AcSDKP treatment significantly improved neurological functional recovery measured by adhesive removal test, foot-fault test, and modified neurological severity score starting at 21d after MCAO compared with saline treated rats. In addition, AcSDKP substantially reduced spatial learning deficits measured by the Morris water maze (41±4 vs 33±3% of time spent in the correct quadrant) 35d after MCAO. AcSDKP treatment significantly reduced vascular and parenchymal fibrin deposition (43±9 vs 92±11/mm 2 ) in the ipsilateral hippocampus and peri-infarct corpus callosum (CC), which was associated with reduction of IL-1 receptor associated kinase-1(IRAK1) immunoreactive vessels (95±13 vs 47±11/mm 2 ) 7d after MCAO (n=4/group). AcSDKP treatment did not reduce infarct volume, but robustly increased myelinated axonal density (14±4 vs 8±3% in hippocampus, 36±6 vs 27±7% in CC) and oligodendrocyte density (71±18 vs 46±16/mm 2 in hippocampus, 526±113 vs 394±107/mm 2 in CC) 35d after MCAO (n=8/group). In vitro, high glucose increased protein level of IRAK1 and NF-κB in primary cerebral endothelial cells, whereas AcSDKP suppressed high glucose-increased IRAK1 and NF-κB protein levels by 50%. Conclusions: AcSDKP promotes ischemic brain repair by reducing neurovascular damage in the hippocampus and white matter, which likely contributes improvement of neurological functional recovery in middle-aged DM rats. IRAK1/NF-κB signals suppressed by AcSDKP may be involved these processes.

Role of ROS in Aβ42 Mediated Activation of Cerebral Endothelial Cells.

IntroductionThere is substantial evidence that the deposition of aggregated amyloid-beta peptide (Aβ) in brain parenchyma and brain vessels is the main cause of neuronal dysfunction and death in Alzheimer’s disease (AD). Aβ exhibits multiple cytotoxic effects on neurons and glial cells and causes dysfunction of the blood brain barrier (BBB). In AD brains, an increased deposition of Aβ in the cerebral vasculature has been found to be correlated with increased transmigration of blood-borne inflammatory cells and neurovascular inflammation. However, regulatory mediators of these processes remain to be elucidated. In this study, we examined the role of ROS in actin polymerization and expression of adhesion molecules (P-selectin) on the surface of the cerebral endothelial cells (CECs) that are activated by Aβ42.Materials and methodsMouse BEnd3 line (ATCC) was used in this research. BEnd3 cells respond to Aβ treatment similarly to human primary CECs and are a common model to investigate CECs’ function. We used immortalized bEnd3 cells as the following: controls; cells incubated with Aβ42 for 10, 30, and 60 minutes; cells incubated with 30 mM of antioxidant N-acetylcysteine (NAC) for 1 hr; and, cells pre-treated with NAC followed by Aβ42 exposure. We measured DHE fluorescence to investigate intracellular ROS production. Immunofluorescent microscopy of anti-P-selectin and oregon green phalloidin was used to quantify the surface P-selectin expression and actin polymerization, and Western blot analysis was used to analyze total P-selectin expression.ResultsThe results of this study have demonstrated a significant time-dependent ROS accumulation after 10 minutes, 30 minutes, and 60 minutes of Aβ42 treatment, while Aβ42 stimulated ROS production in CECs was attenuated by pre-treatment with the NAC antioxidant. We also found that Aβ42 increased P-selectin fluorescence at the surface of bEnd3 cells in a time dependent manner in parallel to ROS elevation. However, total expression levels of P-selectin were not changed following exposure to Aβ42. Pretreatment with NAC attenuated Aβ42 induced P-selectin localization, while NAC alone did not significantly affect P selectin localization. As a positive control, H2O2 also increased P-selectin expression on the cell surface, which peaked after 30 minutes of H2O2 treatment. Exposure of CECs with Aβ42 promoted actin polymerization, which peaked after 10 minutes of Aβ42 treatment, while no significant increase of F-actin intensity was observed when cells were pre-treated with NAC. H2O2 was able to mimic Aβ42 induced oxidative stress, causing increased actin polymerization with similar timing.ConclusionsThe results of our study have indicated that Aβ42 induced accumulation of P-selectin on the surface of bEnd3 cells and promoted actin polymerization, and all these events were correlated with ROS generation. The rapid post-translational cell signaling response mediated by ROS may well represent an important physiological trigger of the microvascular inflammatory responses in AD and requires further investigations.

Abstract 2966: Role of the Cerebral Vascular Toll-Like Receptor Signaling Pathway in Neuroprotection Induced by Combination Therapy of VELCADE and TPA

Background and Purpose: Toll-like receptors (TLRs) and their family members including IL-1 receptor-associated kinase-1 (IRAK1) mediate ischemic cell damage. MicroRNAs (miRNAs) including miR-146a regulate TLRs. The present study investigated whether treatment of stroke in older animals with VELCADE in combination with tPA affects expression of TLRs and miR-146a in cerebral endothelial cells. Methods: Wistar rats at the age of 16-18 months were subjected to right embolic middle cerebral artery occlusion (MCAo). VELCADE (0.1mg/kg) and tPA (5mg/kg) were intravenously administered 2h after MCAo (n=16). Ischemic rats treated with tPA alone (n=14) or saline (n=15) were used as control groups. Levels of TLRs and miR-146a in cerebral endothelial cells were measured. Cultured primary cerebral endothelial cells (PCECs) were used for investigating the direct effect of VELCADE on expression of miR-146a. Results: Quantitative RT-PCR analysis revealed that VELCADE in combination with tPA significantly increased miR-l46a levels (11±3 fold) in the cerebral endothelial cells isolated by laser capture microdissection compared with that in ischemic rats treated with tPA monotherapy (2±1). Concurrently, immunostaining showed that the combination therapy suppressed tPA monotherapy-upregulated TLR2 (44±7 vs 93±8/mm 2 in tPA), TLR4 (39±6vs 91±8), IRAK1 (16±3 vs 46±5), fibrin (17±6 vs 40±3), and NF-kB (9±2 vs 22±3) immunoreactive vessels, leading to reduction of infarct volume (14±2 vs 28±3% in tPA, and 30±3% in saline, p<0.05). These in vivo data indicate that combination therapy-upregulated miR-146a is inversely related with TLRs levels and ischemic cell damage. In vitro, Western blots showed that incubation of PCECs with fibrin at 1.5 µg/ml increased levels of IRAK1 (1.4 fold) and myeloid differentiation factor 88 (MyD88, 1.4 fold). RT-PCR analysis of PCECs showed that fibrin reduced miR-146a levels by 30 %. However, PCECs treated with VELCADE (10 ng/ml) in the presence of fibrin elevated miR-146 levels by 80% and abolished fibrin-elevated IRAK1 and MyD88 proteins. Moreover, overexpression of miR146a in PCECs downregulated IRAK1 protein, a miR-146a target, by 40%. Conclusion: Our data demonstrate that fibrin activates endothelial TLRs and that VELCADE-upregulated miR-146a abolishes tPA-induced TLRs, suggesting that miRNAs and the TLR signaling pathway in cerebral endothelial cells play an important role in the neuroprotective effect of the combination therapy of VELCADE and tPA for acute stroke.

Honokiol traverses the blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-mitochondrion-cytochrome c-caspase protease pathway

Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

Inhibition of HSP90‐dependent telomerase activity in amyloid β‐induced apoptosis of cerebral endothelial cells

Although apoptosis induced by amyloid β (Aβ) has been identified, the effect of Aβ on telomerase activity in relation to apoptosis induction remains unclear. In the present study, Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), reduce the viability of primary cerebral endothelial cells (CECs) in accordance with apoptosis induction. Increases in caspase 3 and PARP protein cleavage with reductions of the Bcl-2/Bax protein ratio accompanied by a loss in the mitochondria membrane potential were identified in Aβ(1-40) and Aβ(25-35)-treated CECs. A significant decrease in intracellular telomerase activity by Aβ(1-40) and Aβ(25-35) was detected; meanwhile, reduced telomerase activity by telomerase reverse transcriptase (TERT) siRNA enhanced the cytotoxic effect of Aβ. The addition of serum might block the Aβ(25-35)-induced cytotoxic effect via elevated telomerase activity in according with stimulating phospho-AKT protein expression, which was blocked by adding AKT inhibitor LY294002. Decreases in heat shock protein 90 (HSP90) and its client proteins including TERT, AKT, p53, CDK4 were observed in Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), -treated CECs. The knockdown of HSP90 gene expression by HSP90 siRNA significantly inhibits telomerase activity with decreasing TERT protein expression. The application of HSP90 activity inhibitor geldanamycin (GA) and radicicol (RD) potentiates the telomerase inhibition and apoptosis induction of Aβ in CECs. An increase in protein ubiquitination by Aβ(25-35), but not Aβ(35-25), treatment was examined, and Aβ-inhibited HSP90 and TERT protein expression and telomerase activity was reversed by adding proteasome inhibitor, MG132. Additionally, increased TERT protein ubiquitination by Aβ(25-35) was detected in CECs via immunoprecipitation/Western blotting analysis. The data of the present study firstly demonstrates that telomerase inhibition contributes to the apoptosis induction of Aβ in CECs.

Responses of Endothelial Cell and Astrocyte Matrix-Integrin Receptors to Ischemia Mimic Those Observed in the Neurovascular Unit

Apposition of endothelial cells and astrocyte foot processes to the basal lamina matrix is postulated to underlie the cerebral microvessel permeability barrier. Focal cerebral ischemia induces rapid loss of select matrix-binding integrins from both cell compartments in the nonhuman primate. This study is the first to examine the conditions underlying integrin loss from these cell-types during ischemia in vitro and their relation to the changes in vivo. The impact of normoxia or standardized oxygen-glucose deprivation on integrin expression by murine primary cerebral endothelial cells and astrocytes grown on matrix substrates (collagen IV, laminin, and perlecan) of the basal lamina were quantitatively assessed by flow cytometry. Endothelial cell expression of the beta1 and alpha 5 subunits significantly increased on all matrix ligands, whereas astrocytes displayed modest significant decreases in alpha 5 and alpha 6 subunits. Oxygen-glucose deprivation produced a further significant increase in subunit beta1 expression by both cell types, but a clear decrease in both alpha1 and alpha 6 subunits by murine astrocytes. Ischemia in vitro significantly increased endothelial cell beta1 expression, which is consistent with the increase in beta1 transcription by microvessels peripheral to the ischemic core. The loss of alpha1 and alpha 6 integrins from murine astrocytes is identical to that seen in the nonhuman primate in vivo. These findings establish both isolated murine cerebral endothelial cells and astrocytes as potential integrin response cognates of microvascular cells of the neurovascular unit in primates, and allow determination of the mechanisms of their changes to ischemia.

Erythropoietin involves the phosphatidylinositol 3‐kinase pathway, 14‐3‐3 protein and FOXO3a nuclear trafficking to preserve endothelial cell integrity

Clinical indications for erythropoietin (EPO) in the vascular system reach far beyond the treatment of anemia, but the development of EPO as a non-toxic agent rests heavily upon the cellular pathways controlled by EPO that require elucidation. We modulated gene activity and examined cellular trafficking of critical pathways during oxidative stress that may work in concert with EPO to protect primary cerebral endothelial cells (ECs) during oxidative stress, namely protein kinase B (Akt1), 14-3-3 protein, the Forkhead transcription factor FOXO3a. Here, we show that preservation of ECs by EPO during oxygen-glucose deprivation (OGD) required the initial activation of the phosphatidylinositol 3-kinase (PI-3K) pathway through Akt1, since specific pharmacological blockade of Akt1 activity or gene silencing of Akt1 prevented EC protection by EPO. EPO subsequently involved a series of anti-apoptotic pathways to activate STAT3, STAT5, and ERK 1/2. Furthermore, EPO maintained the inhibitory phosphorylation and integrity of the 'pro-apoptotic' transcription factor FOXO3a, promoted the binding of FOXO3a to 14-3-3 protein and regulated the intracellular trafficking of FOXO3a. Additionally, gene silencing of FOXO3a during OGD significantly increased EC survival, but did not synergistically improve cytoprotection by EPO, illustrating that EPO relied upon the blockade of the FOXO3a pathway. Our work defines a novel cytoprotective pathway in ECs that involves PI-3 K, STAT3, STAT5, ERK 1/2, 14-3-3 protein and FOXO3a, which can be targeted for the development of EPO as a clinically effective and safe agent in the vascular system.

Direct evidence that polysorbate-80-coated poly(butylcyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles.

[corrected] It has recently been suggested that the poly(butylcyanoacrylate) (PBCA) nanoparticle drug delivery system has a generalized toxic effect on the blood-brain barrier (BBB) (8) and that this effect forms the basis of an apparent enhanced drug delivery to the brain. The purpose of this study is to explore more fully the mechanism by which PBCA nanoparticles can deliver drugs to the brain. Both in vivo and in vitro methods have been applied to examine the possible toxic effects of PBCA nanoparticles and polysorbate-80 on cerebral endothelial cells. Human, bovine, and rat models have been used in this study. In bovine primary cerebral endothelial cells, nontoxic levels of PBCA particles and polysorbate-80 did not increase paracellular transport of sucrose and inulin in the monolayers. Electron microscopic studies confirm cell viability. In vivo studies using the antinociceptive opioid peptide dalargin showed that both empty PBCA nanoparticles and polysorbate-80 did not allow dalargin to enter the brain in quantities sufficient to cause antinociception. Only dalargin preadsorbed to PBCA nanoparticles was able to induce an antinociceptive effect in the animals. At concentrations of PBCA nanoparticles and polysorbate-80 that achieve significant drug delivery to the brain, there is little in vivo or in vitro evidence to suggest that a generalized toxic effect on the BBB is the primary mechanism for drug delivery to the brain. The fact that dalargin has to be preadsorbed onto nanoparticles before it is effective in inducing antinociception suggests specific mechanisms of delivery to the CNS rather than a simple disruption of the BBB allowing a diffusional drug entry.